U.S. patent application number 11/143864 was filed with the patent office on 2006-12-07 for hibiscus anthocyanins for inhibiting cancers.
Invention is credited to Chau-Jong Wang.
Application Number | 20060275521 11/143864 |
Document ID | / |
Family ID | 37494436 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275521 |
Kind Code |
A1 |
Wang; Chau-Jong |
December 7, 2006 |
Hibiscus anthocyanins for inhibiting cancers
Abstract
This invention provides a composition for inhibiting cancer cell
growth comprising Hibiscus anthocyanins extracted from Hibiscus
sabdariffa. This invention also provides a method for treating
cancer comprising administering a patient with an effective amount
of Hibiscus anthocyanins.
Inventors: |
Wang; Chau-Jong; (Taichung,
TW) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 8910
RESTON
VA
20195
US
|
Family ID: |
37494436 |
Appl. No.: |
11/143864 |
Filed: |
June 3, 2005 |
Current U.S.
Class: |
424/778 |
Current CPC
Class: |
H04N 5/23248 20130101;
H04N 5/2253 20130101; A61K 36/185 20130101; A61P 35/04
20180101 |
Class at
Publication: |
424/778 |
International
Class: |
A61K 36/185 20060101
A61K036/185 |
Claims
1. A composition for inhibiting cancer cell growth comprises
Hibiscus anthocyanins.
2. The composition as claimed in claim 1, wherein the Hibiscus is
Roselle (Hibiscus sabdariffa).
3. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins are prepared from the flower or calyx of the
Roselle.
4. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins are characterized by HPLC shown in FIG. 1.
5. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins are extracted by water or organic solvent.
6. The composition as claimed in claim 5, wherein the organic
solvent is alcohol, ester, alkane or halogenated alkane.
7. The composition as claimed in claim 6, wherein the alcohol is
methanol.
8. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins comprise delphindin and cyaniding.
9. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins are prepared by a method comprising: (a) incubating
Roselle with methanol, (b) concentrating the incubation under
reduced pressure, (c) eluting the precipitate with methanol, and
wherein the methanol contains 0.1% HCl.
10. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins induce the apoptosis of the cancer cell.
11. The composition as claimed in claim 10, wherein the cancer cell
is human leukemia cell.
12. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins activate Caspase 8 pathway.
13. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins activate Caspase 3 pathway.
14. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins stimulate cytochrome c release.
15. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins activate p 38 MAP kinase pathway.
16. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins activate c-Jun kinase pathway.
17. The composition as claimed in claim 1, wherein the Hibiscus
anthocyanins activate Bid pathway.
18. The method as claimed in claim 1, further comprises a
pharmaceutical acceptable carrier.
19. A method for treating cancer comprises administering a patient
with an effective amount of Hibiscus anthocyanins.
20. The method as claimed in claim 19, wherein the cancer is
leukemia.
21. The method as claimed in claim 20, wherein the Hibiscus
anthocyanins is characterized by HPLC shown in FIG. 1.
22. The method as claimed in claim 19, wherein the effective amount
is 0.5.about.5.0 mg/ml.
23. The method as claimed in claim 22, wherein the effective amount
is 3.0 mg/ml.
24. The method as claimed in claim 19, wherein the administration
is via injection, oral or external application
25. The method as claimed in claim 23, wherein the administering is
via injection.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a composition for inhibiting
cancers comprising Hibiscus anthocyanins.
DESCRIPTION OF PRIOR ART
[0002] Nutrition should be given a thorough consideration in cancer
prevention because almost a third of all cancer events can be
prevented by a change in diet (Doll and Peto, 1981, J. Natl. Cancer
Inst. 66: 1191; Willett, 1995, Environ. Health Perspect. 103: 165).
Vegetables and fruit are excellent sources of cancer preventive
substances. Strong epidemiological evidences suggest that a diet
rich in vegetables and fruits can notably reduce the risk for
diverse human cancers (Block et al., 1992, Nutr. Cancer 18: 1).
[0003] Recent research has identified food compounds
(phytochemicals) that may have important anticarcinogenic
activities (Mazur and Adlercreutz, 2000, Nutrition 16: 654).
Chemopreventive phytochemicals can suppress the initiation or
reverse the promotion stage in multistep carcinogenesis. They can
also block the progression of precancerous cells into malignant
ones (Surh, 2003, Cancer 3: 768). Anthocyanins, which are bioactive
phytochemicals, are widely distributed in plants. Anthocyanins not
only possess antioxidant ability (Pool-Zobel et al., 1999, Eur. J.
Nutr. 38: 227; Tsuda et al., 2000, Biofactors 13: 133), but also
mediate other physiological functions related to cancer suppression
(Kamei et al., 1995, Cancer Invest. 13: 590; Meiers et al., 2001,
J. Agric. Food Chem. 49: 958; Nagase et al., 1998, Planta Med. 64:
216). There has been increasing interest in the pharmaceutical
function of anthocyanins.
