U.S. patent application number 11/246009 was filed with the patent office on 2006-05-25 for angelicae sinensis extracts useful for treatment of cancers.
This patent application is currently assigned to Buddhist Tzu Chi General Hospital. Invention is credited to Wen-Liang Chang, Yeung-Leung Cheng, Horng-Jyh Harn, Shinn-Zong Lin, Jiann-Kuan Luo, Nu-Man Tsai.
Application Number | 20060110469 11/246009 |
Document ID | / |
Family ID | 35613784 |
Filed Date | 2006-05-25 |
United States Patent
Application |
20060110469 |
Kind Code |
A1 |
Luo; Jiann-Kuan ; et
al. |
May 25, 2006 |
Angelicae sinensis extracts useful for treatment of cancers
Abstract
The invention provides an acetone extract, chloroform extract or
hexane extract of Angelicae sinensis and/or the active components
purified therefrom, such as n-butylidenephthalide, which are
effective in treating cancers.
Inventors: |
Luo; Jiann-Kuan; (Temple
Terrace, FL) ; Harn; Horng-Jyh; (Taipei, TW) ;
Chang; Wen-Liang; (Taipei, TW) ; Lin; Shinn-Zong;
(Taipei, TW) ; Cheng; Yeung-Leung; (Taipei,
TW) ; Tsai; Nu-Man; (Shiu Township, TW) |
Correspondence
Address: |
AKIN GUMP STRAUSS HAUER & FELD L.L.P.
ONE COMMERCE SQUARE
2005 MARKET STREET, SUITE 2200
PHILADELPHIA
PA
19103
US
|
Assignee: |
Buddhist Tzu Chi General
Hospital
|
Family ID: |
35613784 |
Appl. No.: |
11/246009 |
Filed: |
October 7, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60616636 |
Oct 8, 2004 |
|
|
|
Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 36/232 20130101; A61P 35/02 20180101; A61P 35/00 20180101;
A61K 31/34 20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 36/185 20060101
A61K036/185 |
Claims
1. A method for inhibiting cancer cell proliferation and migration
in tumor tissues in a subject in need thereof, comprising
administering to the subject an acetone extract, chloroform extract
or hexane extract of Angelicae sinenses or at least one active
component purified from any of the Angelicae sinenses extracts in
an amount effective for inhibiting cancer cell proliferation in
tumor tissues.
2. A method for inhibiting telomerase activity of cancer cells in a
subject in need thereof, comprising administering to the subject an
acetone extract, chloroform extract or hexane extract of Angelicae
sinenses or at least one active component purified from any of the
Angelicae sinenses extracts in an amount effective for inhibiting
telomerase activity of cancer cells.
3. A method for inducing apoptosis of cancer cells in a subject in
need thereof, comprising administering to the subject an acetone
extract, chloroform extract or hexane extract of Angelicae sinenses
or at least one active component purified from any of the Angelicae
sinenses extracts in an amount effective for inducing apoptosis of
cancer cells.
4. A method for treating cancer in a subject in need thereof,
comprising administering to the subject a medicine comprising an
acetone extract, chloroform extract or hexane extract of Angelicae
sinenses or at least one active component purified from any of the
Angelicae sinenses extracts in an amount effective for at least one
of preventing and treating the cancer.
5. A method for the treatment of cancer in a subject in need
thereof, comprising administering to the subject an acetone
extract, chloroform extract or hexane extract of Angelicae sinenses
or at least one active component purified from any of the Angelicae
sinenses extracts as an adjuvant in combination with one or more
chemotherapy drugs through their activities on cell cycle
regulation or telomerase inhibition.
6. The method according to any one of claims 1 to 5, wherein the
active component purified from any of the Angelicae sinensis
extracts includes n-butylidenephthalide.
7. The method according to any one of claims 1 to 5, wherein the
cancer is selected from the group consisting of human malignant
glioblastoma, colorectal, leukemia, neuroblastoma, hepatoma,
breast, ovarian and lung cancer.
8. The method according to any one of claims 1 to 5, wherein the
Angelicae sinenses extract or the at least one active component
purified from any of the Angelicae sinenses extracts is
administered via an oral, parenteral, intravenous (iv),
intraperitoneal (ip), intravenous (iv), intramuscular (im),
subcutaneous (sc), pulmonary, transdermal, buccal, nasal,
sublingual, ocular, rectal or vaginal route.
9. The method according to claim 8, wherein the Angelicae sinenses
extract or the at least one active component purified from any of
the Angelicae sinenses extract is administered via an oral,
parenteral, or intravenous route.
10. A method for the treatment of cancer in a subject in need
thereof, comprising administering to the subject
n-butylidenephthalide in an amount effective for treatment of the
cancer.
11. A method for the treatment of cancer in a subject in need
thereof, comprising administering to the subject
n-butylidenephthalide in an adjuvant in combination with one or
more chemotherapy drugs through their activities on cell cycle
regulation or telomerase inhibition.
12. The method according to claim 10 or 11, wherein the cancers are
selected from the group consisting of human malignant glioblastoma,
colorectal, leukemia, neuroblastoma, hepatoma, breast, ovarian and
lung cancer.
13. The method according to claim 10 or 11, wherein
n-butylidenephthalide is administered via an oral, parenteral,
intravenous (iv). intraperitoneal (ip), intravenous (iv),
intramuscular (im), subcutaneous (sc), pulmonary, transdermal,
buccal, nasal, sublingual, ocular, rectal or vaginal route.
14. The method according to claim 13, wherein n-butylidenephthalide
is administered via an oral, parenteral, or intravenous route.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/616,636, filed Oct. 8, 2004, the disclosure of
which is hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] The invention mainly relates to a new use of an acetone
extract, chloroform extract or hexane extract of Angelicae sinesis
and the active components purified therefrom in the treatment of
cancers.
[0003] Cancers are abnormal cell proliferations that result from
the accumulation of genetic changes in cells endowed with
proliferative potential. Treatment of cancers has relied mainly on
surgery, chemotherapy, radiotherapy and more recently
immunotherapy. However, new approaches for treating and preventing
cancers are still desired.
[0004] Angelicae sinensis (Dangqui) is one of the most frequently
occurring drugs in the prescriptions of traditional Chinese
medicines. The traditional uses of Angelicae sinensis include those
to promote blood production, protect liver, lower blood pressure,
kill bacteria, ease pain mostly for menstrual disorder in women,
and lower cholesterol (Chinese Herbs, Shanghai Science and
Technology Publication, Inc., Shanghai, China, Vol. 5, p. 893,
1999).
[0005] CN1053747 disclosed that Angelicae sinensis (Oliv) Diels,
ASD, and the ASDP and ASDE as effective components of an adjuvant
was prepared, and could be used as an immunological adjuvant to
genetically-engineered hepatitis B vaccines. It was reported in
CN1109356 that the effective component, lactones (ASDE), extracted
from Angelicae sinensis (oliv) diels, ASD, could be used as an
immunological adjuvant, which can enhance the immunogenicity and
help lower toxicity. Kumazawa et al. provided immunostimulating
polysaccharides separated from a hot water extract of Angelicae
sinensis, which could be used as a potent adjuvant for its
anti-tumor activity as observed in the prolongation of the survival
period of mice bearing Ehrlich ascites cells (Y. Kumazawa, et al.,
Immunology, Vol. 47, p. 75, 1982). However, this prior art
reference provides only a general description of the treatment of
cancers with the polysaccharides separated from Angelicae sinesis
through their immunostimulating activity, without sufficient
evidence regarding the mechanism.
