U.S. patent application number 13/440496 was filed with the patent office on 2012-08-30 for anti-neoplastic compositions comprising extracts of black cohosh.
Invention is credited to Linda Saxe Einbond, Morando Soffritti, Claudia Weinstein, I. Bernard Weinstein, Joan Weinstein, Matthew Weinstein, Tamara Weinstein.
Application Number | 20120219643 13/440496 |
Document ID | / |
Family ID | 40876967 |
Filed Date | 2012-08-30 |
United States Patent
Application |
20120219643 |
Kind Code |
A1 |
Einbond; Linda Saxe ; et
al. |
August 30, 2012 |
ANTI-NEOPLASTIC COMPOSITIONS COMPRISING EXTRACTS OF BLACK
COHOSH
Abstract
A method for treating, preventing or ameliorating breast cancer
is provided by administering a synergistic amount of digitoxin and
either actein or an extract of black cohosh comprising triterpene
glycosides, and optionally another chemopreventive agent which may
be paclitaxel. Methods for treating or preventing a neoplasia using
a synergistic combination, and compositions of a synergistic
combination of a cardiac glycoside and either actein or an extract
of black cohosh comprising triterpene glycosides, and optionally
another chemopreventive agent which may be a taxane are also
provided. The compositions may also be used in a method for
modulating Na.sup.+K.sup.+ATPase activity. In addition, a method
for inhibiting the progression or development of breast cancer in
vivo by administering either actein or an extract of black cohosh
comprising triterpene glycosides and optionally at least one other
chemoprotective agent is provided.
Inventors: |
Einbond; Linda Saxe;
(Crestwood, NY) ; Soffritti; Morando; (Bologna,
IT) ; Weinstein; I. Bernard; (New York, NY) ;
Weinstein; Joan; (New York, NY) ; Weinstein;
Tamara; (Decatur, GA) ; Weinstein; Claudia;
(New York, NY) ; Weinstein; Matthew; (Brooklyn,
NY) |
Family ID: |
40876967 |
Appl. No.: |
13/440496 |
Filed: |
April 5, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12228562 |
Aug 13, 2008 |
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13440496 |
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12221478 |
Aug 4, 2008 |
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12228562 |
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10746960 |
Dec 23, 2003 |
7407675 |
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12221478 |
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60437159 |
Dec 27, 2002 |
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Current U.S.
Class: |
424/725 ;
435/375; 514/26 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61K 36/71 20130101; A61P 35/00 20180101; A61K 45/06 20130101; A61P
35/02 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
36/71 20130101; A61K 31/7048 20130101 |
Class at
Publication: |
424/725 ; 514/26;
435/375 |
International
Class: |
A61K 36/71 20060101
A61K036/71; C12N 5/09 20100101 C12N005/09; C12N 5/071 20100101
C12N005/071; A61P 35/00 20060101 A61P035/00; A61K 31/7048 20060101
A61K031/7048 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under NIH
Grant Nos. 3P50 AT 00090-02S.sub.2 and NIH NCCAM 5K01AT001692-03.
As such, the United States Government has certain rights in this
invention.
Claims
1. A method for treating, preventing or ameliorating breast cancer
comprising administering to a patient in need thereof a composition
comprising a synergistic amount of digitoxin and an extract of
black cohosh comprising a triterpene glycoside, and a
pharmaceutically acceptable carrier, and optionally an effective
amount of at least one additional chemopreventive or
chemotherapeutic agent.
2. A method for treating, preventing or ameliorating breast cancer
comprising administering to a patient in need thereof a composition
comprising a synergistic amount of digitoxin and actein, and a
pharmaceutically acceptable carrier, and optionally an effective
amount of at least one additional chemopreventive or
chemotherapeutic agent.
3. A method for treating or preventing neoplasia in a subject
comprising administering to the subject an amount of an extract of
black cohosh comprising a triterpene glycoside, which amount of the
black cohosh is effective to treat or prevent neoplasia, in
combination with an amount of an a cardiac glycoside which is
effective to treat or prevent the neoplasia, and optionally an
effective amount of at least one additional chemopreventive or
chemotherapeutic agent.
4. The method of claim 3, wherein the extract comprises actein, and
optionally further comprises cimigenol.
5. The method of claim 3, wherein the extract is selected from the
group consisting of an ethyl acetate extract of black cohosh and an
n-butanolic fraction of an EtOH/water extract of black cohosh.
6. A method for treating or preventing neoplasia in a subject
comprising administering to the subject an amount of actein
effective to treat or prevent neoplasia, in combination with an
amount of a cardiac glycoside which is effective to treat or
prevent the neoplasia, and optionally an effective amount of at
least one additional chemopreventive or chemotherapeutic agent.
7. The method according to claim 3 wherein the neoplasia is a
carcinoma.
8. The method according to claims 6 wherein the neoplasia is a
carcinoma.
9. The method of claim 7 wherein the carcinoma is breast
cancer.
10. The method of claim 8 wherein the carcinoma is breast
cancer.
11. The method according to claim 3, wherein the cardiac glycoside
is selected from the group consisting of digitoxin, ouabain,
proscillaridin A, digoxin, lanatoside C, and combinations
thereof.
12. The method according to claim 6, wherein the cardiac glycoside
is selected from the group consisting of digitoxin, ouabain,
proscillaridin A, digoxin, lanatoside C, and combinations
thereof.
13. The method of claim 11, wherein the cardiac glycoside is
digitoxin.
14. The method of claim 12, wherein the cardiac glycoside is
digitoxin.
15. The method according to claim 13 wherein the actein and
digitoxin are in amounts that result in a synergistic
anti-neoplastic effect.
16. The method according to claim 14 wherein the actein and
digitoxin are in amounts that result in a synergistic
anti-neoplastic effect.
17. The method according to claim 4 wherein the amount of actein
that is effective to treat or prevent the neoplasia is from about
0.2 .mu.g/ml to about 40.0 .mu.g/ml.
18. The method according to claim 6 wherein the amount of actein
that is effective to treat or prevent the neoplasia is from about
0.2 .mu.g/ml to about 40.0 .mu.g/ml.
19. The method according to claim 15, wherein the amount of actein
is from about 0.2 .mu.g/ml to about 2 .mu.g/ml and the digitoxin is
in an amount of from about 0.01 .mu.g/ml to about 0.8 .mu.g/ml, or
the amount of actein is from about 2 .mu.g/ml to about 20 .mu.g/ml
and the digitoxin is in an amount of from about 0.0004 .mu.g/ml to
about 5 .mu.g/ml.
20. The method according to claim 16, wherein the amount of actein
is from about 0.2 .mu.g/ml to about 2 .mu.g/ml and the digitoxin is
in an amount of from about 0.01 .mu.g/ml to about 0.8 .mu.g/ml, or
the amount of actein is from about 2 .mu.g/ml to about 20 .mu.g/ml
and the digitoxin is in an amount of from about 0.0004 .mu.g/ml to
about 5 .mu.g/ml.
21. A method for modulating Na.sup.+-K.sup.+-ATPase activity
comprising contacting a cell that expresses Na.sup.+-K.sup.+-ATPase
with an extract of black cohosh comprising a triterpene glycoside
and a cardiac glycoside, and optionally at least one additional
chemopreventive or chemotherapeutic agent.
22. A method for modulating Na.sup.+-K.sup.+-ATPase activity
comprising contacting a cell that expresses Na.sup.+-K.sup.+-ATPase
with actein and a cardiac glycoside, and optionally at least one
additional chemopreventive or chemotherapeutic agent.
23. The method according to claim 21 wherein the cardiac glycoside
is digitoxin.
24. The method according to claim 22 wherein the cardiac glycoside
is digitoxin.
25. A method for modulating a growth inhibitory effect of digitoxin
on a carcinoma, which comprises contacting the carcinoma with
digitoxin and an effective amount of an extract of black cohosh
comprising a triterpene glycoside, which results in a synergistic
effect of the digitoxin on the carcinoma, and optionally an
effective amount of at least one additional chemopreventive or
chemotherapeutic agent.
26. A method for modulating a growth inhibitory effect of digitoxin
on a carcinoma which comprises contacting the carcinoma with
digitoxin and an effective amount of actein, which results in a
synergistic effect of the digitoxin on the carcinoma, and
optionally an effective amount of at least one additional
chemopreventive or chemotherapeutic agent.
27. The method of claim 25 wherein the carcinoma is breast
cancer.
28. The method of claim 26 wherein the carcinoma is breast
cancer.
29. A composition for use in treating or preventing neoplasia
comprising an effective anti-neoplastic amount of an extract of
black cohosh comprising a triterpene glycoside and an effective
anti-neoplastic amount of a cardiac glycoside, and optionally an
effective amount of at least one additional chemopreventive or
chemotherapeutic agent.
30. The composition of claim 29, wherein the extract comprises
actein, and optionally further comprises cimigenol.
31. The composition of claim 29, wherein the extract is selected
from the group consisting of an ethyl acetate extract of black
cohosh and an n-butanolic fraction of an EtOH/water extract of
black cohosh.
32. A composition for use in treating or preventing neoplasia
comprising an effective anti-neoplastic amount of actein and an
effective anti-neoplastic amount of a cardiac glycoside, and
optionally an effective amount of at least one additional
chemopreventive or chemotherapeutic agent.
33. The composition according to claim 31, wherein the cardiac
glycoside is selected from the group consisting of digitoxin,
ouabain, proscillaridin A, digoxin, lanatoside C, and combinations
thereof.
34. The composition according to claim 32, wherein the cardiac
glycoside is selected from the group consisting of digitoxin,
ouabain, proscillaridin A, digoxin, lanatoside C, and combinations
thereof.
35. The composition of claim 33, wherein the cardiac glycoside is
digitoxin.
36. The composition of claim 34, wherein the cardiac glycoside is
digitoxin.
37. The composition of claim 35, wherein actein and digitoxin are
in amounts that result in a synergistic anti-neoplastic effect.
38. The composition of claim 36, wherein actein and digitoxin are
in amounts that result in a synergistic anti-neoplastic effect.
39. The composition according to claim 33, wherein the effective
anti-neoplastic amount of actein is from about 0.2 .mu.g/ml to
about 40.0 .mu.g/ml.
40. The composition according to claim 34, wherein the effective
anti-neoplastic amount of actein is from about 0.2 .mu.g/ml to
about 40.0 .mu.g/ml.
41. The composition according to claim 39, wherein the cardiac
glycoside is digitoxin and the amount of actein is from about 0.2
.mu.g/ml to about 2 .mu.g/ml and the amount of digitoxin is from
about 0.01 .mu.g/ml to about 0.8 .mu.g/ml, or the amount of actein
is from about 2 .mu.g/ml to about 20 .mu.g/ml, and the digitoxin is
in an amount of from about 0.0004 .mu.g/ml to about 5 .mu.g/ml.
42. The composition according to claim 40, wherein the cardiac
glycoside is digitoxin and the amount of actein is from about 0.2
.mu.g/ml to about 2 .mu.g/ml and the amount of digitoxin is from
about 0.01 .mu.g/ml to about 0.8 .mu.g/ml, or the amount of actein
is from about 2 .mu.g/ml to about 20 .mu.g/ml, and the digitoxin is
in an amount of from about 0.0004 .mu.g/ml to about 5 .mu.g/ml.
43. A pharmaceutical composition which comprises a composition
according to claim 37, and a pharmaceutically acceptable
carrier.
44. A pharmaceutical composition which comprises a composition
according to claim 38, and a pharmaceutically acceptable
carrier.
45. The method according to claim 1 wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
46. The method according to claim 2 wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
47. The method according to claim 3 wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
48. The method according to claim 6 wherein the at least one
additional chemopreventive or chemotherapeutic agent is a
taxane.
49. The method according to claim 21 wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
50. The method according to claim 22 wherein the at least one
additional chemopreventive or chemotherapeutic agent is a
taxane.
51. The method of claim 48 wherein the taxane is selected from the
group consisting of paclitaxel, docetaxel, and mixtures
thereof.
52. The method of claim 50 wherein the taxane is selected from the
group consisting of paclitaxel, docetaxel, and mixtures
thereof.
53. The method of claim 51 wherein the taxane is paclitaxel.
54. The method of claim 52 wherein the taxane is paclitaxel.
55. The method according to claim 45 wherein the paclitaxel is in
an amount that results in a synergistic effect with the digitoxin
and the triterpene glycoside.
56. The method according to claim 46 wherein the paclitaxel is in
an amount that results in a synergistic effect with the digitoxin
and the actein.
57. The method according to claim 47 wherein the paclitaxel is in
an amount that results in a synergistic effect with the cardiac
glycoside and the triterpene glycoside.
58. The method according to claim 53 wherein the paclitaxel is in
an amount that results in a synergistic effect with the cardiac
glycoside and the actein.
59. The method according to claim 49 wherein the paclitaxel is in
an amount that results in a synergistic effect with the cardiac
glycoside and the triterpene glycoside.
60. The method according to claim 54 wherein the paclitaxel is in
an amount that results in a synergistic effect with the cardiac
glycoside and the actein.
61. The composition according to claim 30 wherein the at least one
additional chemopreventive or chemotherapeutic agent is
paclitaxel.
62. The composition according to claim 32 wherein the at least one
additional chemopreventive or chemotherapeutic agent is a
taxane.
63. The composition of claim 62 wherein the taxane is selected from
the group consisting of paclitaxel, docetaxel, and mixtures
thereof.
64. The composition of claim 63 wherein the taxane is
paclitaxel.
65. The composition according to claim 61 wherein the paclitaxel is
in an amount that results in a synergistic effect with the cardiac
glycoside and the actein.
66. The composition according to claim 64 wherein the paclitaxel is
in an amount that results in a synergistic effect with the cardiac
glycoside and the actein.
67. A pharmaceutical composition which comprises the composition of
claim 66 and a pharmaceutically acceptable carrier.
68. A method for modulating a growth inhibitory effect of
paclitaxel on a carcinoma which comprises contacting the carcinoma
with paclitaxel and an effective amount of digitoxin, which results
in a synergistic effect of the paclitaxel on the carcinoma, and
optionally an additional chemopreventive or chemotherapeutic agent
which is selected from the group consisting of an ethyl acetate
extract of black cohosh and actein.
69. The method of claim 68 wherein the amount Of digitoxin and
paclitaxel are at least 0.01 .mu.g/ml digitoxin and 1 nM
paclitaxel.
70. The method of claim 68 wherein the amount of digitoxin and
paclitaxel are at least 0.05 .mu.g/ml digitoxin and 0.025 nM
paclitaxel.
71. A method for inhibiting the progression or development of
breast cancer in vivo, comprising administering to a subject a
composition comprising an extract of black cohosh comprising a
triterpene glycoside, and optionally an effective amount of at
least one additional chemopreventive or chemotherapeutic agent.
72. A method for inhibiting the progression or development of
breast cancer in vivo, comprising administering to a subject a
composition comprising actein, and optionally an effective amount
of at least one additional chemopreventive or chemotherapeutic
agent.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application deposited under Express Mail label No. EQ 523728379 US
and having Docket No. IR 1390 CO35795/0225095, on Aug. 4, 2008
(informally designated U.S. Ser. No. 12/221,478), which is a
divisional of U.S. patent application Ser. No. 10/746,960, filed on
Dec. 23, 2003, which claims the benefit of U.S. Provisional
Application Ser. No. 60/437,159, filed on Dec. 27, 2002, and
entitled "ANTICANCER COMPOSITIONS OF EXTRACTS OF BLACK COHOSH". The
contents of these prior applications are incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0003] Black cohosh, Actaea racemosa L. (Cimicifuga racemosa), a
perennial in the buttercup family (Ranunculaceae), is frequently
used to treat gynecological and other conditions. In particular,
the roots and rhizomes of black cohosh have been used to treat a
variety of disorders, including inflammatory conditions, diarrhea,
dysmenorrhea, and rheumatism; they have also been used to stimulate
menstrual flow and to suppress coughs (Foster, S., Black cohosh:
Cimicifuga racemosa. A literature review. HerbalGram, 45:35-49,
1999).
[0004] Additionally, black cohosh has been used as a natural
alternative to hormone-replacement therapy. In fact, American women
are increasingly turning to black cohosh as a "more natural"
alternative to estrogen, in the belief that it has the benefits,
without the risks, of estrogen-replacement therapy. To date, a
standardized black cohosh extract (Remifemin), developed in
Germany, has been studied, both in animals and in short-term
clinical trials of menopausal women. These studies suggest that the
extract alleviates a variety of menopausal symptoms, particularly
hot flashes (Lehmann-Willenbrock and Riedel, Clinical and
endocrinological examinations concerning therapy of climacteric
symptoms following hysterectomy with remaining ovaries. Zent. Bl.
Gynakol., 110:611-18, 1988; Stoll, W., Phytotherapy influences
atrophic vaginal epithelium: double-blind study--cimicifuga vs.
estrogenic substances. Therapeuticum, 1:23-31, 1987). Although most
studies report that black cohosh is free of significant
side-effects, these studies have not been carried out for a length
of time sufficient to ensure the safety of black cohosh with
respect to uterine function and/or the induction or stimulation of
breast cancer growth. Since the population using black cohosh
(i.e., middle-aged females in developed countries) is at a higher
risk for breast cancer, research is needed to clarify whether black
cohosh extracts stimulate or inhibit breast cancer cells. Such
studies could also identify new approaches to breast cancer
prevention and treatment.
[0005] The components of the black-cohosh rhizome have been
examined in several studies. It is known that the rhizome contains
triterpene glycosides, aromatic acids, cinnaminic acid esters,
sugars, tannins, and long-chain fatty acids (Zheng et al., CimiPure
(Cimicifuga racemosa): a standardized black cohosh extract with
novel triterpene glycoside for menopausal women. In Phytochem.
Phytopharm., Shahidi and Ho, eds. (Champaign, Ill.: AOCS Press,
2000) pp. 360-70). However, little is known about the mechanisms by
which these compounds are metabolized in vivo.
[0006] Crude extracts of black cohosh, and several components
present in black cohosh, have been shown to exhibit biological
activity. Fukinolic acid (2-E-caffeoylfukiic acid) exhibited weak
estrogenic activity on MCF7 cells (Kruse et al., Fukic and piscidic
acid esters from the rhizome of Cimicifuga racemosa and the in
vitro estrogenic activity of fukinolic acid. Planta. Med.,
65:763-64, 1999); it also inhibited the activity of neutrophil
elastase, which is involved on the inflammatory process (Loser et
al., Inhibition of neutrophil elastase activity by cinnamic acid
derivatives from Cimicifuga racemosa. Planta. Med., 66:751-53,
2000). Bioactivity-guided fractionation of the methanolic extract
resulted in the isolation of nine antioxidant compounds. Of these,
methyl caffeate was the most active in reducing menadione-induced
DNA damage in cultured S30 breast cancer cells (Burdette et al.,
Black cohosh (Cimicifuga racemosa L.) protects against
menadione-induced DNA damage through scavenging of reactive oxygen
species: bioassay-directed isolation and characterization of active
principles. J. Agric. Food Chem., 50:7022-28, 2002). None of the
compounds was cytotoxic to S30 cells (Burdette et al., Black cohosh
(Cimicifuga racemosa L.) protects against menadione-induced DNA
damage through scavenging of reactive oxygen species:
bioassay-directed isolation and characterization of active
principles. J. Agric. Food Chem., 50:7022-28, 2002).
[0007] Extracts and components purified from black cohosh have also
been shown to exhibit anti-cancer activity, in vitro and in vivo.
Extracts of black cohosh (ethanol extract, 0.1% v/v) inhibited the
growth of serum-stimulated T-47D breast cancer cells (Dixon-Shanies
and Shaikh, Growth inhibition of human breast cancer cells by herbs
and phytoestrogens. Oncol. Rep., 6:1383-87, 1999), and, at doses
starting at 2.5 .mu.g/ml, inhibited the proliferation of the
mammary carcinoma cell line, 435 (Nesselhut et al., Studies on
mammary carcinoma cells regarding the proliferation potential of
herbal medication with estrogen-like effects. Archives of
Gynecology and Obstetrics, 254:817-18, 1993). Furthermore,
isopropanolic extracts of black cohosh inhibited estrogen-induced
proliferation of MCF7 cells, and enhanced the inhibitory effect of
tamoxifen (Bodinet and Freudenstein, Influence of Cimicifuga
racemosa on the proliferation of estrogen receptor-positive human
breast cancer cells. Breast Cancer Research and Treatment, 76:1-10,
2002).
[0008] More recently, it has been shown that cycloartane glycosides
isolated from black cohosh inhibit the growth of human oral
squamous cell carcinoma cells (Watanabe et al., Cycloartane
glycosides from the rhizomes of Cimicifuga racemosa and their
cytotoxic activities. Chem. Pharm. Bull., 50:121-25, 2002).
Additionally, recent studies by Sakurai et al. have indicated that
triterpene glycosides and aglycones--the most active of which is
cimigenol--inhibit Epstein-Barr virus early antigen activation
(induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells
(Sakurai et al., Antitumor agents 220. Antitumor-promoting effects
of cimigenol and related compounds on Epstein-Barr virus activation
and two-stage mouse skin carcinogenesis. Bioorg. Med. Chem.
11:1137-40, 2003). Cimigenol has also been shown to inhibit mouse
skin tumor promotion using DMBA as an initiator and TPA as a
promoter.
[0009] All of the foregoing studies, however, have been limited in
scope, and have not addressed issues of specificity and mechanism
of action.
SUMMARY OF THE INVENTION
[0010] The invention disclosed herein generally relates to the
effects of extracts of black cohosh on the growth and progression
of the cell cycle, and on the expression of proteins involved in
cell-cycle control in cancer-cell lines. More particularly, the
present invention relates to the effects of actein and
triterpene-glycoside extracts of black cohosh on neoplastic
cells--when used alone or in combination with a chemopreventive or
chemotherapeutic agent.
[0011] Accordingly, in one aspect, the present invention provides a
composition for use in treating or preventing neoplasia, comprising
an effective anti-neoplastic amount of an ethyl acetate extract of
black cohosh.
[0012] In another aspect, the present invention provides a
combination of anti-neoplastic agents, comprising an effective
anti-neoplastic amount of an ethyl acetate extract of black cohosh
and an effective anti-neoplastic amount of at least one additional
chemopreventive or chemotherapeutic agent. In one embodiment of the
invention, the combination is a synergistic combination.
[0013] In a further aspect, the present invention provides a
composition for use in treating or preventing neoplasia, comprising
an effective anti-neoplastic amount of actein. In one embodiment,
the composition further comprises an effective anti-neoplastic
amount of at least one additional chemopreventive or
chemotherapeutic agent.
[0014] In yet another aspect, the present invention provides a
method for treating or preventing neoplasia in a subject, by
administering to the subject an amount of an ethyl acetate extract
of black cohosh effective to treat or prevent the neoplasia.
[0015] In still another aspect, the present invention provides a
method for treating or preventing neoplasia in a subject, by
administering to the subject an amount of an ethyl acetate extract
of black cohosh effective to treat or prevent the neoplasia, in
combination with an amount of at least one additional
chemopreventive or chemotherapeutic agent effective to treat or
prevent the neoplasia. In one embodiment of the invention, a
synergistic anti-neoplastic effect results.
[0016] Furthermore, the present invention provides a method for
treating or preventing neoplasia in a subject, comprising
administering to the subject an amount of actein effective to treat
or prevent the neoplasia. In one embodiment, the method further
comprises administering to the subject an amount of at least one
additional chemopreventive or chemotherapeutic agent effective to
treat or prevent the neoplasia.
[0017] Additional aspects of the present invention will be apparent
in view of the description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is an illustration of the methods of the invention
which were used to fractionate black cohosh.
[0019] FIGS. 2A and 2B illustrate the effect of black cohosh
extracts on the growth of MCF7 cells. FIG. 2A shows the effect of
the ethyl acetate extract when MCF7 cells were treated with the
indicated concentrations of the ethyl acetate fraction for
increasing times. FIG. 2B shows the effect of actein when MCF7
cells were treated with the indicated concentrations of actein for
increasing times. In each case, the number of viable cells was
determined using a Coulter Counter, and the control contained 0.08%
DMSO. Bars=SD
[0020] FIG. 3 shows the structures of the triterpene glycoside
compounds of the invention.
[0021] FIG. 4 depicts the effect of actein and the effect of the
ethyl acetate fraction of black cohosh on MCF7 cell-cycle
distribution at 48 h. MCF7 cells were treated with 0, 30, and 60
.mu.g/ml of the ethyl acetate extract, or actein, and then analyzed
at 48 h by DNA flow cytometry. The values indicate the percentage
of cells in the indicated phases of the cell cycle.
[0022] FIG. 5 illustrates the effect of actein on the G1 phase of
the cell cycle in MCF7 cells. MCF7 cells were treated with 10 (14.8
.mu.M), 20, or 40 .mu.g/ml actein, and then analyzed at 24 and 48 h
by DNA flow cytometry. The values indicate the percentage of cells
in the G1 phase of the cell cycle.
[0023] FIGS. 6A-6F show Western-blot analyses of MCF7 cells treated
with actein. The cells were treated with 0, 20, or 40 .mu.g/ml
actein. 20 .mu.g/ml actein is equivalent to 29.6 .mu.M actein.
After 3, 10, and 24 h, extracts were analyzed by Western blotting
with antibodies to: cyclin D1 (FIG. 6A); ppRb (FIG. 6B); cdk4 (FIG.
6C); p21.sup.cip1 (FIG. 6D); EGFR (FIG. 6E); and phospho-EGFR (FIG.
6F). An antibody for .beta.-actin was used as a loading
control.
[0024] FIG. 7 shows the effects of actein alone, and in combination
with paclitaxel, on cell proliferation in MDA-MB-453 (Her2
overexpressing) human breast cancer cells. MDA-MB-453 cells were
treated with all combinations of 3 concentrations of actein and 3
concentrations of paclitaxel, and the solvent control, for 96 h.
The number of viable cells was determined using a Coulter Counter.
Similar results were obtained in two additional studies. The
control contained 0.044% DMSO. bars=SD
[0025] FIG. 8 illustrates a Western-blot analysis of extracts
obtained from MDA-MB-453 cells treated with actein. The cells were
treated with 0, 20, or 40 .mu.g/ml of actein. After 3 and 24 h,
extracts were prepared and analyzed by Western blotting with an
antibody to Her2 or an antibody to phospho-Her2 (p-Her2). An
antibody to .beta.-actin was used as a loading control. The
staining intensities of the visualized blots were quantified using
NIH image software. For each protein, the relative band intensities
were determined by comparing treated samples with untreated
controls. These values were then normalized (fold), using
.beta.-actin as an internal control.
[0026] FIG. 9 presents a reporter promoter analysis of extracts
obtained from MDA-MB-453 cells treated with actein. Using
lipofectin, triplicate samples of MDA-MB-453 breast cancer cells
were co-transfected with DNA of the indicated reporter plasmid,
using 3-gal DNA as an internal control. The cells were then treated
with actein at 0, 20, and 40 .mu.g/ml, in quadruplicate. Luciferase
and .beta.-gal activities were determined, as previously described
(Masuda et al., Effects of epigallocatechin-3-gallate on growth,
epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Luciferase activities were normalized to .beta.-gal
activities. left panel=cyclin D1; right panel=nuclear factor kappa
B (NF-.kappa.B); bars=SD
[0027] FIG. 10 sets forth the effects of the aglycone cimigenol and
the triterpene glycosides cimigenol glycoside, and actein, purified
from black cohosh, on cell proliferation in MDA-MB-453 cells.
MDA-MB-453 cells were exposed to increasing concentrations of the
indicated purified components for 96 h, and the number of viable
cells was determined using a Coulter Counter.
[0028] FIG. 11 shows the effects of butanol fractions from black
cohosh on cell proliferation in MCF7 cells. MCF7 cells were exposed
to increasing concentrations of the indicated purified components,
for 26 or 96 h, and the number of viable cells was determined using
a Coulter Counter.
[0029] FIG. 12 demonstrates the effects of the components ferulic
and isoferulic acid, purified from black cohosh, on cell
proliferation in MCF7 cells. MCF7 cells were exposed to increasing
concentrations of the indicated purified components for 96 hrs, and
the number of viable cells was determined using a Coulter
Counter.
[0030] FIG. 13 illustrates the effects of actein on cyclin D1 mRNA
in MCF7 cells (RT-PCR). MCF7 cells were treated with DMSO or actein
for 3, 10, or 24 h. RNA was isolated and analyzed by RT-PCR, using
primers for cyclin D1 and actin (control). The staining intensities
of the visualized blots were quantified using NIH image software.
The relative band intensities were determined by comparing treated
samples with untreated controls. These values were then normalized
(fold), using .beta.-actin as an internal control.
[0031] FIG. 14 demonstrates the effects of actein on cyclin D1 mRNA
in MDA-MB-453 cells (RT-PCR). MDA-MB-453 cells were treated with
DMSO or actein for 3, 10, or 24 h. RNA was isolated and analyzed by
RT-PCR, using primers for cyclin D1 and actin (control). The
staining intensities of the visualized blots were quantified using
NIH image software. The relative band intensities were determined
by comparing treated samples with untreated controls. These values
were then normalized (fold), using .beta.-actin as an internal
control.
[0032] FIG. 15 illustrates MCF7 cells treated with 0, 20, or 40
.mu.g/ml actein. 20 .mu.g/ml actein is equivalent to 29.6 .mu.M.
After 3, 10, and 24 h, extracts were analyzed by Western blotting
with an antibody to p21. An antibody for .beta.-actin was used as a
loading control.
[0033] FIG. 16 shows the effects of actein on p21 mRNA in
MDA-MB-453 cells (RT-PCR). MDA-MB-453 cells were treated with DMSO
or actein for 3, 10, or 24 h. RNA was isolated and analyzed by
RT-PCR, using primers for cyclin D1 and actin (control). The
staining intensities of the visualized blots were quantified using
NIH image software. The relative band intensities were determined
by comparing treated samples with untreated controls. These values
were then normalized (fold), using .beta.-actin as an internal
control.
[0034] FIG. 17 illustrates MDA-MB-453 cells that were treated with
0, 20, or 40 .mu.g/ml actein. 20 .mu.g/ml actein is equivalent to
29.6 .mu.M. After 3, 10, and 24 h, extracts were analyzed by
Western blotting, with an antibody to ik.beta.. An antibody for
.beta.-actin was used as a loading control.
[0035] FIG. 18 illustrates MDA-MB-453 cells that were treated with
0, 20, or 40 .mu.g/ml actein (20 .mu.g/ml actein is equivalent to
29.6 .mu.M). After 3 and 24 h, extracts were analyzed by Western
blotting, with an antibody to PPAR.gamma.. An antibody for
.beta.-actin was used as a loading control.
[0036] FIG. 19 Results of chemopreventive study: A) Cumulative
number of mammary tumors (per 100 animals) by weeks of age (from 56
to 139 weeks of age at death), observed during clinical examination
(for 35.7 mg/kg b.w, 7.14 mgkg b.w., and 0.714 mg/kg b.w. of an
extract of black cohosh enriched for triterpene glycosides, and
control). B) Survival (as a percentage plotted against age at death
(weeks)). C) Mean body weight (as a percentage plotted against age
at death (weeks)).
[0037] FIG. 20 IHC staining of mammary gland tissue: A) ER; B)
Her2; PC=positive control; MG=mammary gland (magnification:
100.times.). The mammary tissue was positive for ER in the nucleus
as shown in panel A. The mammary tissue was negative for Her2
expression, as shown in panel B.