[0004] "Anthocyanin-rich extracts" or "AREs" are extracts derived
from foods such as fruits and vegetables that are preferably,
semi-purified, purified and/or concentrated such that the water
content, sugar content and acid content are reduced and the
remaining components are mainly the phenolics including
anthocyanins. AREs are known in the art and many are readily
available commercially from sources such as Artemis International,
Inc. (Madera, Calif.). Concentrated and highly concentrated (about
at least 2-3 grams of monomeric anthocyanin per liter or per kg)
AREs obtained using standard separation and purification techniques
and are also readily commercially available in the form of powders
and liquids.
[0005] Recent studies have shown molecular evidence of cancer
chemoprevention by anthocyanins. The mechanisms can be grouped into
three aspects: (i) antioxidation, (ii) molecular mechanisms related
to anticarcinogenesis, and (iii) molecular mechanisms involved in
apoptosis induction in tumor cells (Hou, 2003, Curr. Mol. Med. 3:
149). It has been reported that anthocyanins exhibited inhibitory
effects on the growth of several cancer cells (Kamei et al., 1995,
Cancer Invest. 13: 590; Meiers et al., 2001, J. Agric. Food Chem.
49: 958; Nagase et al., 1998, Planta Med. 64: 216), antioxidative
effects (Pool-Zobel et al., 1999, Eur. J. Nutr. 38: 227; Tsuda et
al., 2000, Biofactors 13: 133), and anticarcinogenic effect in
1,2-dimethylhydrazine-initiated F344/DuCrj rats (Hagiwara et al.,
2002, J Toxicol. Sci. 27: 57). An invention discloses that the
anthocyanin-rich extracts from chockeberry, bilberry and grape can
be used to inhibit colon carcinoma cell (U.S. patent application
No. 2005013880).
[0006] Hibiscus sabdariffa L. belongs to the Malvaceae family. The
calyces of Hibiscus have been used in traditional medicine.
Hibiscus flowers contain gossypetin, glucoside, bibiscin, Hibiscus
anthocyanin, and Hibiscus protocatechuic acid and have the
following effects, choleretic and diuretic functions, decreasing
blood pressure, reducing the viscosity of the blood, and
stimulating intestinal peristalsis (Ali et al., 1991, J
Ethnopharmacol. 31: 249). Thus, the dried flowers of Hibiscus
sabdariffa are a functional natural product with a chemopreventive
capacity.
[0007] Previous studies have shown that Hibiscus anthocyanins (HAs)
(which are extracted from the dried calyx of Hibiscus sabdariffa)
possess antioxidant bioactivity both in vivo and in vitro (Tseng et
al., 1997, Food Chem. Toxicol. 35: 1159; Wang et al., 2000, Food
Chem. Toxicol. 38: 411).
[0008] There remains a need for identifying natural compounds,
phytochemicals and food extracts that are effective as
chemopreventatives against cancer, including leukemia. The present
inventors have discovered that compositions comprising Hibiscus
anthocyanins (HAs) extracts are effective for inhibiting cancer
cell growth, and have further discovered methods for specifically
inhibiting the growth of leukemia cells without inhibiting the
growth of normal leukemia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows delphinidine content of HAs analyzed by HPLC.
In this figure, (A) Standard is delphinidine (0.1 mg/ml; 10 .mu.l);
(B) HAs extract (1 mg/ml; 10 .mu.l); and (C) HAs (1 mg/ml; 5
.mu.l)+delphinidine (0.1 mg/ml;
[0010] FIG. 2 shows induction of cell death by HAs. HL-60 cells
(5.times.10.sup.4 cells/well) treated with various concentrations
(0-4 mg/ml) of HAs for 24 h (A) or treated with 3 mg/ml of HAs for
the indicated times (B). The survival cell number is directly
proportional to formazan, which was measured spectrophotometrically
at 563 nm. The value are means.+-.SD, n=3.
[0011] FIG. 3 shows the effects of HAs on HL-60 cell cycle
distribution. HL-60 cells treated with 3 mg/ml of HAs for indicated
time (A), or with various concentrations (0-3 mg/ml) of HAs for 24
h (B). The DNA content was analyzed using fluorescence flow
cytometry. The position of the sub-G1 peak (hypodiploidy),
integrated by apoptotic cells, and the G0/G1 and G2/M peaks are
indicated. Quantitative assessment of the percentage of HL-60 cells
in the sub-G1 and G0/G1 phases was indicated by propidium iodide
(PI). The experiment was repeated three times and the
representative histograms are shown.
[0012] FIG. 4 shows induction of apoptosis by HAs and the effects
of various MAP kinase inhibitors. HL-60 cells (1.times.10.sup.6
cells/dish) were preincubated for 24 h with various MAP kinase
inhibitors, including SB203580 (SB, 50 AM), SP600125 (SP, 20 AM),
PD98059 (PD, 25 AM), and wortmannin (Wort, 1 AM), and then
incubated for 24 h with HAs (3 mg/ml). DMSO (0.25%) served as
solvent control. Quantitative assessment of the percentage of HL-60
cells in sub-G1 and G0/G1 phases was indicated by propidium iodide
(PI). Quantitative analysis of apoptosis was determined by flow
cytometry assay.