BRIEF SUMMARY OF THE INVENTION
[0006] This invention provides that an acetone extract, chloroform
extract or hexane extract of Angelicae sinensis, or at least one
component purified therefrom, such as n-butylidenephthalide (BP),
can also inhibit telomerase activity of cancer cells and further
induce their apoptosis so that they can be used to treat malignant
neoplasms. Therefore, an acetone extract, chloroform extract or
hexane extract of Angelicae sinenses, and the components purified
therefrom, such as n-butylidenephthalide, are potent for
manufacturing of medicines for the treatment of cancers, and can be
used in combination with chemotherapy drugs through their
activities on cell cycle regulation, and telomerase inhibition.
[0007] Accordingly, one object of the present invention is to
provide a method for inhibiting cancer cell proliferation and
migration in tumor tissues.
[0008] Another object of the present invention is to provide a
method for inhibiting telomerase activity of cancer cells.
[0009] Yet another object of the present invention is to provide a
method for inducing apoptosis of cancer cells.
[0010] Another object of the present invention is to provide the
use of an acetone extract, chloroform extract or hexane extract of
Angelicae sinenses, or at least one component purified therefrom,
such as n-butylidenephthalide, for manufacturing medicine for the
treatment of cancer, and as an adjuvant in combination with
chemotherapy drugs through their activities on cell cycle
regulation and/or telomerase inhibition.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings relating to embodiments
which are presently preferred. It should be understood, however,
that the invention is not limited to the precise embodiments
shown.
[0012] In the drawings:
[0013] FIG. 1a provides the results of the cell cycle analysis,
which demonstrates that treatment with 70 .mu.g/ml AS-C (the
chloroform extract of Angelicae sinensis) enhanced cell cycle
accumulation at G0/G1 phase (>90%) in GBM cells (DBTRG-05M)
(*p<0.05) with a concurrent decrease in S phase. The results on
G5T/VGH were about the same and not shown in the graph.
[0014] FIG. 1b provides the results of the cell cycle analysis,
which demonstrates that the treatment with 400 .mu.M BP enhanced
cell cycle accumulation at G0/G1 phase (>90%) in GBM cells
(DBTRG-05MG) (*p<0.05, **p<0.005) with concurrent decrease in
S phase.
[0015] FIG. 2 shows the effect of BP (n-butylidenephthalide) of 5
to 800 .mu.M, in inducing GBM tumor cell (DBTRG-05MG) apoptosis, as
assessed by TUNEL method, using propidium iodide as a counter
staining (*p<0.05, ** p<0.005, *** p<0.0005).
[0016] FIGS. 3a-3c provide the results of the analyses of apoptosis
pathways induced by AS-C, 70 .mu.g/ml (wherein the DBTRG-05MG cell
line was used.)
[0017] FIGS. 3d-3g provide the results of the analyses of apoptosis
pathways induced by BP, 400 .mu.M (wherein the DBTRG-05MG cell line
was used.)
[0018] FIG. 4 shows the inhibitory effect of the AS-C treatment
(500 mg/kg) on the tumor sizes in mice bearing subcutaneous GBM
tumors (RG-2) (p<0.05).
[0019] FIG. 5 shows that the survival rate of the AS-C treated mice
(dose--500 mg/kg) was significantly prolonged as compared with the
control group (p<0.0001), wherein the DBTRG-05MG cell line was
used.
[0020] FIG. 6 shows the inhibitory effect of AS-C treatment (500
mg/kg) on the growth of in situ GBM tumor (RG2) volume on rats (*
p<0.05, ** p<0.001).
[0021] FIG. 7 shows the inhibitory effect of the AS-C treatment
(500 mg/kg by intra-peritoneal or subcutaneous administration) on
the xenograft tumor growth of mice (p<0.005), wherein the
DBTRG-05MG cell line was used.
[0022] FIG. 8 shows the inhibitory effect of the BP treatment (300
mg/kg) on tumor volume of GBM in situ (RG2) on rats, which was
calculated with MRI imaging using echo-planar imaging capability (*
p<0.05, ** p<0.001).
[0023] FIG. 9 shows the inhibitory effect of BP treatment, at
different dosages (70 to 800 mg/kg), on xenograft tumor growth on
mice (p<0.005), wherein the DBTRG-05MG cell line was used.
[0024] FIG. 10 shows the effect of BP treatment (70 to 800 mg/kg)
on the prolongation of survival period of nude mice with xenograft
tumor (subcutaneous DBTRG-05MG) (p<0.001).
DETAILED DESCRIPTION OF THE INVENTION
[0025] This invention provides that the organic solvent extracts of
Angelicae sinensis, or the components purified therefrom, such as
n-butylidenephthalide (BP), can inhibit telomerase activity of
cancer cells and further induce their apoptosis. Therefore, they
can inhibit cancer cell proliferation and can be used to treat
cancers.
[0026] Preparation of Angelicae sinensis Extracts
[0027] Angelicae sinensis (Dangqui) has long been used in blood
diseases and female diseases. Normally, the dried root of Angelica
sinensis (Oliv.) Diels, belonging to the family of Umbelliferae, is
used. Angelica sinensis (AS) is appreciated by those skilled in
this art. A variety of techniques are well known in the art for
extracting, separating, and/or purifying individual active
components of Angelicae sinensis. The organic solvent extracts of
Angelicae sinensis may be obtained by any standard procedures
commonly used in the field. According to the invention, Angelicae
sinensis is extracted with acetone, chloroform, or hexanes. In one
embodiment of the invention, the dried and powdered rhizomes of
Angelicae sinensis were extracted with acetone as a solvent to give
an extract as AS-A. Furthermore, AS-A was further extracted with
chloroform to give an extract as AS-C; and AS-A was further
extracted with hexanes to give an extract as AS-H.
[0028] Purification of Active Components
[0029] Active components of Angelicae sinensis may be isolated
and/or purified from the organic solvent extracts of Angelicae
sinensis by using any techniques known in the art. The active
components may be purified from Angelicae sinensis in any form,
particularly the rhizomes. Various techniques that may be employed
in the further purification include filtration, selective
precipitation, extraction with organic solvents, extraction with
aqueous solvents, column chromatography, high performance liquid
chromatography (HPLC), etc. According to the invention, some active
components were purified from the organic solvent extracts of
Angelicae sinensis, such as ligustilide and n-butylidenephthalide,
which can induce tumor cell apoptosis. In one embodiment of the
invention, E- and Z-geometrical isomers of n-butylidenephthalide
(BP) were separated with column chromatography and characterized
with HPLC and NMR.
[0030] Mechanisms of Cancer Treatment
[0031] Telomeres, the extremities of eukaryotic chromosomes, are
essential for maintaining the integrity of the genome and are a key
determinant of cellular aging and immortality (N. W. Kim, M. A.
Piatyszek, K. R. Prowse, C. B. Harley, M. D. West, P. L. C. Ho, G.