[0038] FIG. 21 H&E staining of frozen sections of
fibroadenomas: A) control; age detected: 93 weeks; age at death: 95
weeks; B) treated with black cohosh extract at 7.14 mg/kg; age
detected: 89 weeks; age at death: 101 weeks; C) treated with black
cohosh extract at 35.7 mg/kg; age detected: 89 weeks, age at death:
96 weeks. Treatment panels B and C show a decrease in the
proportion of glandular tissue in treated versus control in panel
C. (In color, glands are shown as blue; connective tissue as pink;
white as undefined, empty space or filled with secretory material
or blood vessels). Also shown is IHC of Fibroadenomas: D) IHC
cyclin D1; Fibroadenoma, control, age detected 93 weeks, age at
death 95 weeks; E) IHC cyclin D1: Fibroadenoma, 7.14 mg/kg, age
detected: 89 weeks, age at death: 101 weeks; F) IHC cyclin Ki67;
Fibroadenoma, control, age detected 93 weeks, age at death 95
weeks; G) IHC Ki67 Fibroadenoma, 7.14 mg/kg, age detected: 89
weeks, age at death: 101 weeks. A significant difference is seen
comparing Ki67 (panels F and G) and cyclin D1 (panels D and E)
staining for rats treated with 7.14 mg/kg black cohosh extract.
[0039] FIG. 22 A) IHC of rat liver tissue (24 hr): EGFR;
PC=positive control. IHC staining indicated the presence of EGFR in
the nucleus; B) H&E staining of frozen sections of rat liver
with control (untreated) and treated with black cohosh 35.7 mg/kg.
Mild toxicity was displayed; and C) H&E staining showing
periportal localization of lipid accumulation in control and
treated rat liver.
[0040] FIG. 23 Pathway map of the Mitochondrial Oxidative
Phosphorylation pathway. Genes represented by probe sets on the
array are shown as colored circles (p>0.05) or diamonds
(p<0.05). Red indicates upregulation while green indicates
downregulation of the gene.
[0041] FIG. 24 Zoomed view of hierarchical clustering heat map
(UPGMA, Pearson's correlation) of 668 liver experiments across
impact against 135 DrugMatrix pathways. Statistical analysis of the
treatments in the cluster using the hypergeometric distribution
revealed a significant representation of treatments with
anti-proliferative compounds, specifically tubulin binding vinca
alkaloids (3 experiments, p=0.0017) and DNA alkylators (4
experiments, p=0.029).
[0042] FIG. 25 Real-time RT-PCR of rat liver after treating with
black cohosh extract (35.7 mg/kg), which confirmed that black
cohosh suppressed the expression of cyclin D1 and ID3.
[0043] FIG. 26 Chemical structures of A) actein, and B) digitoxin.
Shown in C) is a schematic diagram of pathways linking
Na.sup.+-K.sup.+-ATPase with the ERK and Akt pathways (adapted from
Haas et al., J. Biol. Chem. 275 (2000) 27832-27837, and Lavoie et
al., J. Biol. Chem. 271 (1996) 20608-16).
[0044] FIG. 27 Inhibition of Na.sup.+-K.sup.+-ATPase activity in
response to increasing concentrations of ouabain or actein. The
Na.sup.+-K.sup.+-ATPase assay was performed as described in
Materials and Methods. The DMSO controls contained 3.3% DMSO. Bars:
SD of triplicate assays (a).
[0045] FIG. 28 Effects of increasing concentrations of actein alone
or in combination with increasing concentrations of digitoxin on
Na.sup.+-K.sup.+-ATPase activity or cell growth.
Na.sup.+-K.sup.+-ATPase activity: A) x-axis, actein concentration;
B) x-axis, digitoxin concentration. Cell proliferation in
MDA-MB-453 breast cancer cells: C) x-axis, actein concentration; D)
x-axis, digitoxin concentration. The DMSO controls contained
3.3.degree. A) DMSO (A, B) or 0.33% DMSO(C, D). Similar results (A,
B) were obtained in an additional experiment. Bars: SD of
triplicate assays (C, D).
[0046] FIG. 29 Effect of actein on targets downstream of
Na.sup.+-K.sup.+-ATPase. All assays were performed on MDA-MB-453
cells as described in Materials and Methods. A) Western blot
analysis of p-Src, following cell exposure to actein or digitoxin
for 30 minutes (80 ug/ml); fold relative to .beta.-actin; B) Growth
inhibitory effects of actein (20 .mu.g/ml for 48 h) on cells
pretreated with siRNA to ERK2 for 24 h (p=0.0665); C) Western blot
analysis of proteins obtained from cells 3, 8 or 24 hours after
treatment with 0, 20 or 40 .mu.g/mL of actein; D) luciferase
promoter activity of NE-KB following treatment of cells actein at
20 or 40 .mu.g/ml for 24 h.
[0047] FIG. 30 Hierarchical clustering of differentially expressed
genes analyzed on U1332.0A Affymetrix chips after treating
MDA-MB-453 wells with digitoxin at 0.1, 0.2 or 1.0 .mu.g/mL for 6
or 24 hours. Clustering was performed with the Program Cluster 3.0
(Khatri et al. 2005). Probesets were restricted to those that
corresponded to an absolute value of M (log fold)>2.0 for at
least one of the conditions. The threshold for color in the
hierarchical clustering map is M>3 log fold. Fold change
indicates relative expression in. digitoxin versus DMSO control
cells. To pick the blowup region, the area containing a specific
gene was expanded to include a well-defined expression pattern.
Shown is the full hierarchical clustering map, which contains 4706
probesets A) upregulated gene region amplified for ATF3, B) down
regulated gene region amplified for CDC16; C) upregulated gene
region amplified for EGR1; red, upregulated; green, down
regulated.
[0048] FIG. 31 A, B, C) Real-time RT-PCR analysis after treating
MDA-MB-453 cells with digitoxin at 0.1 .mu.g/mL for 6 or 24 hours.
The cells were treated with 0.1 .mu.g/mL of digitoxin and, after 6
or 24 hours, extracts were prepared and analyzed by Real-time
RT-PCR, as described in Materials and Methods. Fold change
indicates relative expression in digitoxin versus DMSO control
cells. * indicates p<0.05. A, B and C display different patterns
of gene expression. D, E, F) Real-time RT-PCR analysis after
treating MCF7 cells with digitoxin at 1 .mu.g/mL for 6 or 24 hours,
as described in Materials and Methods. Fold change indicates
relative expression in digitoxin versus DMSO control cells. All p
values were <0.05.
[0049] FIG. 32. A) Effects of digitoxin on the level of ATF3, EGR1
protein. Western blot analysis of extracts obtained from MDA-MB-453
cells treated with digitoxin. Cells were treated with 0, 0.1, 0.2
or 1 .mu.g/mL of digitoxin and after 1, 6 or 24 hours extracts were
prepared and analyzed by Western blotting; an antibody to
.beta.-actin was used as a loading control. B) siRNA to Erk2. Cell
were pretreated with control and MAPK1 (Erk2) siRNA for 24 hours,
exposed to digitoxin (0.4 .mu.g/ml) for 24 h and percent inhibition
of cell growth determined by the MIT assay. Positive=Mapk1; Erk2;
Negative=Control siRNA. Percentages are normalized to DMSO.
[0050] FIG. 33 Effects of digitoxin alone or in combination with
TAX on cell proliferation in MDA-MB-453 breast cancer cells: A)
x-axis, TAX concentration, B) Table of Combination Index (CI)
Values, C) x-axis, digitoxin concentration. The DMSO control
contained <0.1% DMSO; Bars: SD.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The Examples below provide the first detailed examination of
the effects on human breast cancer cells of extracts and purified
compounds present in black cohosh. In these studies, the roots and
rhizomes of black cohosh were extracted with MeOH/H.sub.2O, and
fractionated by solvent-solvent partitioning to yield three
fractions: hexane, ethyl acetate (EtOAc), and H.sub.2O. The EtOAc
fraction exhibited the greatest growth-inhibitory activity. This
fraction inhibited growth of both the ER.sup.+MCF7 and ER.sup.-
/Her2+MDA-MB-453 human breast cancer cell lines, with IC.sub.50
values of about 18 .mu.g/ml and 10 .mu.g/ml, respectively. The
normal human mammary epithelial cell line, MCF10F, was much less
sensitive to growth inhibition by this extract (with an IC.sub.50
value of 46 .mu.g/ml). It is possible that the greater sensitivity
of the malignant cells may reflect, in part, the difference in
growth rates of the malignant and non-malignant cells.
[0052] The inventors tested the effects of crude extracts, methanol
and ethanol, as well as ethanol extracts provided by Pure World,
native and plus expedient: the IC.sub.50 values for these extracts
after 96 hours of treatment were: methanol: 100 .mu.g/ml; ethanol:
>200 .mu.g/ml; Pure World native: 175 .mu.g/ml; and Pure World
plus expedient: 195 .mu.g/ml. To partition the phytochemicals
according to polarity, the water portion was also partitioned
sequentially with hexane and n-butanol (n-BuOH). The n-BuOH
fraction was tested for its effect on the growth of MDA-MB-453
breast cancer cells. The IC50 value after 96 hours of treatment
was: 40 .mu.g/ml.
[0053] The inventors also examined the effects of the EtOAc
fraction of black cohosh on SW480 human colon cancer cells. The
IC50 values after 48 hours of incubation using the MTT assay were:
SW480: 42 .mu.g/ml; MCF7: 38 .mu.g/ml (Luo et al., PM-3, a
benzo-g-pyran derivative isolated from propolis, inhibits growth of
MCF-7 human breast cancer cells. Anticancer Res 21: 1665-1672,
2001).
[0054] The inventors further demonstrated that the EtOAc fraction
of black cohosh induced cell-cycle arrest in MCF7 human breast
cancer cells at G1 at 30 .mu.g/ml, and at G2/M at 60 .mu.g/ml. The
triterpene glycoside fraction that was obtained by polyamide column
chromatography, and the specific triterpene glycosides (actein,
23-epi-26-deoxyactein, and cimiracemoside A), inhibited growth of
MCF7 human breast cancer cells and induced cell-cycle arrest at G1.
At 60 .mu.g/ml, actein induced a less-pronounced G1 arrest.
Therefore, it is likely that, at high concentrations, actein and
related compounds affect proteins that regulate later phases in the
cell cycle.
[0055] Because the triterpene glycosides induced cell-cycle arrest
at G1, the inventors decided to ascertain the effect of the most
potent compound, actein, on cell-cycle proteins that control G1
cell-cycle progression. As discussed below, actein decreased the
level of cyclin D1, cdk4, and the hyperphosphorylated form of pRb,
and increased the level of the cdk inhibitory protein,
p21.sup.cip1, in MCF7 cells--changes that may contribute to the
arrest in G1. The inventors also found that actein reduced the
level of cyclin D1 mRNA within 3 h of treatment, and significantly
reduced the level at 24 h, suggesting an effect at the level of
transcription. The level of the EGFR was not altered after
treatment with actein; nor was there a consistent effect on the
level of the phosphorylated form of the EGFR (p-EGFR), which
reflects its state of activation. Thus, the EGFR did not appear to
be a direct target for actein. This result is in contrast to the
effect of another plant-derived compound--a flavonol,
epigallocatechin-gallate--which is the active component in green
tea (Masuda et al., Effects of epigallocatechin-3-gallate on
growth, epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Previous studies have also indicated that micromolar
concentrations of the aglycone compounds, cyanidin and delphinidin,
inhibited activation of the EGFR and cell proliferation in the
human vulva carcinoma cell line, A431, whereas the corresponding
glycosides had a minimal effect (Meiers et al., The anthocyanidins
cyanidin and delphinidin are potent inhibitors of the epidermal
growth-factor receptor. J. Agric. Food Chem., 49:958-62, 2001). The
inventors tested the effects of the aglycone cimigenol on the
growth of human breast cancer cells. Cimigenol was less active than
cimigenol glycoside.
[0056] Triterpene molecules are structurally related to steroids,
and have been present in the plant kingdom for millions of years.
Some may have evolved to become ligands for receptors on animal
cells (Sporn and Suh, Chemoprevention of cancer. Carcinogenesis,
21:525-30, 2000). However, the mode of action triterpene glycosides
is not well understood. Studies by Haridas et al. (Avicins:
triterpenoid saponins from Acacia victoriae (Bentham) induce
apoptosis by mitochondrial perturbation. Proc. Natl. Acad. Sci.
USA, 98:5821-26, 2001) indicate that avicins--triterpenoid saponins
from the plant Acacia victoriae (Bentham)--are potent inhibitors of
the transcription factor, nuclear factor kappa B (NE-KB), and act
by inhibiting its translocation to the nucleus and its capacity to
bind DNA--perhaps by altering sulfhydryl groups critical for
NF-.kappa.B activation. Betulinic acid, a pentacyclic triterpene
present in the bark of white birch trees, is a selective inhibitor
of human melanoma (Pisha et al., Discovery of betulinic acid as a
selective inhibitor of human melanoma that functions by induction
of apoptosis. Nat. Med., 1:1046-51, 1995). It induces apoptosis in
neuroectodermal tumors by a direct effect on mitochondria (Fulda
and Debatin, Betulinic acid induces apoptosis through a direct
effect on mitochondria in neuroectodermal tumors. Med. Pediatr.
Oncol., 35:616-18, 2000).
[0057] Suh et al. (Novel triterpenoids suppress inducible nitric
oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in mouse
macrophages. Cancer Res., 58:717-23, 1998) have generated a series
of derivatives of the triterpenes, oleanic and ursolic acids, that
are highly potent in suppressing the expression of inducible nitric
oxide synthase and cyclooxygenase-2 in primary mouse macrophages.
Indeed, the derivative, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic
acid (CDDO), is 1000 times more potent than oleanic acid in this
cell system (Sporn and Suh, Chemoprevention of cancer.
Carcinogenesis, 21:525-30, 2000). Suh et al. also found that CDDO
displays potent differentiating, anti-proliferative, and
anti-inflammatory activities (Suh et al., A novel synthetic
oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic
acid, with potent differentiating, antiproliferative, and
anti-inflammatory activity. Cancer Res., 59:336-41, 1999).
[0058] CDDO further induces apoptosis by a caspase-8-dependent
mechanism (Ito et al., The novel triterpenoid CDDO induces
apoptosis and differentiation of human osteosarcoma cells by a
caspase-8 dependent mechanism. Mol. Pharmacol., 5:1094-99, 2001;
Pedersen et al., The triterpenoid CDDO induces apoptosis in
refractory CLL B cells. Blood, 8:2965-72, 2002), and inhibits
NF-.kappa.B-mediated gene expression, following translocation of
the activated form to the nucleus (Stadheim et al., The novel
triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)
potently enhances apoptosis induced by tumor necrosis factor in
human leukemia cells. J. Biol. Chem., 19:16448-55, 2002). It is a
ligand for the peroxisome proliferator activated receptor-.gamma.
(PPAR-.gamma.) (Wang et al., A synthetic triterpenoid,
2-cyano-2,12-dioxooleana-1,9-dien-1-28-oic acid (CDDO), is a ligand
for the peroxisome proliferator-activated receptor gamma. Mot
Endocrinol., 14:1550-56, 2000), but the specific cellular target of
CDDO and related compounds, for mediating the above biologic
effects, is not known.
[0059] The triterpene glycoside, actein, and the fraction of black
cohosh enriched for triterpene glycosides (which are selective for
human breast cancer versus normal mammary epithelial cells),
synergize with several classes of chemotherapy agents. For example,
the inventors have demonstrated that actein has synergy with the
taxane, paclitaxel; the antimetabolite, 5-fluorouracil (5-FU); the
Her2 antibody, herceptin; the anthracycline antibiotic,
doxorubicin; and the platinum analog, cisplatin. Additionally, the
inventors have shown that black cohosh extracts have synergy with
paclitaxel and doxorubicin. Because it is easier to prepare
enriched extracts, the extracts of black cohosh might represent the
preferred sources to be used in combination with such
chemotherapeutic agents.
[0060] In view of the foregoing, the present invention provides
methods for treating and preventing neoplasia in a subject. The
subject is preferably a mammal (e.g., humans, domestic animals, and
commercial animals, including cows, dogs, monkeys, mice, pigs, and
rats). More preferably, the subject is a human.
[0061] As used herein, "neoplasia" refers to the uncontrolled and
progressive multiplication of cells under conditions that would not
elicit, or would otherwise cause cessation of, the multiplication
of normal or non-neoplastic cells. Neoplasia results in the
formation of a neoplasm, which is any new and abnormal growth,
particularly a new growth of tissue, in which the growth is
uncontrolled and progressive. Malignant neoplasms are distinguished
from benign in that the former show a greater degree of anaplasia,
or loss of differentiation and orientation of cells, and have the
properties of invasion and metastasis. Thus, neoplasia includes
"cancer", which refers herein to a proliferation of cells having
the unique trait of loss of normal controls, resulting in
unregulated growth, lack of differentiation, local tissue invasion,
and metastasis (Beers and Berkow, eds., The Merck Manual of
Diagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, N.J.:
Merck Research Laboratories, 1999) 973-74, 976, 986, 988, 991).
[0062] Neoplasias which may be treated and/or prevented by the
methods of the present invention include, without limitation,
carcinomas, particularly those of the bladder, breast, cervix,
colon, head, kidney, lung, neck, ovary, prostate, and stomach;
lymphocytic leukemias, particularly acute lymphoblastic leukemia
and chronic lymphocytic leukemia; myeloid leukemias, particularly
acute monocytic leukemia, acute promyelocytic leukemia, and chronic
myelocytic leukemia; malignant lymphomas, particularly Burkitt's
lymphoma and Non-Hodgkin's lymphoma; malignant melanomas;
myeloproliferative diseases; sarcomas, particularly Ewing's
sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, peripheral
neuroepithelioma, and synovial sarcoma; and mixed types of
neoplasias, particularly carcinosarcoma and Hodgkin's disease.
Preferably, the methods of the present invention are used to treat
or prevent breast cancer, colon cancer, leukemia, lung cancer,
malignant melanoma, ovarian cancer, or prostate cancer. More
preferably, the cancer is breast cancer.
[0063] The method of the present invention comprises administering
to the subject an ethyl acetate extract of black cohosh or a
composition comprising actein. It is well known that black cohosh
is a medicinal plant from the genus Cimicifuga (or Actea) and the
species racemosa. The ethyl acetate extract of black cohosh is a
partially-purified extract, enriched for triterprene glycosides. It
is a safe and effective extract, with few side effects. The ethyl
acetate extract of black cohosh may be prepared in any suitable
manner that maintains or enriches the triterpene glycosides
component in the extract. By way of example, the method of
extraction may comprise a first extraction of the rhizome of black
cohosh, with an aqueous solution of a lower alkyl alcohol, followed
by partitioning of the aqueous alcohol layer with a lower alkyl
acetate. A preferred lower alkyl alcohol is methanol, and a
preferred lower alkyl acetate is ethyl acetate. The resultant
extract comprises triterpene glycoside compounds and cinnamic acid
esters. Of interest to the invention are the triterpene glycosides,
which can be separated from the cinnamic acid esters by
purification of the ethyl acetate extract. Such triterpene
glycosides include, without limitation, actein, cimifugoside,
cimigenol glycoside, cimiracemoside A, 23-epi-26-deoxyactein and
the aglycone cimigenol. Preferably, the triterpene glycoside
compound is actein.
[0064] The individual triterpenoid components in the ethyl acetate
extract of black cohosh can be individually separated by
purification. The ethyl acetate extract may be maintained in any
form, provided that the activity of the triterpene glycosides, and
of each component therein, is maintained. Activity of the
triterpene glycosides may be assayed by reference to the Examples
presented below. Furthermore, actein and related triterprene
glycosides may be modified to increase their activity, while
retaining their selectivity for neoplastic cells.
[0065] In accordance with the method of the present invention, the
ethyl acetate extract of black cohosh may be administered to the
subject in an anti-neoplastic amount, which is an amount that is
effective to treat or prevent neoplasia in the subject. As used
herein, "anti-neoplastic" includes the ability to inhibit or
prevent the development or spread of a neoplasm, and the ability to
limit, suspend, terminate, or otherwise control the development,
maturation, and proliferation of cells in a neoplasm. As further
used herein, an amount of the ethyl acetate extract of black cohosh
that is "effective to treat or prevent the neoplasia" is an amount
that is effective to ameliorate or minimize the clinical impairment
or symptoms of the neoplasia, or to inhibit their development. For
example, the clinical impairment or symptoms of the neoplasia may
be ameliorated or minimized by diminishing any pain or discomfort
suffered by the subject; by extending the survival of the subject
beyond that which would otherwise be expected in the absence of
such treatment; by inhibiting or preventing the development or
spread of the neoplasm; or by limiting, suspending, terminating, or
otherwise controlling the development, maturation, and
proliferation of cells in the neoplasm.
[0066] Exemplary doses of actein, administered intraperitoneally,
may be between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml, and
preferably, between about 1 .mu.g/ml and about 3.0 .mu.g/ml.
However, the amount of actein effective to treat or prevent
neoplasia or other disorders in a subject will vary depending on
the particular factors of each case, including the target molecule,
the type of neoplasia, the stage of neoplasia, the subject's
weight, the severity of the subject's condition, and the method of
administration. These amounts can be readily determined by the
skilled artisan, based upon known procedures, including analysis of
titration curves established in vivo, dose-response experiments
analogous to those provided in the Examples, and methods and assays
disclosed herein.
[0067] The ethyl acetate extract of black cohosh or the actein
composition may be administered to a human or animal subject by
known procedures, including, without limitation, oral
administration, parenteral administration, transdermal
administration, and by way of catheter. Preferably, the ethyl
acetate extract of black cohosh or the actein composition is
administered parenterally, by epifascial, intracapsular,
intracranial, intracutaneous, intrathecal, intramuscular,
intraorbital, intraperitoneal, intraspinal, intrasternal,
intravascular, intravenous, parenchymatous, subcutaneous, or
sublingual injection.
[0068] For oral administration, a formulation comprising the ethyl
acetate extract of black cohosh or the actein composition may be
presented as capsules, tablets, powders, granules, or as a
suspension. The formulation may have conventional additives, such
as lactose, mannitol, corn starch, or potato starch. The
formulation also may be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch, and
gelatins. Additionally, the formulation may be presented with
disintegrators, such as corn starch, potato starch, and sodium
carboxymethylcellulose. The formulation also may be presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation may be presented with lubricants, such as
talc and magnesium stearate.
[0069] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal), the
ethyl acetate extract of black cohosh or the actein composition may
be combined with a sterile aqueous solution that is preferably
isotonic with the blood of the subject. Such a formulation may be
prepared by dissolving a solid active ingredient in water
containing physiologically-compatible substances, such as sodium
chloride, glycine, and the like, and having a buffered pH
compatible with physiological conditions, so as to produce an
aqueous solution, then rendering said solution sterile. The
formulation may be presented in unit or multi-dose containers, such
as sealed ampoules or vials. The formulation may be delivered by
any mode of injection, including, without limitation, epifascial,
intracapsular, intracranial, intracutaneous, intrathecal,
intramuscular, intraorbital, intraperitoneal, intraspinal,
intrasternal, intravascular, intravenous, parenchymatous,
subcutaneous, and sublingual.
[0070] For transdermal administration, the ethyl acetate extract of
black cohosh or the actein composition may be combined with skin
penetration enhancers, such as propylene glycol, polyethylene
glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and
the like, which increase the permeability of the skin to the ethyl
acetate extract of black cohosh, and permit the ethyl acetate
extract of black cohosh to penetrate through the skin and into the
bloodstream. The ethyl acetate extract of black cohosh, or the
actein composition, /enhancer composition also may be further
combined with a polymeric substance, such as ethylcellulose,
hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl
pyrrolidone, and the like, to provide the composition in gel form,
which may be dissolved in a solvent, such as methylene chloride,
evaporated to the desired viscosity, and then applied to backing
material to provide a patch.
[0071] In accordance with the method of the present invention, the
ethyl acetate extract of black cohosh or the actein composition
also may be administered to a subject by way of a pharmaceutical
composition for use in treating or preventing neoplasia. The
pharmaceutical composition of the present invention comprises an
effective anti-neoplastic amount of the ethyl acetate extract of
black cohosh or an effective amount of the actein composition and a
pharmaceutically-acceptable carrier. The
pharmaceutically-acceptable carrier must be "acceptable" in the
sense of being compatible with the other ingredients of the
composition, and not deleterious to the recipient thereof. The
pharmaceutically-acceptable carrier employed herein is selected
from various organic or inorganic materials that are used as
materials for pharmaceutical formulations, and which may be
incorporated as analgesic agents, buffers, binders, disintegrants,
diluents, emulsifiers, excipients, extenders, glidants,
solubilizers, stabilizers, suspending agents, tonicity agents,
vehicles, and viscosity-increasing agents. If necessary,
pharmaceutical additives, such as antioxidants, aromatics,
colorants, flavor-improving agents, preservatives, and sweeteners,
may also be added. Examples of acceptable pharmaceutical carriers
include carboxymethyl cellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methyl cellulose, powders,
saline, sodium alginate, sucrose, starch, talc, and water, among
others.
[0072] In the pharmaceutical composition of the present invention,
the ethyl acetate extract of black cohosh is provided in an
effective anti-neoplastic amount. For example, where the ethyl
acetate extract comprises actein, the actein may be present in an
amount between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml.
Preferably, the actein is present in an amount between about 1.0
.mu.g/ml and about 3.0 .mu.g/ml.
[0073] The pharmaceutical composition of the present invention may
be prepared by methods well-known in the pharmaceutical arts.
Actein may be obtained from plant extracts or by chemical
synthesis. For example, the ethyl acetate extract of black cohosh
may be brought into association with a carrier or diluent, as a
suspension or solution. Optionally, one or more accessory
ingredients (e.g., buffers, flavoring agents, surface active
agents, and the like) also may be added. The choice of carrier will
depend upon the route of administration.
[0074] Since multiple genetic and epigenetic targets are altered in
the process of carcinogenesis, combination chemoprevention and
chemotherapy are generally optimal. Accordingly, the present
invention further provides a method for treating or preventing
neoplasia in a subject, by administering to the subject an amount
of an ethyl acetate extract of black cohosh or the actein
composition, as described above, in combination with an amount of
at least one additional chemopreventive or chemotherapeutic agent
effective to treat or prevent the neoplasia. As used herein, the
term "effective" also covers the dosages at which the
chemopreventive or chemotherapeutic agent by itself does not have
any significant effect on neoplasia but may significantly promote
or enhance the anti-neoplastic effects of the ethyl acetate extract
of black cohosh, and vice-versa.
[0075] Examples of additional chemopreventive or chemotherapeutic
agents for use in the method of the present invention include,
without limitation cisplatin, docetaxel, doxorubicin,
5-fluorouracil (5-FU), herceptin, paclitaxel, tamoxifen, and
vinblastine, and any fragments, analogues, and derivatives thereof.
In a preferred embodiment, the chemopreventive or chemotherapeutic
agent is paclitaxel. Ethyl acetate extracts of black cohosh, and
additional chemopreventive or chemotherapeutic agents, are referred
to herein as "anti-neoplastic agents."
[0076] By way of example, the term "paclitaxel" includes a natural
or synthetic functional variant of paclitaxel which has paclitaxel
biological activity, as well as a fragment of paclitaxel having
paclitaxel biological activity.
[0077] As used herein, the term "paclitaxel biological activity"
refers to paclitaxel activity which interferes with cellular
mitosis by affecting microtubule formation and/or action, thereby
producing antimitotic and anti-neoplastic effects. Methods of
preparing paclitaxel and its analogues and derivatives are
well-known in the art, and are described, for example, in U.S. Pat.
Nos. 5,569,729; 5,565,478; 5,530,020; 5,527,924; 5,484,809;
5,475,120; 5,440,057; and 5,296,506. Paclitaxel and its analogues
and derivatives are also available commercially. For example,
synthetic paclitaxel can be obtained from Bristol-Myers Squibb
Company, Oncology Division (Princeton, N.J.), under the registered
trademark Taxol.TM.. Moreover, paclitaxel may be synthesized in
accordance with known organic chemistry procedures that are readily
understood by one skilled in the art. Taxol for injection may be
obtained in a single-dose vial, having a concentration of 30 mg/5
ml (6 mg/ml per 5 ml) (Physicians' Desk Reference, 54.sup.th ed.
(Montvale, N.J.: Medical Economics Company, Inc., 2000) 307,
682).
[0078] Paclitaxel and its analogues and derivatives have been used
successfully to treat leukemias and tumors. In particular,
paclitaxel is useful in the treatment of breast, lung, and ovarian
cancers. Since paclitaxel is frequently utilized in the treatment
of human cancers, a strategy to enhance its utility in the clinical
setting, by combining its administration with that of an ethyl
acetate extract of black cohosh, may be of great benefit to many
subjects suffering from malignant neoplasias, particularly advanced
cancers.
[0079] In the method of the present invention, administration of an
ethyl acetate extract of black cohosh "in combination with" one or
more additional chemopreventive or chemotherapeutic agents refers
to co-administration of the anti-neoplastic agents.
Co-administration may occur concurrently, sequentially, or
alternately. Concurrent co-administration refers to administration
of the anti-neoplastic agents at essentially the same time. For
concurrent co-administration, the courses of treatment with the
ethyl acetate extract of black cohosh, and with the one or more
additional chemopreventive or chemotherapeutic agents, may be run
simultaneously. For example, a single, combined formulation,
containing both an amount of the ethyl acetate extract of black
cohosh and an amount of the additional chemopreventive or
chemotherapeutic agent, in physical association with one another,
may be administered to a subject. By way of example, the single,
combined formulation may consist of a liquid mixture, containing
amounts of both anti-neoplastic agents, which may be injected into
a subject, or an oral formulation, containing amounts of both
anti-neoplastic agents, which may be orally administered to a
subject.
[0080] It is also within the confines of the present invention that
an amount of the ethyl acetate extract of black cohosh, and an
amount of the one or more additional chemopreventive or
chemotherapeutic agents, may be administered concurrently to a
subject, in separate, individual formulations. Accordingly, the
method of the present invention is not limited to concurrent
co-administration of the anti-neoplastic agents in physical
association with one another.
[0081] In the method of the present invention, the ethyl acetate
extract of black cohosh, and the one or more additional
chemopreventive or chemotherapeutic agents, also may be
co-administered to a subject in separate, individual formulations
that are spaced out over a period of time, so as to obtain the
maximum efficacy of the combination. Administration of each drug
may range in duration from a brief, rapid administration to a
continuous perfusion. When spaced out over a period of time,
co-administration of the anti-neoplastic agents may be alternate or
sequential. For alternate co-administration, partial courses of
treatment with the ethyl acetate extract of black cohosh may be
alternated with partial courses of treatment with the one or more
additional chemopreventive or chemotherapeutic agents, until a full
treatment of each drug has been administered. For sequential
co-administration, one of the anti-neoplastic agents is separately
administered, followed by the other. For example, a full course of
treatment with the ethyl acetate extract of black cohosh may be
completed, and then may be followed by a full course of treatment
with the one or more additional chemopreventive or chemotherapeutic
agents. Alternatively, for sequential co-administration, a full
course of treatment with the one or more additional chemopreventive
or chemotherapeutic agents may be completed, then followed by a
full course of treatment with the ethyl acetate extract of black
cohosh.
[0082] The anti-neoplastic agents of the present invention (i.e.,
the ethyl acetate extract of black cohosh or the actein composition
and the one or more additional chemopreventive or chemotherapeutic
agents, either in a single, combined formulation, or in separate,
individual formulations) may be administered to a human or animal
subject by known procedures, including, but not limited to, oral
administration, parenteral administration, and transdermal
administration, as described above. Preferably, the anti-neoplastic
agents of the present invention are administered orally or
intravenously. For oral administration, the formulations of the
ethyl acetate extract of black cohosh or the actein composition and
the one or more additional chemopreventive or chemotherapeutic
agents (whether individual or combined) may be presented as
capsules, tablets, powders, granules, as a suspension, or in any
other form described herein. For parenteral administration, the
formulations of the ethyl acetate extract of black cohosh or the
actein composition and the one or more additional chemopreventive
or chemotherapeutic agents (whether individual or combined) may be
combined with a sterile aqueous solution which is preferably
isotonic with the blood of the subject. Such formulations may be
prepared in accordance with methods described herein. For
transdermal administration, the formulations of the ethyl acetate
extract of black cohosh or the actein composition and the one or
more additional chemopreventive or chemotherapeutic agents (whether
individual or combined) may be combined with skin penetration
enhancers, such as propylene glycol, polyethylene glycol,
isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the
like, and prepared in accordance with methods described herein.