[0013] FIG. 5 shows HAs activating MAP kinases in a time-dependent
manner. HL-60 (1.times.10.sup.6 cells/dish) was incubated with HAs
(3 mg/ml) for various durations (0-360 min). Protein extracts were
prepared at the incubated time points to assess the activation of
MAP kinases. The levels of phosphorylated MAP kinases (p38, ERK1/2,
and c-Jun) were determined by Western blotting using specific
antibodies. Actin, load controls. This figure is a representative
of three independent experiments with similar results.
[0014] FIG. 6 shows time course of HAs-induced Fas and FasL
activation in HL-60 cells. HL-60 cells were treated with HAs (3
mg/ml) for the time indicated and assayed for Fas, FasL activation,
and mRNA expression. (A) Untreated control cells were run in
parallel in the same gel. The proteins were analyzed using Western
blotting. .alpha.-Tubulin, load controls. (B) Total RNA was
extracted at each time point, Fas and FasL mRNA expressions were
analyzed by RT-PCR. M, molecular weight marker. GAPDH, loading
controls. This figure is a representative of three independent
experiments with similar results.
[0015] FIG. 7 shows involvement of caspase-8 and caspase-3 in
HAs-induced apoptosis. The levels of cleaved caspase-8/-3 and
pro-caspase-8/-3 proteins in HL-60 cells with HAs treatments were
determined. Cells were treated with HAs (360 min) for the indicated
concentration and analyzed using immunoblotting with
anti-cleaced-caspase-8/-3 and pro-caspase-8/-3 antibody. Actin and
.alpha.-tubulin were the loading controls. This figure is a
representative of three independent experiments with similar
results.
[0016] FIG. 8 shows effect of HAs-induced Bid activation and
cytochrome c release. (A) HL-60 cells were treated with HAs (360
min) for the indicated concentration and analyzed by immunoblotting
with anti-Bcl-2, Bax, and Bid antibody. The arrows indicate the
position of full-length Bid (p23) and the p15 truncated form of
active Bid (tBid). Actin was the loading control. (B) HL-60 cells
were treated with HAs (3 mg/ml) for the indicated times and
analyzed by immunoblotting with anti-Bid antibody. (C) HL-60 cells
were treated with HAs (360 min) for the indicated concentration,
and the expression of cytochrome c in the cytosol and the
mitochondria of the untreated and HAs-treated HL-60 cells was
assayed by immunoblotting. (D) HL-60 cells were treated with HAs (3
mg/ml) for the indicated times, and the expression of cytochrome c,
cytochrome oxidase subunit IV(COX4), a mitochondrial marker served
as a protein loading control, was determined. This figure is a
representative of three independent experiments with similar
results.
[0017] FIG. 9 shows effect of p38 MAPK inhibitor (SB203580) on
HAs-induced apoptosis in HL-60 cells. Cultured cells were treated
in the absence or presence of SB203580 (50 .mu.M, 24 h
preincubation) with HAs (3 mg/ml) for 6 h, and phospho-c-Jun, Fas,
and FasL expressions were analyzed by Western blotting. Actin was
used as the loading control. This figure is a representative of
three independent experiments with similar results.
[0018] FIG. 10 shows the model showing pathways that mediate
HAs-induced apoptosis in HL-60 cells. HAs were been shown to be
capable of inducing HL-60 cell apoptosis. p38 signaling activation
involves mitochondrial membrane alterations resulting in the
release of cytochrome c and caspases activation.
[0019] FIG. 11 shows pathological analysis of rat liver using
H&E stain. (a) normal control, (b) the animal were treated with
NMU alone (35 mg/kg), (c) NMU and HAs (0.1%), and (d) NMU and HAs
(0.2%). The arrow indicates leukemia cells invaded in liver tissue.
The amplification factor is
[0020] FIG. 12 shows pathological analysis of rat spleen was using
H&E stain. (a) normal control, (b) the animal were treated with
NMU alone (35 mg/kg), (c) NMU and HAs (0.1%), (d) NMU and HAs
(0.2%). The leukemia cells invaded in spleen red pulp tissue. The
amplification factor is 100.times..
[0021] FIG. 13 shows pathological analysis of rat liver was using
Myeloperoxidase stain. (a) normal control, (b) the animal were
treated with NMU alone (35 mg/kg), (c) NMU and HAs (0.1%), (d) NMU
and HAs (0.2%). The amplification factor is 100.times..
[0022] FIG. 14 shows morphology analysis of rat whole blood was
using Liu's stain. (a) normal control, (b) the animal were treated
with NMU alone (35 mg/kg), (c) NMU and HAs (0.1%), (d) NMU and HAs
(0.2%), (e) normal control, and (f) the animal were treated with
NMU alone (35 mg/kg). The amplification factor of (a) to (d) is
40.times., and the amplification factor of (e) and (f) is
100.times..