M. Coviello, W. E. Wright, S. L. Weinrich, J. W. Shay, Science,
266, 2011-2015 (1994)). Telomere length and the rate of its
reduction vary among organs and individuals. Large interchromosomal
variation in telomere length exists in mice and humans and an
aberration of a telomere in a single chromosome can lead to
abnormal chromosomal segregation (L. L. Sandell, V. A. Zakian,
Cell, 75, 729-739 (1993)). Therefore, it is concluded that the
regulation and maintenance of telomere length variation play an
important role in cancer development. Apparently cells have a
system to protect against both critical shortening and abnormal
elongation of the telomere. Telomerase has been identified as one
of the telomere length regulators (G. B. Morin, Cell, 59, 521-529
(1989)). Hence, any compound or substance having a selective
inhibiting telomerase activity can inhibit tumor cell growth and
thus further induce cell apoptosis of tumor cells.
[0032] Apoptosis is another mechanism of cancer therapy, which has
become one of the newest areas of cell biology research.
[0033] The activation of the apoptosis program is regulated by
various signals from both intracellular and extracellular stimuli.
Indeed, in recent years evidence is beginning to accumulate that
many (and perhaps all) agents of cancer chemotherapy kill tumour
cells by launching the mechanisms of apoptosis. New drugs
associated with apoptosis are expected to be most effective against
tumours with high proliferation rates. Many such candidates are
being screened for use in the treatment of cancer (Ricardo
Perez-Tomas, Beatriz Montaner, Esther Llagostera, Vanessa
Soto-Cerrato, Biochemical Pharmacology, 66, 1447-1452 (2003)).
[0034] Apoptosis is monitored by the analysis of two commonly used
endpoints--the morphological changes of cells (condensation of
nuclear chromatin, formation of apoptotic bodies) and DNA
fragmentation into large fragments (300 and 50 kbp) and then to
oligonucleosomesized fragments (multiples of 200 bp), which appear
as a "ladder" of DNA bands upon agarose gel electrophoresis.
Although observation of these endpoints is an indicator of
apoptosis, quantification of the percentage of apoptotic cells in a
population by such an assay is impossible. For this purpose, we
also used the TUNEL assay during which fluorescently-biotinylated
nucleotides were added to the ends of DNA fragments within fixed
cells (Jacques Piettea, Cedric Volantia, Annelies Vantieghemb,
Jean-Yves Yvette Habrakena, Patrizia Agostinis, Biochemical
Pharmacology, 66, 1651-1659 (2003)).
[0035] The relative contribution of the receptor and the
mitochondrial pathways to drug-induced apoptosis has been a subject
of controversy. It depends on the type of the cytotoxic drug
itself, the dose and kinetcs or on differences between certain cell
types, which affects the cell type dependent signaling in the
Fas/FasL pathway.
[0036] Apoptosis pathways can be initiated through different sites,
such as the plasma membrane, by way of death receptor mediated
signaling (receptor pathway; Fas/FasL/caspase-8/caspase-3 pathway),
the mitochondria (mitochondrial pathway;
Bax/AIF/caspase-9/caspase-3 pathway), and cell cycle regulation
(including p53, Rb tumor suppressors, p16 and p21 cyclin kinase
inhibitors and cyclin/cdk cell cycle check points) (Simone Fulda,
Matroulea, Klaus-Micheal Debatin, Cancer Letter, 197, 131-135
(2003)).
[0037] It has been reported in the literature that acetone extract,
chloroform extract or hexane extract of Angelicae sinensis and its
active components have anti-angina, anti-agglutination and certain
other activities on the cardiovascular system.
[0038] Surprisingly, we found in this invention that the acetone
extract, chloroform extract or hexane extract of Angelicae
sinensis, and the active components purified therefrom, such as
n-butylidenephthalide, have anti-cancer activities.
[0039] According to this invention, the growth of several cancer
cell lines were tested against the acetone extract, chloroform
extract or hexane extract of Angelicae sinensis, the active
components purified therefrom, such as n-butylidenephthalide. It
was found that they were cytotoxic to cancer cells; they inhibited
telomerase activity of cancer cells (as shown in Example 7); they
suppressed cancer cell proliferation (as shown in Example 2) and
they also induced cancer cell apoptosis (as shown in Examples 3 and
4). Furthermore, animal studies also showed they were effective in
suppressing cancer growth (as shown in Examples 5 and 6). They are
therefore potent for treating cancers, particularly human malignant
glioblastoma, colorectal cancer, leukemia, neuroblastoma, hepatoma,
breast, ovarian and lung cancers.
[0040] Pharmaceutical Compositions
[0041] The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may be administered
by any conventional route of administration including, but not
limited to, oral, parenteral, intraperitoneal (ip), intravenous
(iv), intramuscular (im), subcutaneous (sc), pulmonary,
transdermal, buccal, nasal, sublingual, ocular, rectal, vaginal or
other routes. It will be readily apparent to those skilled in the
art that any dosage or frequency of administration that provides
the desired therapeutic effect is suitable for use in the present
invention. In a preferred embodiment of the invention, they are
administered by oral delivery, using methods known to those skilled
in the art of drug or food delivery.
[0042] For the purposes of therapeutic administration, the acetone
extract, chloroform extract or hexane extract of Angelicae
sinensis, active components purified therefrom, and the derivatives
may be in the form of a tablet, pill, capsule, granule, gel,
powder, sterile parenteral solution or suspension, metered aerosol
or liquid spray, or suppository, depending on the administration
route. To prepare a pharmaceutical composition of the present
invention, the organic solvent extracts of Angelicae sinensis,
active components purified therefrom, or the derivatives are
admixed with a pharmaceutically acceptable carrier according to
conventional pharmaceutical compounding techniques, wherein the
carrier may take a wide variety of forms depending on the form of
preparation desired for administration. Suitable pharmaceutically
acceptable carriers are well known in the art. Descriptions of some
pharmaceutically acceptable carriers may be found in The Hand Book
of Pharmaceutical Excipients, published by the American
Pharmaceutical Association and the Pharmaceutical Society of Great
Britain. For instance, the tablets, capsules, gels, solutions or
suspensions may also include the following components: a
pharmaceutically acceptable excipient or carrier, which is a
non-toxic, inert solid or semi-solid, diluent, encapsulating
material, a gel base or formulation auxiliary of any type. The
solutions and suspensions may contain auxiliaries, such as water
for injection, saline solution, polyethylene glycols, glycerin,
propylene glycol or other synthetic solvents; proteins such as
serum albumin to enhance solubility; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; buffers such as acetates, citrates or
phosphates and agents for the adjustment of tonicity such as sodium
chloride or dextrose. The solution or suspension preparations can
be enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0043] The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may also be
administered as an adjuvant in combination with chemotherapy drugs,
such as actinomycin, adriamycin, Ara-C, bleomycin, carmustin,
cisplatin, cyclophosphamide, daunomycin, mitomycin, taxol,
vinblastine, etc.
[0044] The acetone extract, chloroform extract or hexane extract of
Angelicae sinensis, active components purified therefrom, and the
derivatives according to the present invention may also be
formulated in any dietary compositions by using any techniques
known to those skilled in the art.