[0083] Additionally, in accordance with the method of the present
invention, the ethyl acetate extract of black cohosh or the actein
composition and the one or more additional chemopreventive or
chemotherapeutic agents are administered to a subject in amounts
effective to treat or prevent neoplasia and other disorders in the
subject. As discussed above, exemplary doses of actein may range
from about 0.5 .mu.g/ml to about 40.0 .mu.g/ml; exemplary doses of
paclitaxel, for example, may range from 0.5 nM to about 5.0 nM.
However, the amounts of the ethyl acetate extract of black cohosh,
or the actein composition, and the one or more additional
chemopreventive or chemotherapeutic agents, that are effective to
treat or prevent neoplasia in a subject will vary depending on the
particular factors of each case, including the type and stage of
disorders (e.g. neoplasia), the subject's weight, the severity of
the subject's condition, and the method of administration. These
amounts can be readily determined by the skilled artisan, based
upon known procedures, including analysis of titration curves
established in vivo, dose-response experiments analogous to those
provided in the Examples, and methods and assays disclosed
herein.
[0084] In one embodiment of the present invention, an ethyl acetate
extract of black cohosh or the actein composition is administered
to a subject in combination with at least one additional
chemopreventive or chemotherapeutic agent, such that a synergistic
anti-neoplastic effect is produced. As used herein, a "synergistic
anti-neoplastic effect" refers to a greater-than-additive
anti-neoplastic effect which is produced by a combination of two
drugs, and which exceeds that which would otherwise result from
individual administration of either drug alone.
[0085] In the method of the present invention, combination therapy
using an ethyl acetate extract of black cohosh or the actein
composition and at least one additional anti-neoplastic agent
preferably results in an anti-neoplastic effect that is greater
than additive, as determined by any of the measures of synergy
known in the art. One measure of synergy between two drugs is the
fractional inhibitory concentration (FIC) (Hall et al., The
fractional inhibitory concentration (FIC) index as a measure of
synergy. J. Antimicrob. Chemother., 11(5):427-33, 1983). This
fractional value is determined by expressing the IC.sub.50 of a
drug acting in combination, as a function of the IC.sub.50 of the
drug acting alone. For two interacting drugs, the sum of the FIC
value for each drug represents the measure of synergistic
interaction. Where the FIC is less than 1, there is synergy between
the two drugs. An FIC value of 1 indicates an additive effect. The
smaller the FIC value, the greater the synergistic interaction.
[0086] Another measurement of synergy is the combination index (CI)
method of Chou and Talalay (Quantitative analysis of dose-effect
relationships: the combined effects of multiple drugs or enzyme
inhibitors. Adv. Enzyme Regul., 22:27-55, 1984), which is based on
the median-effect principle. This method calculates the degree of
synergy, additivity, or antagonism between two drugs at various
levels of cytotoxicity. Where the CI value is less than 1, there is
synergy between the two drugs. Where the CI value is 1, there is an
additive effect, but no synergistic effect. CI values greater than
1 indicate antagonism. The smaller the CI value, the greater the
synergistic effect.
[0087] As the inventors have demonstrated herein, administration of
an ethyl acetate extract of black cohosh, in combination with at
least one additional chemopreventive or chemotherapeutic agent,
frequently results (unexpectedly) in a synergistic anti-neoplastic
effect, by providing greater efficacy than would result from use of
either of the anti-neoplastic agents alone. In these cases, the
ethyl acetate extract of black cohosh enhances the effects of the
additional chemopreventive or chemotherapeutic agent; therefore,
lower doses of one or both of the anti-neoplastic agents may be
used in treating and preventing neoplasias, resulting in increased
chemotherapeutic/chemopreventive efficacy, and decreased
side-effects.
[0088] By way of example, the ethyl acetate fraction of black
cohosh (2 .mu.g/ml) may be combined with doxorubicin (0.2 .mu.g/ml;
0.34 .mu.M) or paclitaxel (4 nM) for a synergistic effect.
Furthermore, actein (2 .mu.g/ml; 3.0 .mu.M) may be combined with
5-FU (0.002 .mu.g/ml, 0.015 .mu.M) for a synergistic effect; actein
(0.2 or 2 .mu.g/ml) may be combined with herceptin (8 .mu.g/ml; 54
nM) for a synergistic effect; actein (1 .mu.g/ml) may be combined
with paclitaxel (1 nM) for a synergistic effect; actein (2
.mu.g/ml; 3.0 .mu.M) may be combined with doxorubicin (0.2
.mu.g/ml; 0.34 .mu.M) for a synergistic effect; actein (2 .mu.g/ml;
2.8 .mu.M) may be combined with tamoxifen (2 .mu.g/ml; 5.4 .mu.M)
for a synergistic effect; actein (2 .mu.g/ml; 3.0 .mu.M) may be
combined with cisplatin (2 .mu.g/ml; 6.7 .mu.M) for a synergistic
effect; and actein (2 .mu.g/ml; 3.0 .mu.M) may be combined with
vinblastine (4 .mu.g/ml; 4.4 .mu.M) for an additive effect. In a
preferred embodiment of the present invention, actein (e.g., about
0.5 .mu.g/ml to about 5.0 .mu.g/ml) is administered to a subject in
combination with paclitaxel (e.g., about 0.5 nM to about 5.0
nM).
[0089] As shown herein, administration of the ethyl acetate extract
of black cohosh (particularly the extract containing one or more
triterpene glycosides, such as actein, cimifugoside, cimigenol
glycoside, cimiracemoside A, and 23-epi-26-deoxyactein), or the
actein composition, in combination with one or more additional
chemopreventive or chemotherapeutic agents (particularly the
anti-neoplastic agents, cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and vinblastine),
may unexpectedly result in a synergistic anti-neoplastic effect by
providing greater efficacy than would result from use of either of
the anti-neoplastic agents alone. Accordingly, it is also within
the confines of the present invention that a formulation of the
ethyl acetate extract of black cohosh or the actein composition and
a formulation of the one or more additional chemopreventive or
chemotherapeutic agents (whether individual or combined) may be
further associated with a pharmaceutically-acceptable carrier,
thereby comprising a combination of anti-neoplastic agents. In one
embodiment of the invention, the combination of anti-neoplastic
agents is a synergistic combination. As used herein, a "synergistic
combination" of anti-neoplastic agents refers to a combination of
anti-neoplastic agents that achieves a greater anti-neoplastic
effect than would otherwise result if the anti-neoplastic agents
were administered individually.
[0090] The formulations of the combination of the present invention
may be prepared by methods well-known in the pharmaceutical arts
and described herein. Exemplary acceptable pharmaceutical carriers
have been discussed above. An additional carrier, Cremophor.TM.,
may be useful, as it is a common vehicle for Taxol.
[0091] In the combination of the present invention, the relative
proportions of the ethyl acetate extract of black cohosh (including
the triterpene glycoside compounds) or the actein composition and
the one or more chemopreventive or chemotherapeutic agents will
depend on the specific application of the combination. Thus, while
certain proportions may be beneficial in treating one type of
tumor, entirely different proportions may be beneficial in treating
other tumors. Such a determination can be made by a person skilled
in the art, in accordance with methods known in the art and
described in the Examples provided below. Some preferred
combinations, containing at least one triterpene glycoside compound
in the ethyl acetate extract of black cohosh, and at least one
additional chemopreventive or chemotherapeutic agent, may be
formulated such that the amount of the triterpene glycoside is
selected synergistically to enhance the effect of the
chemopreventive or chemotherapeutic agents, while alleviating
unwanted side effects attributable to such agents. Exemplary
combinations comprising the ethyl acetate extract of black cohosh,
and at least one additional chemopreventive or chemotherapeutic
agent, are described above. In a preferred embodiment of the
present invention, the combination comprises actein (e.g., about
0.5 .mu.g/ml to about 5.0 .mu.g/ml) and paclitaxel (e.g., about 0.5
nM to about 5.0 nM).
[0092] In the combination of anti-neoplastic agents of the present
invention, the ethyl acetate extract of black cohosh, or the actein
composition, and the one or more additional chemopreventive or
chemotherapeutic agents, may be combined in a single formulation,
such that the extract is in physical association with the agent.
This single, combined formulation may consist of a liquid mixture,
containing amounts of both the extract and the agent, which may be
injected into a subject, or an oral formulation, containing amounts
of both the extract and the agent, which may be orally administered
to a subject.
[0093] Alternatively, in the combination of the present invention,
a separate, individual formulation of the extract may be combined
with a separate, individual formulation of the agent. For example,
an amount of the extract may be packaged in a vial or unit dose,
and an amount of the agent may be packaged in a separate vial or
unit dose. A combination of the extract and the agent then may be
produced by mixing the contents of the separate vials or unit doses
in vitro. Additionally, a synergistic combination of the extract
and the agent may be produced in vivo by co-administering to a
subject the contents of the separate vials or unit doses, according
to the methods described above. Accordingly, the combination of the
present invention is not limited to a combination in which amounts
of the extract and the agent are in physical association with one
another in a single formulation.
[0094] It is also within the confines of the present invention for
the ethyl acetate extract of black cohosh, or the actein
composition, and the one or more additional chemopreventive or
chemotherapeutic agents, to be co-administered in combination with
radiation therapy or an anti-angiogenic compound (either natural or
synthetic). Examples of anti-angiogenic compounds with which the
anti-neoplastic agents may be combined include, without limitation,
angiostatin, thalidomide, and thrombospondin.
[0095] The combination of anti-neoplastic agents of the present
invention comprises an effective anti-neoplastic amount of the
ethyl acetate extract of black cohosh and an effective
anti-neoplastic amount of the one or more additional
chemopreventive or chemotherapeutic agents. As used herein, an
"effective anti-neoplastic amount" of the extract or the agent is
an amount of the extract or the agent that is effective to
ameliorate or minimize the clinical impairment or symptoms of
neoplasia in a subject, in either a single or multiple dose.
[0096] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention, and should not be construed to limit in any way the
scope of the invention as defined in the claims which follow
thereafter.
EXAMPLES
Example 1
Chemicals and Reagents
[0097] Polyamide resin SC6<0.07 mm was purchased from Alltech
Associates, Inc. (Deerfield, Ill.). RP.sub.18CC silica gel (40
.mu.M) was obtained from J. T. Baker (Phillipsburg, N.J.), and the
RP.sub.18F.sub.254 plate (1-mm layer thickness) was obtained from
EM Science (Darmstadt, Germany). Actein, 27-deoxyactein
(23-epi-26-deoxyactein) (Zheng et al., CimiPure (Cimicifuga
racemosa): a standardized black cohosh extract with novel
triterpene glycoside for menopausal women. In Phytochem.
Phytopharm., Shahidi and Ho, eds. (Champaign, Ill.: AOCS Press,
2000) pp. 360-70), cimifugoside, and cimiracemoside A were obtained
from ChromaDex (Laguna Hills, Calif.), and 27-deoxyactein was also
obtained from Herbstandard (Chesterfield, Mo.). Tamoxifen,
5-fluorouracil (5-FU), doxorubicin, cisplatin, and paclitaxel were
purchased from Sigma (St. Louis, Mo.). Herceptin was obtained from
Genentech (CA). Cimigenol and cimigenol glycoside were obtained
from Dr. WC Ye (Department of Phytochemistry, China Pharmaceutical
University, Nanjing 210009, China).
[0098] Black cohosh extracts and purified components were dissolved
in dimethylsulfoxide (DMSO) (Sigma Chemical Co.). Water (H.sub.2O)
was distilled and deionized. All solvents and reagents were reagent
grade.
Example 2
Plant Material
[0099] Black cohosh roots and rhizomes (GFP) were obtained from
PureWorld Botanicals (South Hackensack, N.J.; lot number
9-2677).
Example 3
Separation of the Ethyl Acetate Extract
[0100] As shown in FIG. 1, black cohosh roots and rhizomes were
extracted with 80% methanol (MeOH)/H.sub.2O, and partitioned with
n-hexane. Two layers were obtained: a water layer and an n-hexane
layer. N-hexane was used to extract the non-polar phytochemicals,
respectively, with yields of 0.05% hexane, 0.73% ethyl acetate, and
1.69% water. The water layer was partitioned with ethyl acetate,
and two fractions were obtained: a water layer and an ethyl acetate
layer. Ethyl acetate was used to extract the mid-polar and polar
phytochemicals. The ethyl acetate layer was dried and evaporated to
yield an ethyl acetate extract. The triterpene glycosides and
cinnamic acid esters were separated from the ethyl acetate extract
by polyamide chromatography (Kruse et al., Fukic and piscidic acid
esters from the rhizome of Cimicifuga racemosa and the in vitro
estrogenic activity of fukinolic acid. Planta. Med., 65:763-64,
1999).
Example 4
Cell Cultures
[0101] MDA-MB-453 human breast cancer cells (HER2 overexpressing,
ER negative), MCF7 cells (ER positive, HER2 low), MDA-MB-231 cells
(ER negative, HER2 low), MCF10F cells (normal mammary epithelial
cells), and SW480 colon cancer cells were obtained from ATCC
(Manassas, Va.). BT474 clone Sc-1 cells (ER positive, Her2
overexpressing) were the kind gift of Dr. S. Friedman (Incyte
Pharmaceuticals). Cells were grown in Dulbecco's Modified Eagle
medium (DMEM) (Gibco BRL Life Technologies, Inc., Rockville, Md.)
containing 10% (v/v) fetal bovine serum (FBS) (Gibco BRL), at
37.degree. C. and 5% CO.sub.2. The medium was supplemented with
bovine insulin (0.01 mg/ml) for the growth of BT474 cells.
Example 5
Cell-Growth Assays
[0102] Cell cultures were treated with increasing concentrations of
extracts and/or purified compounds for increasing times and
cytoxicity (for SW480 cells) measured using the MTT
{3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolilum bromide}
(Dojindo, Tokyo, Japan) method (Luo et al., PM-3, a benzo-g-pyran
derivative isolated from propolis, inhibits growth of MCF-7 human
breast cancer cells. Anticancer Res 21: 1665-1672, 2001) and
inhibition of cell proliferation by performing cell counts using a
Coulter Counter (Lim et al., 1999, supra). For the cell count
assay, breast cancer cells were seeded, in triplicate, at
2.times.10.sup.4 cells per well, in 24- or 96-well plates. Two or 3
days later, the medium was replaced with fresh medium--with or
without black cohosh extracts or purified compounds--and the number
of attached viable cells was counted at increasing times (or, to
determine IC.sub.50 values, at 48 or 96 h), using a Coulter
Counter, model Z.sub.F (Coulter Electronics Inc., Hialeah, Fla.)
(Lim et al., Sulindac derivatives inhibit growth and induce
apoptosis in human prostate cancer cell lines. Biochem. Pharmacol.,
58:1097-107, 1999).
[0103] For the MTT assay, cells were seeded at 3.times.10.sup.3
cells per well in 96-well plates; 24 hours later the medium was
replaced with fresh medium containing black cohosh extracts or
components and assayed with MIT reagents at 48 hours.
[0104] To determine the combination index (CI) for potential
combination therapies, the inventors treated the breast cancer
cells with all combinations of 3 concentrations of the black cohosh
component and 3 concentrations of the chemotherapy agent, using a
solvent control. Surviving cells were counted using the Coulter
Counter (Masuda et al., Effects of epigallocatechin-3-gallate on
growth, epidermal growth factor receptor signaling pathways, gene
expression, and chemosensitivity in human head and neck squamous
cell carcinoma cell lines. Clinical Cancer Research, 7:4220-29,
2001). Data that were obtained were analyzed for possible
synergistic effects using previously-described methods (e.g., the
median-effect plot method of Chou and Talalay (Quantitative
analysis of dose-effect relationships: the combined effects of
multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22:27-55,
1984)). The CIs were calculated using the index-isobologram method
(Soriano et al., Synergistic effects of new chemopreventive agents
and conventional cytotoxic agents against human lung cancer cell
lines. Cancer Res., 59:61, 78-84, 1999) based on the median-effect
principle of Chou and Talalay, 1984, supra.
Example 6
Statistical Analysis
[0105] The inventors were interested in determining the effects of
combinations of actein and paclitaxel concentrations on MDA-MB-453
cells. In calculating statistical significance, a Two-Way Analysis
of Variance (ANOVA) was performed to test whether the effects of
paclitaxel and actein concentrations were independent, or were
related, or "interacted" with each other (alpha=0.05; significant
difference=p<0.05; very significant difference=p<0.01). If
the F-test showed that the interaction was significant, the Least
Significant Difference method (LSD) was then used for multiple
comparisons, to clarify the significance between the different
combinations of paclitaxel and actein concentrations.
Example 7
Cell-Cycle Analysis
[0106] To obtain exponential cultures of breast cancer cells,
3.times.10.sup.5 cells were plated onto 10-cm dishes, and grown for
2-3 days; the medium was then replaced with fresh medium containing
black cohosh extracts or purified components alone and in
combination with chemotherapy agents. Synchrony: To synchronize the
cells, 3.times.10.sup.5 cells were plated onto 10 cm dishes and
grown for 2 days in DMEM supplemented with 10% fetal bovine serum.
The medium was then replaced with DMEM containing 0.25% FBS (Imoto
et al., 1997) and black cohosh extracts or purified components.
[0107] After incubation for 1-3 days, the supernatant was
collected, and the cells were trypsinized, collected, and washed
with phosphate buffered saline (PBS) containing 5% FBS. Cell
pellets were re-suspended in 1 ml of PBS plus 5% FBS. Thereafter, 5
ml of 70% ethanol were added drop-wise, while vortexing the tube,
and the mixture was stored at 4.degree. C. Cells were centrifuged,
washed with PBS plus 5% FBS, and re-suspended in 400 .mu.l of
propidium iodide (0.1 mg/ml) (Sigma Chemical Co.). 400 .mu.l (2
mg/ml) of RNase (Sigma Chemical Co.) were added, and the cells were
incubated in the dark at room temperature for 30 min. The
suspension was filtered through a 41-.mu.M spectra/mesh filter
(Spectrum Medical Industries, CA), and analyzed with a FACScalibur
instrument (Becton Dickinson, Franklin Lakes, N.J.) equipped with
Cell Quest software (Becton Dickinson). The percentage of cells in
different cell-cycle phases was then calculated (Lim et al.,
Sulindac derivatives inhibit growth and induce apoptosis in human
prostate cancer cell lines. Biochem. Pharmacol., 58:1097-107, 1999;
Luo et al., PM-3, a benzo-g-pyran derivative isolated from
propolis, inhibits growth of MCF7 human breast cancer cells.
Anticancer Research, 21:1665-72, 2001; Soh et al., Cyclic GMP
mediates apoptosis induced by sulindac derivatives via activation
of c-Jun NH2-terminal kinase 1. Clin. Cancer Res., 10:4136-41,
2000).
Example 8
Western-Blot Analysis
[0108] Cells were treated for increasing times with approximately
the IC.sub.50 concentration, or twice the IC.sub.50 concentration,
of actein. The cells were harvested, washed with PBS, and sonicated
in extraction buffer according to the procedure of Han et al. (Han
et al., Stable overexpression of cyclin D1 in a human mammary
epithelial cell line prolongs the S-phase and inhibits growth.
Oncogene, 10:953-61, 1995). The lysates were subjected to
electrophoresis on a 10% or 12.5% SDS-polyacrylamide gel, and then
transferred to a polyvinylidene difluoride (PVDF) membrane. The
membrane was blocked with milk protein, and incubated with a
solution containing the primary antibody against the following:
cyclin D1 (Upstate Biotechnology, Lake Placid, N.Y.), p21.sup.ciP1
(Oncogene Research Products, Darmstadt, Germany), ppRb (ser 780,
Medical and Biological Laboratories, Nagoya, Japan), cdk4 (Upstate
Biotechnology, Lake Placid, N.Y.), EGFR (clone-74, Transduction
Laboratories, Lexington, Ky.), p-EGFR (phospho (Y1173)-EGFR) (Cell
Signaling, Beverly, Mass.), actin (Sigma, St. Louis, Mo.),
Her-2/neu (Cell Signaling, Beverly, Mass.), or
phospho-(Y1248)-Her-2/neu (Cell Signaling, Beverly, Mass.)),
I.kappa.B (Sant Cruz Biotechnolgy, Santa Cruz, Calif.),
I.kappa..kappa.B (Sigma, St. Louis, Mo.) and PPAR.gamma. (Santa
Cruz biotechnology, Santa Cruz, Calif.) (Masuda et al.,
Epigallocatechin-3-gallate inhibits activation of HER-2/neu and
downstream signaling pathways in human head and neck and breast
carcinoma cells. Clin. Cancer Res., 9: 3486-91, 2003). The membrane
was washed, and incubated with horseradish peroxidase conjugated
secondary antibody.
[0109] Protein bands were visualized with the ECL-enhanced
chemiluminescence system, according to the manufacturer's
directions (Amersham Pharmacia Biotech) (Sgambato et al.,
Overexpression of p27 (Kip1) inhibits the growth of both normal and
transformed human mammary epithelial cells. Cancer Research,
58:3448-54, 1998). The staining intensities of the visualized blots
were quantified using NIH image software. For each protein, the
relative band intensities were determined by comparing treated
samples with untreated controls. These values were then normalized,
using .beta.-actin as an internal control.
Example 9
Thin-Layer Chromatography Analysis
[0110] Extracts were tested for triterpene glycosides and cinnamic
acid esters using silica gel 60 F.sub.254 plates (0.25-mm layer
thickness) and RP.sub.18F.sub.254 plates (1-mm layer thickness)
from EM Science (Darmstadt, Germany). The solvent system for the
silica gel thin-layer chromatography (TLC) was chloroform-MeOH
(9:1); the solvent system for the RP.sub.18 plates was
MeOH--H.sub.2O (9:1). After development, the compounds were
visualized under UV, and visualized by spraying with vanillin in
10% (v/v) H.sub.2SO.sub.4 in ethanol (EtOH).
Example 10
Polyamide Chromatography
[0111] Polyamide SC6 resin (1.5 gm), pre-conditioned with MeOH (15
min) and H.sub.2O (10 min), was packed under pressure in a 12-ml
syringe (approximately 3.3 cm in height, with a column volume of
4.5 ml); the syringe was then rinsed with water. The black cohosh
ethyl acetate extract (100 mg) was dissolved in 1 ml of
H.sub.2O/MeOH (1:1), and adsorbed to the polyamide column for 20
min before elution. The column was then eluted sequentially, twice,
with 6 ml of H.sub.2O/MeOH (50:50), H.sub.2O/MeOH (75:25), MeOH,
EtOH, and EtOH+0.1% TFA, to yield 10 fractions (Kruse et al., Fukic
and piscidic acid esters from the rhizome of Cimicifuga racemosa
and the in vitro estrogenic activity of fukinolic acid. Planta.
Med., 65:763-64, 1999).
Example 11
Cyclin D1 Reporter Assay
[0112] The cyclin D1 promoter luciferase reporter plasmid,
1745CD1LUC, was prepared by Dr. R. Pestell (Albert Einstein Cancer
Center, New York, N.Y.). The method used for transient transfection
reporter assays was previously described (Soh et al., Novel roles
of specific isoforms of protein kinase C in activation of the c-fos
serum response element. Molecular and Cellular Biology, 19:1313-24,
1999). Using lipofectin, triplicate samples of MDA-MB-453 breast
cancer cells (1.times.10.sup.5 cells in 35-mm plates) were
co-transfected using DNA of the indicated reporter plasmid (1
.mu.g) and the (3-gal plasmid as an internal control (10 .mu.g of
the pCMV-b-gal plasmid) in opti-MEM 1 medium (Life Technologies,
Inc.). After 24 h, the medium was replaced with serum-free medium
containing the indicated concentrations of actein. After 24 h,
cells were harvested, and luciferase activity was determined with
the luciferase assay system (Promega Corp. Madison, Wis.);
.beta.-gal activities were determined with the .beta.-gal enzyme
assay system (Promega). Luciferase activities were normalized to
.beta.-gal activities, to correct for differences in transfection
efficiency.
Example 12
Bioactivity-Guided Fractionation
[0113] Open chromatography techniques were used to fractionate the
extracts further. The stationary phases used included Diaion HP-20,
Sephadex, normal and reversed-phase silica, and polyamide.
[0114] 1. Alcoholic black cohosh powder extract was redissolved in
MeOH/H.sub.2O, and evaporated to dryness, leaving the water
portion.
[0115] 2. To partition the phytochemicals according to polarity,
the water portion was partitioned sequentially with .sub.hexane and
n-butanol (n-BuOH). The three resulting fractions--hexane, n-BuOH,
and water--were evaporated to dryness, and tested for their effects
on the growth of MCF7 breast cancer cells. The n-BuOH extract
showed high activity (FIG. 11).
[0116] 3. The n-BuOH extract was further separated using Diaion
HP-20 as a stationary phase, and eluting sequentially with
MeOH/H.sub.2O (1:1), MeOH, and acetone. By thin-layer
chromatography, the MeOH/H.sub.2O (1:1) contained mostly
UV-absorbing compounds (aromatic acid derivatives), while the MeOH
contained mostly triterpenoids (FIG. 11).
[0117] 4. Further separation of the MeOH/H.sub.2O fraction, over
silica gel, RP.sub.18, and polyamide columns, yielded isoferulic
acid, ferulic acid, and caffeic acid. Preliminary experiments
indicated that isoferulic, the more potent, and ferulic acids were
active in suppressing the growth of MCF7 human breast cancer cells
(FIG. 12).
[0118] Summarized below are results obtained by the inventors in
connection with the experiments described in Examples 1-12:
Effects of Extracts of Black Cohosh on the Growth of Human Breast
Cancer Cells
[0119] Black cohosh roots and rhizomes were extracted with
MeOH/H.sub.2O, and fractionated by solvent-solvent partitioning to
yield three fractions: hexane, ethyl acetate (EtOAc), and H.sub.2O
(FIG. 1). These fractions were assayed for growth inhibition on
human breast cancer cell lines. By TLC, it was determined that
triterpene glycosides are present at the highest level in the EtOAc
extract; low levels were detected in the hexane and water
extracts.
[0120] The effects of increasing amounts of the three black cohosh
fractions on the growth of the (ER+) human breast cancer cell line,
MCF7, were determined after exposure of the cells for 96 h. The
results, expressed as IC.sub.50 values (i.e., the concentration
that causes approximately 50% inhibition of growth), are set forth
in Table 1. The results indicate that the EtOAc extract was the
most active fraction.
[0121] The inventors tested the effects of crude extracts, methanol
and ethanol, as well as ethanol extracts provided by Pure World,
native and plus expedient: the IC.sub.50 values for these extracts
after 96 hours of treatment of MDA-MB-453 cells were: methanol: 100
.mu.g/ml; ethanol: >200 .mu.g/ml; PW native 175 .mu.g/ml: and PW
expedient: 195 .mu.g/ml.
[0122] To partition the phytochemicals according to polarity, the
water portion was also partitioned sequentially with hexane and
n-butanol (n-BuOH). The n-BuOH fraction was tested for its effect
on the growth of MDA-MB-453 breast cancer cells. The IC50 value
after 96 hours of treatment was: 40 .mu.g/ml.
[0123] The inventors also examined the effects of the EtOAc
fraction of black cohosh on SW480 human colon cancer cells. The
IC50 values after 48 hours of incubation using the MTT assay were:
SW480: 42 .mu.g/ml; MCF7: 38 .mu.g/ml (Luo et al., PM-3, a
benzo-g-pyran derivative isolated from propolis, inhibits growth of
MCF-7 human breast cancer cells. Anticancer. Res. 21: 1665-1672,
2001).
TABLE-US-00001 TABLE 1 Effects of black cohosh extracts on MCF7
cells. IC.sub.50 Values (.mu.g/ml) Black Cohosh Extracts H.sub.2O
extract 150 ethyl acetate extract 18 hexane extract 28 Purified
Components Actein 14 (21 .mu.M) 23-cpi-26-deoxyactein 21 (32 .mu.M)
Cimifugoside 22 (36 .mu.M) cimiracemoside A 41 (61 .mu.M)
[0124] The effects of two concentrations of the EtOAc fraction on
the growth of MCF7 cells were examined at increasing times.
Exposure to 20 .mu.g/ml of the EtOAc fraction led to partial
inhibition of cell proliferation as early as 24 h after addition;
40 .mu.g/ml resulted in complete inhibition and cell death after 72
h (FIG. 2A), while 60 .mu.g/ml resulted in cell death at 24 h.
[0125] Two major signaling pathways in breast cancer cells are the
ER-mediated signaling pathway (exemplified in the
estrogen-dependent human breast cancer cell line, MCF7) and the
HER2-mediated signaling pathway (exemplified in the
estrogen-independent human breast cancer cell line, MDA-MB-453,
which overexpresses HER2 (erb2, c-neu), a membrane-associated
tyrosine kinase receptor (p185 HER2)). Clinical studies indicate
that a reciprocal relationship often occurs in the expression of
the two pathways in primary human breast cancers (Tsutsui et al.,
Prognostic value of c-erbB2 expression in breast cancer. J. Surg.
Oncol., 79:216-33, 2002). It was important, therefore, for the
inventors to determine if black cohosh extracts have different
effects on the two cell types. Accordingly, the following three
breast cancer cell lines were tested: MCF7 (ER positive, HER2 low),
MDA-MB-231 (ER negative, HER2 low), and MDA-MB-453 (HER2
overexpressing, ER negative).
[0126] Treatment with the EtOAc fraction for 48 h inhibited the
growth of all three cell lines, with IC.sub.50 values in the range
of 20-40 .mu.g/ml (Table 2). The Her2 overexpressing cells were the
most sensitive. It is of interest that the normal human mammary
epithelial cell line, MCF10F, was considerably less sensitive, with
an IC.sub.50 value of 85 .mu.g/ml.
TABLE-US-00002 TABLE 2 Effects of black cohosh extracts on breast
cancer cells. Cells Receptors Expressed IC.sub.50 (.mu.g/ml)
MDA-MB-453 ER-/HER2+ 18 MCF7 ER+/HER2- 35 MDA-MB-231 ER+/HER2- 39
MCF10F Normal Mammary 85 Epithelial Cells (ER-)
[0127] Observed over a 48-h period, the approximate doubling times
for the malignant cells were 36 h for MDA-MB-453, 32 h for MCF7,
and 30 h for MDA-MB-231; the approximate doubling time for the
non-malignant MCF10F cells was 48 h. It is possible that the
greater sensitivity of the malignant cells may reflect, in part,
the difference in growth rates. The IC.sub.50 values were less when
the cells were treated for 96 h: 18 .mu.g/ml for MCF7 cells, 10
.mu.g/ml for MDA-MB-453 cells, and 46 .mu.g/ml for MCF10F cells.
Based upon these results, it can be concluded that the EtOAc
fraction of black cohosh does not act specifically through the ER
or the Her2 receptors.
[0128] Characterization of the Active Components in the Ethyl
Acetate Extract
[0129] As the ethyl acetate extract of black cohosh contains many
components, it was important for the inventors to identify the
specific active compounds and their modes of action.
[0130] To separate the triterpene glycosides from the aromatic
acids and esters, the ethyl acetate extract was fractionated on a
polyamide SC6 column (Kruse et al., Fukic and piscidic acid esters
from the rhizome of Cimicifuga racemosa and the in vitro estrogenic
activity of fukinolic acid. Planta. Med., 65:763-64, 1999). The
first four fractions (water/methanol--50:50; 75:25), which are
enriched for triterpene glycosides, suppressed the growth of MCF7
cells. Incubation with fraction 1 (5.7 .mu.g/ml) resulted in 25%
cell death; incubation with fraction 2 (23 .mu.g/ml) resulted in
67% cell death; and incubation with fraction 3 (30 .mu.g/ml)
resulted in 73% cell death. In view of these results, it appears
that the triterpene glycosides are among the active components in
the ethyl acetate extract.