[0023] FIG. 15 shows the body weight of the animals in experiment.
Every 30 days record one times. *p<0.05; *p<0.005, compared
with NMU-treated group. Final survival number of control group was
12 rats; NMU only group was 7 rats; NMU+HAs 0.1% group was 9 rats;
and NMU+HAs 0.2% group was 11 rats.
SUMMARY OF THE INVENTION
[0024] This invention provides a composition for inhibiting cancer
cell growth comprises Hibiscus anthocyanins (HAs).
[0025] This invention also provides a method for treating cancer
comprising administering a patient with an effective amount of
Hibiscus anthocyanins.
DETAILED DESCRIPTION OF THE INVENTION
[0026] This invention provides a composition for inhibiting cancer
cell growth comprising Hibiscus anthocyanins (HAs). In a preferred
embodiment, the Hibiscus is Roselle, Hibiscus sabdariffa. The plant
tissue such as leaf, stem, flower or fruit can be used to extract
HAs. Because the flower and calyx of Roselle contain high
concentration of HAs, the HAs are prepared from the flower or calyx
of the Roselle in this invention. The HAs are characterized by HPLC
shown in FIG. 1.
[0027] Several methods for extracting anthocyanins are well known,
such as supercritical fluid, water extraction, solvent extraction
etc. In a preferred embodiment, the HAs are extracted from the
flower or calyx of the Roselle by methanol. In this invention, the
anthocyanins-rich extract from the Roselle is analyzed by HPLC, and
the result shows that the HAs comprise delphindin and cyaniding, at
least.
[0028] The Hibiscus anthocyanins in the invention could be
extracted by water or organic solvent. The organic solvent includes
but is not limited to alcohol (such as CH.sub.3OH,
C.sub.2H.sub.5OH, C.sub.3H.sub.7OH), ester (such as acetyl
acetate), alkane (such as hexane) and halogenated alkane (such as
CH.sub.3C.sub.1, C.sub.2H.sub.2Cl.sub.2). The preferred organic
solvent is ethanol or alcoholic solvent without causing any side
effect of human. The most preferred organic solvent is
methanol.
[0029] The subject accepting the mixture of the invention includes
but is not limited to human, mammal, mouse, rat, horse, pig,
chicken, duck, dog and cat.
[0030] The HAs preparation method is not limited by the following
description. The known methods can be used to extract HAs. In this
invention, the HAs are prepared by a method comprising: (a)
incubating Roselle with methanol, (b) concentrating the incubation
under reduced pressure, and (c) eluting the precipitate with
methanol, wherein the methanol contains 0.1% HCl. In a preferred
embodiment, the step (a) is at low temperature such as 4, and the
step (a) is at room temperature such as 25.
[0031] The HAs can induce the apoptosis of the cancer cell in
accordance with this invention. The cancer cells include abnormal
cancer cells associated with lymphoma, leukemia, plasma cell
dyscrasias, multiple myeloma, amylodosis, also as known as
hematopoietic tumors, colorectal cancer, ovarian cancer, bone
cancer, renal cancer, breast cancer, gastric cancer, pancreatic
cancer, or melanoma. Preferably, the cancer cell is leukemia cell,
and the growth and cell cycle progression of the leukemia cells are
inhibited by the composition of this invention. In a preferred
embodiment, the leukemia cell is human leukemia cell.
[0032] In this invention, the human promyelocytic leukemia cells
(HL-60) are used as the target cells, served as a useful model for
testing antileukemic or general antitumoral compounds (Suh et al.,
1995, Anticancer Res. 15: 233). The HL-60 cells exhibit the
strongest HAs cytotoxicity potency. The mechanism by which HAs
caused apoptotic death in human myeloid leukemic HL-60 cells is
also elucidated. The Hibiscus anthocyanins induce HL-60 apoptosis,
which is via p38-FasL signaling pathway and Bid pathway. Further,
the p38 signaling pathway acts at an early step prior to the
cytochrome c release and caspase activation.
[0033] The Hibiscus anthocyanins can be used to inhibit cancer cell
growth. In particular, the HAs can be used to induce apoptosis of
cancer cells. There are several signaling pathways involved in the
HAs-inducing apoptosis mechanisms. In this invention, the HAs
activate the Caspase 8 pathway, Caspase 3 pathway, p38 MAP kinase
pathway, c-Jun kinase pathway, or Bid pathway. The HAs can
stimulate the cytochrome c release from mitochrondrial of cancer
cell. These pathways can act alone or combined.
[0034] This invention also provides a method for treating cancer
comprising administering a patient with a composition comprising
HAs. The composition further comprises a pharmaceutical acceptable
carrier. In a preferred embodiment, the cancer is leukemia. The HAs
are characterized by HPLC shown in FIG. 1. For treating leukemia,
the effective amount of the composition Hibiscus anthocyanins is
0.5.about.5.0 mg/ml. In a preferred embodiment, the effective
amount is 3.0 mg/ml.