[0045] The following Examples are given for the purpose of
illustration only and are not intended to limit the scope of the
present invention.
[0046] Materials and Methods
[0047] Preparation of Angelicae sinensis extracts and Compounds
[0048] The roots of Angelicae sinensis (Oliv.) were supplied from
Chung-Yuan Co., Taipei, Taiwan and were identified by Professor
Han-Ching Lin. A voucher of herbarium specimen was deposited at the
School of Pharmacy, National Defense Medical Center. The dried and
powdered rhizomes of Angelicae sinensis (12 kg) were extracted 3
times with acetone (24 L/time) to give an acetone extract called
AS-A. AS-A was then subjected to chloroform extraction 3 times (24
L/times). The latter extracts were concentrated under reduced
pressure to yield 31.67 g of chloroform extract, called AS-C (from
100 g of acetone extract). A hexane extract (AS-H) was obtained by
extracting AS-A with hexanes. n-Butylidenephthalide (BP) was
purchased from Lancaster Synthesis Ltd. (Newgate, Morecambe, UK)
and used without further purification. E- and Z-forms of BP were
separated with column chromatography and characterized with HPLC
and NMR. They were dissolved in DMSO, incubated with shaking at
25.degree. C. for 1 hour and stored at 4.degree. C. before each in
vitro experiment.
[0049] Cell Proliferation Assay in vitro
[0050] Human umbilical vascular endothelial cells (HUVECs) were
purchased from Cascade Biologics, Inc. (USA). HUVECs were
maintained in Medium 200 (Cascade Biologics, Inc. USA) supplemented
with 10% fetal bovine serum (FBS; Gibco BRL) and low serum growth
supplement (LSGS; Cascade Biologics, Inc. USA). Human dermal
fibroblasts (HDFs) were purchased from Cascade Biologics, Inc.
(USA). HDFs were maintained in Medium 106 (Cascade Biologics, Inc.
USA) supplemented with 10% FBS and low serum growth supplement
(LSGS; Cascade Biologics, Inc. USA). The human colon adenocarcinoma
cell lines HT-29 were purchased from the ATCC (Manassas, Va. USA).
HT-29 were maintained in Dubecco's Modified Eagle Medium (DMEM;
Gibco) supplemented with 10% FBS and 100 ng/ml penicillin and 100
ng/ml streptomycin (Life Technologies, Inc., Grand Island, N.Y.,
USA). For the proliferation assay of cell in log growth (G1), the
cells were harvested at 60-80% of confluence. 100 .mu.l of cell
suspensions were dispensed onto a Falcon 96-well plates The
densities were 6.times.10.sup.3 cells/well in Medium 200 (for
HUVECs), and 9.times.10.sup.3 cells/well in Medium 106 (for HDFs),
and 5.times.10.sup.3 cells/well in DMEM (for HT-29) supplemented
with 10% FBS. The cells were pre-incubated for 24 hours in an
incubator (Humidified atmosphere, e.g., at 37.degree. C., 5%
CO.sub.2). 10 .mu.l of toxicant of various concentrations were
added into the culture medium of the plate. The cell cultures were
incubated for 48 hours. 10 .mu.l of the Cell Counting Kit-8 (CCK-8;
DOJINDO) solution were added and the cell cultures incubated for
1.about.4 hours in the incubator. The absorbance was measured at
450 nm, using a microplate reader with a reference wavelength at
600 nm or over.
[0051] Extraction of RNA from HUVECs
[0052] Total RNA was isolated from the cultured stromal cells using
a modified guanidium isothiocyanate method (Trizol;
Invitrogen).
[0053] Homogenization: The cells were lysed directly in a 6 well
culture plate by adding 1 ml of trizol reagent to a 3.5 cm diameter
well, and passing the cell lysate several times through a
pipette.
[0054] Phase separation: The homogenized samples were incubated for
5 minutes at 15 to 30.degree. C. to permit the complete
dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform
(Riedel-de-haen) per 1 ml of trizol reagent was added. Sample tubes
were capped securely. The tubes were vigorously shaken by hand for
15 seconds and incubated at 15 to 30.degree. C. for 2 to 3 minutes.
The samples were centrifuged at no more than 12,000.times.g for 15
minutes at 2 to 8.degree. C. Following centrifugation, the mixture
separated into a lower red, phenol-chloroform phase, an interphase,
and a colorless upper aqueous phase. RNA remained exclusively in
the aqueous phase.
[0055] RNA precipitation: The aqueous phase was transferred to a
fresh tube. The RNA was precipitated from the aqueous phase by
mixing with isopropyl alcohol (Fluka). 0.5 ml of isopropyl alcohol
was added for every 1 ml of trizol reagent which was added for
homogenization initially. The samples were incubated at 15 to
30.degree. C. for 10 minutes and centrifuged at no more than
12,000.times.g for 10 minutes at 2 to 8.degree. C.
[0056] RNA wash: The supernatant was removed. The RNA pellet was
washed once with 75% ethanol, using at least 1 ml of 75% ethanol
for every 1 ml of trizol reagent, which was added for the
homogenization initially. The sample was mixed by vortexing and
centrifuged at no more than 7,500.times.g for 5 minutes at 2 to
8.degree. C.
[0057] Re-dissolving the RNA: The supernatant was removed, and then
the RNA pellet was dried. The RNA was dissolved in RNase-free
water, and incubated for 10 minutes at 55 to 60.degree. C. The RNA
can be stored at -70.degree. C.
[0058] Cell Lines
[0059] Human tumor cell lines (MCF-7, CL1-5, HT-29, Caco-2), human
umbilical vascular endothelial cells (HUVEC), and human dermal
fibroblasts (HDF) were tested for sensitivity on Angelicae sinensis
extracts, ligustilide, n-butylidenephthalide and its derivatives in
vitro. The DBTRG-05MG, BCM, HL-60 and J5 cells were grown in
RPMI-1640 medium containing 10% fetal calf serum and 100 ng/ml
penicillin and 100 ng/ml streptomycin at 37.degree. C. in a
humidified atmosphere with 5% CO.sub.2. The G5T/VGH, RG2, N18, SVEC
and Balb/3T3 cells were cultured in DMEM with 10% fetal calf serum
and 100 ng/ml penicillin and 100 ng/ml streptomycin at 37.degree.
C. in 5% CO.sub.2. Mycoplasma infection of the culture cells was
excluded by PCR screening methods before each experiment.