Effects of Triterpene Glycoside Fraction and Pure Components on
Cell Proliferation
[0131] To ascertain the nature of the triterpene glycosides of
black cohosh, the purified triterpene glycosides (set forth in FIG.
3) were tested for growth inhibition on MCF7 cells (FIG. 2B and
Table 1). Actein, which has an hydroxyl group on the C-26 position
of 23-epi-26-deoxyactein (Chen et al., Isolation, structure
elucidation, and absolute configuration of 26-deoxyactein from
Cimicifuga racemosa and clarification of nomenclature associated
with 27-deoxyactein. J. Nat. Prod., 65:601-05, 2000)), had an
IC.sub.50 of 21 .mu.M; it was approximately 1.5-fold more potent
than 23-epi-26-deoxyactein or cimifugoside, and approximately 3
times more potent than cimiracemoside A, in inhibiting the growth
of MCF7 cells (Table 1). The substitution of an hydroxyl on the
aglycone moiety can significantly alter this inhibitory
activity.
[0132] The effects of two concentrations of actein on the
proliferation of MCF7 cells were examined at increasing times.
Treatment with actein (15 .mu.g/ml) resulted in partial inhibition
of growth, within 24 h after addition of the compound, while
treatment with actein at 30 .mu.g/ml resulted in complete
inhibition of growth (FIG. 2B). In additional studies, it was found
that MCF7 cells were approximately three times more sensitive to
growth inhibition by actein than the MCF10F normal mammary
epithelial cells; the respective IC.sub.50 values were 14 .mu./ml
vs. 42 .mu.g/ml, when measured at 96 h of exposure. The mean of the
MCF7 cells that were alive (38.0%.+-.3.0) after 96 h of treatment
with actein (20 .mu.g/ml) was significantly less than the mean of
the MCF10F cells that were alive (63.8%.+-.1.4) after 96 h of
treatment with actein (20 .mu.g/ml) (p<0.01). As was the case
for the EtOAc fraction, the MDA-MB-453 cells were the most
sensitive to treatment with actein--with an IC.sub.50 value of
approximately 8 .mu.g/ml at 96 h.
Effects of the EtOAc Extract and Purified Components of Black
Cohosh on Cell-Cycle Kinetics
[0133] The ability of an extract or purified compound to affect
specific phases of the cell cycle may provide clues to its
mechanism of action (Weinstein, I. B., Disorders of cell circuitry
during multistage carcinogenesis: the role of homeostasis.
Carcinogenesis, 5:857-64, 2000). To determine the effects of black
cohosh on the cell cycle, MCF7 cells were treated with 30 and 60
.mu.g/ml of the EtOAc fraction of black cohosh, or 30 and 60
.mu.g/ml of actein, for 48 h. The cells were then stained with
propidium iodide, and analyzed by DNA flow cytometry (FIG. 4).
After exposure to 30 .mu.g/ml of the EtOAc fraction, there was an
increase of cells in G1 (from 70% to 82%) when compared to the DMSO
solvent control, and a concomitant decrease of cells in S (9% to
3%) and G2/M (19% to 12%). After treatment with 60 .mu.g/ml of the
EtOAc fraction, there was a decrease of cells in G1 (68% to 58%)
and an increase of cells in G2/M (21% to 31%).
[0134] The above results indicate that the extract contains more
than one component, with the more active or abundant component
inducing G1 arrest, and the less active component inducing G2/M
arrest, and/or that individual components in the extract exert
different effects at different concentrations. To distinguish
between these possibilities, cells were treated with the purified
compound, actein, at 30 and 60 .mu.g/ml. Exposure to actein at 30
.mu.g/ml also resulted in an increase of cells in G1 (70% to 82%)
and a decrease of cells in G2/M (19% to 12%). After exposure to 60
.mu.g/ml of actein, there was also an increase of cells in G1 (68%
to 77%), and a decrease of cells in G2/M (21% to 18%). Thus, with
60 .mu.g/ml of actein, the inventors did not observe the increase
in G2/M cells that was seen with 60 .mu.g/ml of the EtOAc extract
(FIG. 4).
[0135] To examine in greater detail the effects of actein on
cell-cycle progression, MCF7 cells were treated with 0, 10, 20, or
40 .mu.g/ml of actein, and analyzed at 0, 24, and 48 h by DNA flow
cytometry. FIG. 5 summarizes the results obtained with respect to
the percent of cells in G1. When cells were treated with 10
.mu.g/ml of actein, the percentage of cells in G1 increased from
64% at time zero to 75% at 24 h, and to 77% at 48 h. With 20
.mu.g/ml of actein, the respective values were 64%, 77%, and 87%;
with 40 .mu.g/ml of actein, the respective values were 64%, 74%,
and 79%. These increases in the G1 population were associated with
decreases in both the S and G2/M populations of cells. Indeed, the
maximal increase in the G1 population occurred at about 20 .mu.g/ml
actein. Therefore, it is possible that, at high concentrations,
actein and related compounds affect proteins that regulate later
phases in the cell cycle. The triterpene glycoside fraction of
black cohosh (polyamide eluate, fraction 3), 23-epi-26-deoxyactein,
and cimiracemoside A also induced cell-cycle arrest at G1, when
tested at about 40 .mu.g/ml.
[0136] Treatment with the EtOAc fraction at 30 .mu.g/ml induced a
small amount of apoptosis for 48 h (1.3%); at 60 .mu.g/ml, there
was a further increase in apoptosis (3.2%), as determined by the
sub G1 fraction (FIG. 4). When the cells were exposed to 20
.mu.g/ml actein for 48 h, approximately 1.4% of the population
displayed apoptosis; at 72 h, this value was 3.6%, when assessed by
the size of the sub G1 peak.
Effects of Actein on the Expression of Specific Proteins Involved
in Cell-Cycle Control and Apoptosis
[0137] Since actein induces cell-cycle arrest at G1, the inventors
examined the effect of actein on proteins which control the
progression of the cell cycle. Cyclin D1 was of particular
interest, since it plays a critical role in mediating the
transition from G1 to S, is overexpressed in approximately 50-60%
of primary human breast carcinomas (Joe et al., Cyclin D1
overexpression is more prevalent in non-Caucasian breast cancer.
Anticancer Res., 21:3535-39, 2001), and is overexpressed in several
human breast cancer cell lines (Han et al., Effects of sulindac and
its metabolites on growth and apoptosis in human mammary epithelial
and breast carcinoma cell lines. Breast Cancer Res. Treat.,
48:195-203, 1998). Therefore, the inventors monitored possible
changes in cellular levels of cyclin D1 by Western-blot analysis of
extracts obtained from control and actein-treated cells.
[0138] Treatment of MCF7 cells with 40 .mu.g/ml of actein for 3 or
10 h resulted in a partial decrease, and treatment for 24 h caused
a marked decrease, in the cellular level of cyclin D1, when
compared to comparable time points in the control (untreated)
cells. Indeed, after treatment with 40 .mu.g/ml for 24 h, there was
almost a complete loss of this protein (FIG. 6A). The MCF10F normal
mammary epithelial cells did not express an appreciable level of
cyclin D1. Thus, the inventors could not assess the effect of
actein on cyclin D1 in these cells.
[0139] Cyclin D1 binds to and activates the cyclin dependent
kinases, cdk4 and cdk6; the resulting complexes phosphorylate and
inactivate pRb (retinoblastoma protein), thereby preventing pRb
from inhibiting the transcription factor, E2F, and allowing the
cells to progress from G1 to S (Weinstein, I. B., Disorders of cell
circuitry during multistage carcinogenesis: the role of
homeostasis. Carcinogenesis, 5:857-64, 2000). The inventors
examined the effect of actein on the cellular level of the
inactivated, hyperphosphorylated form of Rb (designated ppRb).
After treatment with actein, the intensities of the ppRb bands
relative to the .beta.-actin bands were: 1.51 (3 h, 20 .mu.g/ml),
1.59 (3 h, 40 .mu.g/ml), 0.61 (10 h, 20 .mu.g/ml), 0.64 (10 h, 40
.mu.g/ml), 0.80 (24 h, 20 .mu.g/ml), and 0.43 (24 h, 40 .mu.g/ml).
The inventors found that there was a increase in the level of ppRb
at 3 hours and a decrease at 10 hours after treating MCF7 cells
with 20 or 40 .mu.g/ml actein; there was a marked decrease at 48
hours after exposure to 40 .mu.g/ml actein (FIG. 6B). The inventors
also observed a decrease in the level of cdk4 at 10 hours after
treatment with 20 or 40 .mu.g/ml actein and a pronounced decrease
at 24 hours after exposure to 40 .mu.g/ml actein (FIG. 6C).
[0140] The cdk inhibitory protein p21.sup.cip1 negatively regulates
the activity of the cyclin D1/cdk4 complex. Therefore, the
inventors examined the effect of actein on this protein. After
exposure to actein, the intensities of the p21.sup.cip1 bands
relative to the .beta.-actin bands were: 1.47 (3 h, 20 .mu.g/ml),
1.17 (3 h, 40 .mu.g/ml), 1.75 (10 h, 20 .mu.g/ml), 1.37 (10 h, 40
.mu.g/ml), 0.94 (24 h, 20 .mu.g/ml), and 0.78 (24 h, 40 .mu.g/ml).
Thus treatment of MCF7 cells with 20 or 40 .mu.g/ml of actein
induced an increase in p21.sup.cip1 within 3 hours and this
increase persisted at 10 hours. The increase was more pronounced
after treatment with 20 .mu.g/ml. However, this increase was not
seen with the 20 or 40 .mu.g/ml dose at. 24 hours (FIG. 6D).
[0141] In view of the foregoing, the ability of actein to arrest
cells in G1 (FIG. 5) may be due to the decreased expression of
cyclin D1 and cdk4, and the increased expression of
p21.sup.cip1-both of which result in a decrease in the level of the
hyperphosphorylated form of pRb.
[0142] The level of the epidermal growth factor receptor (EGFR),
which is overexpressed in various cancers (Masuda et al., Effects
of epigallocatechin-3-gallate on growth, epidermal growth factor
receptor signaling pathways, gene expression, and chemosensitivity
in human head and neck squamous cell carcinoma cell lines. Clinical
Cancer Research, 7:4220-29, 2001), was not significantly affected
by treatment with actein (FIG. 6E). There was also not a consistent
effect of actein on the phosphorylated and activated form of EGFR
(p-EGFR). However, the inventors did observe a significant decrease
with the 40 .mu.g/ml dose at 24 h (FIG. 6F).
The Effects of Actein and the Ethyl Acetate Extract of Black
Cohosh--Alone and in Combination with Chemotherapy Agents--on the
Proliferation of Human Breast Cancer Cells
[0143] It was essential for the inventors to explore the effects of
actein (h e structure of which is set forth in FIG. 3) and extracts
from black cohosh on Her2 overexpressing breast cancer cells, such
as MDA-MB-453 cells, because these cells appeared to be more
sensitive to inhibition by the black cohosh components, and because
Her2 overexpressing breast cancers have a poorer clinical
prognosis. To determine the interaction of black cohosh with
chemotherapeutic drugs, actein was combined with several different
classes of drugs. Among the chemotherapy drugs tested were the
taxane, paclitaxel (Taxol); the selective estrogen receptor
modulator (SERM), tamoxifen; the anthracycline antibiotic,
doxorubicin; the anti-Her2 monoclonal antibody, herceptin (rhuMab
Her2); the antimetabolite, 5-fluorouracil; the platinum analog,
cisplatin; and the vinca alkaloid, vinblastine. The SERM,
tamoxifen, was tested on ER+MCF7 cells; the Her2 antibody and the
remainder of the agents were tested on MDA-MB-453 cells. The
combinations of actein with herceptin and the EtOAc extract with
doxorubicin were also tested on BT474 human breast cancer cells,
which form xenografts in athymic mice.
[0144] The results for the combination of actein and Taxol are
shown in FIG. 7. IC.sub.50 values obtained from the graphs were
used to calculate the combination index (CI) (Table 3). The
inventors found that actein (2 .mu.g/ml) potentiates the effect of
Taxol at concentrations of 1 and 4 .mu.M. These concentrations are
reported to be attainable in the blood after treatment with
Taxol.
TABLE-US-00003 TABLE 3 Combination index values for the combination
of actein and paclitaxel on MDA-MB-453 cells. Actein (ug/mL) Taxol
(nM) 0.1 1 10 0.25 2.10 -- 1.70 -- 1.00 +/- 1 1.15 - 0.75 ++ 0.05
+++ 4 1.10 +/- 0.70 ++ 0.00 +++ Symbols: CI -- >1.3 antagonism -
1.1-1.3 moderate antagonism +/- 0.9-1.1 additive effect + 0.8-0.9
slight synergism ++ 0.6-0.8 moderate synergism +++ <0.6
synergism IC.sub.50 values determined from the graphs in FIG. 7
were used to obtain combination index values: CI = {IC.sub.50
(actein + paclitaxel)/IC.sub.50 (actein alone)} +{IC.sub.50
(paclitaxel + actein)/IC.sub.50 (actein alone)}.
Results of Statistical Analyses
[0145] In the two-way ANOVA analysis, the F-test showed very
significant differences (p values approaching to zero) among the
paclitaxel concentrations, among the actein concentrations, and
among the combinations of actein and paclitaxel concentrations
(i.e., there were very significant interactions between the actein
and paclitaxel concentrations).
[0146] The LSD t-test indicated that, under the fixed paclitaxel
concentrations, 0 and 0.25 nM, there were very significant
differences (p<0.01) among the four different actein
concentrations. Under the paclitaxel concentration, 1 nM, there
were very significant differences between the actein
concentrations, 0 and 1 .mu.g/ml, and between the actein
concentrations, 0.1 and 1 .mu.g/ml. The addition of 0.1 .mu.g/ml
actein to 1 nM paclitaxel did not produce a significant effect;
however, the addition of 1 .mu.g/ml did.
TABLE-US-00004 TABLE 4 2-way ANOVA. 1 2 3 average DMSO 236742
255918 216444 236368 1 actein .1 169636 175420 179339 174798.3 2
actein 1 147900 148206 148502 148202.7 3 actein 10 110402 105501
104339 106747.3 4 tax .25 189954 205308 199987 198416.3 1 actein .1
tax .25 165780 168440 164526 166248.7 2 actein 1 tax .25 145603
159404 152503.5 3 actein 1 tax .25 107455 98838 107202 104498.3 4
tax 1 125602 119850 110420 118624 1 actein .1 tax 1 126449 125399
125924 2 actein 1 tax 1 105302 100944 92668 99638 3 actein 10 tax 1
76402 72726 76398 75175.33 4 tax4 42388 35462 33332 37060.67 1
actein .1 tax 4 40902 35448 35962 37437.33 2 actein 1 tax 4 48694
50033 49363.5 3 actein 10 tax 4 46204 50002 48033 48079.67 4
Two-way ANOVA V.R DF SS MS F p-value A 3 1.09E+11 3.64E+10 753.6035
1.35E-27 B 3 2.55E+10 8.51E+09 176.1761 1.04E-18 A .times. B 9
1.92E+10 2.13E+09 44.18546 1.53E-14 Error 29 1.4E+09 48318909 Total
44 1.55E+11 t-test (Least Significant Difference Method; LSD)
Combination Average t-value p-value DMSO 236368 actein .1 174798.3
10.8481 1.01E-11 actein 1 148202.7 15.53406 4.685953 1.36E-15
6.0678E-05 actein 10 106747.3 22.83817 11.99007 7.304113 4.37E-20
9.2448E-13 4.8E-08 tax .25 198416.3 actein .1 tax .25 166248.7
5.667697 3.98E-06 actein 1 tax .25 152503.5 7.23546 2.166118
5.75E-08 0.03866239 actein 1 tax .25 104498.3 16.54763 10.87994
7.565194 2.6E-16 9.4171E-12 2.44E-08 tax1 118624 actein .1 tax 1
125924 1.150416 0.259371 actein 1 tax 1 99638 3.345188 4.142443
0.002284 0.00027139 actein 10 tax 1 75175.33 7.655323 7.997545
4.310135 1.93E-08 8.0625E-09 0.000171 tax4 37060.67 actein .1 tax 4
37437.33 0.066366 0.947542 actein 1 tax 4 49363.5 1.938819 1.879459
0.06231 0.07026431 actein 10 tax 4 48079.67 1.941464 1.875098
0.202321 0.061975 0.07088192 0.841079 Factor A is tax
concentration; Factor B is actein concentration; A .times. B is
combination Bold numbers represent significant t-value and
p-value.
t = x 1 - x 2 MS ( error ) .times. ( 1 n 1 + 1 n 2 )
##EQU00001##
[0147] Similar experiments were performed on the combination of
actein with herceptin, doxorubicin, cisplatin, 5-fluorouracil, and
vinblastine on MD-MBA-453 cells, and on the combination of actein
plus tamoxifen on MCF7 cells. The same method was used to obtain
the CI values for these classes of chemotherapy agents (Tables 5a
and 5b).
[0148] Actein at concentrations achievable in vivo (0.2 or 2
.mu.g/ml) potentiates the effects of several chemotherapy agents at
clinically-relevant drug concentrations (Table 5a). Actein at 2
.mu.g/ml (2.8 .mu.M) enhances the effects of 5-FU (0.002-0.2
.mu.g/ml; 1.54 .mu.M), doxorubicin (0.2 .mu.g/ml; 0.34 .mu.M),
cisplatin (2 .mu.g/ml; 6.7 .mu.M) and tamoxifen (2 .mu.g/ml, 5.4
.mu.M). Actein at 0.2 or 2 .mu.g/ml enhances the effect of
herceptin (8 .mu.g/ml, 54 nM). At 2 .mu.g/ml, actein has an
additive effect on vinblastine (4 .mu.g/ml).
[0149] When black cohosh was extracted with MeOH, and partitioned
with EtOAc, hexane, and water, the triterpene glycosides were
present primarily in the EtOAc extract. When the EtOAc extract was
combined with doxorubicin or paclitaxel, synergy occurred with 2
.mu.g/ml actein, and with 0.02-0.2 .mu.g/ml (0.34 .mu.M)
doxorubicin or 4 nM paclitaxel (Table 5b).
TABLE-US-00005 TABLE 5 Combination index values for the combination
of: (a) actein with various chemotherapy drugs; herceptin,
tamoxifen, doxorubicin, cisplatin, 5-FU or vinblastine; and (b)
EtOAc fraction with doxorubicin or paclitaxel. (a) Actein
(.mu.g/mL) 0.2 2 20 5-FU (.mu.g/mL) 0.002 1.75 -- 0.51 +++ 0.23 +++
0.02 1.69 -- 0.45 +++ 0.17 +++ 0.2, 0.15 uM 1.69 -- 0.45 +++ 0.17
+++ herceptin (.mu.g/mL) 0.08 1.15 - 1.12 - 1.13 - 0.8 1.20 - 1.17
- 1.18 - 8, 54 nM 0.35 +++ 0.32 +++ 0.33 +++ tamoxifen (.mu.g/mL)
0.5 1.47 -- 1.22 - 0.94 +/- 5 1.15 - 0.90 + 0.61 ++ 50, 134 uM 1.07
+/- 0.82 + 0.54 +++ cisplatin (.mu.g/mL) 0.2 3.33 -- 1.93 -- 1.44
-- 2 2.11 -- 0.71 ++ 0.22 +++ 20, 67 uM 2.04 -- 0.64 ++ 0.15 +++
vinblastine (.mu.g/mL) 0.4 4.40 -- 4.45 -- 4.08 -- 4 0.95 +/- 1.00
+/- 0.63 ++ 40, 44 uM 0.95 +/- 1.00 +/- 0.63 ++ (b) EtOAc
(.mu.g/mL) 0.2 2 20 doxorubicin (.mu.g/mL) 0.002 1.13 - 1.21 - 0.75
++ 0.02 0.43 +++ 0.51 +++ 0.05 +++ 0.2, 0.34 uM 0.43 +++ 0.50 +++
0.04 +++ taxol (nM) 0.25 1.89 -- 1.86 -- 1.86 -- 1 1.08 +/- 1.05
+/- 1.05 +/- 4 0.79 ++ 0.76 ++ 0.76 ++ Symbols: CI -- >1.3
antagonism - 1.1-1.3 moderate antagonism +/- 0.9-1.1 additive
effect + 0.8-0.9 slight synergism ++ 0.6-0.8 moderate synergism +++
<0.6 synergism IC.sub.50 values were determined from the
combination of 3 concentrations of actein and 3 concentrations of
the specific chemotherapy agent and the solvent control, as
illustrated for the combination of actein and paclitaxel in Table
3.
TABLE-US-00006 TABLE 6 Combination index values for the combination
of actein with the EtOAc fraction and cimigenol with paclitaxel.
EtOAc (.mu.g/ml) actein (.mu.g/ml) EtOAc .2 EtOAc 2 EtOAc 20 actein
.2 3.8166 -- 3.8166 -- 3.96666 -- actein 2 3.65 -- 3.65 -- 3.8 --
actein 20 0.15126 +++ 0.15126 +++ 0.30126 +++ Cimigenol (.mu.g/ml)
Taxol (.mu.g/ml) cimi .2 cimi 2 cimi 20 tax .25 1.5023 -- 1.5023 --
1.4665 -- tax 1 1.85714 -- 1.85714 -- 1.82142 -- tax 4 0.85714 +
0.85714 + 0.82142 +
TABLE-US-00007 TABLE 7 Combination index values for the combination
of actein with herceptin and the EtOAc fraction of black cohosh
with doxorubicin on BT474 human breast cancer cells. Actein
(.mu.g/mL) herceptin (.mu.g/mL) 0.2 2 20 0.8, 5.4 nM 3.14 -- 3.06
-- 3.06 -- 8 0.08 +++ 0 +++ 0 +++ 32 0.08 +++ 0 +++ 0 +++ EtOAc
(.mu.g/mL) doxorubicin (.mu.g/mL) 0.2 2 20 0.002 1.45 -- 1.79 --
0.79 ++ 0.02 0.67 ++ 1 +/- 0 +++ 0.2, 0.34 uM 0.67 ++ 1 +/- 0 +++
Symbols: CI -- >1.3 antagonism - 1.1-1.3 moderate antagonism +/-
0.9-1.1 additive effect + 0.8-0.9 slight synergism ++ 0.6-0.8
moderate synergism +++ <0.6 synergism IC.sub.50 values were
determined from the combination of 3 concentrations of actein and 3
concentrations of the specific chemotherapy agent and the solvent
control.
[0150] To further understand the effect of actein and the EtOAc
fraction of black cohosh, the investigators tested the effects on
BT474 human breast cancer cells (ER.sup.+, her2 overexpressing,
25-fold), which can form tumors in athymic mice. The investigators
obtained strong synergy when actein (0.2 or 2 .mu.g/ml) was
combined with herceptin (0.8 or 8 .mu.g/ml) and additive effects
when the EtOAc fraction (2 .mu.g/ml) was combined with doxorubicin
(0.02 .mu.g/ml, 34 nM).
[0151] Actein, or the fraction enriched for triterpene glycosides,
could be used in combination with agents, in single use (including
paclitaxel, herceptin, and tamoxifen), to treat breast cancer. If
actein or the triterpene glycoside fraction is free of significant
side effects, they could be used in combination with herceptin for
long-term treatment of patients with metastatic disease.
Effects of Actein, in Combination with Chemotherapy Agents on the
Distribution of Cells in the Cell Cycle
[0152] To understand the nature of the interaction of actein with
the different classes of chemotherapy agents, we determined the
effect of actein in combination with various chemotherapy agents on
the distribution of cells in the cell cycle. When the cells were
synchronized by serum starvation followed by serum stimulation,
treatment with actein induced a dose dependent increase in the
percent of cells in G1 at 48 hours (Table 8a). When actein (2 or 20
.mu.g/ml) was combined with paclitaxel (1 nM), or when actein (20
.mu.g/ml) was combined with doxorubicin (0.1 .mu.g/ml, nM) or 5 FU
(0.02 .mu.g/ml, nM), there was a synergistic increase in the
percent of cells in the subG.sub.1 phase at 48 hours, an indicator
of apoptosis (Table 8b, c).
[0153] In the case of doxorubicin and 5 FU, the addition of actein
to the chemotherapy agent resulted in an increase in cells in the
G1 phase of the cell cycle (Table 8). The inventors' results
indicate that it may be better to give the chemotherapy agents
before actein, in order to retain the block at S or G2/M that is
induced by some chemotherapy agents.
TABLE-US-00008 TABLE 8 Effect of actein alone and in combination
with chemotherapy agents on cell cycle distribution in MDA-MB-453
cells. Sub G1 (%) G1 (%) S (%) G2/M (%) (a) The cells were grown in
DMEM + 0.25% FBS for 48 hrs and then treated with actein at 20
.mu.g/ml or 40 .mu.g/ml and analyzed at 48 hrs by DNA flow
cytometry. The values indicate the % of cells in the indicated
phases of the cell cycle. The control contains 0.08% DMSO. dmso,
0.08% 2.1 74.3 10.5 13.6 actein, 20 .mu.g/ml 1.8 79.6 8.5 10.0
actein, 40 .mu.g/ml 2.2 83.8 4.9 9.0 (b) The cells were treated
with 0, 2 or 20 .mu.g/ml (29.6 .mu.M) actein alone and in
combination with paclitaxel (1 nM) and analyzed at 48 hrs by DNA
flow cytometry. The values indicate the % of cells in the indicated
phases of the cell cycle. The control contains 0.044% DMSO. Dmso
1.0 70.6 11.8 17.0 Actein 2 .mu.g/ml 0.9 69.8 11.0 18.6 Acetin 20
.mu.g/ml 1.6 70.8 9.7 18.2 Taxol 1 nM 1.0 71.0 10.8 17.0 Taxol 1 nM
+ 1.8 69.2 10.6 18.5 Actein 2 .mu.g/mL Taxol 1 nM + 2.8 70.1 8.6
18.9 Acetin 20 .mu.g/mL c. The cells were treated with 0 or 20
.mu.g/ml (29.6 .mu.M) aetein alone and in combination with
doxorubicin (0.1 .mu.g/ml, 0.17 .mu.M), 5-FU (0.02 .mu.g/ml, 0.15
.mu.M) and analyzed at 48 hrs by DNA flow cytometry, The values
indicate the % of cells in the indicated phases of the cell cycle.
The control contains 0.08% DMSO. dmso, 0.08% 3.0 59.0 10.0 28.0
Acetin 20 .mu.g/mL 2.7 59.2 8.6 29.5 Doxorubicin, 2.5 30.1 5.6 61.7
0.1 .mu.g/mL Doxorubicin + Acetin, 5.3 39.2 9.6 46.0 20 .mu.g/mL 5-
FU, 0.02 .mu.g/mL 3. 28.0 47.6 20.3 5- FU + Acetin, 6.8 38.0 34.1
21.2 20 .mu.g/mL
Effects of Actein on Proteins Involved in Carcinogenesis
[0154] The inventors' previous results indicated that actein
decreased the level of cyclin D1, cdk4, and the hyperphosphorylated
form of the pRB protein, and increased the level of p21.sup.cip1 in
MCF7 cells--changes that may contribute to the arrest in G1. The
level of the epidermal growth factor receptor (EGFR), which is
overexpressed in various cancers (Suzui et al., Growth inhibition
of human hepatoma cells by acyclic retinoid is associated with
induction of p21 (CIP1) and inhibition of expression of cyclin D1.
Cancer Research, 62:3997-4006, 2002), was not altered after
treatment with actein. There also was no consistent effect of
actein on the phosphorylated and activated form of EGFR (p-EGFR).
However, the inventors did see a significant decrease of p-EGFR
with the 40 .mu.g/ml dose at 24 h. Thus, the EGFR did not appear to
be a direct target for actein.
[0155] Since the Her2 overexpressing cells were the most sensitive
to growth inhibition by black cohosh extracts and components, the
inventors tested the effect of actein on the Her2 receptor and on
the phosphorylation and activation of the Her2 receptor (p-Her2)
(FIG. 8) in MDA-MB-453 human breast cancer cells (which express
both Her2 and p-Her2 at high levels). Actein at 20 .mu.g/ml caused
a slight decrease in the level of the Her2 protein at 3 and 24 h.
After exposure to actein at 20 or 40 .mu.g/ml, there was a small
effect on p-Her2 at 3 h. The inventors found that actein at 20 or
40 .mu.g/ml induced a dose-dependent decrease in the level of the
p-Her2 receptor at 24 h. It is not clear how actein inhibits
phosphorylation. For example, it is not clear whether actein binds
to and directly inhibits the kinase activity of the Her2 receptor,
analogous to the action of Iressa (Masuda et al.,
Epigallocatechin-3-gall-ate inhibits activation of HER-2/neu and
downstream signaling pathways in human head and neck and breast
carcinoma cells. Clin. Cancer Res., 9: 3486-91, 2003), or whether
it inhibits activation of the other component of the heterodimeric
complex.
[0156] As the synthetic triterpenoid
2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a ligand
for PPAR-.gamma. (Wang et al., 2000; Lapillonne et al, 2003), the
inventors next tested the effect of actein on PPAR-.gamma. (FIG.
18). After treatment with actein, the intensities of the
PPAR-.gamma. bands relative to the .beta.-actin bands were: 1.39 (3
h, 20 .mu.g/ml), 0.93 (3 h, 40 .mu.g/ml), 1.3 (24 h, 20 .mu.g/ml),
and 0.67 (24 h, 40 .mu.g/ml). Thus actein 20 .mu.g/ml increased the
level of PPAR.gamma. at 3 and 24 hours. This anti-inflammatory
protein is therefore among the targets of actein.
Effects of Actein on Transcriptional Control of Specific Genes
[0157] To further determine the nature of the target of actein, the
inventors tested the effect of actein on molecules, such as cyclin
D1, that function downstream of active Her2-containing
heterodimers. Since the inventors found that actein induces
cell-cycle arrest at G1, it was of interest to examine the effects
of this compound on cellular levels of proteins that control
cell-cycle progression. Cyclin D1 was of particular interest,
because it plays a critical role in mediating the transition from
G1 to S, is overexpressed in about 50-60% of primary human breast
carcinomas (Joe et al., Resveratrol induces growth inhibition,
S-phase arrest, apoptosis, and changes in biomarker expression in
several human cancer cell lines. Clin. Cancer Res., 8:893-903,
2002), and is overexpressed in several human breast cancer cell
lines (Soh et al., Novel roles of specific isoforms of protein
kinase C in activation of the c-fos serum response element. Mol.
Cel. Bio., 19:1313-24, 1999).
[0158] Actein suppressed the level of cyclin D1 protein in
MDA-MB-453 cells. After treatment with actein, the intensities of
the cyclin D1 bands relative to the .beta.-actin bands were: 3 hr,
40 .mu.g/ml: 0.93; 24 hr, 20 .mu.g/ml: 1.3; 40 .mu.g/ml: 0.44. The
inventors further show that actein at 40 .mu.g/ml reduced the level
of cyclin D1 mRNA at 24 hours, 0.66-fold in MCF7 cells (FIG. 13)
and 0.56-fold in MDA-MB-453 cells (FIG. 14). The inventors next
examined the effect of actein on cyclin D1 transcriptional promoter
activity in MDA-MB-453 cells, using transient transfection reporter
assays (Soh et al., Novel roles of specific isoforms of protein
kinase C in activation of the c-fos serum response element. Mol.
Cell. Biol., 19:1313-24, 1999; Soh et al., Cyclic GMP mediates
apoptosis induced by sulindac derivatives via activation of c-Jun
NH2-terminal kinase 1. Clin. Cancer Res., 10:4136-41, 2000; Masuda
et al., Effects of epigallocatechin-3-gallate on growth, epidermal
growth factor receptor signaling pathways, gene expression, and
chemosensitivity in human head and neck squamous cell carcinoma
cell lines. Clin. Cancer Res., 7:4220-29, 2001). To accomplish
this, the inventors used luciferase promoter sequences that were
1745 bp upstream of the cyclin D1 gene. At 24 hours after exposure
to actein at 20 (0.87 fold) or 40 .mu.g/ml (0.093 fold), there was
a dose dependent decrease in promoter activity, compared to
.beta.-gal as a control (FIG. 9). This result, in addition to the
inventors' Western-blot data, suggests that actein inhibits the
expression of cyclin D1 at the level of transcription.