[0035] The administration route of HAs to a patient is via
injection, oral or external application. In a preferred embodiment
for treating leukemia, the administration route to a patient is via
injection.
EXAMPLE
[0036] The following examples are offered by way of illustration
and not by way of limitation.
Materials Used in this Invention
[0037] SB203580
(4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]-1H-imidazole-
), PD098059 (2-[2-amino-3-methoxyphenyl]-4H-1-benzophyran-4-one),
SP600125 (1,9-pyrazoloanthrone), wortmannin were purchased from
Sigma (St. Louis, Mo., USA). These inhibitors were stored in
dimethyl sulfoxide (DMSO) and added to the culture medium to a
final concentration as described in the figure legends. However,
the amount of DMSO in the cell culture medium did not exceeded 0.3%
upon drug treatment. Polyclonal antibody against phospho-p38 MAP
kinase (Thr180/Tyr182) and phospho-c-Jun (Ser-73) were purchased
from Cell Signaling Technology (Beverly, Mass., USA). Antibody
against p-ERK (E-4), FAS (FL-335), FASL (C-178), caspase-8 (H-134),
caspase-3 (H-277), BID (C-20), Bcl-2 (N-19), Bax (P-16), and
cytochrome c (A-8) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Monoclonal mouse antibody with reactivity to
human cytochrome oxidase subunit IV was purchased from Molecular
Probes (Eugene, Oreg., USA). Horseradish peroxidase-conjugated
anti-mouse secondary antibodies were purchased from NEN Life
Science Products, Inc. (Boston, Mass., USA). Anti-rabbit,
anti-actin, and anti-a-tubulin secondary antibodies were purchased
from Sigma.
Example 1
Preparation of Hibiscus Anthocyanins
[0038] HAs were prepared from the dried flower of Hibiscus
sabdariffa L. (20 g) with methanol (2 l) containing 1% HCl for 1
day at 4. The extract was filtered and then concentrated under
reduced pressure at 25. The precipitate was collected, stood on an
Amberlife Diaion HP-20 resin column for 24 h, and then cleaned with
distilled water (5 l) containing 0.1% HCl solution and eluted with
methanol. The filtrate was collected and then lyophilized to obtain
approximately 2 g of HAs and stored -20 before use.
Example 2
HPLC Assay for HAs
[0039] Total anthocyanins were extracted using the Fuleki and
Francis (1968, J Food Sci. 33: 78) method. In particular, 100 Al of
HAs (10 mg/ml) were diluted to 3 ml with the pH 1.0 and 4.5
buffers, respectively. The O.D. of the sample was measured at 535
nm, using distilled water as blank. The difference in O.D. was
obtained by subtracting the total O.D. at pH 4.5 from that at pH
1.0. Both values were calculated from the O.D. readings using the
appropriate dilution and calculation factors. For the
standardization of HAs, delphinidine in HAs were determined by HPLC
using a symmetry shield RP18 column (3.5 .mu.m, 4.6.times.150 mm)
and a UV/visible detector (monitored at 530 nm). The mobile phase
was consisted of H.sub.2O and 10% formic acid/methanol (65/35,
v/v). The sample (1 mg) was dissolved in 1 ml acidic methanol
(HCl--CH.sub.3OH 1:1, v/v) and boiled at 95 for 30 min, and 10
.mu.l of which was injected into chromatography. The flow rate was
set at 1 ml/min. The result was evaluated with the commercially
available standard delphinidine.
[0040] Spectrophotometer analysis of HAs showed that the purity of
HAs were approximately 85-95%. For HAs standardization,
delphinidine contained in the HAs was determined using HPLC. Pure
delphinidine showed a retention time of 18.83 min (FIG. 1A). HPLC
analysis of HAs exhibited a peak at 18.42 min (FIG. 1B), which was
merged with that of delphinidine standard at 18.36 min (FIG. 1C).
The data confirmed that delphinidine is the major component in the
Hibiscus sabdariffa L. anthocyanin, consisting of approximately
3-4% of HAs in each analysis.
Example 3
(A) Cell Line and Cell Culture
[0041] Human gastric carcinoma AGS was maintained in F-12 Nutrient
Mixture medium; mouse NIH/Swiss embryo cells, hepatocellular
carcinoma Hep 3B, and colorectal adenocarcinoma Caco-2 were
maintained in DMEM; and hepatoblastoma HepG2, adenocarcinoma MCF-7,
and human oral epidermoid carcinoma KB were maintained in MEM.
HL-60 cell line, a model of human promyelocytic leukemia (Collins
et al., 1977, Nature 270: 347), was cultured in suspension in RPMI
1640 medium (Gibco, Grand Island, N.Y., USA) supplemented with 10%
fetal calf serum (FBS) and antibiotics (100 units/ml of penicillin
and 100 .mu.g/ml of streptomycin). Incubation was carried out at 37
in a humidified atmosphere of 5% CO.sub.2 and 95% air. Cells were
passaged thrice a week to maintain logarithmic growth. Cell
viability, as determined by trypan blue exclusion, was greater than
95%.