EXAMPLE 1
Analysis of Cell Cytotoxicity
[0060] The effects on cell viability after the treatments with
different concentrations of the Angelicae sinensis extracts or the
active components purified therefrom were evaluated by modified MTT
assay in triplicate. Briefly, the cells (5.times.10.sup.3) were
incubated into 96-well plates containing 100 .mu.l of a growth
medium. The cells were permitted to adhere for 24 hours, then
treated with 100 .mu.l of the herbal extracts or the active
components dissolved in the medium. The control contained DMSO of
.ltoreq.0.02% (v/v). After 24, 48 and 72 h incubation, the
drug-containing medium was replaced by 50 .mu.l of fresh medium,
and cells in each well were incubated in 50 .mu.l of 400 .mu.g/ml
MTT for 6-8 h. The medium and MTT were removed later and 100 .mu.l
of DMSO was added to each well and to the control, to dissolve the
soluble components. Absorbance at 550 nm of the solutions was
measured with MRX Microtiter Plate Luminometer (DYNEX, USA). The
absorbance of untreated cells was considered as 100%. To evaluate
the effects of the extracts or the active components on cell growth
rate of GBM cells, 5.times.10.sup.3 exponentially growing cells
were treated with different concentrations for 24, 48, or 72 h. The
cytotoxicity of each test substance was determined as an IC50
value, which represents the drug concentration required to cause
50% inhibition. All experiments in this study were performed in
triplicate. TABLE-US-00001 TABLE 1 Cytotoxicity (IC50) of the
different Angelicae sinensis organic solvent extracts, BP and its
derivatives on different cells lines. Cytotoxicity, IC.sub.50
Normal Cells Cancer cells HUVEC HDF Caco-2 MCF-7 RG2 Extract AS-A
91.85 >100 >100 92.06 6.83 (.mu.g/ml) AS-C 44.19 85.08 51.17
>100 7.22 AS-H >100 66.55 >100 62.42 30.49 BP >100 300
>20 >20 1.4 (>532 .mu.M) (1596 .mu.M) (>106 .mu.M)
(>106 .mu.M) (7.45 .mu.M) BP-E form >100 >100 >20
(>532 .mu.M) (>532 .mu.M) (>106 .mu.M) BP-Z form >100
65.25 >20 (>532 .mu.M) (347 .mu.M) (>106 .mu.M) HUVEC:
Human umbilical vascular endothelial cells HDF: Human dermal
fibroblasts Caco-2: Human colon adenocarcinoma MCF-7: Human breast
carcinoma RG2: Rat malignant glioma SVEC: SV40 transformed mouse
lymph node endothelial cell Balb/3T3: Mouse fibroblast cell
[0061] TABLE-US-00002 TABLE 2 Cytotoxicity (IC.sub.50) of the
organic solvent extracts of Angelicae sinensis, and BP on different
cell lines. AS-A AS-C AS-H BP (.mu.g/ml) (.mu.g/ml) (.mu.g/ml)
(.mu.g/ml) A549 90-110 8-12 (42.6-63.8 .mu.M) AT12 100 10-15
(53.2-79.8 .mu.M) J5 70-90 15-20 (79.8-106.4 .mu.M) HCT15 80-100
25-30 (133.0-160.0 .mu.M) HT-29 21-94 20 30 15-97 (79.8-516.0
.mu.M) CL1-5 29 22 28 >100 (>532 .mu.M) DBTRG-05MG 40-110 44
>400 7-10 (37.2-53.2 .mu.M) G5T/VGH 50-223 46-60 N18 111 35 BCM
300 142 >400 HL-60 367 173 RG2 35 30 1.4 (7.45 .mu.M) SVEC 76 86
Balb/3T3 38 >400 >400 >300 (>1596 .mu.M) A549, AT12:
Human lung adenocarcinoma cell lines (AT12 and A549 are
taxol-resistant subclones) J5: Human hepatoma cell line HCT15:
Human colon adenocarcinoma cell line HT-29: Human colon
adenocarcinoma cell line CL1-5: Human lung adenocarcinoma
DBTRG-05MG: Human glioblastoma multiform cell line G5T/VGH: Human
glioblastoma multiform cell line N18: Neuroblastoma BCM: Human
breast carcinoma HL-60: Human promyelocytic leukemic cell RG2: Rat
maglinant glioma SVEC: SV40 transformed mouse lymph node
endothelial cell Balb/3T3: Mouse fibroblast cell
[0062] TABLE-US-00003 TABLE 3 Test results of ETS-1, MMP-2, cell
migration and tube formation of HUVEC Inhibition RT-PCR Tube ETS-1
MMP-2 Migration Formation Extract AS-A 1 1 30 30 (.mu.g/ml) AS-C 30
30 1 10 AS-H 100 100 30 10 BP 53.13 159.4 531.3 (282.6 .mu.M)
(847.9 .mu.M) (2826 .mu.M) BP-E form 159.4 -- -- BP-Z form 159.4
531.3 159.4
[0063] Both the n-butylidenephthalide (BP) and the Angelicae
sinensis extracts (AS-A, AS-C, AS-H,) generally displayed a lower
value of IC.sub.50 to a number of human tumor cell lines (Table 1
and Table 2) in comparison with the normal cells (HUVEC and HDF).
BP, in particular, exhibited strong cytotoxic effect on human brain
tumor cells. BP was also cytotoxic to two taxol-resistant human
lung adenocarcinoma cells, a human hepatoma cell and two human
colon adenocarcinoma cells.
[0064] The IC.sub.50 of AS-C and BP to brain tumor cell lines were
35.about.60 .mu.g/ml and 1.4.about.10 .mu.g/ml, respectively, while
those to the normal cell line (HDF) were 85.about.300 .mu.g/ml,
(p<0.0001).
[0065] Among the normal cells, the vascular endothelial cells
(IC.sub.50=44.2.+-.0.1 .mu.g/ml) were more sensitive than
fibroblast cells (IC.sub.50=85.1 .mu.g/ml) to AS-C (p<0.05).
[0066] The inhibition effects of carmustin (BCNU) and Taxol were
also tested and compared. The results showed that GBM tumors were
not sensitive to carmustin (IC.sub.50>100 .mu.g/ml) but
DBTRG-05MG and G5T/VGH GBM cells were sensitive to Taxol
(IC.sub.50=61.0.+-.3.3 .mu.g/ml and IC.sub.50<0.1 .mu.g/ml,
respectively). However, Taxol induced a very high cytotocixity
(IC.sub.50<0.1 .mu.g/ml), which is much greater than that
induced by AS-C and BP, in vascular endothelia cells. After
treatment with AS-C or BP, the GBM cells (DBTRG-05MG) were seen to
be detached and floating in the media at different points of time
within a 72-hour period of observation. The extent of GBM cell
detachment and flotation was found to increase with time, and with
the increase in dosage (in the case of BP when an observation was
made at 3 hours).
[0067] The GBM cell detachment and flotation after treatment with
AS-C or BP, can be attributed to morphology change in the tumor
cells. In the above experiment, BCNU (carmustin) was used as the
system control.
EXAMPLE 2
AS-C and BP Enhance the Cell Cycle Arrest at G.sub.0/G.sub.1 Phase
in GBM Cells
[0068] Brain tumor cell lines DBTRG-05MG and G5T/VGH were cultured
in the growth medium with a diluent. For each test and control,
DMSO was added, and the content is less than 0.02% (v/v). For the
AS-C and BP treatment, 70 .mu.g/ml of AS-C and 400 .mu.M of BP were
added, respectively. All were cultured for 48 hours. The analysis
of cell cycle distribution was performed by DNA staining with
propidium iodide (PI). Briefly, 2.times.10.sup.6 adherent cells
were detached by trypsinization. The detached cells and the
floating dead cells were centrifuged and washed twice with 10 ml of
cold 1.times.PBS (Life Technologies, Inc.). Supernatant was
aspirated, cells were re-suspended in 0.8 ml of 1.times.PBS, and
then 200 .mu.l of PI solution (50 .mu.g/ml PI+0.05 mg/ml RNase A;
Sigma Chemical Co.) was added, and the cells were refrigerated at
4.degree. C. overnight. The cells were incubated while protected
from light at room temperature for at least 2 h before DNA
analysis. After staining, DNA was detected and quantified on 20,000
total cells using a FACScan (Becton Dickinson Immunocytometry
Systems, San Jose, Calif., USA) and CellQuest analysis software.