[0159] Since NF-kB is instrumental in controlling cell
proliferation, the inventors then explored the effect of actein on
NF-kB promoter activity. Actein at 20 .mu.g/ml induced an increase
(1.59 fold) and, at 40 .mu.g/ml, a decrease (0.12 fold), in NF-kB
promoter activity (FIG. 9). To understand the basis for this
effect, the inventors checked the effect of actein on the level of
the related proteins, I.kappa.B and I.kappa..kappa.B. After
treatment with actein, the intensities of the I.kappa.B bands
relative to the .beta.-actin bands were: 1.2 (3 h, 20 .mu.g/ml),
1.09 (3 h, 40 .mu.g/ml), 0.81 (24 h, 20 .mu.g/ml), and 0.53 (24 h,
40 .mu.g/ml) (FIG. 17). After treatment with actein, the
intensities of the I.kappa..kappa.B bands relative to the
.beta.-actin bands were: 1.79 (3 h, 20 .mu.g/ml), 1.78 (3 h, 40
.mu.g/ml), 0.48 (10 h, 20 .mu.g/ml), 0.59 (10 h, 40 .mu.g/ml), 1.06
(24 h, 20 .mu.g/ml), and 0.95 (24 h, 40 .mu.g/ml).
[0160] In summary, the EtOAc fraction of black cohosh: (1) inhibits
cell proliferation at .about.20 and 10 .mu.g/ml, in ER+ and ER-
human breast cancer cell lines, respectively; and (2) induces
cell-cycle arrest at G1 at low concentrations (.about.IC.sub.50),
and at G2/M at high concentrations (.about.3.times.IC.sub.50).
[0161] The triterpene glycoside fraction of black cohosh, and the
triterpene glycosides--actein, 23-epi-26-deoxyactein, cimifugoside,
and cimiracemoside A--inhibit the growth of human breast cancer
cells, and induce cell cycle arrest at G1.
[0162] In MCF7 cells, actein decreases the level of cyclin D1,
cdk4, and ppRb and increases the level of p21 and p27-changes which
lead to G1 arrest. It reduces the level of cyclin D1 mRNA and
promoter activity, thereby acting at the level of transcription.
Actein does not affect the level of EGFR, and, therefore, does not
specifically act through the estrogen receptor, the Her2 receptor,
or the EGFR receptor. Actein is capable of enhancing the effects of
tamoxifen on MCF7 breast cancer cells.
[0163] In MDA-MB-453 cells, actein decreases the level of p-Her2
and the level of cyclin D1 mRNA and promoter activity at 24 h. It
increases the level of p21 mRNA at 24 h. Its effects on the level
of NF-.kappa.B promoter activity is complex: actein increase the
level of NF-.kappa.B promoter activity at 20 .mu.g/ml while
decreases the level at 40 .mu.g/ml at 24 h. Actein is capable of
enhancing the effects of paclitaxel, herceptin, 5-FU, doxorubicin,
and cisplatin.
TABLE-US-00009 TABLE 9 Summary. molecule assay 0 3 hr, - 20 ug/ml
40 ug/ml 10 hr, - 20 ug/ml 40 ug/ml 24 hr, - 20 ug/ml 40 ug/ml
cyclin D1 promoter MDA-MB-453 1 0.87 0.093 CD1 RNA RT-PCR
MDA-MB-453 1 0.88 1 1 1.18 1.02 1 0.98 0.56 CD1 RNA RT-PCR MCF7 1
0.91 0.94 1 1.1 0.93 1 0.84 0.66 cyclin D1 MDA-MB-453 1 0.93 1 1.3
0.44 p-Her2 WB MDA-MB-453 1 0.71 0.86 1 0.81 0.53 p21 WB MCF7 1
1.47 1 1 1.75 1.37 1 0.94 0.78 RT-PCR MDA-MB-453 1 0.92 1.03 1 1.06
1.19 1 0.92 1.46 ppRB WB MCF7 1 1.51 1.59 1 0.61 0.64 1 0.8 0.43
PPAR-g 1 1.39 0.92 1 1.3 0.67 NF-kB promoter MDA-MB-453 1 1.59 0.12
ikb WB MDA-MB-453 1 1.2 1.09 1 0.81 0.53 ikkb WB MDA-MB-453 1 1.79
1.78 1 0.48 0.59 1 1.06 0.95
[0164] In an embodiment of the present invention, a composition is
provided for use in treating or preventing neoplasia, comprising an
effective anti-neoplastic amount of an ethyl acetate extract of
black cohosh. In the composition, the ethyl acetate extract
preferably comprises at least one triterpene glycoside compound.
The triterpene glycoside compound is preferably selected from the
group consisting of actein, cimifugoside, cimigenol glycoside,
cimiracemoside A, and 23-epi-26-deoxyactein or mixtures thereof.
The triterpene glycoside compound more preferably is actein or
mixtures thereof.
[0165] In an embodiment, the effective anti-neoplastic amount of
actein is between about 0.5 .mu.g/ml and about 40.0 .mu.g/ml. In a
preferred embodiment, the effective anti-neoplastic amount of
actein is between about 1.0 .mu.g/ml and about 3.0 .mu.g/ml.
[0166] In a further embodiment, the ethyl acetate extract of the
composition comprises at least one aglycone. In a preferred
embodiment, at least one aglycone is cimigenol.
[0167] In another embodiment of the present invention, a
composition of anti-neoplastic agents is provided, comprising an
effective anti-neoplastic amount of an ethyl acetate extract of
black cohosh and an effective anti-neoplastic amount of at least
one additional chemopreventive or chemotherapeutic agent. In a
preferred embodiment, the composition is a synergistic
combination.
[0168] In an embodiment of the composition of anti-neoplastic
agents, the anti-neoplastic agents are combined in a single
formulation. In an alternative embodiment, a separate, individual
formulation of the ethyl acetate extract of black cohosh is
combined with a separate, individual formulation of the at least
one additional chemopreventive or chemotherapeutic agent.
[0169] In a preferred embodiment of the composition of
anti-neoplastic agents, the ethyl acetate extract comprises a
triterpene glycoside compound. The triterpene glycoside compound is
preferably selected from the group consisting of actein,
cimifugoside, cimigenol glycoside, cimiracemoside A, and
23-epi-26-deoxyactein, or mixtures thereof. The triterpene
glycoside compound is more preferably actein or mixtures
thereof.
[0170] In an embodiment of the composition of anti-neoplastic
agents, the ethyl acetate extract comprises at least one aglycone.
In a preferred embodiment, at least one aglycone is cimigenol.
[0171] In an embodiment of the composition of anti-neoplastic
agents, the at least one additional chemopreventive or
chemotherapeutic agent is selected from the group consisting of
adriamycin, cisplatin, docetaxel, doxorubicin, 5-fluorouracil,
herceptin, paclitaxel, tamoxifen, and vinblastine. In a preferred
embodiment, the at least one additional chemopreventive or
chemotherapeutic agent is paclitaxel.
[0172] In an embodiment of the composition of anti-neoplastic
agents, the ethyl acetate extract of black cohosh comprises actein
and the at least one additional chemopreventive or chemotherapeutic
agent is paclitaxel. In a further embodiment, the effective
anti-neoplastic amount of actein is between about 0.5 .mu.g/ml and
about 40.0 .mu.g/ml, and the effective anti-neoplastic amount of
paclitaxel is between about 0.5 nM and about 5.0 nM.
[0173] In a further embodiment of the present invention, a
composition for use in treating or preventing neoplasia is
provided, comprising an effective anti-neoplastic amount of actein.
The neoplasia may be, but is not limited to, a carcinoma, a
lymphocytic leukemia, a myeloid leukemia, a malignant lymphoma, a
malignant melanoma, a myeloproliferative disease, a sarcoma, a
brain tumor, a childhood tumor, or a mixed type of neoplasia.
[0174] In a further embodiment thereof, the composition comprises
an effective anti-neoplastic amount of at least one additional
chemopreventive or chemotherapeutic agent.
[0175] In another embodiment of the present invention, a
composition is provided for use in treating or preventing disorders
caused by or related to the abnormality of at least one factor
selected from the group consisting of cyclin D1, cdk4, Her2,
I.kappa.B, I.kappa..kappa.B, NE-.kappa.B, p21, p27, PPAR.gamma.,
and ppRb, wherein the composition comprises an effective amount of
actein.
[0176] Pharmaceutical compositions of each of the compositions are
provided, which have a pharmaceutically acceptable carrier.
[0177] In another aspect of the present invention, a method for
treating or preventing neoplasia in a subject is provided,
comprising administering to the subject an amount of an ethyl
acetate extract of black cohosh effective to treat or prevent the
neoplasia. The neoplasia may be, but is not limited to, a
carcinoma, a lymphocytic leukemia, a myeloid leukemia, a malignant
lymphoma, a malignant melanoma, a myeloproliferative disease, a
sarcoma, a brain tumor, a childhood tumor, or a mixed type of
neoplasia. The carcinoma may be, but is not limited to, breast
cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer,
bladder cancer, uterine cancer, or skin cancer.
[0178] In an embodiment of a method for treating or preventing
neoplasia, the ethyl acetate extract comprises at least one
triterpene glycoside compound. In a preferred method, the
triterpene glycoside compound is actein or mixtures thereof.
[0179] In another embodiment of a method for treating or preventing
neoplasia, the effective amount of actein is between about 0.5
.mu.g/ml and about 40.0 .mu.g/ml.
[0180] In a further embodiment of a method for treating or
preventing neoplasia, the ethyl acetate extract comprises at least
one aglycone. In a preferred embodiment the at least one aglycone
is cimigenol.
[0181] In an embodiment of the present invention, a method for
treating or preventing neoplasia in a subject is provided,
comprising administering to the subject an amount of an ethyl
acetate extract of black cohosh effective to treat or prevent the
neoplasia, in combination with an amount of at least one additional
chemopreventive or chemotherapeutic agent effective to treat or
prevent the neoplasia. In a preferred embodiment, the method
results in a synergistic anti-neoplastic effect.
[0182] In an embodiment of the method, administration is
concurrent. In an alternative embodiment, administration is
sequential. In another embodiment, administration is alternate.
[0183] In a further embodiment of the method, the ethyl acetate
extract comprises actein.
[0184] In a further embodiment of the method, the at least
additional chemopreventive or chemotherapeutic agent is selected
from the group consisting of cisplatin, docetaxel, doxorubicin,
5-fluorouracil, herceptin, paclitaxel, tamoxifen, and vinblastine.
In a preferred embodiment, the at least one additional
chemopreventive or chemotherapeutic agent is paclitaxel.
[0185] In an embodiment of a method for treating or preventing
neoplasia, the ethyl acetate extract of black cohosh comprises
actein and the at least one additional chemopreventive or
chemotherapeutic agent is paclitaxel. Preferably, the effective
amount of actein is between about 0.5 .mu.g/ml and about 40.0
.mu.g/ml, and the effective amount of paclitaxel is between about
0.5 nM and about 5.0 nM.
[0186] In an embodiment of the present invention, a method for
treating or preventing neoplasia in a subject is provided,
comprising administering to the subject an amount of actein
effective to treat or prevent the neoplasia. The neoplasia may be,
but is not limited to a carcinoma, a lymphocytic leukemia, a
myeloid leukemia, a malignant lymphoma, a malignant melanoma, a
myeloproliferative disease, a sarcoma, a brain tumor, a childhood
tumor, or a mixed type of neoplasia. The carcinoma may be, but is
not limited to, breast cancer, colon cancer, lung cancer, ovarian
cancer, prostate cancer, bladder cancer, uterine cancer, or skin
cancer.
[0187] In a further embodiment of a method for treating or
preventing neoplasia, the method further comprises administering to
the subject an amount of at least one additional chemopreventive or
chemotherapeutic agent effective to treat or prevent the neoplasia.
In a preferred embodiment, at least one additional chemopreventive
or chemotherapeutic agent is selected from the group consisting of
cisplatin, docetaxel, doxorubicin, 5-fluorouracil, herceptin,
paclitaxel, tamoxifen, and vinblastine.
[0188] In a further embodiment of the present invention, a method
for treating or preventing disorders in a subject caused by or
related to the abnormality of at least one factor is provided,
wherein the factor is selected from the group consisting of cyclin
D1, cdk4, Her2, I.kappa.B, I.kappa..kappa.B, NF-.kappa.B, p21, p27,
PPAR.gamma., and ppRb. The method comprising administering to the
subject an amount of actein effective to treat or prevent the
disorder.
[0189] It has been reported that extracts of black cohosh and
isolated components inhibit the growth of human breast cancer cells
(Einbond, et al., Gene expression analysis of the mechanisms
whereby black cohosh inhibits human breast cancer cell growth,
Anticancer Res., 2007. 27(2): p. 697-712; Einbond, et al., The
growth inhibitory effect of actein on human breast cancer cells is
associated with activation of stress response pathways, Int J
Cancer, 2007. 121(9): p. 2073-83; Einbond, et al., Growth
inhibitory activity of extracts and purified components of black
cohosh on human breast cancer cells, Breast Cancer Res Treat, 2004
83(3): p. 221-31), but the precise mechanism of action of this
natural product is not known. Gene expression has been used to
characterize the nature of the inhibition, in vitro. The results
indicated that the growth inhibitory effect of actein (Einbond, et
al., Int J Cancer, 2007. 121(9): p. 2073-83) or the MeOH extract
(Einbond, et al., Anticancer Res., 2007. 27(2): p. 697-712) on
human breast cancer cells is associated with activation of stress
response pathways (Benjamin, I. J., et al., Viewing a stressful
episode of ER: is ATF6 the triage nurse? Circ Res, 2006. 98(9): p.
1120-2; Wu, Y. et al., Endoplasmic reticulum stress signal
mediators are targets of selenium action, Cancer Res, 2005. 65(19):
p. 9073-9). Both actein and the MeOH extract induced 2 phases of
the integrated stress response, either the survival or the
apoptotic phase, depending on the duration of treatment; for actein
the results indicated that it also depends on the dose of
treatment.
[0190] Recent case control and animal studies substantiate the in
vitro findings of black cohosh's anticancer and chemopreventive
potential. Rebbeck et al. (A retrospective case-control study of
the use of hormone-related supplements and association with breast
cancer, Int. J. Cancer, 2007. 120(7): p. 1523-1528) used a
population-based case control study of women to show that black
cohosh extracts and Remifemin appear to reduce the incidence of
breast cancer, in particular PR positive tumors. The recent
pharmacoepidemiologic observational retrospective cohort study of
Zepelin et al. (Isopropanolic black cohosh extract and
recurrence-free survival after breast cancer, Clin Pharmacol Ther,
2007. 45: p. 143-54) indicates that use of isoproanolic extracts,
Remifemin and Remifemin plus were associated with prolonged
recurrence-free survival after breast cancer. Studies of Sakurai et
al. (2005) indicated that cimigenol and cimigenol-3,15-dione have
antitumor initiating activity commensurate with EGCG (Sakurai, et
al., Cancer preventive agents. Part 1: chemopreventive potential of
cimigenol, cimigenol-3,15-dione, and related compounds, Bioorg Med
Chem, 2005. 13(4): p. 1403-8), suggesting a chemopreventive role
for these compounds. The studies of Seidlova-Wuttke et al,
(Inhibitory Effects of a Black Cohosh (Cimicifuga racemosa) Extract
on Prostate Cancer, Planta Med, 2006. 72(6): p. 521-26), indicated
that the Cimicifuga racemosa extract BNO 1055 inhibited
development, proliferation and malignancy of tumors induced by
subcutaneous inoculation of LNCaP cells in immunodeficient
mice.
[0191] To assess chemopreventive utility, considerations include
whether, following oral administration, sufficient blood and tissue
levels can be achieved, and whether this extract/compound exerts
significant toxicity. The studies of Johnson, et al., (In vitro
formation of quinoid metabolites of the dietary supplement
Cimicifuga racemosa (black cohosh), Chem Res Toxicol, 2003. 16: p.
838-46) indicated that black cohosh catechols can be converted to
(by metabolism or chemicals) to electrophilic quinones, in vitro,
but these were not detected in the urine of women who ingested up
to 256 mg of a standardized black cohosh extract (70% ethanol
extract, prepared by Pure World Botanicals). The catechols do not
appear to be absorbed across the intestinal epithelium, whereas the
triterpenoids are absorbed.
[0192] It may be of concern that triterpene glycosides from black
cohosh have been shown to inhibit thymidine transport into
phytohemagglutinin-stimulated lymphocytes and cimifugoside appears
to be immunosuppressive in PHA-stimulated lymphocytes in vitro.
(Hemmi, et al., Inhibition of thymidine transport into
phytohemagglutinin-stimulated lymphocytes by triterpenoids from
Cimicifuga species, J. Pharmacobio-Dyn, 1979. 2: p. 339-349). Black
cohosh may interact with CYP2D6 substrates (Gurley, et al., In vivo
effects of goldenseal, kava kava, black cohosh, and valerian on
human cytochrome P450 1A2, 2D6, 2E1, and 3A4/5 phenotypes, Clin.
Pharmacol. Ther., 2005. 77: p. 415-26). Studies indicate that
commercially available black cohosh, whose active constituents were
identified as triterpene glycosides, inhibited CYP3A4 in intestinal
epithelium (Tsukamoto, et al. Isolation of CYP3A4 Inhibitors from
the Black Cohosh (Cimicifuga racemosa). Evidence-based
complementary and alternative medicine: eCAM, 2005. 2: p. 223-6).
Black cohosh may thus increase the bioavailability of a variety of
CYP3A4 substrates.
[0193] Animal studies indicate that extracts do not induce toxic,
mutagenic or carcinogenic effects (Foster, et al., Black cohosh:
Cimicifuga racemosa, A literature review. HerbalGram, 1999. 45: p.
35-49). Wistar rats given 5 g/kg of Remifemin granulate for 26
weeks did not show any organ or chemical toxicity (Liske, et al.,
Therapeutic efficacy and safety of Cimicifuga racemosa for
gynecologic disorders, Adv. Ther., 1998. 15: p. 45-53). Nor was
toxicity observed in dogs given 400 mg/kg/day for 26 weeks
(Johnson, et al., Chem Res Toxicol, 2003. 16: p. 838-46). The
results of the Ames test for a 40% 2-propanol extract were
negative. The LD50 of a black cohosh preparation in mice was 7.7
mg/kg (intragastric) and 1.1 g/kg (intravenous).
[0194] Although black cohosh appears to be safe at doses higher
than the human therapeutic dose, these results may or may not apply
to the use of partially purified fractions of black cohosh or to
purified components from black cohosh. It is also of concern that
Davis et al. found (in an unpublished study) an increase in the
incidence of lung metastases in a mouse MMTV neu model. (Davis et
al. Black Cohosh, Breast Cancer, and Metastases to Lung: Data from
the Mouse Model. Workshop on the Safety of Black Cohosh in Clinical
Studies. 2004. Bethesda, Md.: National Institutes of Health).
[0195] Studies have yielded conflicting results on the effect of
black cohosh on lipids. In a double blind study with placebo and CR
extract BNO 1055 at 40 mg for three months, Wuttke et al. (Effects
of black cohosh (Cimicifuga racemosa) on bone turnover, vaginal
mucosa, and various blood parameters in postmenopausal women: a
double-blind, placebo-controlled, and conjugated
estrogens-controlled study, Menopause, 2006. 13(185-196)), induced
a statistically significant increase in TTG, but no effects on
total, LDL or HDL cholesterol. (Spangler et al., The effects of
black cohosh therapies on lipids, fibrinogen, glucose and insulin,
Maturitas, 2007. 57: p. 195-204), who conducted a randomized,
placebo controlled trial using the same extract at a higher dose,
120 mg/kg, for twelve weeks; agreed in that they found no effect on
cholesterol, but disagreed in that they did not find a difference
in TTG. In another study, however, black cohosh (40 mg) for 52
weeks increased TTG and cholesterol (HDL-cholesterol) and lowered
LDL-cholesterol (Raus et al., 2006; First-time proof of endometrial
safety of the special black cohosh extract (Actaea or Cimicifuga
racemosa extract) CR BNO 1055, Menopause 13, 678-91).
[0196] It is believed that there are no reports of gene expression
profiles of the effects of black cohosh obtained in vivo.
[0197] There is evidence that the bioactive triterpene glycoside
actein is selective for malignant cells and able to synergize at
low concentrations with different classes of chemotherapy agents to
inhibit breast cancer cell growth: (K. Watanabe, et al.,
Cycloartane glycosides from the rhizomes of Cimicifuga racemosa and
their cytotoxic activities, Chem Pharm Bull. (Tokyo) 50 (2002)
121-5; L. S. Einbond, et al.), Growth inhibitory activity of
extracts and purified components of black cohosh on human breast
cancer cells, Breast Cancer Res Treat. 83 (2004) 221-31; and L. S.
Einbond, et al., Actein and a fraction of black cohosh potentiate
antiproliferative effects of chemotherapy agents on human breast
cancer cells, Planta Med. 72 (2006) 1200-6). Actein's growth
inhibitory effects may be related to the altered expression of
genes involved in calcium homeostasis and stress response pathways,
particularly the unfolded protein response and cell cycle control
genes. (L. S. Einbond, et al. The growth inhibitory effect of
actein on human breast cancer cells is associated with activation
of stress response pathways, Int J Cancer 121 (2007) 2073-83).
Actein's downregulation of cyclin D1, CDK4, pEGFR and the
hyperphosphorylated form of pRb and upregulation of the CDK
inhibitory protein p21.sup.cip1 in MCF7 cells may contribute to its
ability to arrest cells in G1. (L. S. Einbond, et al., Growth
inhibitory activity of extracts and purified components of black
cohosh on human breast cancer cells, Breast Cancer Res Treat. 83
(2004) 221-31).
[0198] Actein is structurally related to the cardiac glycosides.
(FIG. 26A, B). Both are members of the saponin group of glycosides,
in which there are neutral steroidal saponins (such as the cardiac
glycosides digitoxin and ouabain) and acid triterpenoid saponins
(such as actein). As early as 1832, it was reported that the
medical effects of black cohosh resembled, but were not as strong
as, those of digitalis. (R. Upton, Black Cohosh Rhizome, American
Herbal Pharamacopoeia and Therapeutic Compendium. American Herbal
Pharmacopoeia. (2002)). Cardiac glycosides have been more highly
studied than actein, and knowledge of their mode of action may
provide insights to the mechanism of action of actein.
[0199] Cardiac glycosides bind to the alpha subunit of the
Na.sup.+-K.sup.+-ATPase, an oligomeric complex with two
non-covalently linked .alpha. (catalytic) and .beta. subunits and a
third subunit comprised of seven FXYD transmembrane proteins. The
Na.sup.+-K.sup.+-ATPase and partners are present in caveolae
(membrane microdomains) (J. Liu, et al., Ouabain-induced
endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells
requires caveolin-1, Kidney Int. 67 (2005) 1844-54), and the
Na.sup.+-K.sup.+-ATPase alpha subunit contains two conserved
caveolin-1-binding motifs. Ouabain potently inhibits the enzyme's
active transport of Na.sup.+ and K.sup.+ across cell membranes,
leading to a small increase in intracellular Na.sup.+ and a large
increase in intacellular Ca.sup.2+. In heart muscle, this enhances
the force of contraction. (Kometiani P, et al. Digitalis-induced
signaling by Na.sup.+/K.sup.+-ATPase in human breast cancer cells,
Mol Pharmacol 67 (2005) 929-36). The binding of ouabain to this
ATPase also converts the enzyme to a signal transducer (J. Tian, et
al., Binding of Src to Na.sup.+/K.sup.+-ATPase forms a functional
signaling complex, Mol Biol Cell 17 (2006) 317-26), by releasing
and activating Src, which has been shown to subsequently
phosphorylate effectors such as the epidermal growth factor
receptor, leading to assembly and activation of multiple signaling
cascades (FIG. 26c). Z. Li, et al., The Na/K-ATPase/Src complex and
cardiotonic steroid-activated protein kinase cascades, Pflugers
Arch. February 19; [Epub ahead of print] (2008)).
[0200] There is evidence that cardiac glycosides also have
antitumor activity. Breast cancers from women on digitalis have
more benign characteristics, and the rate of recurrence after 5
years following a mastectomy is 9.6% times less in patients on
digitalis. (Lopez-Lazaro M, et al. Anti-tumour activity of
Digitalis purpurea L. subsp. heywoodii. Planta Med. 69(8):701-4,
2003). In animal models, digitoxin has been shown to inhibit both
two-stage carcinogenesis of mouse skin papillomas induced by
7,12-dimethylbenzanthracene (DMBA) and
12-O-tetradecanoylphorbol-13-acetate (TPA), and mouse pulmonary
tumors induced by 4-nitroquinoline-N-oxide (4NQO) and glycerol.
(Inada A et al. Anti-tumor promoting activities of natural
products. II. Inhibitory effects of digitoxin on two-stage
carcinogenesis of mouse skin tumors and mouse pulmonary tumors.
Biol Pharm Bull. 16(9):930-1, 1993.)
[0201] The therapeutic range for digitoxin in the treatment of
heart failure is narrow; it has a therapeutic plasma concentration
greater than 10 ng/ml (13 nM), but is toxic at concentrations above
35 ng/ml (46 nM). Though it was initially thought that only toxic
doses of digitoxin could be useful for anticancer activity, recent
studies indicate that low doses of digitoxin induce apoptosis in
malignant cell lines. (McConkey D et al. Cardiac glycosides
stimulate Ca.sup.2+ increases and apoptosis in
androgen-independent, metastatic human prostate adenocarcinoma
cells. Cancer Res. 60(14):3807-12, 2000.) Crude extracts and
several components present in foxglove appear to inhibit the growth
of serum-stimulated breast cancer cells. Digitoxin is 7.2 fold more
active than the aglycone on MCF7 human breast cancer cells.
(Lopez-Lazaro M, et al. Planta Med. 69(8):701-4, 2003).
[0202] The growth inhibitory activity of cardiac glycosides may be
related to their inhibition of the Na.sup.+-K.sup.+-ATPase, a
member of evolutionarily conserved enzymes that couple ATP
hydrolysis to ion translocation across cellular membranes. (Skou
J., The Na,K-pump. Methods Enzymol. 156:1-25, 1988; Kaplan J.,
Biochemistry of Na,K-ATPase. Annu Rev Biochem. 71:511-35, 2002;
Lingrel J, Kuntzweiler T. Na.sup.+,K(+)-ATPase. J Biol Chem
269(31):19659-62, 1994.) When cardiac glycosides bind to the alpha
subunit of the Na.sup.+-K.sup.+-ATPase, they potently inhibit the
active transport of Na.sup.+ and K.sup.+ across cell membranes,
(Goodman G, editor. Goodman and Gilman's The Pharmacological Basis
of Therapeutics. 9 ed; 1996), leading to a small increase in
intracellular Na.sup.+ and resulting in a large increase in
intacellular Ca.sup.2+, which, as noted enhances the force of
contraction in heart muscle. (Kometiani P, Liu L, A. A. Mol
Pharmacol 67(3):929-36, 2005.) Inhibition of the enzyme also
releases and activates Src; which subsequently transactivates
epidermal growth factor receptor, leading to assembly and
activation of multiple signaling cascades such as Ras/Raf/ERK1/2
and phospholipase C-/protein kinase C pathways and mitochondrial
ROS production. (Li Z, Xie Z. The Na/K-ATPase/Src complex and
cardiotonic steroid-activated protein kinase cascades. Pflugers
Arch. 2008. [Epub ahead of print]).
[0203] Cardiotonic steroids have been reported to exert growth
regulatory effects at nano- and sub-nanomolar concentrations that
do not inhibit cellular Na.sup.+-K.sup.+-ATPase pumping activity.
(McConkey D et al., Cancer Res. 60(14):3807-12, 2000; Li Z, Xie Z.
The Na/K-ATPase/Src complex and cardiotonic steroid-activated
protein kinase cascades. Pflugers Arch. 2008. [Epub ahead of
print]; Liu L, Askari A., Cell Molec Biol (Noisy-le-grand).
52(8):28-30, 2006.) Apoptosis may be induced by the cardiac
glycosides' downstream Src-mediated effects involving NE-KB.
(Winnicka K, et al. Cardiac glycosides in cancer research and
cancer therapy. Acta Pol Pharm. 63(2):109-15, 2006.) Digitoxin has
been shown to block phosphorylation of the NF-.kappa.B inhibitor
I.kappa.B.kappa. in cystic fibrosis lung epithelial cells
(Srivastava M, et al. Digitoxin mimics gene therapy with CFTR and
suppresses hypersecretion of IL-8 from cystic fibrosis lung
epithelial cells. Proc Natl Acad Sci USA 101:7693-8, 2004), and
inhibit TNF-.alpha./NF-.kappa.B signaling by blocking recruitment
of TNF receptor-associated death domain (TRADD) to the TNF
receptor. (Yang Q, et al. Cardiac glycosides inhibit
TNF-alpha/NF-kappaB signaling by blocking recruitment of TNF
receptor-associated death domain to the TNF receptor. Proc Natl
Acad Sci USA 102(27):9631-6, 2005.)
[0204] The risks of digitoxin administration in humans are well
known.
[0205] In an embodiment of the present invention, a method for
treating, preventing or ameliorating breast cancer is provided,
comprising administering to a patient in need thereof a composition
comprising a synergistic amount of digitoxin and an extract of
black cohosh comprising a triterpene glycoside, and a
pharmaceutically acceptable carrier, and optionally an effective
amount of at least one additional chemopreventive or
chemotherapeutic agent. In an alternative embodiment of the present
invention, a method for treating, preventing or ameliorating breast
cancer is provided comprising administering to a patient in need
thereof a composition comprising a synergistic amount of digitoxin
and actein, and a pharmaceutically acceptable carrier, and
optionally an effective amount of at least one additional
chemopreventive or chemotherapeutic agent.
[0206] In a further embodiment a method for treating or preventing
neoplasia in a subject is provided, comprising administering to the
subject an amount of an extract of black cohosh comprising a
triterpene glycoside effective to treat or prevent neoplasia, in
combination with an amount of an a cardiac glycoside which is
effective to treat or prevent the neoplasia, and optionally an
effective amount of at least one additional chemopreventive or
chemotherapeutic agent. In a preferred embodiment, the extract
comprises the triterpene glycoside actein. The extract optionally
further comprises an aglycone which is preferably cimigenol.
[0207] In another preferred embodiment, the extract of black cohosh
comprising a triterpene glycoside is selected from the group
consisting of an ethyl acetate extract of black cohosh and an
n-butanolic fraction of an EtOH/water extract of black cohosh.
Preferred is the n-butanolic fraction of an EtOH/water extract of
black cohosh. Black cohosh extract that is enriched for triterpene
glycosides preferably has at least 15% triterpene glycosides. More
preferably, the extract has at least 20% triterpene glycosides.
Most preferred is an extract having about 27% triterpene
glycosides.
[0208] In an alternative embodiment, a method for treating or
preventing neoplasia in a subject is provided, comprising
administering to the subject an amount of actein effective to treat
or prevent neoplasia, in combination with an amount of a cardiac
glycoside which is effective to treat or prevent the neoplasia, and
optionally an effective amount of at least one additional
chemopreventive or chemotherapeutic agent.
[0209] In any of the methods of treating or preventing neoplasia,
the neoplasia is preferably a carcinoma. In a further preferred
embodiment, the carcinoma is breast cancer.
[0210] Also, in any of the methods of treating or preventing
neoplasia, the cardiac glycoside may be selected from the group
consisting of digitoxin, ouabain, proscillaridin A, digoxin,
lanatoside C, and combinations thereof. Preferably, the cardiac
glycoside is digitoxin.