(B) Cytotoxicity of HAs on HL-60 Cells
[0042] Cells were seeded at a density of 5.times.10.sup.4
cells/well and cultured with various concentrations (0, 0.05, 0.1,
0.2, 0.5, 1, 3, 4 mg/ml) of HAs for 24 h or treated with HAs (3
mg/ml) for various periods of time (0, 12, 24, 48 h). Thereafter,
the medium was changed and
3-(4,5-dimethylthiazol-zyl)-2,5-diphenyltetrazolium bromide (MTT;
0.1 mg/ml) (Sigma) was added for 4 h. The viable cell number is
directly proportional to the production of formazan, which was
dissolved in isopropanol and measured spectrophotometrically at 563
nm. The percentage of viable cells estimated by comparison with
untreated control cells.
[0043] Cell viability was assayed in cultures exposed to 0.05-4.0
mg/ml HAs for 24 h and showed a concentration dependent inhibitory
effect on the growth of NIH3T3, Hep G2, MCF-7, KB, Caco-2, Hep 3B,
HL-60, and AGS cells. The strongest cytotoxicity of HAs was found
in human leukemia HL-60 cells. The results also showed less
cytotoxicity toward normal cells (NIH3T3 cells).
[0044] The influence of HAs on the human leukemia HL-60 cell growth
process is investigated by using the MTT assay. HL-60 cells were
incubated with 0.05-4.0 mg/mil HAs for 24 h, or with 3 mg/ml of HAs
for various periods of time (0, 12, 24, 48 h). The concentration of
HAs inhibiting 50% of HL-60 cell viability (IC50) was around 2.49
mg/ml (FIG. 2A). Treatment with HAs (3 mg/ml) for 0-48 h, an
approximate 75% of decrease in cell number was observed at 24 h
(FIG. 2B). Morphological examination showed that HAs-treated cells
expressed typical apoptosis characteristics, including membrane
blebbing, cell shrinkage, and apoptotic bodies.
Example 4
(A) Analysis of Cell Cycle and Quantification of Apoptosis
[0045] Flow cytometric analysis of HL-60 cells was performed on the
24-h cultures using a FACScan. The cells of harvest were
centrifuged at 1000 rpm for 5 min at room temperature. Thereafter,
cells were washed once with Tris-buffered saline (TBS). Then cell
suspension was centrifuged again (1000 rpm, 5 min) and resuspended
in 70% ethanol. After incubation at -20 for at least 24 h, the
cells were resuspended in 1 ml of cell cycle assay buffer [50
.mu.g/ml propidium iodide (PI), 50 .mu.g/ml RNase A, and 0.1%
Triton X-100], and it was incubated for 15 min in darkness.
Propidium iodide (Sigma) was excited at 488 nm, and fluorescence
signal was subjected to logarithmic amplification with PI
fluorescence (red) being detected above 600 nm. Cell cycle
distribution is presented as the number of cells versus the amount
of DNA as indicated by the intensity of fluorescence, and the
extent of apoptosis was determined by counting cells of DNA content
below the G0/G1 peak.
(B) Dose- and Time-Dependent HAs-Induced Apoptosis in HL-60
Cells
[0046] As shown in FIG. 2, HAs have presented an inhibitory effect
on the growth of HL-60 cells. The exposure HAs caused a clear
cellular ladder-like pattern of DNA fragments, a characteristic of
apoptosis (data not shown). The HAs-induced apoptotic effect was
confirmed with flow cytometry. When cells were treated with HAs (3
mg/ml) for various periods of time (0, 12, 24, and 48 h), an
apparent accumulation of cells in the sub-G1 phase (from 0.95% to
66.39%) (hypodiploid phase) was observed (FIG. 3A). It was found
that HL-60 cells exposed to 0, 1, 2, and 3 mg/ml HAs for 24 h
demonstrated 0.97%, 1.33%, 5.53%, and 51.46% apoptosis,
respectively. This was more than a 50% increase in apoptotic cells
(FIG. 3B). Therefore, HAs have stimulated a time- and
concentration-dependent increase in HL-60 cells. These data
indicated that HAs were cytotoxic to HL-60 cells.