The G.sub.0/G.sub.1 phases were gated in M1 (.times.2); G2/M phases
were gated in M2 (.times.2); the total cells were gated in M3; S
phase was M3-(M1(.times.2)+M2(.times.2)); Sub G1 phase (apoptosis
cells) was gated in M4.
[0069] The cell cycle analysis demonstrated that both 70 .mu.g/ml
of AS-C and 400 .mu.M of BP enhanced the cell cycle accumulation at
G.sub.0/G.sub.1 phase (>90%) in GBM cells. FIGS. 1a and 1b
showed that both AS-C and BP enhanced a significant G0/G1 phase
arrest with a concurrent decrease of S phase after treatment for 12
hours to 48 hours (p<0.05,p<0.005).
EXAMPLE 3
AS-C and BP Induce GBM Cells Apoptosis
[0070] Apoptotic cell death was analyzed using In Situ Cell Death
Detection Kit, POD (Roche, Germany). Changes in DNA chromatin
morphologic features were used for quantification. The procedures
were performed in accordance with the manufacturer's instructions.
Briefly, cells were cultured on culture dish and analyzed 72 hours
after treatment with AS-C (70 .mu.g/ml) and BP (5.about.800
.mu.g/ml), respectively. In AS-C and BP-treated groups, the
suspended cells were collected. In the control group, adherent and
floating cells were collected. Then, the cells were fixed with 3.7%
formaldehyde at room temperature for 15 min. on saline coated
slides, washed once in 1.times.PBS, and incubated in cold
permeabilization solution (0.1% Triton X-100+0.1% sodium citrate)
after reducing activity of endogenous peroxidase with 3%
H.sub.2O.sub.2. The cells were washed with 1.times.PBS again, and
incubated with terminal deoxynucleiotidyl transferase
(TdT)-mediated dUTP nicks labeling (TUNEL) reaction mixture for 60
minutes at 37.degree. C. Then, the cells were washed with
1.times.PBS, counterstained with propidium iodide (PI) for cell
counting. For quantification of apoptosis, the results were viewed
under fluorescence microscopy (Nikon, Kawasaki, Japan).
[0071] When compared to untreated cells, almost all detached GBM
cells in the AS-C and BP-treated groups were found to have
undergone apoptosis. The apoptotic cells with AS-C treatment were
detected by fluorescence microscopy (400.times.), with In Situ
TUNEL staining and propidium iodide cell counterstaining, and
observed under light field. Likewise, BP induced apoptosis in GBM
cells was assessed by TUNEL method and using propidium iodide as a
counter stain. The GBM cells were exposed to BP (5 to 800 .mu.M)
for 48 hours before assessment. The results were shown in FIG. 2.
It was found that as compared to the untreated cells (control), the
apoptosis rates of GBM cells in the BP treatment group, were much
higher.
EXAMPLE 4
AS-C and BP Induce Apoptosis through the Activation of Multiple
Pathways
[0072] Western blot analysis of apoptosis molecules
[0073] DBTRG-05MG cells (human GBM cells) were treated with AS-C
(70 .mu.g/ml) for 0, 6, 12, 24 and 48 hours. In another test,
DBTRG-05MG cells were treated with BP (400 .mu.M) for 0, 1.5, 3, 6,
12, 24 and 48 hours. The cell pellets were re-suspended in lysis
buffer (10 nM Tris-HCl, pH 7.5, 1 mM EGTA, 0.5% CHAPS, 10% (v/v)
glycerol, 5 mM , .beta.-2-mercaptoethanol and 0.1 mM
phenylmethylsulfonyl fluoride) and incubated on ice for 30 min, and
then centrifuged at 13000.times.rpm at 4.degree. C. for 20 minutes.
The protein concentration of whole cell lysates was measured with
BCA protein assay kit (Pierce, Rockford, Ill.) following the
manufacturer's instructions. The cell lysates (20 .mu.g/lane) were
electrophoresed on 10-12% SDS-PAGE (Bio-Rad, Hercules, Calif.).
Proteins were transferred to polyvinyldenefluoride (PVDF) membranes
(Amersham Lifesciences, Piscataway, N.J.). The membranes were
masked for 1 hour at room temperature with 5% skim milk as the
blocking agent, and incubated with the respective antibodies of Fas
(FL-335), Fas-L (C-178), caspase 3 (H-277), caspase 8 (H-134),
caspase 9 (H-170), Bax (B-9), p16 (F-12), p21 (F-5), p53 (DO-1;
1/100 dilution) (Santa Cruz Biotechnology Inc., Calif., U.S.A.),
phospho-p53 (Ser15; 1/2000 dilution) and phospho-Rb (Ser795; 1/2000
dilution) (Cell Signaling Technology, Mass., USA) for 2 hours at
room temperature. Antibody recognition was detected, by incubating
the membranes with the respective anti-mouse, anti-rabbit,
anti-goat IgG secondary antibodies ( 1/1000 dilution; Santa Cruz
Biotechnology Inc., Calif., U.S.A.) conjugated to horseradish
peroxidase, for 1 hour at room temperature, and visualized with the
ECL Plus chemiluminescence system (Amersham, Arlington Heights,
Ill.). For system control, SDS-PAGE gels, for every test sample,
were prepared in duplicate, containing the same amount of protein;
and the control gel was stained with coomassie blue. The other gel
was used for Western Blot analysis. The intensity of the bands was
analyzed by densitometry with a GS-800 Calibrated Imaging
Densitometer (Quantity One 4.0.3 software; Bio-Rad). The results
showed that AS-C significantly increased Fas expression of GBM
cells (1 to 159 fold) but not Fas-L expression. In addition, the
activation of the death receptor-induced apoptosis-related
caspases-8 was monitored. The results indicated that the amount of
procaspase-8 was only slightly increased at 6 h after AS-C
treatment, whereas the amount of the activated caspase-8 was
greatly increased at 6 h after AS-C treatment (see FIG. 3a).
[0074] The phosphorylations of p53 and Rb proteins were monitored
and the results showed the AS-C increased phosphorylated p53
protein at 6 h after treatment. Furthermore, the amount of total
p53 protein was increased as well at 6 h and then gradually
decreased. However, phosphorylated Rb protein was seen to decrease
at 6 h, and became undetectable at 12 h after AS-C treatment. These
results indicated that AS-C could trigger the cell cycle checkpoint
machinery. The amounts of p16, p21 and Bax in AS-C treated GBM
cells were consequently measured and all of these three proteins
were found to increase after treatment with AS-C (see FIG. 3b).
[0075] Finally, the activations of procaspase-9 and procaspase-3
were also determined. Both procaspase-9 and procaspase-3 were
highly activated at 6 h after AS-C treatment (FIG. 3c).