[0211] In any embodiment of the methods of the present invention in
which actein and digitoxin are administered, actein and digitoxin
are preferably in amounts that result in a synergistic
anti-neoplastic effect.
[0212] In an embodiment of any of the methods, the effective
anti-neoplastic amount of actein used is from about 0.2 .mu.g/ml to
about 40.0 .mu.g/ml. In a further embodiment the effective
anti-neoplastic amount of actein is from about 0.2 .mu.g/ml to
about 20.0 .mu.g/ml.
[0213] In another embodiment of any of the methods, the amount of
actein is from about 0.2 .mu.g/ml to about 2 .mu.g/ml and the
digitoxin is in an amount of from about 0.01 .mu.g/ml to about 0.8
.mu.g/ml. Alternatively, the amount of actein is from about 2
.mu.g/ml to about 20 .mu.g/ml and the digitoxin is in an amount of
from about 0.0004 .mu.g/ml to about 5 .mu.g/ml.
[0214] In another embodiment, a method for modulating
Na.sup.+-K.sup.+-ATPase activity is provided comprising contacting
a cell that expresses Na.sup.+-K.sup.+-ATPase with an extract of
black cohosh comprising a triterpene glycoside and a cardiac
glycoside, and optionally at least one additional chemopreventive
or chemotherapeutic agent. In an alternative embodiment, a method
for modulating Na.sup.+-K.sup.+-ATPase activity is provided
comprising contacting a cell that expresses Na.sup.+-K.sup.+-ATPase
with actein and a cardiac glycoside, and optionally at least one
additional chemopreventive or chemotherapeutic agent. In any of
these methods, the cardiac glycoside is preferably digitoxin.
[0215] In another embodiment, a method for modulating a growth
inhibitory effect of digitoxin on a carcinoma is provided which
comprises contacting the carcinoma with digitoxin and an effective
amount of an extract of black cohosh comprising a triterpene
glycoside, which results in a synergistic effect of the digitoxin
on the carcinoma, and optionally an effective amount of at least
one additional chemopreventive or chemotherapeutic agent. In an
alternative embodiment, a method for modulating a growth inhibitory
effect of digitoxin on a carcinoma is provided which comprises
contacting the carcinoma with digitoxin and an effective amount of
actein, which results in a synergistic effect of the digitoxin on
the carcinoma, and optionally an effective amount of at least one
additional chemopreventive or chemotherapeutic agent. In a
preferred embodiment of any of these methods, the cardiac glycoside
is digitoxin. In another preferred embodiment, the carcinoma is
breast cancer.
[0216] A further embodiment is a composition for use in treating or
preventing neoplasia comprising an effective anti-neoplastic amount
of an extract of black cohosh comprising a triterpene glycoside and
an effective anti-neoplastic amount of a cardiac glycoside, and
optionally an effective amount of at least one additional
chemopreventive or chemotherapeutic agent. In a preferred
embodiment, the extract comprises the triterpene glycoside actein.
The extract optionally further comprises an aglycone which is
preferably cimigenol. In another preferred embodiment, the extract
is selected from the group consisting of an ethyl acetate extract
of black cohosh and an n-butanolic fraction of an EtOH/water
extract of black cohosh.
[0217] In an alternative embodiment a composition for use in
treating or preventing neoplasia is provided comprising an
effective anti-neoplastic amount of actein and an effective
anti-neoplastic amount of a cardiac glycoside, and optionally an
effective amount of at least one additional chemopreventive or
chemotherapeutic agent. The cardiac glycoside is preferably
selected from the group consisting of digitoxin, ouabain,
proscillaridin A, digoxin, lanatoside C, and combinations thereof.
More preferably, the cardiac glycoside is digitoxin.
[0218] In an embodiment of any composition of the present invention
comprising actein and digitoxin, the actein and digitoxin are in
amounts that result in a synergistic anti-neoplastic effect. More
preferably, the composition is a pharmaceutical composition
comprising a pharmaceutically acceptable carrier.
[0219] In an embodiment of compositions of the present invention,
the effective anti-neoplastic amount of actein is from about 0.2
.mu.g/ml to about 40.0 .mu.g/ml. In a further embodiment, the
effective anti-neoplastic amount of actein is from about 0.2
.mu.g/ml to about 20.0 .mu.g/ml.
[0220] In a further embodiment of a composition, the cardiac
glycoside is digitoxin and the amount of actein is from about 0.2
.mu.g/ml to about 2 .mu.g/ml and the amount of digitoxin is from
about 0.01 .mu.g/ml to about 0.8 .mu.g/ml. Alternatively, the
amount of actein is from about 2 .mu.g/ml to about 20 .mu.g/ml, and
the digitoxin is in an amount of from about 0.0004 .mu.g/ml to
about 5 .mu.g/ml.
[0221] In an embodiment of each of the methods of the present
invention that optionally provide an effective amount of at least
one additional chemopreventive or chemotherapeutic agent,
particularly in a method for treating, preventing or ameliorating
breast cancer, the at least one additional chemopreventive or
chemotherapeutic agent is paclitaxel.
[0222] In an embodiment of each of the methods of the present
invention that optionally provide an effective amount of at least
one additional chemopreventive or chemotherapeutic agent, the at
least one additional chemopreventive or chemotherapeutic agent is a
taxane. Preferably, the taxane is selected from the group
consisting of paclitaxel, docetaxel, or mixtures thereof. More
preferably, the taxane is paclitaxel.
[0223] In an embodiment of any of the methods of the present
invention in which paclitaxel is used, the paclitaxel is in an
amount that results in a synergistic effect with the digitoxin and
one or more triterpene glycoside, preferably actein or mixtures
with actein.
[0224] In preferred embodiments of compositions comprising
paclitaxil and actein, the paclitaxel is in an amount that results
in a synergistic effect with the digitoxin and one or more
triterpene glycoside, preferably actein or mixtures with actein.
Preferably, such a composition is a pharmaceutical composition
which comprises the composition and a pharmaceutically acceptable
carrier.
[0225] In a further embodiment, a method for modulating a growth
inhibitory effect of paclitaxel on a carcinoma is provided which
comprises contacting the carcinoma with paclitaxel and an effective
amount of digitoxin, which results in a synergistic effect of the
paclitaxel on the carcinoma, and optionally an additional
chemopreventive or chemotherapeutic agent which is selected from
the group consisting of an extract of black cohosh comprising a
triterpene glycoside and actein. In an embodiment thereof, the
amount of digitoxin and paclitaxel are at least 0.01 .mu.g/ml
digitoxin and 1 nM paclitaxel. In another embodiment thereof, the
amount of digitoxin and paclitaxel are at least 0.05 .mu.g/ml
digitoxin and 0.025 nM paclitaxel.
[0226] In another aspect of the present invention, a method for
inhibiting the progression or development of breast cancer in vivo
is provided, comprising administering to a subject a composition
comprising an extract of black cohosh comprising a triterpene
glycoside, and optionally an effective amount of at least one
additional chemopreventive or chemotherapeutic agent. In an
alternative embodiment thereof, a method for inhibiting the
progression or development of breast cancer in vivo is provided,
comprising administering to a subject a composition comprising
actein, and optionally an effective amount of at least one
additional chemopreventive or chemotherapeutic agent.
[0227] The aglycone cimigenol is poorly soluble and tends to
precipitate. Reporting an IC50 for such a compound with confidence
in the value can be tenuous. A derivative of the aglycone
cimigenol, 25-acetyl-7,8-didehydrocimigenol
3-O-.beta.-D-xylopyranoside, however, has been found to be
sufficiently soluble to obtain an IC50 with confidence to report.
The IC50 of the derivative was determined in testing cell
proliferation in MDA-MB-453 (Her2 overexpressing) human breast
cancer cells. As provided in Einbond, et al., Phytomedicine 15
(2008) 504-511, the IC50 for the derivative is 3.2, which is more
potent than actein. It is believed that the aglycone cimigenol is
comparably potent to its derivative. It is contemplated that the
present invention encompasses embodiments of methods and
compositions as provided herein in which the aglycone cimigenol or
the derivative, 25-acetyl-7,8-didehydrocimigenol
3-O-.beta.-D-xylopyranoside, is used in place of actein.
Example 13
Chemopreventive Potential of Black Cohosh
Materials and Methods
Materials
[0228] All solvents and reagents were reagent grade; H.sub.2O was
distilled and deionized. Naturex, Inc. (South Hackensack, N.J.)
generously provided the black cohosh extract containing 27%
triterpene glycosides. Black cohosh raw material was collected in
the United States in 1998 from natural habitat, dried naturally by
air and identified by Dr. Scott Mori from the New York Botanical
Garden. Each lot of the raw material was compared with the
authentic samples using HPLC. Black cohosh roots and rhizomes (Lot
number 9-2677; South Hackensack, N.J.) were extracted with 75%
EtOH/water as noted below. A voucher sample (9-2677) was deposited
in Naturex's herbarium.
Black Cohosh Enriched for Triterpene Glycosides
[0229] The black cohosh fraction provided by Naturex was extracted
and isolated from black cohosh as reported in Einbond et al.
(Phytomedicine 15 (2008) 504-511), as follows. The black cohosh
roots and rhizomes were extracted with 75% EtOH/water. The ethanol
was removed at 45-55.degree. C. under reduced pressure. The
concentrated extract was partitioned between methylene chloride and
water, which provided a fraction of 15% triterpene glycosides (TG)
from methylene chloride and 1% TG from water. The methylene
chloride was removed at 45-55.degree. C. under reduced pressure.
The concentrated fraction was further partitioned between n-butanol
and water and a fraction was obtained from the n-butanol phase. The
resulting n-butanolic extract of the EtOH/water extract of black
cohosh was enriched for triterpene glycosides. The black cohosh
enriched for triterpene glycosides had about 27% TG.
Animal Treatment And Data Collection
[0230] Experimental Animals:
[0231] The experimental animals were female Sprague-Dawley rats, 56
weeks old at the start of the experiment. This strain belongs to
the colony used for over 30 years in the laboratory of the Cancer
Research Centre (CRC) of the Ramazzini Foundation (RF); wide
information dealing with normeoplastic and neoplastic pathologies
is available on over 15,000 controls.
[0232] Treatment with Black Cohosh Extract:
[0233] Four groups of 99 females were treated with 35.7, 7.14,
0.714 or 0 mg/kg of body weight (b.w.) of the black cohosh enriched
for triterpine glycosides (27%) by intragastric tube, from 56 to 96
weeks of age (the window of age for higher risk of mammary cancer
in this strain of rats). A sample of each mammary tumor was
collected, frozen in liquid nitrogen and kept at -70.degree. C.
Samples for studies of pharmacokinetics, pharmacodynamics and gene
expression analysis of different organ and tissues were collected
from two groups of 12 female rats treated with 35.7 or 0 mg/kg b.w.
of black cohosh.
[0234] Necropsy:
[0235] During the necropsy, portions from the liver from the last 4
animals of both treated (with 35.7 mg/kg b.w.) and control groups
(sacrificed 6 and 24 hours after the start of the experiment) were
collected for analysis. Four portions of about 100 mg each were
collected from the main lobe of the liver. Each portion was
individually retained in a cryovial, frozen and stored at
-70.degree. C. until use. (Actein was used for pharmacokinetics and
gene expression profile analysis.)
Analyses
[0236] Analysis by Microscopy:
[0237] Histopathological examination of H&E and H&E/Oil Red
0 stained sections of control and treated tissues obtained 6 or 24
hours after treatment were performed. Tissues were embedded in OCT
(optimal cutting temperature compound to enable cryosectioning of
the sample).
[0238] IHC staining:
[0239] Mammary Tissue:
[0240] Cyclin D1 antibody concentration was 1:600, incubation 90
min, at room temperature, PBS Wash, secondary reagent: anti-mouse
cytomation Envision+system. labeled with HRP 30 min(DAKO). PBS
wash, DAB 1 min. 2. Ki67 concentration was 1:200, 90 min
incubation. Secondary antibody: Goat anti rabbit 1:200, (vector) 30
min incubation. PBS wash, ABC 30 min (vector), DAB 2 min.
[0241] Mammary Tissue:
[0242] ER;
[0243] Liver Tissue:
[0244] EGFR.
[0245] Lipid Analysis:
[0246] Hepatic lipids were extracted by homogenization of the liver
followed by addition of choloroform:methanol (2:1). After vortex
and centrifugation for 10 min, the organic phase was collected and
dried under nitrogen. The dried lipids were dissolved in 1% Triton
X-100 in water and sonicated. Extracted hepatic lipids and plasma
lipids were measured by cholesterol and triglyceride enzymatic
assay kits from Infinity (Louisville, Colo.) according to the
manufacturer's instruction. Free fatty acids were measured by
Enzymatic assay using NEFA C kit from Wako Chemicals (Richmond,
Va.). Tissue lipids were normalized by protein concentration.
[0247] Gene Expression Analysis:
[0248] Labeled cDNA was generated from liver tissue from each study
animal and hybridized to Affymetrix RG230-2 rat whole genome arrays
following standard Affymetrix protocols at Columbia University.
Analyses were performed using 2 approaches: 1) Analysis was
performed using the AffyLimmaGUI package in the open-source
Bioconductor suite. All samples were normalized to remove
chip-dependent regularities using the GCRMA method of Irizarry et
al. (Speed, Summaries of Affymetrix GeneChip probe level data,
Nucleic Acids Res, 2003. 31(4): p. e15). The statistical
significance of differential expression was calculated using the
empirical Bayesian LIMMA (LI Model for MicroArrays) method of Smyth
et al. (Use of within-array replicate spots for assessing
differential expression in microarray experiments, Bioinformatics,
2005. 21(9): p. 2067-75). A cut-off B>0 was used for the
statistical significance of gene expression, as previously
described (Einbond et al., 2007). 2) Array data was transmitted to
Iconix Pharmaceuticals as CEL files and uploaded into the Iconix
database (DrugMatrix.RTM.) for Drug Signature and pathway analysis,
as previously described (Natsoulis, et al., Classification of a
large microarray data set: algorithm comparison and analysis of
drug signatures, Genome Res, 2005. 15(5): p. 724-36). Relative
log.sub.10 expression ratios were generated for each probe set on
the array by dividing the log.sub.10 of the average MAS5 normalized
signal for the 3 black cohosh treated animals by the log.sub.10 of
the average MAS5 normalized signal of 2 of the 3 control animals.
Reproducibility of the data between replicate animals in the group
was assessed, then the impact of the unknown compound on Iconix
Drug Signatures was analyzed.
[0249] The database has been extensively mined for gene
expression-based biomarkers called Drug Signatures.RTM. predictive
of pharmacologic, toxicologic, and pathologic effects (Natsoulis,
et al., Genome Res, 2005. 15(5): p. 724-36; Ganter, et al.,
Development of a large-scale chemogenomics database to improve drug
candidate selection and to understand mechanisms of chemical
toxicity and action, J Biotechnol, 2005. 119(3): p. 219-44). The
approach employed to derive Drug Signatures is based on a Sparse
Linear Programming (SPLP) classification algorithm that
mathematically identifies specific gene expression markers for
accurate sample classification.
[0250] Log.sub.10 ratio data for the black cohosh array data set
was compared to the Iconix collection Drug Signatures.RTM.. The
version of DrugMatrix.RTM. used contains 29 Drug Signatures.RTM.
derived on the RG230-2 array platform for liver. These signatures
describe a variety of pharmacological and toxicological endpoints
including steatosis, hepatic necrosis, glucocorticoid and
mineralocorticoid receptor agonism and estrogen receptor
agonism.
[0251] Pathway analysis using the curated pathways within the
database was used to identify particular biological processes
perturbed by exposure to the unknown compound. Gene expression
changes were overlaid onto visual maps of up to 110 pathways, which
illustrate semi-quantitatively which proteins in the pathway have
mRNA levels altered by treatment. Gene lists generated from the
perturbed pathways were then subjected to unsupervised hierarchical
clustering and statistical analysis. Statistical evaluation was
performed using a hypergeometric distribution calculation of the
probability that the number of genes from a given pathway
(DrugMatrix contains 135 curated pathways) that were perturbed in a
given experiment could have happened by chance.
[0252] Real-time RT-PCR analysis: Real-time quantitative RT-PCR
methods were used to determine the nature of the RNA induced by
treatment with black cohosh extract, as previously described
(Einbond et al., 2007).
[0253] mRNA sequences were obtained from the public GeneBank
database (www.ncbi.nlm.nih.gov), and primers were designed using
Primer3 software from The Massachusetts Institute of Technology
(frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). All primers
were synthesized by Invitrogen Company, and quality confirmed in
pre-PCR by no primer dimer and only one peak in dissociation curve,
and by only one band in agarose gel electrophoresis. All PCR
products were in the 140-180 bp range.
Results
Chemoprevention Study: Tumor Incidence And Animal Wellness
[0254] Black cohosh did not have any deleterious effects on the
health of the Sprague-Dawley rats as assessed by weight gain, water
consumption or survival (FIG. 19). Black cohosh reduced both the
incidence of mammary tumors and the number of tumor-bearing
animals. It was determined that 40 weeks of treatment with an
extract of black cohosh (7.14 mg/kg) reduced breast cancer
incidence in the rats with a protective index of 20.45%. (FIG. 19;
Table 10).
TABLE-US-00010 TABLE 10 Protective effect of black cohosh on
Sprague-Dawley rats. tumor bearing Total tumors protection No dose
no % No per 100 index.sup.a 99 35.7 mg/kg 37 37.37 42 42.42 4.55 99
7.14 32 32.32 35 35.35 20.45 99 0.71 31 31.31 41 41.41 6.82 99
control 39 39.39 44 44.44 total 396 139 35.1 162 40.91
.sup.aprotection index = {total tumors (control) - total tumors
(treated)}/total tumors (control) .times. 100
Histopathological Examination of Mammary Tissues and Tumors
[0255] Histopathological examination of IHC stained sections of
mammary tissues and fibroadenomas was performed. The mammary tissue
was positive for ER in the nucleus (FIG. 20A) and negative for Her2
expression (FIG. 20B). These findings lend insight to the signaling
pathways to explore in the gene expressed studies.
H and E Staining of Frozen and Paraffin Sections and IHC Staining
of Paraffin Sections
[0256] The fibroadenomas from rats treated with 7.14 mg/kg (FIG.
21B) or 35.7 mg/kg (FIG. 21C) black cohosh displayed a decrease in
the proportion of glandular tissue and an increase in the
proportion of connective tissue in treated versus control samples
(FIG. 21A) (3 each); whereas one fibroadenoma from rats treated
with the lowest dose (0.714 mg/kg) exhibited an increase in the
proportion of glandular tissue (data not shown).
[0257] Immunohistochemical staining (IHC) was used to examine the
fibroadenomas from Sprague-Dawley rats treated with black cohosh.
Ten fields were counted on each slide and a significant difference
was found in Ki67 and cyclin D1 staining for rats treated with the
middle dose of black cohosh (7.14 mg/kg) versus water (control).
For Ki67: the control was <5%. While treated with 7.14 mg/kg was
-1%. (FIG. 21B) For cyclin D1: the control was 20%; while treated
with 7.14 mg/kg was 1% (FIG. 21D, E, F and G).
Histopathological Examination
[0258] Rat liver treated with black cohosh samples were stained
with Oil Red 0 for lipids and counterstained with H&E
(Hematoxylin stains nucleus, Eosin stains cytoplasm). Lipid
accumulation was not as obvious in control liver tissue as in the
treated sample. The localization in the treated tissue occurred
between the central veins (Periportal--closer to the portal triad
area) and the droplets were small, diffuse, lobular,
subendothelial, and perivenule. The samples displayed mild
toxicity; the lipid droplets were microvesicular (FIG. 22B, C). IHC
staining for EGFR indicated the presence of EGFR in the nucleus and
cytoplasm (FIG. 22A).
[0259] Analysis of the lipid content of the livers revealed a 3.9
(p=1.14xE-5) and 4.6-fold (p=0.00131) increase in the free fatty
acid and triglyceride content, respectively, of the treated livers
compared to the controls at 24 h.
[0260] For the rat kidney: in the treated sample there was the
presence of a lymphoid, inflammatory infiltrate under the lining of
the urinary tract that is not seen in the control. There was no
tubular, no glomeruli damage, tissue inflammation or vascular
damage; it was not a type of toxic injury since glomeruli were
similar in both treated and control.
Chemogenomic Analysis of Black Cohosh Extract
[0261] A dataset derived from the livers of female rats treated
with an extract of black cohosh (35.7) mg/kg for 24 h was
analyzed.
AFFY LIMMA Analysis
[0262] After exposure for 24 h, Affy-Limma analysis indicated that
the extract altered the expression of 2 genes (B>0), the
mitochondrial gene BZRP and the transcription factor F-box only
protein 30.
Drug Matrix Analysis
Pathway Analysis
[0263] Considering both up and down-regulated genes in the
analysis, the highest impact was observed on the Mitochondrial
Oxidative Phosphorylation pathway (Table 11).
TABLE-US-00011 TABLE 11 Top 5 impacted pathways for black cohosh,
35.7 mg/kg, 24 h treatment. Both up and down-regulated genes
(filtered for p < 0.05) were considered. PATHWAY Impact Score
Mitochondrial Oxidative Phosphorylation 4.01 Urea & Aspartate
Cycle 1.93 P450 Family 1.77 Apoptosis 1.75 Hemes from
Protoporphyrin IX 1.64
[0264] When the expression data for the genes in this pathway are
overlaid on a map of the pathway (FIG. 23) it is clear that there
is a profound downregulation of genes in this pathway in response
to black cohosh exposure.
[0265] A general decrease in genes involved in urea and aspartate
metabolism was also observed, including the mitochondrial carbamoyl
phosphate synthase 1, argininosuccinate synthase, glycine amidino
transferase and creatine kinase, possibly also reflecting
mitochondrial damage.
[0266] When upregulated genes only were considered, phospholipid
biosynthesis and remodeling, PI3-Kinase and sphingosine signaling
were observed to be impacted. This was driven largely by an
upregulation of several isoforms of phospholipase C (which is
involved in all 3 pathways). Diacyl glycerol kinase beta was also
significantly upregulated.
Comparative Analysis
[0267] A direct comparison of the liver gene expression profiles
following black cohosh to other liver treatments in the database
(DrugMatrix.RTM. contains data on the RG230-2 platform for >660
compound-dose-time combinations) revealed no similarities using
Pearson's correlation across the top 1000 most variable genes (no
similar experiments were found with a correlation coefficient of
>0.1).
[0268] A hierarchical clustering of pathway impact scores of all
RG230-2 liver experiments in the database was performed alongside
black cohosh (FIG. 24). Black cohosh formed part of a cluster of 51
treatments having a Pearson's correlation coefficient of 0.58.
Statistical analysis of the treatments in the cluster using the
hypergeometric distribution revealed a significant representation
of treatments with anti-proliferative compounds, specifically
tubulin binding vinca alkaloids (3 experiments, p=0.0017) and DNA
alkylators (4 experiments, p=0.029). The repression of cyclin D1
previously reported (Le et al., 2004) was also observed in this
experiment. There was a mixed effect on the apoptosis pathway, with
caspase 9 and IAP5 upregulation (pro-apoptotic) but cytochrome C
and BAX downregulation (anti-apoptotic).
Real-Time RT-PCR
[0269] The more sensitive RT-PCR was used to confirm the microarray
results that black cohosh suppressed the expression of cyclin D1
and ID3 (FIG. 25).
Discussion
[0270] The effect of an extract of black cohosh enriched for
triterpene glycosides on the development of spontaneous mammary
tumors in Sprague-Dawley rats was examined. It was found that
treatment with an extract of black cohosh enriched for triterpene
glycosides (27%) for 40 weeks decreased the incidence of
spontaneous mammary tumors and the number of tumor-bearing
animals.
[0271] Further, fibroadenomas obtained from rats treated with 7.14
mg/kg of the black cohosh extract displayed a decrease in the
proportion of glandular and increase in that of connective tissue
and, in addition, a decrease in the level of cyclin D1 and Ki67
protein, by IHC. Black cohosh reduced the proliferative rate and
thus the malignant potential of the tumors. These findings are in
agreement with the results of Seidlova-Wuttke et al. (Planta Med,
2006. 72(6): p. 521-26) that the Cimicifuga racemosa extract BNO
1055 (aqueous ethanol) reduced the incidence, proliferation, size
and malignancy of prostate tumors induced by injection of LNCaP
cells in immunodeficient mice. The treated animals developed
smaller tumors and less overall tumor tissue, which was mostly
confined to connective tissue. The tumors in the treated animals
thus appeared to be less malignant than those in the untreated
animals, indicating that black cohosh components may inhibit the
progression, as well as the development of tumors.
[0272] Since the rats appeared healthy and lived for more than 40
weeks during treatment, the extract at 35.7 mg/kg was not toxic. It
was, however, found that the extract induced modest liver damage
and increased the level of lipids (TTG and FFA) at 24 h after
treatment. Although a cause for concern, the dose was, however,
50.times. the normal human dose.
[0273] Gene expression profiling was used to gain an understanding
of the alterations of rat liver gene expression induced by actein
or an extract of black cohosh. Sprague-Dawley rats were treated
with an extract of black cohosh enriched for triterpene glycosides
(27%) (at 35.7 mg/kg), and liver samples were obtained for gene
expression analysis at 6 and 24 h. The gene expression data was
analyzed using an unbiased informatics approach and Iconix Drug
Matrix Drug Signature mapping and pathway analysis. To assess the
intrasample variation, 3 replicate treated and control samples were
analyzed for each treatment. The results were confirmed with
real-time RT-PCR for 2 genes.
[0274] When the data were analyzed using Iconix Drug Signature.RTM.
matching, no matches were obtained. Despite the observed lipid
accumulation and reported menopausal benefits of black cohosh, no
match to any of the 29 signatures was observed. This could be due
to the fact that the Iconix database was derived for small
molecular weight purified components using juvenile (8-10) week old
animals, whereas older female rats (56 weeks old) were treated with
an extract of black cohosh that contained many components.
[0275] Another tool for analysis is pathway analysis. Considering
both up and down-regulated genes in the pathway analysis, the
highest impact was observed on the Mitochondrial Oxidative
Phosphorylation pathway (Table 11). Actein also altered this
pathway (data not shown). This downregulation of mitochondrial
genes suggests that black cohosh may cause mitochondrial damage. A
disruption of mitochondrial energy generation could explain the
observed lipid accumulation in the hepatocytes and also presents a
potential mechanism of the hepatitis occasionally observed in human
black cohosh users. Several isoforms of phospholipase C (PLC) which
catalyse the cleavage of phosphatidylinositol-4,5-bisphosphate to
generate the messengers DAG (diacylglycerol) and IP3 (inositol
1,4,5-trisphosphate) were upregulated. IP3, in turn, is required to
activate the ER IP3 receptor which releases Ca2+from the ER. The
upregulation of PLC could thus result in a release of calcium from
the ER which could account for the induction of stress response
genes. Diacyl glycerol kinase beta was also significantly
upregulated, suggesting a possible activation of GPCR-signaling
cascades.
[0276] A hierarchical clustering of pathway impact scores of all
RG230-2 liver experiments in the database was performed alongside
black cohosh (FIG. 22). The finding that black cohosh clustered
with anti-proliferative compounds, specifically tubulin binding
vinca alkaloids and DNA alkylators is interesting in relation to
its antiproliferative effects on human breast cancer cells in
vitro. Einbond, et al. Repression of cyclin D1 was also observed,
suggesting the potential for inducing cell cycle arrest at the G1/S
boundary. The effects on CCND1 and ID3 were confirmed using the
more sensitive technique real-time RT-PCR analysis.
[0277] There was a mixed effect on the apoptosis pathway, with
caspase 9 and IAP5 upregulation (pro-apoptotic), but cytochrome C
and BAX downregulation (anti-apoptotic). This mixed response
probably reflects a mixed early mitogenic and apoptotic response
among the hepatocytes in the liver.
[0278] In support of the findings, a study of the hepatic effects
of black cohosh indicated that an ethanolic extract of black cohosh
given to female Wistar rats (at doses greater than 500 mg/kg)
induced hepatic mitochondrial toxicity; this was evidenced by
microvesicular steatosis, inhibition of p-oxidation and the
respiratory chain and resulting apoptosis. Modest effects on liver
mitochondria were observed after treatment with doses as low as 10
mg/kg. (Lude et al., Hepatic effects of Cimicifuga racemosa extract
in vivo and in vitro, Cell Mol Lide Sci.). Black cohosh reduced
mitochondrial .beta.-oxidation starting at 10 .mu.g/ml in freshly
isolated rat liver mitochondria; since this effect was pronounced,
and was detected at a lower dose than other effects on
mitochondria, it could be the primary effect. Blockage of
.beta.-oxidation can result in the accumulation of long-chain acyl
CoA's, which may induce liver damage and apoptosis. Indeed, the
extract induced a dose dependent increase in early apoptotic cells.
Blood levels of black cohosh used for treatment of menopausal
symptoms, 1.5-3 .mu.g/ml, are slightly lower than the dose that
inhibits .beta.-oxidation, 10 .mu.g/ml. However, the dose required
to cause microvesicular steatosis in rats was significantly higher
than human doses, suggesting that black cohosh is safe.
[0279] In sum, the chemopreventive potential of an extract of black
cohosh on Sprague-Dawley rats was examined based on 3 sets of
analyses: 1) the ability of black cohosh to reduce the incidence of
mammary tumors; 2) histological examination of fibroadenomas from
affected rats, and 3) alterations of rat liver gene expression
induced by an extract of black cohosh. Sprague-Dawley rats were
treated with an extract of black cohosh enriched in triterpene
glycosides (27%) at 0, 0.714 (equivalent to a normal human dose for
symptoms of menopause), 7.14 and 35.7 mg/kg, the maximum tolerated
dose for 40 weeks, and the incidence of mammary tumors, benign and
malignant in the lifetime of the animals was determined.
Sprague-Dawley rats were also treated with 0, 7.14 or 35.7 mg/kg
black cohosh extract and liver tissue samples for lipid and gene
expression analysis at 6 and 24 h and serum samples were obtained
for chemistry analysis.
[0280] In the chemopreventive, study, the intermediate dose, 7.14
mg/kg, reduced the incidence of mammary tumors by 21%. Black cohosh
decreased the amount of glandular tissue and increased the
connective tissue in fibroadenoma samples from rats treated with
black cohosh compared to samples from the control rats. IHC
analysis indicated that black cohosh reduced KI67 and cyclin D1
protein expression in fibroadenomas. In the study of rat liver and
serum, treatment with the black cohosh extract increased the level
of FFA and TTG content in the rat liver at 24 h. Black cohosh
extract downregulated mitochondrial phosphorylation genes and
upregulated several isoforms of PLC, by microarray analysis.
Microarray and RT-PCR analysis indicated that the extract reduced
the expression of the cell cycle gene cyclin D1 and the inhibitor
of differentiation ID3.
[0281] Treatment of Sprague-Dawley rats with an extract of black
cohosh enriched for triterpene glycosides resulted in a
downregulation of the mitochondrial oxidative phorphorylation
pathway, cyclin D1 and ID3 (at 6 and 24 h) and an increase in the
liver content of FFA and TTG at 24 h. Black cohosh extract reduced
the incidence of spontaneous mammary tumors and the proliferative
rate of fibroadenomas.
Example 14
Actein and Digitoxin Combinations: Effect on the Activity of
Na.sup.+-K.sup.+-ATPase and Effect on Growth of Human Breast Cancer
Cells
Abbreviations
Combination Index: CI
[0282] Dulbecco's Modified Eagle's medium: DMEM
Fetal Bovine Serum: FBS
Methods
[0283] Materials.
[0284] All solvents and reagents were reagent grade; H.sub.2O was
distilled and deionized. Actein (ChromaDex, Laguna Hills, Calif.,
lot number 01355-101, purity 89% by HPLC; Planta Analytica,
Danbury, Conn., lot number PA-A-037, purity >95% by HPLC),
digitoxin and ouabain (Sigma, St. Louis, Mo.) were dissolved in
dimethylsulfoxide (DMSO) (Sigma) prior to addition to cell
cultures.