Example 5
(A) Western Blot Analysis
[0047] To analyze of the expression of proteins, HAs (0, 1, 2, 3
mg/ml) were added to the culture for 24 h, or cells were treated
with HAs (3 mg/ml) for various periods of time (0, 15, 30, 60, 180,
360 min). The medium was removed, and the cells were rinsed with
TBS at room temperature. Then 500 .mu.l of cold RIPA buffer (1%
NP-40, 50 mM Tris-base, 0.1% SDS, 0.5% deoxycholic acid, and 150 mM
NaCl, pH 7.5) with three kinds of proteinase freshly added (10
.mu.g/ml leupeptin, 10 .mu.g/ml PMSF, and 17 .mu.g/ml sodium
orthovanadate) were used. After sonication for 1 h at 4.degree. C.,
a centrifugation (10,000.times.g) of cell lysate was performed for
10 min at 4.degree. C. The supernatant (50 .mu.g protein) was mixed
with an equal volume of electrophoresis sample buffer and boiled
for 10 min, which was then subjected to SDS-polyacrylamide gel
electrophoresis using a 10% running gel, and electroblotted to
nitrocellulose membranes (Millipore, Bedford, Mass., USA).
Nonspecific binding was blocked by incubation of the membrane with
TBS containing 1% (W/V) nonfat dry milk and 0.1% (v/v) Tween-20
(TBST) for more than 2 h. Membranes were washed with TBST three
times for 10 min and incubated with appropriate dilution of primary
antibody in TBST for 2 h. Membranes were then extensively washed
with TBST before being incubated with appropriate horseradish
peroxidase-conjugated secondary antibody for 1 h. After washing the
membrane three times, 10 min each in TBST, detection was performed
using ECLWestern blotting detection reagents (Amersham, Arlington
Heights, Ill., USA).
(B) MAP Kinase Inhibitor Effect on the Growth of HL-60 Cells
[0048] JNK and p38 kinase activation is known to be associated with
the stress-induced apoptosis process, such as anticancer drugs, UV
irradiation, and Fas (Butterfield et al., 1997, J. Biol. Chem. 272:
10110; Juo et al., 1997, Mol. Cell. Biol. 17: 24; Seimiya et al.,
1997, J. Biol. Chem. 272: 4631). In order to determine which
pathway is involved in the HAs-induced apoptosis in HL-60 cells,
the effects of three highly specific MAP kinase inhibitors, PD98059
(MEK inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK
inhibitor) were determined, in HAs-induced apoptosis. In addition,
wortmannin, a PI-3K inhibitor, was used and shown to inhibit
apoptosis in neoplastic cells (Cantrell, 2001, J Cell. Sci. 114:
1439). The FACScan analysis was employed to assess the ability of
these inhibitors to repress the HAs-induced apoptosis. HL-60 cells
treated with 50 AM SB203580 (Kim et al., 2002, Cell Imunol. 220:
96) followed by a 24-h exposure to HAs (3 mg/ml) displayed a
significant reduction, .about.50%, in the sub-G1 phase as compared
to that of HAs alone (from 53.21% to 27.39%) (FIG. 4). Inhibitors,
PD98059 (25 AM) (Della Ragione et al., 2002, J Natl. Cancer Inst.
66: 1191), SP600125 (20 AM) (Gajate et al., 2002, J Biol. Chem.
277: 41580), and wortmannin (1 AM) (Liu et al., 1998, J Immunol.
160: 1393) did not reduce the cytotoxic action of HAs. This result
implies that p38 MAP kinase activated by HAs could play a critical
role in inducing apoptotic cell death in HL-60.
(C) HAs Effect on MAP Kinase Family Proteins
[0049] To elucidate the involvement of various p38, c-Jun, and ERK
signaling components, the expression of candidate signaling
molecules was measured upon HAs stimulation. Incubation of HL-60
cells with HAs (3 mg/ml) led to a time-dependent phosphorylation of
p38. Phosphorylated p38 could be detected 15 min after HAs addition
and remained elevated up to 360 min at 2.68-fold (FIG. 5, upper
panel). In addition, the data showed increased phosphorylation of
c-Jun in HL-60 cells after stimulation with HAs (3 mg/ml) that was
occurred at 30 min and reached the maximal level (.about.2.59-fold
of control) at 360 min (FIG. 5, middle panel). ERK1/2, a kinase
been suggested to play a role in survival pathway, therefore showed
no phosphorylation upon exposure to HAs (FIG. 5, bottom panel).
These data suggested that the activation of p38 and c-Jun kinases
was a primary effect during HAs-induced apoptosis in HL-60
cells.