[0076] In the case of BP, the results showed that BP 400 .mu.M
greatly increased the expression of Fas (from 5.2 times at 1.5 h to
27.9 times at 48 h), while suppressing the expression of the Fas
Ligand on the GBM cells (see FIG. 3d).
[0077] It is also observed that BP enhanced the activation of
caspase 8, which was increased to 137.9 by the 48 h, while
procaspase 8 declined (see FIG. 3d).
[0078] The study of the role of mitochondrial pathway in BP-induced
apoptosis showed that BP induced Bax and AIF expression, which
increased to 16 times and 2.4 times respectively, by the 48 h, and
activated caspase-9 by 25.8 times at the 48 h while procaspase-9
declined (see FIG. 3e). Caspase-3 was also observed to increase
while procaspase-3 declined (FIG. 3d).
[0079] The study of the role of cell cycle pathway in BP-induced
apoptosis showed that BP increased p53, p21 and p16 expression by
1.4, 2.3 and 3.1 times respectively at 48 h. It also increased p53
phosphorylation by 5.2 times at 1.5 h and 9.2 times at 48 h, but
decreased Rb phosphorylation by 0.2 times at 48 h (FIG. 3f).
Beta-actin was used as an internal control in this study. In FIG.
3g, BP was seen to decrease also cdk2, cdk4, cdk6 cyclins D1 and
cycline E.
[0080] In conclusion, we theorize a schematic model of the
apoptosis signal transduction pathways induced by BP stress, which
consists of death receptor, mitochondrial and cell cycle
pathways.
EXAMPLE 5
Animal Studies
[0081] The RG2 cells (rat GBM) and DBTRG-05MG cells (human GBM)
were used in animal experiments to monitor the anti-tumor
activities of AS-C and BP. Male F344 rat (230-260 g) and male Foxn1
nu/nu mice (10-12 weeks) were obtained from National Laboratory
Animal Center (Taipei, Taiwan). All procedures were performed in
compliance with the Standard Operation Procedures of the Laboratory
Animal Center of Tzu Chi University (Hualien, Taiwan). Animals were
kept under pathogen-free conditions and fed a standard laboratory
diet. The DBTRG-05MG cells (human GBM) and RG2 cells (rat GBM) were
prepared for nude mice xenografts and rat allogenics,
respectively.
[0082] For AS-C treated group
[0083] Experiment 1--Effect of AS-C administered by subcutaneous
injection on the survival rate and tumor size of rats bearing
subcutaneous GBM tumor
[0084] Syngeneic F344 rats in two groups (6/group) were implanted
subcutaneously on the back with 1.times.10.sup.6 RG2 cells. The
animals were administered by subcutaneous injection either with
AS-C (500 mg/kg/day) (treatment group), or with the vehicle (50
mg/ml propylene glycol and 100 mg/ml Tween-80 in distilled water;
Standard Chem. & Pharm., Tainan, Taiwan) (control group), at a
spot distant from the inoculated tumor sites (>2 cm), on day 3,
6 and 9, after tumor cell implantation. Tumor sizes were measured
using a caliper and the volume was calculated as
L.times.H.times.W.times.0.52. The animals were sacrificed when
tumor volume exceeded 25 cm.sup.3 and the day of sacrifice was
assumed as the final survival day for the animals.
[0085] The results showed that AS-C treatment had a significant
inhibitory effect on tumor growth when compared with the untreated
(control) group (p<0.05) (FIG. 4). The average tumor size at day
26 was 20.7.+-.1.5 cm.sup.3 for the control group and 11.5.+-.0.7
cm.sup.3 for the treatment group, respectively. Survival of rats in
the AS-C treated group was significantly prolonged, compared with
those in the control group (40.+-.2.7 days vs 30.+-.2.1;
p<0.0001) (FIG. 5).
[0086] With a dose of 500 mg/kg subcutaneous injection of AS-C, no
drug related toxicities were observed in the rats as evidenced by
the results of the body weights and histological analysis of the
vital organs.
[0087] Experiment 2--Comparative effects of AS-C administered by
subcutaneous injection and by intra-peritoneal injection on the
tumor size of mice bearing subcutaneous human GBM tumor
[0088] Nude mice, in two groups (6 /group), were implanted s.c.
with 5.times.10.sup.6 DBTRG-05MG cells, and administered with AS-C
(i.p. 500 mg/kg/day), AS-C (s.c. 500 mg/kg/day) or vehicle (s.c.)
on day 5 after tumor cell implantation. Tumor sizes were measured
using a caliper and the volume was calculated as
L.times.H.times.W.times.0.52. The animals were sacrificed when
tumor volume exceeded 1000 mm.sup.3 in mice, and the day of
sacrifice was assumed as the final survival day for the
animals.
[0089] The results showed that there were significant suppressions
of tumor growth in the AS-C i.p. (500 mg/kg) and AS-C s.c. (500
mg/kg)-treated groups compared with the untreated group
(p<0.005). The mean values of tumor sizes at day 38 were
849.9.+-.150.1 mm.sup.3 in the control group, 295.5.+-.25.3
mm.sup.3 in AS-C i.p. (500 mg/kg) treated group and 155.1.+-.56.4
mm.sup.3 in AS-C s.c. (500 mg/kg) treated group. The results were
shown in FIG. 7.
[0090] Experiment 3--Effect of AS-C administered by subcutaneous
injection, on the tumor size of rats bearing in situ GBM tumor
(intra-cranial allogenic GBM).
[0091] The cytotoxic effect of AS-C on in situ tumor was determined
with RG2 cells. Syngeneic rats in two groups (6/group), were
implanted i.c. (striatum) with 5.times.104 RG2 cells, and treated
with AS-C (500 mg/kg/day) or vehicle s.c. at day 4, 5, 6, 7 and 8
after tumor cell implantation. Tumor volumes were measured and
calculated by 3-T unit MRI (General Electric, Wis., USA) with
echo-planar imaging capability (Signa LX 3.8, General Electric,
Wis., USA) in Buddhist Tzu Chi General Hospital (Hualien, Taiwan).
Briefly, rats were anesthetized with chloral hydrate (400 mg/ml, 1
ml/100 g). Functional MRI scanning was conducted with a fast spin
echo, echo-planar acquisition sequence in which the repetition time
was 6000 msec, the echo time was 102 msec, the matrix image was 256
by 256, the field of view was 5 by 5 cm, and the in-plane
resolution was 80 .mu.m. Twenty slices, each 1.5 mm thick, were
obtained every 19.5 seconds for 6.5 minutes for each rat.
[0092] Significant declines of tumor volume in the treated group
were observed in the MRI image data, compared with the untreated
group (p<0.05) (FIG. 6). The mean tumor volumes at day 14 and
day 16 were 70.+-.4.8 mm.sup.3 and 126.4.+-.11.1 mm.sup.3 in the
control group versus 46.2.+-.3.6 mm.sup.3 and 99.5.+-.9.5 mm.sup.3
in AS-C treated group.
[0093] Experiment 4--Cytotoxicities of AS-C administered by
subcutaneous injection, on the tumor size of mice bearing xenograft
human GBM tumor
[0094] In this experiment, the tumor was allowed to grow to a
larger size to simulate a clinical condition where surgical removal
of tumor was not an acceptable option.