[0285] Cell Culture.
[0286] MDA-MB-453 (ER negative, Her2 overexpressing), MCF7 (ER
positive, Her2 low), HCC1569 (ER negative, Her2 overexpressing)
cells (ATCC, Manassas, Va.) were grown in Dulbecco's Modified
Eagle's medium (DMEM) (Gibco BRL Life Technologies, Inc.,
Rockville, Md.) containing 10% (v/v) fetal bovine serum (FBS)
(Gibco BRL) at 37.degree. C., 5% CO.sub.2. BT-474 cells (Incyte
Pharmaceuticals, Wilmington, Del.) were grown in DMEM plus 0.01
mg/mL bovine insulin.
Cell Growth Assays
[0287] Coulter Counter Assay:
[0288] MDA-MB-453 and BT474 cells were seeded at 4.times.10.sup.4
cells per well in 24 well plates (0.875 cm diameter), and attached
viable cells were counted 96 hours later using a Coulter Counter
model Z.sub.F (Coulter Electronics Inc., Hialeah, Fla.) as
previously described. (L. S. Einbond, et al., Breast Cancer Res
Treat. 83 (2004) 221-31).
[0289] MTT Assay:
[0290] The MTT assay was used to determine the sensitivity of
MDA-MB-453, HCC1569 and MCF7 cells to actein or digitoxin.
Following exposure to the various agents for 96 hours, the percent
viable cells was assayed using the MTT method, as previously
described in L. S. Einbond, et al. (Growth inhibitory activity of
extracts and compounds from Cimicifuga species on human breast
cancer cells, Phytomedicine (2007) [Epub ahead of print]).
[0291] Enzymatic Assay of Adenosine 5'-triphosphatase.
[0292] The enzymatic assay of ATPase (adenosine 5'-triphosphatase,
EC 3.6.1.3) followed the Sigma Prod. No. A-7510 protocol
(Sigma-Aldrich, St. Louis, Mo., USA). Actein, digitoxin, or ouabain
were pipetted with ATPase (0.05 ml, 0.5 unit/ml, Sigma-Aldrich, St.
Louis, Mo., USA), mixed and equilibrated for 5 min at 37.degree. C.
[P] was determined by the Taussky-Shorr method.
[0293] Calculating the Combination Index.
[0294] To determine the Combination Index (CI), 1) the
Na.sup.+-K.sup.+-ATPase enzyme or 2) MDA-MB-453 cells were exposed
to all combinations of 3, 4 or 5 concentrations of each of the
agents tested and a solvent control. (L. S. Einbond, et al., Planta
Med. 72 (2006) 1200-6). The results of the enzymatic assay of
ATPase or MTT assay were analyzed for possible synergistic effects
using the median effect principle. Variable ratios of drugs were
employed and mutually exclusive equations were assumed. (L. S.
Einbond, et al., Planta Med. 72 (2006) 1200-6).
[0295] RNAi-Mediated Gene Knockdown.
[0296] To test the functional relevance of ERK2 (p42/44MAPK
pathway), the growth inhibitory effects of actein on MDA-MB-453
cells using the model system RNAi-mediated gene knockdown was
examined. Cells were pretreated with siRNA to ERK2 (Hs/Mm MAPK1
siRNA) (Qiagen, Valencia, Calif.) for 24 h, then treated with
actein at 20 .mu.g/ml for 48 h, and the percent surviving cells was
assayed. Western blot analysis was performed to confirm the ERK2
knockdown.
[0297] Western Blot Analysis.
[0298] Cells were treated in media containing serum for increasing
times with approximately the IC.sub.50 and twice the IC.sub.50
concentration, measured at 48 h, of actein. To assay activation of
p-Src, cells were allowed to attach for 24 h, incubated in media
without serum for 24 h; the medium was replaced with media without
serum with DMSO, actein or digtioxin.
[0299] Western blot analysis was performed as previously described.
(L. S. Einbond, et al., Breast Cancer Res Treat. 83 (2004) 221-31).
The membrane was incubated with the primary antibodies, cyclin D1
(Santa Cruz Biotechnology; Santa Cruz, Calif.), p-Src, Akt, p-Akt,
ERK2 or p-Erk2 (Cell signaling, Beverly, Mass.); .beta.-actin was
used as a loading control.
[0300] NF-.kappa.B Reporter Assay.
[0301] The NE-KB promoter luciferase reporter plasmid was from Dr.
Jae Won Soh. The method for transient transfection reporter assays
was as previously described. (S. M. Masuda M, et al., Effects of
epigallocatechin-3-gallate on growth, epidermal growth factor
receptor signaling pathways, gene expression, and chemosensitivity
in human head and neck squamous cell carcinoma cell lines, Clin
Cancer Res. 7 (2001) 4220-9).
[0302] Statistical Analysis.
[0303] The data are expressed as mean+/-SD. Control and treated
cells were compared using the student's t-test, p<0.05.
Results
Inhibition of ATPase Activity
[0304] To determine whether the Na.sup.+-K.sup.+-ATPase is involved
in the mechanism of action of actein, the ability of actein to
inhibit the in vitro activity of the purified enzyme was assayed,
and 50% inhibition was found at a concentration of 11.2 .mu.M (FIG.
27). In the assay this value is about 10.times. the IC.sub.50
(concentration that causes 50% inhibition) value of ouabain (0.94
.mu.M) (FIG. 27), and of digitoxin (0.8 .mu.M). When combining
increasing concentrations of actein with increasing concentrations
of digitoxin (FIGS. 28A and B, and Table 12), moderate synergy (Cl
2+) was seen with as little as 0.8 .mu.M actein and 0.2 .mu.M
digitoxin, and strong synergy (Cl 3+) with 0.8 .mu.M actein and 0.8
.mu.M digitoxin. ATPase activity decreased from 87.8% after
treatment with digitoxin alone to 68.2% after treatment with 0.2
.mu.M digitoxin plus 0.8 .mu.M actein, p<0.01 (actein alone:
95.1%). Addition of 0.8 .mu.M actein to 0.8 .mu.M digitoxin
decreased ATPase activity from 53.6% to 29.1% (p<0.01).
TABLE-US-00012 TABLE 12 Combination indices* of actein and
digitoxin on Na.sup.+--K.sup.+-ATPase inhibition Digitoxin
Digitoxin Digitoxin 0.2 .mu.M 0.8 .mu.M 4 .mu.M Actein 0.8 .mu.M
0.76 0.38 0.33 Actein 4 .mu.M 0.68 0.30 0.25 Actein 16 .mu.M 0.43
0.05 0 *Combination Index Effect >1.3 antagonism 1.1-1.3
moderate antagonixm 0.9-1.1 additive effect 0.8-0.9 slight
synergism 0.6-0.8 moderate synergism <0.6 synergism
Growth Inhibitory Activity of Actein and Digitoxin on Human Breast
Cancer Cells
[0305] Of the cells that were previously tested, Her2
overexpressing human breast cancer cells appeared to be the most
sensitive to growth inhibition by actein and extracts of black
cohosh. (L. S. Einbond, et al., Breast Cancer Res Treat. 83 (2004)
221-31). Therefore, the effects of actein and digtoxin on the
proliferation of MDA-MB-453 Her2 overexpressing human breast cancer
cells were compared. The IC.sub.50's for actein and digitoxin were
about 5 .mu.g/ml (7.4 .mu.M) and 0.025 .mu.g/ml (0.033 .mu.M),
respectively (FIG. 28C, D). Thus, digitoxin is about 200-fold more
potent than actein in inhibiting the growth of these cells. The
IC.sub.50 ratio for growth inhibition of HCC1569 (ER-, Her2
overexpressing) by actein and digitoxin was similar (25/0.12=213)
to that of the MDA-MB-453 cells, whereas the ratio was
significantly lower for MCF7 (ER.sup.+Her2 low) human breast cancer
cells (20.7/2.61=79) and higher for BT474 (ER.sup.+Her2
overexpressing) cells (23.6/0.039=605). The effect on
Na.sup.+-K.sup.+-ATPase may be similar in different cell lines, but
the downstream signaling targets appear to differ in cells with
different receptors and signaling pathways.
[0306] When comparing the compounds' IC.sub.50's for ATPase
inhibition to the IC.sub.50's for growth inhibition of MDA-MB-453
human breast cancer cells, actein's effect on ATPase activity was
amplified about 2-fold (16: 7.4) and digitoxin's effect was
amplified about 20-fold (0.8: 0.04). The effect of actein was not
amplified for the other cells lines, and the amplification of
digitoxin was about the same for BT474 (0.8/0.039=20.5-fold) and
smaller for MCF7 (0.8/0.26=3.1-fold) and HCC1569 cells
(0.8/0.12=6.8-fold) compared to MDA-MB-453 cells.
Synergistic Growth Inhibitory Effects of Actein and Digitoxin on
Human Breast Cancer Cells
[0307] When combining increasing concentrations of actein with
increasing concentrations of digitoxin (FIGS. 28C and D, and Table
13), moderate synergy (Cl 2+) was seen with as little as 0.2
.mu.g/mL of actein and 0.01 .mu.g/ml digitoxin, and strong synergy
(Cl 3+) with 2 .mu.g/mL actein and 0.01 .mu.g/ml digitoxin. For the
former combination, the percent viable cells decreased from 62.8%
after treatment with digitoxin alone to 52.8% after treatment with
digitoxin plus actein, p<0.01 (actein alone: 97.8%). Addition of
actein (2 .mu.g/ml) to digitoxin (0.01 .mu.g/ml) decreased cell
survival from 62.8% to 47.2% (p<0.01) (actein alone: 75.8%).
TABLE-US-00013 TABLE 13 Combination indices* of actein and
digitoxin on inhibition of MDA-MB-453 cell proliferation Actein
Actein Actein Actein 0.2 .mu.M 2 .mu.M 5 .mu.M 20 .mu.M Digitoxin
0.004 .mu.M 1.32 0.96 0.72 0.72 Digitoxin 0.01 .mu.M 0.72 0.36 0.12
0.12 Digitoxin 0.04 .mu.M 0.6 0.24 0 0 Digitoxin 0.2 .mu.M 0.6 0.24
0 0 Digitoxin 0.5 .mu.M 0.6 0.24 0 0 *Combination Index Effect
>1.3 antagonism 1.1-1.3 moderate antagonixm 0.9-1.1 additive
effect 0.8-0.9 slight synergism 0.6-0.8 moderate synergism <0.6
synergism
Actein's Effect on Proteins Downstream of
Na.sup.+-K.sup.+-ATPase
[0308] The effects of actein and digitoxin on Src were tested and
then the effects of actein on stress response and cell cycle
pathways downstream of the ATPase-Src signaling complex were
assayed.
[0309] Treatment of serum-starved MDA-MB-453 cells with actein at
80 .mu.g/ml (118 .mu.M) or digitoxin at 80 .mu.g/ml (105 .mu.M) for
30 min increased the level of p-Src by 1.5-fold for both treatments
(FIG. 29A).
[0310] For the survival proteins Akt and Erk, actein appeared to
induce a biphasic response; actein upregulated the activated forms
at 20 and 40 .mu.g/ml at 3 hours and at 20 .mu.g/ml at 8 and 24
hours, but downregulated these protein levels at 40 .mu.g/ml at 24
hrs (FIG. 29C). Since ERK/MAPKs have been shown to regulate cyclin
D1 promoter activity and protein expression (Lavoie J N, et al.,
Cyclin D1 expression is regulated positively by the p42/p44MAPK and
negatively by the p38/HOGMAPK pathway, J Biol Chem. 271 (1996)
20608-16), the effects of actein were tested on cyclin D1. While
actein at 20 .mu.g/ml slightly increased the level of cyclin D1
protein at 3 and 24 h, actein at 40 .mu.g/ml decreased the level of
cyclin D1 protein at 3, 8 and 24 hours in MDA-MB-453 cells (FIG.
29C).
[0311] A biphasic response was observed on the promoter activity of
NE-KB, which is instrumental in controlling cell proliferation and
may be activated by Akt. (Rivas M A, et al., TNF alpha acting on
TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and
NF-kappa B-dependent pathways, Exp Cell Res. 314 (2008) 509-29).
Actein induced a 1.59 fold increase in NF-.kappa.B promoter
activity at 20 .mu.g/ml and a 0.12 fold decrease at 40 .mu.g/ml, at
24 h (FIG. 29D).
RNAi-Mediated Gene Knockdown
[0312] Pretreatment with siRNA to ERK2 (MAPK1) reduced the percent
growth inhibition from 100% (control siRNA) to 79.6% (ERK2 siRNA)
(p=0.0665) in response to actein treatment (20 .mu.g/ml) for 48 h
compared to the control. ERK2 may therefore be involved in the
survival phase of the digitoxin-induced stress response (FIG. 29B).
Western blot analysis indicated that the ERK2 siRNA reduced the
quantity of ERK2 protein by about 50%.
Discussion
[0313] The primary molecular target of actein has not been
identified, but the ubiquitous Na.sup.+-K.sup.+-ATPase is a good
candidate; the enzyme mediates many stress response and
proliferation pathways that are affected by actein. The
Na.sup.+-K.sup.+-ATPase enzyme is a known target of cardiac
glycosides such as digitoxin and ouabain, which, like actein, are
saponins. The enzyme also is important in the action of the
chemotherapy agent thapsigargin, an inhibitor of Serca. (J. Tian,
et al., Binding of Src to Na.sup.+/K.sup.+-ATPase forms a
functional signaling complex, Mol Biol Cell. 17 (2006) 317-26).
This study demonstrated that actein inhibits
Na.sup.+-K.sup.+-ATPase activity and subsequently phosphorylates
downstream proteins (FIG. 26C), and showed that actein and
digitoxin synergize with each other to inhibit the enzyme's
activity and proliferation of MDA-MB-453 human breast cancer
cells.
[0314] Inhibition of the enzyme has been shown to be related to a
compound's ability to interact with the enzyme's lipid-rich
environment. (Gorshkova I A, et al., Two different modes of
inhibition of the rat brain Na.sup.+, K(+)-ATPase by triterpene
glycosides, psolusosides A and B from the holothurian Psolus
fabricii., Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 122
(1999) 101-8). An explanation is suggested by a comparison to the
triterpene glycosides psolusosides (Ps) A and B from the
holothurian Psolus PsA is highly lipophilic and three-fold more
active on rat brain Na.sup.+-K.sup.+-ATPase than PsB. PsA, but not
PsB, binds to cholesterol and generates ion channels. It was
suggested that PsA may alter the protomers of the enzyme and the
lipid environment, while PsB may primarily affect the protomers.
(Gorshkova I A, et al., Comp Biochem Physiol C Pharmacol Toxicol
Endocrinol. 122 (1999) 101-8). Actein's weaker inhibition of ATPase
suggest a protomer-dependent mode of action similar to that of PsB,
while digitoxin's 10-fold greater potency for ATPase inhibition is
consistent with its highly lipophilic character and suggests that
it may have a mode of action similar to that of PsA.
[0315] The amplification of ATPase inhibition that was observed for
both actein and digitoxin is consistent with reports of cardiotonic
steroids exerting growth inhibition in cultured cells and animal
models (J. Tian, et al., Mol Biol Cell. 17 (2006) 317-26), at low
doses without inhibiting cellular Na.sup.+-K.sup.+-ATPase pumping
activity. (Z. Li, et al., The Na/K-ATPase/Src complex and
cardiotonic steroid-activated protein kinase cascades, Pflugers
Arch. February 19; [Epub ahead of print] (2008)). The extent of
amplification may also be related to lipid affinity, which may in
turn account for a compound's ability to activate Src and its
downstream signaling cascades. Ouabain has been shown to increase
the translocation of cytosolic Src to a triton-insoluble fraction
and stimulate Src activity in cultures of cardiac myocytes. (Z. Li,
et al., Pflugers Arch. February 19; [Epub ahead of print] (2008)).
The signal amplification that was observed may be due to
interaction of the Na.sup.+-K.sup.+-ATPase protomers in the cell
membrane and the induction of clustering of ATPase with neighboring
proteins in caveolar microdomains. (M. Haas, et al., Involvement of
Src and Epidermal Growth Factor Receptor in the Signal-transducing
Function of Na.sup.+/K.sup.+-ATPase, J. Biol. Chem. 275 (2000)
27832-27837). GRP78, which is expressed on the cell surface and is
involved in ouabain-induced endocytosis of the
Na.sup.+-K.sup.+-ATPase in LLC-PK1 cells (R. Kesiry, et al.,
GRP78/BIP is involved in ouabain-induced endocytosis of the
Na/K-ATPase in LLC-PK1 cells, Front Biosci. 10 (2005) 2045-55), is
also activated by actein (L. S. Einbond, et al., Weinstein, Gene
expression analysis of the mechanisms whereby black cohosh inhibits
human breast cancer cell growth, Anticancer Res. 27 (2007)
697-712), and may therefore be instrumental in actein or
digitoxin-mediated ATPase inhibition. It is important to note,
however, that although cardiac glycosides have been highly studied,
it is not certain that the Na.sup.+-K.sup.+-ATPase signaling
cascade is their primary mechanism of cell growth inhibition.
(Arispe N, et al., Heart failure drug digitoxin induces calcium
uptake into cells by forming transmembrane calcium channels, Proc
Natl Acad Sci USA. 105 (2008) 2610-5).
[0316] Downstream of Na.sup.+-K.sup.+-ATPase inhibition, actein's
upregulation of ERK2 resembled the effects of paclitaxel (TAX) in
human ovarian SKOV3 cells. (Seidman R, et al., The role of ERK 1/2
and p38 MAP-kinase pathways in taxol-induced apoptosis in human
ovarian carcinoma cells, Exp Cell Res. 268 (2001) 84-92). At low
concentrations (1-100 nM), TAX activated ERK1/2 within 0.5-6 h,
whereas the activation was reversed at 24 hours or at high
concentrations (1-10 .mu.M). High concentrations (1-10 .mu.M) of
TAX activated p38 within 2 h, and this activation continued for
over 24 hours. The decrease in ERK activation and the increase in
p38 activation coincided with the transition from inhibition of
proliferation to apoptosis.
[0317] The present study demonstrates a synergistic relationship
between actein and digitoxin. Actein potentiated digitoxin's
inhibition of ATPase activity, and at lower concentrations
potentiated digitoxin's inhibition of cell proliferation. The
concentrations required for the latter synergy are within the
therapeutic range for digitoxin (13-46 nM) and achievable in vivo
for actein, as the bioavailability studies on Sprague-Dawley rats
indicated a peak serum level of about 2.4 .mu.g/ml at 6 h after
treatment with actein at 35.7 mg/kg (data not shown).
[0318] The observed synergy of actein and digitoxin suggests that
the two compounds may bind to different active sites on the ATPase,
or the binding of one compound may enhance binding of the second
agent. Triterpene glycosides from holothurians have been shown to
potentiate the inhibitory effect of ouabain on
Na.sup.+-K.sup.+-ATPase activity without altering the specific
binding of ouabain to the ATPase. (I. Gorshkova, et al., Inhibition
of rat brain Na.sup.+-K.sup.+-ATPase by triterpene glycosides from
holothurians, Toxicon. 27 (1989) 927-36). The synergistic effects
on growth inhibition may also be due, in part, to the fact that
actein and digitoxin inhibit different phases of the cell cycle;
actein induces G1 arrest (L. S. Einbond, et al., Planta Med. 72
(2006) 1200-6), while digitoxin induces G2 arrest Actein may alter
additional targets not altered by digitoxin.
[0319] In sum, the Na.sup.+-K.sup.+-ATPase is a known target of
cardiac glycosides such as digitoxin and ouabain. The enzyme is
also a target of the structurally-related triterpene glycoside
actein, present in the herb black cohosh. Actein's inhibition of
Na.sup.+--K.sup.+-ATPase activity was less potent than that of
digitoxin, but actein potentiated digitoxin's inhibitory effect on
Na.sup.+-K.sup.+-ATPase activity and MDA-MB-453 breast cancer cell
growth. Different degrees of signal amplification were observed for
the two compounds. Actein's inhibitory effect on ATPase activity
was amplified two-fold for cell growth inhibition, whereas
digitoxin's signal was amplified twenty-fold. Actein induced a
biphasic response in proteins downstream of ATPase: low dose and
short duration of treatment upregulated NF-.kappa.B promoter
activity, p-ERK, p-Akt and cyclin D1 protein levels, whereas higher
doses and longer exposure inhibited these activities.
[0320] The results indicate that actein inhibits the activity of
the Na.sup.+-K.sup.+-ATPase and enhances the growth inhibitory
effect of digitoxin on human breast cancer cells. The synergy
demonstrated indicates the utility of combinations of digitoxin and
actein to prevent and treat breast cancer, but suggests that there
may be safety issues for cardiac patients who are prescribed
digitalis compounds and simultaneously take black cohosh to
alleviate menopausal symptoms. Synergistic combinations of
digitoxin and actein or an extract of black cohosh comprising
triterpene glycosides of the present invention, preferably
digitoxin and actein, have clinically advantageous utility,
permitting the use of therapeutic or lower doses of digitoxin and
thus reducing the risk of adverse effects.
Example 15
Effects of Digitoxin on Gene Expression and Effects of Digitoxin
and Paclitaxel Combinations on Cell Proliferation
Materials and Methods
Materials
[0321] All solvents and reagents were reagent grade; H.sub.2O was
distilled and deionized. Digitoxin and paclitaxel were obtained
from Sigma (St. Louis, Mo.). These agents were dissolved in
dimethylsulfoxide (DMSO) (Sigma; St. Louis, Mo.) prior to addition
to the cell cultures.
Cell Culture, Proliferation Assays and Cell Cycle Analysis
[0322] MDA-MB-453 (ER negative, Her2 overexpressing) and MCF7 (ER
positive, Her2 low) cells were obtained and cultured as set forth
above in Example 4.
[0323] Cell proliferation was determined using: 1) the Coulter
Counter assay or 2) the MIT
{3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolilum bromide}
(Dojindo; Tokyo, Japan) cell proliferation assay system, according
to the manufacturer's instructions (Roche Diagnostic, Mannheim,
Germany). For the Coulter counter assay, cells were seeded at
2.times.10.sup.4 cells per well in 24 well plates (0.875 cm
diameter) as described previously. (Einbond L S, et al. Growth
inhibitory activity of extracts and purified components of black
cohosh on human breast cancer cells. Breast Cancer Res Treat.
83(3):221-31, 2004.) For the MTT assay, cells were seeded at
1.times.10.sup.4 cells/well in 96-well plates and allowed to attach
for 24 hours. The medium was replaced with fresh medium containing
DMSO or digitoxin. The cells were treated for 96 hours after which
the cells were incubated with MTT reagents and the absorbance was
read at 575 and 650 nm. Control and treated cells were compared
using the student's t-test (p<0.05). For cell cycle analysis the
cells were plated (3.times.10.sup.5) onto 6 cm dishes and allowed
to attach for 24 hours. Then the medium was replaced with DMEM
containing 10% FBS and DMSO or digitoxin. After 24 hours the cells
were analyzed by DNA flow cytometry, as described previously.
(Einbond L S, et al., Breast Cancer Res Treat. 83(3):221-31,
2004.)
[0324] Calculating the Combination Index.
[0325] To determine the Combination Index (CI), we exposed
MDA-MB-453 cells to all combinations of 4 concentrations of each of
the agents tested and a solvent control (Einbond L S et al. Actein
and a fraction of black cohosh potentiate antiproliferative effects
of chemotherapy agents on human breast cancer cells. Planta Med.
October; 72(13):1200-6, 2006.). The results of the MTT assay were
analyzed as indicated in the "Calculating the Combination Index"
section of Example 14.
[0326] RNAi-Mediated Gene Knockdown
[0327] The procedure in the "RNAi-mediated gene knockdown" section
of Example 14 was followed, using digitoxin at 0.4 .mu.g/ml for 24
hours.
RNA Isolation and Oligonucleotide Microarray Analysis
[0328] RNA was isolated as previously described. (Einbond L S, et
al. The growth inhibitory effect of actein on human breast cancer
cells is associated with activation of stress response pathways.
Int J. Cancer. November 1; 121(9):2073-83, 2007.) Total cellular
RNA was extracted using Trizol (Invitrogen; Carlsbad, Calif.)
according to the manufacture's protocol with minor modifications,
and then purified twice with Qiagen's RNeasy column as previously
described. Total RNA (8 .mu.g) was reverse transcribed with
T7-(dT)24 primer and Super Script III reverse transcriptase
(Invitrogen). After purification, cDNA was in vitro transcribed
into biotin labeled antisense cRNA with the BioArray high yield RNA
transcript labeling kit (Enzo Life Sciences; Farmingdale, N.Y.),
according to a modified Affymetrix protocol. cRNA (15 .mu.g) was
fragmented into the final probe and hybridized to human U133A 2.0
gene chips (Affymetrix, Inc.; Santa Clara, Calif.), comprised of
more than 22,000 probe sets. The Institute for Cancer Genetics Core
Facility at the Columbia Genome Center performed the
hybridization.
Real-time Quantitative RT-PCR and Western Blot Analysis
[0329] We treated MDA-MB-453 cells with 20 ng/ml, 0.1, 0.2 or 1
.mu.g/mL (26 nM, 0.13, 0.26 and 1.3 .mu.M) of digitoxin for 6 or 24
hours and performed real-time RT-PCR analysis on 2 technical
replicates of at least 2 biological sample replicates. (Tsutsumi S
et al. Celecoxib upregulates endoplasmic reticulum chaperones that
inhibit celecoxib-induced apoptosis in human gastric cells.
Oncogene. February 16; 25(7):1018-29, 2006.) Primer sequences used
in qPCR are listed in Table 14.
[0330] For Western blot analysis, cells were treated for increasing
times with approximately the IC.sub.50 and twice the IC.sub.50
concentration, measured at 48 hours, of digitoxin. Western blot
analysis was performed as described previously. (Einbond L S, et
al., Breast Cancer Res Treat. 83(3):221-31, 2004.) The membrane was
incubated with the primary antibodies to ATF3, EGR1 (Santa Cruz
Biotechnology, Santa Cruz, Calif.), and ERK2 (Cell signaling,
Beverly, Mass.); .beta.-actin was used a loading control.
TABLE-US-00014 TABLE 14 Designed primer sequence used in RT-PCR.
Symbol Forward sequence Reverse sequence GAPD ggcctccaaggagtaagacc
aggggtctacatggcaactg ATF3 tgggaggactccagaagatg gacagctctccaatggcttc
EGR1 gagaaggtgctggtggagac tgggttggtcatgctcacta GDF15
ctccgaagactccagattcc agagatacgcaggtgcaggt CDKN1A
gcctggactgttttctctcg attcagcattgtgggaggag HSF2 atgggaaccctgcttcttct
ttgggttggttctgggtcta DNAJB4 ccggacaagaacaaatctcc
cctcctttcaacccttcctc HMGCR gacctttccagagcaagcac
agctgacgtacccctgacat HMGCS1 ccccagtgtggtaaaattgg
tggcctggacttaacattcc INSIG1 gacagtcacctcggagaacc
caccaaaggcccaaagatag ATF4 ccaacaacagcaaggaggat gtgtcatccaacgtggtcag
GADD34 ggaggctgaagacagtggag cctctagggacactggttgc CDC16-
cgatggctgcttacttcaca cagagcttggctgaagaacc Primers were designed
using Primer3 software from the Massachusetts Institute of
Technology (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).
Gene Expression Analysis
[0331] MDA-MB-453 breast cancer cells were treated with digitoxin
at 0.1, 0.2 and 1 .mu.g/mL and RNA was collected at 6 and 24 hours
for gene expression analysis, and with 20 ng/ml digtoxin for 24
hours. Microarray analysis and an unbiased informatics approach was
used to find the genes and signaling pathways whose expression was
altered by exposure of the cells to digitoxin. Two or three
replicates of each microarray were performed to determine
intrasample variation. To bolster the robustness of the analysis,
the effects of 4 doses for 2 time periods were examined.
[0332] All analyses were performed using the AffyLimmaGUI package
in the open-source Bioconductor suite, as previously described.
(Einbond L S, et al., Int J. Cancer. November 1; 121(9):2073-83,
2007.) All samples were normalized to remove chip-dependent
regularities using the GCRMA method of Irizarry et al. (Irizarry R
A et al. Summaries of Affymetrix GeneChip probe level data. Nucleic
Acids Res. 31(4):e15, 2003.) The statistical significance of
differential expression was calculated using the empirical Bayesian
LIMMA (LI Model for MicroArrays) method of Smyth et al. (Smyth G K
et al. Use of within-array replicate spots for assessing
differential expression in microarray experiments. Bioinformatics.
21(9):2067-75, 2005.) A cut-off B>0 was used for the statistical
significance of gene expression. Variability is reported in terms
of a p-value representing the probability that differences between
treated and untreated could occur by chance. The p-value takes into
account both the variability within groups (in this case the groups
are treated and control) and the variability between groups.
M = log 2 ( Intensity : Treatment Intensity : Control ) = log 2 ( [
Treatment ] [ Control ] ) ##EQU00002##
P-value=The Bayesian P-value (This will not equal the conventional
frequentist P-value since the Bayesian method used by LIMMA pools
variance across genes to increase statistical power). This P-value
was corrected by the Benjamini-Hochberg method to give the number
of false discoveries. B=Log.sub.e(Odds of differential expression).
The Bayesian natural (base e) log of the odds that the genes are
differentially expressed. B>0 Odds>1 implies that the genes
are differentially expressed for the high-throughput analysis, with
especially important genes being validated by PCR.
[0333] The genes that displayed significant changes in levels of
expression were assigned to Gene Ontology categories and KEGG
Pathways. (Khatri P, Draghici S. Ontological analysis of gene
expression data. In Encyclopedia of Genetics, Proteomics and
Bioinformatics. New York: John Wiley and Sons Inc.; 2005.)
Intersections between treatments were calculated using the Gene
Traffic program. Clustering was performed with the Program Cluster
3.0. (Einbond L S, et al., Int J. Cancer. November 1;
121(9):2073-83, 2007; de Hoon M J, et al. Open source clustering
software. Bioinformatics. 20(9):1453-4, 2004.)
Results
The Effects of Digitoxin on Breast Cancer Cell Growth
[0334] After treating Her2 overexpressing, ER low MDA-MB-453 breast
cancer cells with increasing concentrations of digitoxin for 96
hours, digitoxin's effects were assessed by the MTT assay and it
was found that the concentration of digitoxin that caused 50%
inhibition of cell proliferation, the IC.sub.50 value, was about
0.025 .mu.g/mL (0.04 .mu.M). The Coulter counter assay indicated
that digitoxin inhibited growth of the MDA-MB-453 and ER.sup.+BT474
breast cancer cells, with IC.sub.50 values of 0.04 .mu.g/ml (0.05
.mu.M) and 0.03 .mu.g/ml (0.04 .mu.M), respectively. The IC.sub.50
values (0.025 to 0.04 .mu.g/mL) are within the therapeutic range,
10-35 ng/mL (13-46 nM). Digitoxin was less potent on ER positive,
Her2 low MCF7 breast cancer cells, with an IC.sub.50 value of 0.2
.mu.g/ml.
Effects of Digitoxin on Cell Cycle Distribution in MDA-MB-453 Human
Breast Cancer Cells
[0335] The effects on cell cycle distribution at 24 hours after
exposing MDA-MB-453 cells to 0, 0.2 or 2 .mu.g/mL (0, 0.26 or 2.6
.mu.M) are shown in Table 15 below. After treatment with digitoxin
at 0.2 or 2 .mu.g/mL, there was an increase in the subG1 peak,
which may indicate apoptosis. Digitoxin induced a dose-dependent
increase in the percent of cells in G2 and a decrease in the
percent of cells in G1, and, at the higher dose decreases in G1 and
S phases.
TABLE-US-00015 TABLE 15 Effect of digitoxin on cell cycle
distribution in MDA- MB-453 cells treated with 0.2 or 2 mg/ml of
digitoxin and analyzed at 24 hours by DNA flow cytometry. Treatment
SubG1 G1 S G2 0 .mu.g/mL 1.93 (1.02) 57.75 (1.20) 26.90 (0.99)
11.45 (4.74) 0.2 .mu.g/mL 6.77 (1.06) 46.50 (1.98) 27.65 (0.64)
19.05 (0.07) 2.0 .mu.g/mL 5.78 (1.02) 41.90 (2.26) 17.80 (0.42)
32.10 (1.27) The values indicate the percent of cells in the
indicated phases of the cell cycle. The control contained 0.01%
DMSO. Standard deviations are indicated in parentheses.
Alterations in Gene Expression Induced by a Nontoxic Dose of
Digitoxin
[0336] When the effect of a therapeutic dose of digitoxin (20
ng/ml) was examined on gene expression patterns at 24 hours, it was
found that digitoxin significantly altered the expression of 22
genes. The 11 upregulated genes included corneodesmosin, keratin 23
(histone deacetylase inducible), Desmoplakin, and cysteine-rich
secretory protein LCCL domain containing 2; the 11 downregulated
genes included calmegin, chromosome 9 open reading frame 127, and
ubiquitin specific protease 34 baculoviral IAP repeat-containing 1
(Table 16). Of the 22 genes, 4 were also activated after treatment
with a 5-fold higher dose. These included genes involved in
response to stress. It is worth noting that several genes were
activated by Src mediated pathways: GRB7 is phosphorylated in
response to EGF stimulation. RPS6KA5 is activated by ERK. RAB15 is
a member of the RAS oncogene family and involved in GTP binding.
BIRC1 is anti-apoptotic. Genes involved in regulating the cell
cycle are HSF2, which bookmarks DNA during mitosis, and C9orf127.
KCNAB2 has a role in mediating potassium voltage-gated
channels.
TABLE-US-00016 TABLE 16 Differentially expressed genes after
treating MDA-MB-453 cells with digitoxin at 20 ng/ml for 24 hours,
B > 0. Category ID Symbol Name M P. Value B apoptosis 206192_at
CDSN corneodesmosin 2.255 1.34E-05 4.96 218963_s_at KRT23 keratin
23 (histone deacetylase inducible) 2.231 0.199 1.03 204860_s_at
BIRC1 baculoviral IAP repeat-containing 1 -0.55 0.262 0.52 stress
204635_at RPS6KA5 ribosomal protein S6 kinase, 90 kDa, polypeptide
5 -0.87 0.0215 2.57 protein 200606_at DSP desmoplakin 1.176 0.24
0.71 221541_at CRISPLD2 cysteine-rich secretory protein LCCL domain
0.842 0.24 0.71 containing 2 215339_at NKTR natural killer-tumor
recognition sequence -0.42 0.154 1.26 212980_at USP34 ubiquitin
specific protease 34 -1.1 0.271 0.39 205830_at CLGN calmegin -1.47
0.282 0.3 transcription 207839_s_at C9orf127 chromosome 9 open
reading frame 127 -1.12 1.34E-05 4.91 221810_at RAB15 RAB15, member
RAS onocogene family 0.403 0.24 0.63 ion 203402_at KCNAB2 potassium
voltage-gated channel, shaker-related 0.383 0.262 0.49 subfamily,
beta member 2 210486_at ANKMY1 ankyrin repeat and MYND domain
containing 1 0.199 0.121 1.57 signal transduction 210222_s_at RTN1
reticulon 1 0.172 0.24 0.66 nucleotide 201766_at ELAC2 elaC homolog
2 (E. coli) -0.16 0.282 0.29 212913_at MSH5 mutS homolog 5 (E.
coli) -0.64 0.327 0.16 Function unknown 205796_at FLJ11336 NA -0.19
0.213 0.93 222307_at LOC282997 NA -0.34 0.262 0.45 215364_s_at
KIAA0467 NA -0.42 0.0122 3.05 219054_at FLJ14054 NA 1.105 0.144
1.38 221843_s_at KIAA1609 NA 0.916 0.015 2.84 Fold-change (log) is
the mean of the ratio of hybridization signals in digitoxin treated
versus DMSO control treated cells. NA designates function not
known.
Alterations in Gene Expression Induced by Various Treatments with
Digitoxin
[0337] Since only a few genes were altered after treatment with the
nontoxic dose at 24 hours, MDA-MB-453 breast cancer cells were
treated with digitoxin at three higher concentrations, 0.1, 0.2 and
1 .mu.g/mL (0.13, 0.26 and 1.3 .mu.M), RNA was collected at 6 and
24 hours for gene expression analysis in order to maximize the
cells' response to digitoxin and delineate its mechanism of action.
The number of genes impacted by the individual treatments of
digitoxin (IMP>0, p<0.05) increased in a dose- and
time-dependent manner. Thus, treatment with 0.1 .mu.g/mL for 6
hours or 24 hours altered 2 and 8 genes respectively; 0.2 .mu.g/mL
for 6 and 24 hours altered 6 and 88 genes, respectively; 1 .mu.g/mL
for 6 and 24 hours altered 87 and 1491 genes, respectively. Under
all treatment conditions at 6 hours (B>0, p<0.05, IMP>0),
more genes were induced than repressed by a factor of about 1.0 to
2.5-fold, but the inverse (0.5-0.7-fold) was true at 24 hours.
[0338] Using the program Gene Traffic to identify commonly
perturbed genes amongst the 3 doses of digitoxin and 2 time
periods, no commonly perturbed genes were found at 0.1 .mu.g/mL for
6 or 24 hours, 2 commonly perturbed genes at 0.2 .mu.g/mL, and 61
genes or 61/87 genes (at 6 hours) at 1 .mu.g/mL. Thus, the lower
doses of digitoxin altered different sets of genes at 6 and 24
hours, while the highest dose altered similar sets of genes at the
two timepoints.
[0339] Affymetrix Netaffx analysis showed a significant effect on
stress response genes after treatment with digitoxin at 1 .mu.g/ml
for 6 hours (see Table 17 below). Among the early effects were
upregulation of stress (EGR1, NAB2), apoptotic (IHPK2, ARID5B),
lipid biosynthetic (SC5DL), transcription regulation
(NR4A1,ZNF297B, RORA), anti-proliferation (BTG1) and RNA processing
(DDX26) genes and downregulation of cell cycle (C10orf7),
replication (POLR3B) and transcription (EIF2B1) genes. As predicted
(Li Z, Xie Z. The Na/K-ATPase/Src complex and cardiotonic
steroid-activated protein kinase cascades. Pflugers Arch. 2008.
[Epub ahead of print]), digitoxin altered the response of genes
involved in calcium metabolism (IHPK2, NR4A1).
TABLE-US-00017 TABLE 17 Differentially expressed genes after
treating MDA-MB-453 cells with digitoxin at 1.0 .mu.g/ml for 6
hours, B > 0, M > 3. Category ID Symbol Name M P. Value B
transcription 36711_at MAFF v-maf musculoaponeurotic fibrosarcoma
oncogene 8.68 0.00805 7.23 homolog F (avian) 201693_s_at EGR1 early
growth response 1 6.25 0.000282 10.08 216017_s_at NAB2 NGFI-A
binding protein 2 (EGR1 binding protein 2) 5.75 0.000221 10.26
205193_at MAFF v-maf musculoaponeurotic fibrosarcoma oncogene 5.16
0.00103 9.03 homolog F (avian) 202340_x_at NR4A1 nuclear receptor
subfamily 4, group A, member 1 3.35 0.00269 8.22 201725_at C10orf7
chromosome 10 open reading frame 7 -1.24 0.00712 7.35 214185_at
KHDRBS1 KH domain containing, RNA binding, signal 2.22 0.00366 7.95
transduction associated 1 DNA binding 210426_x_at RORA RAR-related
orphan receptor A 0.679 0.000998 9.06 212614_at ARID5B AT rich
interactive domain 5B (MRF1-like) 1.24 0.0033 8.04 219459_at POLR3B
polymerase (RNA) III (DNA directed) polypeptide B -1.92 0.0036 7.96
protein binding 203002_at AMOTL2 angiomotin like 2 2.84 0.00266
8.23 204182_s_at ZNF297B zinc finger protein 297B 2.58 4.53E-06
12.76 221890_at ZNF335 zinc finger protein 335 2.47 0.00767 7.28
78330_at ZNF335 zinc finger protein 335 0.62 0.00393 7.88
201823_s_at RNF14 ring finger protein 14 -1.13 0.00784 7.26
209630_s_at FBXW2 F-box and WD-40 domain protein 2 -1.93 0.00777
7.27 218819_at DDX26 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide
26 1.39 0.00504 7.66 cell growth (-), 218192_at IHPK2 inositol
hexaphosphate kinase 2 2.83 0.00379 7.91 apopotosis (+),
200920_s_at BTG1 B-cell translocation gene 1, anti-proliferative
1.72 0.00121 8.9 oxidative stress 200797_s_at MCL1 myeloid cell
leukemia sequence 1 (BCL2-related) 1.86 0.00378 7.92 214056_at MCL1
myeloid cell leukemia sequence 1 (BCL2-related) 1.76 0.00634 7.45
phase 2 221906_at TXNRD3 thioredoxin reductase 3 -0.755 0.00626
7.46 protein 210592_s_at SAT spermidine/spermine
N1-acetyltransferase 2.66 0.00478 7.71 metabolism 202140_s_at CLK3
CDC-like kinase 3 1.45 0.00155 8.69 201632_at EIF2B1 eukaryotic
translation initiation factor 2B, subunit 1 -1.21 0.00449 7.76
alpha, 26 kDa lipid 211423_s_at SC5DL sterol-C5-desaturase (ERG3
delta-5-desaturase 1.98 0.00453 7.75 metabolism, homolog,
fungal)-like biosynthesis Function 215150_at DKFZp451J1719 NA 2.75
0.00592 7.51 unknown 219397_at FLJ13448 NA 1.59 0.00557 7.57
219016_at FLJ13149 NA 1.3 0.000151 10.54 Fold-change (log) is the
mean of the ratio of hybridization signals in digitoxin treated
versus DMSO control treated cells.
Hierarchical Clustering of Alterations in Gene Expression after
Treating Cells with Digitoxin
[0340] Hierarchical clustering was used to reveal genes that are
coordinately controlled (FIG. 30B). Probesets were restricted to
those that corresponded to an absolute value of M (log fold)
greater than 2.0 for at least one of the treatment conditions. The
threshold for color in the hierarchical clustering map is M greater
than 3 log fold. FIG. 30B shows the full hierarchical clustering
map, which contains 4706 probesets. FIG. 30 A, B and C are expanded
displays of specific subcategories of these probesets.
[0341] FIG. 30 panel A contains a cluster of genes, which, like
ATF3, were mainly activated after treatment with digitoxin at 1
.mu.g/mL, for either 6 or 24 hours, although some of these genes
were also activated after treatment with 0.1 or 0.2 .mu.g/mL for 6
or 24 hours. These included additional stress response (GADD34,
IER2, HSF2) genes. FIG. 30 panel B displays genes that were
downregulated by treatment with digitoxin. They include the cell
cycle control gene CDC16 and replication gene ORC3, which were
repressed after treatment with digitoxin at 1 .mu.g/mL for 6 hours,
further repressed at 24 hours, and slightly repressed after
treatment with digitoxin at 0.2 .mu.g/mL for 6 or 24 hours. The
cluster of genes displayed in FIG. 30 panel C contains the gene
EGR1 which was upregulated after treatment with 0.1, 0.2 or 1
.mu.g/mL of digitoxin for 6 hours, but this did not persist at 24
hours. This cluster also contained lipid biosynthetic genes
(INSIG1). A fourth region, expanded for CDKN1A, showed a
progressive increase in expression after treatment with digitoxin
at 0.1, 0.2 and 1 .mu.g/ml for 6 or 24 hours, with a more
pronounced increase at 24 hours. This cluster also contained the
stress gene DNAJB4 and the apoptotic gene GDF15.
The Effects of Digitoxin on Expression of Specific mRNAs Determined
by Real-Time RT-PCR
[0342] To verify some of the digitoxin-induced changes observed in
gene expression detected by microarray analysis, MDA-MB-453 cells
were also treated with 0.1, 0.2 or 1 .mu.g/mL of digitoxin for 6 or
24 hours, and real-time RT-PCR analysis was performed on 12 genes
related to the stress response or cell cycle control. The RT-PCR
results (FIG. 31 and Tables 18 and 19 as set forth below) were
consistent with those obtained in the microarray analysis. To show
how the data varied, p-values are indicated for all microarray and
PCR genes in the tables.
TABLE-US-00018 TABLE 18 Comparison of the effects of digitoxin on
selected genes by real-time PCR and microarray analysis after
treating MDA-MB-453 cells with digitoxin at 0.1, 0.2 or 1.0
.mu.g/mL for 6 or 24 hours. Fold-change relative to DMSO fold
change (p value) Digitoxin treatment Digitoxin treatment Digitoxin
treatment Affymetrix (6 h, 0.1 .mu.g/mL) (6 h, 0.2 .mu.g/mL) (6 h,
1.0 .mu.g/mL) Categories Gene Number RT-PCR Microarray RT-PCR
Microarray RT-PCR Microarray Stress hsf2 211220_s_at 0.41 (0.06)
1.266 (1.0) 0.905 * 1.93 (0.35) 1.34 * 2.45 0.049 response ATP1A1
220948_s_at 0.188 (0.17) 0.158 (1.0) 0.62 (0.088) 0.223 (1.0) 0.415
0.032 0.241 (1.0) ATF3 202672_s_at -0.25 (0.38) 0.16 (1.0) 1.15 *
1.66 (1.0) 3.99 * 6.91 (0.10) DNAJB4 202887_s_at 0.09 (0.48) 0.80
(1.0) 0.30 * 0.88 (1.0) 1.43 * 2.52 (.1.0) EGR-1 211936_at 1.18
(0.13) 1.68 (1.0) 3.26 * 3.43 (1.0) 5.15 * 5.05 (1.0) INSIG1
201625_s_at 1.53 * 1.71 (1.0) 2.29 * 2.33 (1.0) 2.77 * 2.85 (0.88)
ATF4 200779_at -0.49 (-0.003) -0.38 (1.0) -0.12 (0.26) -0.19 (1.0)
0.97 * 0.56 (1.0) GADD34 37028_at 1.32 (0.07) 1.78 (1.0) 2.48 *
3.42 (1.0) 4.05 * 5.49 (0.02) Promote CDKN1A 209383_at 0.73 (0.09)
1.22 (1.0) 1.62 * 2.06 (1.0) 2.61 * 3.41 (0.34) apoptosis GDF15
221577_x_at 0.14 (0.68) 0.19 (1.0) 0.60 (0.12) 0.73 (1.0) 1.34 *
1.75 (1.0) Cell Cycle CDC16 209658_at -0.51 * -0.83 (1.0) -0.67 *
-1.55 (1.0) -1.31 * -2.82 (0.05) Cholesterol/ HMGCS1 205822_s_at
1.20 0.01 1.09 (1.0) 2.04 * 1.69 (1.0) 2.75 * 2.53 (1.0) fatty acid
biosynthesis
TABLE-US-00019 TABLE 19 24 hours. Fold-change relative to DMSO fold
change (p value) Digitoxin treatment Digitoxin treatment Digitoxin
treatment Affymetrix (24 h, 0.1 .mu.g/mL) (24 h, 0.2 .mu.g/mL) (24
h, 1.0 .mu.g/mL) Categories Gene Number RT-PCR Microarray RT-PCR
Microarray RT-PCR Microarray Stress hsf2 211220_s_at 0.4 (0.062)
1.17 (0.54) 0.63 0.013 1.64 (1.0) 2.32 * 3.23 * response ATP1A1
220948_s_at -0.099 (0.38) 0.35 (1.0) 0.0025 (0.98) 0.5 (1.0) -0.028
(0.78) 0.67 (1.0) ATF3 202672_s_at -1.04 * -0.10 (1.0) 0.62 0.13
0.85 (1.0) 5.32 * 8.42 (0.02) DNAJB4 202887_s_at -0.49 0.15 0.20
(1.0) 0.35 0.18 2.15 (1.0) 3.51 * 6.00 * EGR-1 211936_at -2.00 *
-2.40 (1.0) -3.38 * -4.07 (1.0) -2.30 * -2.49 (1.0) INSIG1
201625_s_at -0.58 0.15 -0.50 (1.0) -1.51 * -1.31 (1.0) -1.08 0.02
-0.61 (1.0) ATF4 200779_at -1.54 * -1.08 (1.0) -0.66 * -0.49 (1.0)
2.71 * 1.65 (1.0) GADD34 37028_at 0.96 (0.04) 0.69 (1.0) 1.64 *
1.80 (1.0) 4.36 * 5.50 (0.02) Promote CDKN1A 209383_at 0.73 (0.09)
1.52 (1.0) 1.62 * 2.76 (1.0) 2.61 * 5.22 (0.01) apoptosis GDF15
221577_x_at 0.97 0.01 1.41 (1.0) 2.35 0.01 2.94 (1.0) 3.20 * 4.25
(1.0) Cell cycle CDC16 209658_at -0.33 (0.12) -0.87 (1.0) -0.44
(0.19) -1.71 (1.0) -1.58 * -4.93 * Cholesterol/ HMGCS1 205822_s_at
-1.06 (0.07) -0.54 (1.0) -1.89 * -1.88 (1.0) -2.01 * -1.01 (1.0)
fatty acid biosynthesis Exponentially dividing cultures of
MDA-MB-453 cells were treated with digitoxin at 0.1, 0.2 and 1.0
.mu.g/ml, and then collected for RNA extraction at 6 or 24 hours.
Microarray analysis was performed as described in Materials and
Methods. Fold-change (log) is the mean of the ratio of
hybridization signals in digitoxin treated versus DMSO control
treated cells. Real-time RT-PCR was performed as previously
described (Einbond et al. 2007a, b); p values are < 0.01, unless
indicated in parentheses
[0343] Consistent with the hierarchical clustering results, there
were four main patterns of expression: (1) mRNAs for the ER stress
gene EGR1 and the lipid genes INSIG1 and HMGCS1 increased at 6
hours and decreased at 24 hours, (FIG. 31C); (2) the integrated
stress response (ISR) genes ATF4, ATF3, PPP1R15A and DNAJB4-B and
HSF2 displayed complex expression patterns after treatment with the
3 doses; at 0.1 .mu.g/ml the expression of ATF3, ATF4 and DNAJB4
decreased from 6 to 24 hours and the expression of HSF2 and
PPP1R15A increased and then leveled off (FIG. 31A) (after treatment
with 1 .mu.g/ml, they showed a progressive increase in mRNA
expression; (3) expression of the apoptotic genes GDF15-A, CDKN1A
and the Na.sup.+-K.sup.+-ATPase ATP1A1 progressively increased
after treatment with digitoxin at all doses for 6 or 24 hours (FIG.
31B, Tables 18 and 19); and 4) the expression of CDC16 decreased at
6 hours and then leveled off (FIG. 31C). Thus, digitoxin activated
early response, cholesterol biosynthetic and integrated stress
response genes, depending on the dose and the duration of
treatment, and progressively induced the expression of apoptotic
genes.
The Effects of Digitoxin on Expression of ATF3 and EGR1Protein in
MDA-MB-453 Cells
[0344] Digitoxin significantly upregulated the expression of the
transcription factor ATF3 and the early response genes EGR1, both
in the microarray analysis studies and in the RT-PCR analysis.
Western blot analysis confirmed that when MDA-MB-453 cells were
treated with digitoxin, EGR1 protein was induced after treating
with digitoxin at 0.1, 0.2 or 1.0 .mu.g/mL for 1 hour, whereas the
ATF3 protein was induced after treating with digitoxin at 1.0
.mu.g/mL for 24 hours (FIG. 32A). Treatment of serum-starved
MDA-MB-453 cells with digitoxin at 80 .mu.g/ml (105 .mu.M) for 30
min yielded a 1.5-fold increase in the level of pSrc.
RNAi-Mediated Gene Knockdown
[0345] To clarify the effects of digitoxin on the ISR survival and
apoptotic responses, the growth inhibitory effects of digitoxin
were examined using the model system RNAi-mediated gene knockdown.
Pretreating cells with siRNA to MAPK1 before treating with
digitoxin (0.4 .mu.g/ml) for 24 hours resulted in a de'crease in
cell proliferation from 96.0% to 76.9%, indicating that MAPK1 is
involved in the survival phase of the digitoxin-induced stress
response (FIG. 32B). Western blot analysis confirmed that ERK2
siRNA did, in fact, reduce the quantity of ERK2 protein by about
50%.
Gene Expression Analysis of the Effects of Digitoxin on MCF7
cells
[0346] To determine whether other cell lines would react similarly
to digitoxin, the effect of digitoxin was tested at 1 .mu.g/mL for
6 or 24 hours on ER positive MCF7 cells, using real-time RT-PCR
analysis. Patterns of expression were observed similar to those
found with MDA-MB-453 cells. Thus,: 1) mRNAs for the ER stress gene
EGR1 and the lipid gene INSIG1 increased at 6 hours and decreased
at 24 hours; 2) the stress genes ATF4, ATF3 and GADD34 showed a
progressive increase in expression of the related mRNAs after
treatment with digitoxin at 1 .mu.g/mL for 6 or 24 hours; and 3)
the cell cycle gene CDC 16 showed a progressive decrease in
expression (FIG. 31 D, E, F). All p values were <0.05.
Digitoxin in Combination Therapy
[0347] The effects of digitoxin on cell proliferation in
combination with the chemotherapy agent paclitaxel on the
MDA-MB-453 cell line were explored. The data obtained when
increasing concentrations of paclitaxel were combined with
increasing concentrations of digitoxin are shown in FIGS. 33 A and
C. In these studies the two test agents were added simultaneously
to the cells. These data were analyzed for the respective
Combination indices (CI) (FIG. 33B). Additive effect was seen with
as little as 0.01 .mu.g/ml digitoxin and 0.5 nM of taxol, and
moderate synergy with 0.01 .mu.g/ml digitoxin and 1 nM of
paclitaxel. With the former combination, the percent viable cells
decreased from 90.6% after treatment with paclitaxel alone to 42.9%
after treatment with paclitaxel plus digitoxin, p<0.01
(digitoxin alone: 73.0%). Addition of digitoxin (0.01 .mu.g/ml) to
paclitaxel (1 nM) decreased cell survival from 63.2% to 30.3%
(p<0.01).
[0348] Gene expression profiles were used to identify the genes and
signaling pathways whose expression was altered by exposure of the
cells to digitoxin in order to obtain insights into mechanisms by
which the cardiac glycoside digitoxin inhibited the growth of
breast cancer cells. The studies indicated that digitoxin at low
dose activated the expression of Src-mediated signaling pathways
and enhanced the effect of the chemotherapy agent paclitaxel;
higher doses of digitoxin activated the expression of stress
response and apoptotic genes.
[0349] A significant impact on stress response genes was
consistently found following exposure to low and high doses for 6
and 24 hours. Digitoxin at a nontoxic dose activated the expression
of Src pathway genes, induced the expression of apoptotic genes and
repressed the expression of replication genes at 24 hours. At
higher doses, digitoxin induced expression of the ISR transcription
factors ATF4, ATF3, ISR genes PPP1R15A and DNAJB4, apoptotic genes
EGR1, CDKN1A and GDF15 and lipid related genes (Table 17).
Real-time RT-PCR validated these findings (Tables 18 and 19).
[0350] Digitoxin altered the expression of several genes involved
in calcium homeostasis, including EGR1, IHPK2 and NR4A1 (Table 17).
Microarrays, hierarchical clustering, and PCR analysis showed
several potential intermediaries of the growth inhibitory effects
of digitoxin, including increased expression of EGR-1, ATF3 and
p21, and decreased expression of the cell cycle related gene CDC16
and replication gene POLR3B.
[0351] The results are consistent with the finding that the
induction of ATF3 occurs via EGR1 downstream of pSrc and ERK1/2.
(Bottone F G, Jr. et al. Transcriptional regulation of activating
transcription factor 3 involves the early growth response-1 gene. J
Pharmacol Exp Ther. 315(2):668-77, 2005.) To test the functional
relevance of ERK, we employed MDA-MB-453 cells and RNAi-mediated
gene knockdown; ERK upstream of EGR-1 appears to mediate the
survival response. These results are consistent with the finding
that digitoxin inhibits Na.sup.+-K.sup.+-ATPase in cardiac
myocytes, thereby activating Src and downstream ERK signaling
cascades that eventually inhibit cell proliferation.
[0352] Digitoxin's upregulation of lipid biosynthetic genes 6 hours
after treatment with 0.1, 0.2 or 1.0 .mu.g/mL of digitoxin may be
due to the ability of ERK to activate gene transcription mediated
by sterols in HepG2 liver cancer cells [Kotzka J et al. Sterol
regulatory element binding proteins (SREBP)-1a and SREBP-2 are
linked to the MAP-kinase cascade. Journal of Lipid Research
41:99-108, 2000.]. This finding may be a cause for concern and
requires additional research. Since digitoxin altered very
different sets of genes after treatment with various dose and time
combinations, doses must be carefully monitored for optimal
clinical outcomes.
[0353] The effects of digitoxin on the expression of genes related
to the ISR are not limited to the MDA-MB-453 cell line; though the
MCF7 cell line was less sensitive to the growth inhibitory effect
of digitoxon, it exhibited increased expression of ATF4, DDIT3,
GDF15, SLC7A11 and CYP1A1 in response to digitoxin treatment. The
results of real-time RT-PCR analysis were remarkably similar
between the two lines (FIG. 31 and Tables 18 and 19): digitoxin
activated early response, cholesterol biosynthetic and integrated
stress response genes, depending on the duration of treatment, and
progressively induced the expression of apoptotic genes.
[0354] To further explore the anticancer potential of nontoxic
concentrations of digitoxin, the effect of nontoxic doses of
digitoxin combined with the chemotherapy agent paclitaxel was
tested and a strong synergy was found. Paclitaxel and digitoxin
inhibit the in vitro activity of purified
Na.sup.+-K.sup.+-ATPtpase. The percent inhibition for paclitaxel
and digitoxin at 20 .mu.M were 21.7 and 35.2%, respectively.
Results indicate that paclitaxel is a potent inhibitor of the
Na.sup.+-K.sup.+-ATPase (data not shown). Consistent with these
findings, paclitaxel has been shown to competitively inhibit ATP
binding activity of the NTPase/helicase of hepatitis C virus with
an IC.sub.50 of about 16 .mu.M (Borowski P. et al., Biochemical
properties of a minimal functional domain with ATP-binding activity
of the NTPase/helicase of hepatitis C virus. Eur J. Biochem.
266(3):715-23, 1999.). It is possible that digitoxin and paclitaxel
alter different sites on the Na.sup.+-K.sup.+-ATPase or have
different molecular targets. The ability of low concentrations of
digitoxin to potentiate the effects of paclitaxel permits the use
of lower doses of this toxic chemotherapy agent in cancer
treatment.
[0355] It is also noted that actein inhibited the in vitro activity
of purified Na.sup.+-K.sup.+-ATPtpase, although not to the extent
of the inhibition seen with either of digitoxin or paclitaxel. The
activities of digitoxin and actein for ATPase inhibition appeared
to be correlated with their activities for cell growth
inhibition.
[0356] The risks, pharmacokinetics and pharmacodynamics of
digitoxin administration are well known in humans, (Lopez-Lazaro M
et al. Digitoxin inhibits the growth of cancer cell lines at
concentrations commonly found in cardiac patients. J Nat. Prod.
68(11):1642-5, 2005). Digitoxin activated transcription of
apoptotic factors and repressed cell cycle related genes and at low
concentrations enhanced the growth inhibitory effect of paclitaxel
on human breast cancer cells.
[0357] In sum, an unbiased informatics approach was used to
characterize the genes and pathways perturbed by digitoxin in
breast cancer cells. Her2 overexpressing, ER low MDA-MB-453 human
breast cancer cells were treated with digitoxin at 4 doses (20
ng/ml to 1 .mu.g/ml; 26 nM to 1.3 .mu.M) RNA was collected at 6
hours and 24 hours. At doses that inhibited cell proliferation,
digitoxin activated the expression of Src-mediated genes. To reveal
primary effects, digitoxin's effect was examined 6 hours after
treatment with the highest dose, 1 .mu.g/ml. Upregulation of the
stress response genes EGR-1 and NAB2, lipid biosynthetic genes and
the tumor suppressor gene p21 was found, and downregulation of the
mitotic cell cycle gene CDC16 and the replication gene PoIR3B was
found. Hierarchical clustering and real-time RT-PCR assays
confirmed four expression patterns: 1) the induction of the early
genes and lipid genes did not increase with time or concentration;
2) ISR genes displayed a complex response depending on the dose and
duration of exposure; 3) the induction of the apoptotic genes
increased with time and dose; and 4) the expression of the cell
cycle gene CDC16 decreased at 6 hours. Thus, digitoxin appears to
inhibit cell growth by activating the transcription of apoptotic
factors (p21, EGR-1, DNAJB4) and repressing cell-cycle-related
genes (CDC16), depending on the dose and the duration of treatment.
Low concentrations of digitoxin enhanced the growth inhibitory
effects of the chemotherapy agent paclitaxel.
[0358] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
Sequence CWU 1
1
26120DNAArtificialsynthetic construct; GAPD forward 1ggcctccaag
gagtaagacc 20220DNAArtificialsynthetic construct; gapd reverse
2aggggtctac atggcaactg 20320DNAArtificialSynthetic construct;
atf3-forward 3tgggaggact ccagaagatg 20420DNAArtificialSynthetic
construct; atf3-reverse 4gacagctctc caatggcttc
20520DNAArtificialSynthetic construct; egr1-forward 5gagaaggtgc
tggtggagac 20620DNAArtificialSynthetic construct; egr1-reverse
6tgggttggtc atgctcacta 20720DNAArtificialSynthetic construct;
gdf15-forward 7ctccgaagac tccagattcc 20820DNAArtificialSynthetic
construct; gdf15-reverse 8agagatacgc aggtgcaggt
20920DNAArtificialSynthetic construct; cdkn1a-forward 9gcctggactg
ttttctctcg 201020DNAArtificialSynthetic construct; cdkn1a-reverse
10attcagcatt gtgggaggag 201120DNAArtificialSynthetic construct;
hsf2 forward 11atgggaaccc tgcttcttct 201220DNAArtificialSynthetic
construct; hsf2-reverse 12ttgggttggt tctgggtcta
201320DNAArtificialSynthetic construct; dnajb4-forward 13ccggacaaga
acaaatctcc 201420DNAArtificialSynthetic construct; dnajb4-reverse
14cctcctttca acccttcctc 201520DNAArtificialSynthetic construct;
hmgcr-forward 15gacctttcca gagcaagcac 201620DNAArtificialSynthetic
construct; hmgcr-reverse 16agctgacgta cccctgacat
201720DNAArtificialSynthetic construct; hmgcs1 17ccccagtgtg
gtaaaattgg 201820DNAArtificialSynthetic construct; hmgcs1-reverse
18tggcctggac ttaacattcc 201920DNAArtificialSynthetic construct;
insig1 forward 19gacagtcacc tcggagaacc 202020DNAArtificialSynthetic
construct; insig1 reverse 20caccaaaggc ccaaagatag
202120DNAArtificialSynthetic construct; atf4 forward 21ccaacaacag
caaggaggat 202220DNAArtificialSynthetic construct; atf4-reverse
22gtgtcatcca acgtggtcag 202320DNAArtificialSynthetic construct;
gadd34 forward 23ggaggctgaa gacagtggag 202420DNAArtificialSynthetic
construct; gadd34 reverse 24cctctaggga cactggttgc
202520DNAArtificialSynthetic construct; cdc16-forward 25cgatggctgc
ttacttcaca 202620DNAArtificialsynthetic construct; cdc16-reverse
26cagagcttgg ctgaagaacc 20
* * * * *