[0095] As described above, DBTRG-05 MG cells (5.times.10.sup.6)
were implanted s.c. on the backs of nude mice. The tumor-bearing
mice were treated with single dose of AS-C (500 mg/kg) or vehicle
(s.c.) only when the tumor volumes were .gtoreq.250 mm.sup.3. The
mice were sacrificed to determine the cytotoxicities in tumors by
H&E tissue staining at day 10 after treatment of AS-C. The
tissue sections were observed and photographed under a light
microscope at magnifications of 50.times. and 400.times..
[0096] The photographs of histology analysis showed that AS-C had
induced a nucleic degradation, a cavity cytosol and tumor cell
death in the tumor cell mass. In contrast, the control tumor were
growing very well and the cytotoxic effects as seen in the AS-C
treated group, were not found in the tumor mass.
[0097] For BP treated group
[0098] Experiment 1--Effects of BP on intracranial (i.c.) rat
allogenics GBM tumors in F344 male rat.
[0099] The rats in two groups (6/group) were implanted i.c.
(striatum) with 5.times.104 RG2 cells, and randomly treated with BP
(300 mg/kg/day) or vehicle s.c. in the hind flank region after
tumor cell implantation at day 4, 5, 6, 7 and 8 for five dosages.
Tumor volume was measured and calculated by MRI. MRI was performed
with a 3-T unit (General Electric, Wis., USA) with echo-planar
imaging capability (Signa LX 3.8, General Electric, Wis., USA).
Briefly, rats were anesthetized with chloral hydrate (400 mg/ml, 1
ml/100 g). Functional MRI scanning was conducted with a fast spin
echo, echo-planar acquisition sequence in which the repetition time
was 6000 msec, the echo time was 102 msec, the matrix image was 256
by 256, the field of view was 5 by 5 cm, and the in-plane
resolution was 80 .mu.m. Twenty slices (1.5 mm thick each) were
obtained every 19.5 seconds for 6.5 minutes for each rat. Finally,
the whole tumor sizes (mm.sup.3) were measured and calculated for
each group.
[0100] The results indicated that there was a significant reduction
of tumor volume in the BP treated group as compared with the
untreated group (p<0.05), as calculated using MRI scanning and
Echo-planar as described above (FIG. 8). Each column in FIG. 8
represents a mean.+-.SE (*: p<0.05; **: p<0.001). The mean of
tumor volumes at day 14 and day 16 were 69.9.+-.4.81 mm3 and
126.43.+-.11.07 mm.sup.3 for the control group respectively; and
46.6.+-.1.8 mm.sup.3 and 91.68.+-.8.3 mm.sup.3 for the BP-treated
group respectively. MRI image data showed that the tumor volume in
situ of BP-treated group had a smaller region than control
group.
[0101] Experiment 2: Effects of BP on supressing the growth of s.c.
xenograft human GBM tumors in nude mice, and on the survival rate
of the mice.
[0102] Animals (Foxn1 nude mice) in six groups (6/group) were
implanted subcutaneously (s.c.) with 1.times.10.sup.6 DBTRG-05MG
cells, and randomly treated with BP s.c. (70, 150, 300, 500, 800
mg/kg/day) or vehicle s.c. at a site remote (>2 cm) from the
incubated tumor after tumor cell implantation, at day 4, 5, 6, 7
and 8 for all five dosages. Tumor size was measured every 2 days
and tumor volume was calculated. Animals were sacrificed when tumor
exceeded 1000 mm.sup.3. Tumor growth was monitored for 3 months for
those not sacrificed. Survival rate was followed for up to 200
days.
[0103] The results showed that there were significant suppressions
of tumor growth in the BP-treated groups compared with the
untreated group (FIG. 9; p<0.005 for the 300 mg/kg group), and
the degree of tumor growth inhibition is dose dependent.
[0104] Log-rank (Mantel-Cox) comparison of survival plots given in
FIG. 10 indicated that the survival period of nude mice with
xenograft subcutaneous human GBM was prolonged up to 200 days upon
treatment with BP.
EXAMPLE 7
Effects of AS-C on Telomerase Activity of Human Malignant Tumor
Cell
[0105] Assay for telomerase activity by TRAP
[0106] The telomerase activity was measured by the modified
telomere repeat amplification protocol (TRAP) assay as described in
the literature. The pelleted cells were lysed with 200 .mu.l of
ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.5%
CHAPS, 10% [v/v] glycerol, 5 mM .beta.-2-mercaptoethanol, and 0.1
mM phenylmethylsulfonyl fluoride) and incubated on ice for 30
minutes, and then centrifuged at 13,000.times.g at 4.degree. C. for
20 minutes. The supernatant extracts were quantified for protein
using a BCA Protein Assay Kit (Pierce, Ill., USA). TRAP assay was
performed using a TRAPeze Telomerase Detection Kit (Intergen Co.,
Purchase, N.Y., USA) and the procedures were followed in accordance
with the manufacturer's protocol. In brief, a volume of the extract
containing 0.5 .mu.g of protein was added to 50 .mu.l of reaction
mixtures containing 0.1 .mu.g of substrate oligonucleotide (TS)
primer (5'-AATCCGTCGAGCAGAGTT-3'), 0.1 .mu.g of TSK1 template
(internal control), 0.1 .mu.g of reverse oligonucleotide primer
(RP), 2 U of Takara Taq DNA polymerase (Takara Shuzo Co., Japan),
20 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% (v/v) Tween
20, 1 mM EGTA, 50 .mu.M each of deoxynucleotide triphosphate
(dNTPs), and 1.25 .mu.Ci of (.gamma.32P) ATP, 3000 Ci/mmol
(PerkinElmer Life Science, Boston, Mass., USA). The reaction
mixtures were incubated at 94.degree. C. for 2.5 minutes and then
amplified for 30 cycles of polymerase chain reaction (PCR)
amplification at 94.degree. C. for 30 seconds, 59.degree. C for 30
seconds, and 72.degree. C. for 90 seconds in a DNA thermal cycler
(GeneAmp PCR System 2400, PerkinElmer Co., Norwalk, Conn., USA).
The TRAP products were resolved by 12.5% (w/v) non-denaturing
polyacrylamide gel electrophoresis (PAGE) in a buffer containing 54
mM Tris-HCl, pH 8.0, 54 mM boric acid, and 1.2 mM EDTA. The gel was
dried on filter paper for 1 hour and exposed on the X-film (Bio-Max
MR, Kodak Rochester, N.Y., USA) at -80.degree. C. for 6 hours with
an intensifying screen. The signal intensity of the TRAP assay DNA
ladder products was quantified by the Bio-Profil Biolight imaging
analysis software, V2000.01 (Vulber Lourmat, France) and
compared.
[0107] Statistics
[0108] Data were expressed as the mean.+-.SD or SE. Statistical
significance was analyzed by Student's t-test. A p value of
<0.05 was considered significant. Survival was compared by
log-rank (Mantel-Cox) test. Median survival time was estimated from
Kaplan-Meier analysis.
[0109] While embodiments of the present invention have been
illustrated and described, various modifications and improvements
can be made by persons skilled in the art. It is intended that the
present invention is not limited to the particular forms as
illustrated, and that all the modifications not departing from the
spirit and scope of the present invention are within the scope as
defined in the appended claims.
* * * * *