Example 6
(A) Reverse Transcription Polymerase Chain Reaction (RT-PCR)
[0050] Total RNA was isolated from cells using a guanidium chloride
procedure. cDNA synthesis and PCR amplification were performed as
previously described (Hsieh et al., 2004, Arthroscopy 20: 482). For
reverse transcription, 4 .mu.g of total cellular RNA were used as
templates in a 20-[I reaction containing 4 .mu.l dNTPs (2.5 mM),
2.5 .mu.l Oligo dT (10 pmole/.mu.l), and RTase (200 U/.mu.l), and
the reaction was performed at 42.degree. C. for 1 h. Afterwards,
5-.mu.l cDNA was used as templates in PCR amplifications together
with appropriate primers. The Fas primers were Forward:
5'-CAAGTGACTGACATCAACTCC-3' and Reverse: 5'-CTATTTTGG
CTTCATTGACACC-3', which amplified a fragment of 727 bp. Each PCR
cycle for human Fas consisted of denaturation at 94.degree. C. for
1 min, annealing at 60.degree. C. for 1 min, and elongation at
72.degree. C. for 2 min for a total of 30 cycles. The FasL primers
were Forward: 5'-GGAT TGGGCCTGGGGATG=T]CA-3' and Reverse:
5'-TTGTGGCTCAG GGGCAGGTTGTTG-3', which amplified a fragment of 344
bp. Each PCR cycle for human FasL consisted of denaturation at
94.degree. C. for 1 min, annealing at 63.degree. C. for 1 min, and
elongation at 72.degree. C. for 2 min for a total of 25 cycles.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA served as an
internal control. The GAPDH primers were Forward:
5'-CGGAGTCAACGGATTTGGTCGTAT-3' and Reverse: 5'-AGCCT TCTCCATGGT
TGGTGAAGAC-3', which amplified a fragment of 304 bp. Each PCR cycle
for GAPDH consisted of denaturation at 94.degree. C. for 1 min,
annealing at 65.degree. C. for 1 min, and elongation at 72.degree.
C. for 2 min for a total of 25 cycles. The PCR products were
visualized on 2% agarose gels stained with ethidium bromide.
(B) HAs-Induced Apoptosis in HL-60 Cells Involves the Release of
Apoptosis-Related Proteins from the Mitochondria
[0051] The involvement of Fas receptor/ligand system has been
proposed to mediate apoptosis induced by many anticancer drugs
(Friesen et al., 1996, Nat. Med. 2: 574). It was first analyzed
whether HAs-induced apoptosis could involve the expression of Fas
receptor and its natural ligand. Western blotting analysis showed
that Fas expression increased to about 1.78-fold of control after
HAs exposure (3 mg/ml) for 360 min. The same treatment condition
also induced the expression of the ligand to about 2.38-fold (FIG.
6A). Accordingly, it was suspected that p38 could regulate Fas and
FasL transcription via c-Jun. Therefore, the mRNA levels of Fas and
FasL were measured by using RT-PCR analysis. The data showed that
HAs markedly induced cellular expression of Fas and FasL mRNA that
reached the maximal level of about 2.15- and 3.45-fold within 360
min, respectively (FIG. 6B).
[0052] Caspases play a pivotal role during apoptosis after
treatment with an inducing agent. Immunoblot analysis was used to
understand if caspases were activated by HAs in HL-60 cells.
Caspase-8 and caspase-3 expression was examined next. The level of
cleaved caspase-8 protein increased to 1.78-fold upon the treatment
of 3 mg/ml HAs. In contrast, the pro-caspase-8 level decreased (to
about 0.62-fold) (FIG. 7). Caspase-3, a downstream effector of
caspase-8, also revealed an activation, as shown by the appearance
of the p17/20 active form that was increased to 2.93-fold after the
exposure of HAs (3 mg/ml) for 360 min. This induction in capase-3
was accompanied by a decrease in pro-caspase-3 (about 0.59-fold)
(FIG. 7). These data suggested that caspase-8 mediated the
HAs-induced caspase-3 activation.
Example 7
(A) Preparation for Cytosolic and Mitochondrial Fraction
[0053] Subcellular fractionation was performed as follows. The
cells were washed with TBS, and then cold buffer A (20 mM Tris,
0.03 mM Na.sub.3VO.sub.4, 2 mM MgCl.sub.2.times.6H.sub.2O, 2 mM
EDTA, 0.5 mM EGTA, 2 mM PMSF, 1 mM DTT, 250 mM sucrose, 10 .mu.g/ml
leupeptin) was added. The cells were harvested and carefully
homogenized using a homogenizer (Eyela Nazelax) on ice. The lysate
was centrifuged at 50,000 rpm, 4.degree. C. for 1 h. The
supernatant was collected and used as the cytosol fraction. The
pellet was solubilized with buffer B (20 mM Tris, 0.03 mM
Na.sub.3VO.sub.4, 5 mM MgCl.sub.2.times.6H.sub.2O, 2 mM EDTA, 0.5
mM EGTA, 2 mM PMSF, 1 mM DTT, 5 mM NaF, 10 .mu.g/ml leupeptin, and
0.1% Triton X-100) at 4.degree. C. and mixed by homogenizer (Eyela
Nazelax) on ice approximately 10 min to extract mitochondria
proteins. Samples were recentrifuged at 50,000 rpm, 4.degree. C.
for 1 h, and the supernatant was used as the mitochondrial
fraction. Cytochrome c proteins in the two fractions were analyzed
with immunoblot and densitometer.
(B) Bid Mediates Release of Mitochondrial Cytochrome c into the
Cytosol
[0054] Bcl-2 family proteins have exhibited a complex network that
regulates apoptosis in multiple biological systems. The cellular
levels of Bcl-2, Bax, and Bid in HL-60 cells treated with HAs for
24 h were examined. The expression of Bcl-2 and Bax proteins showed
no changes (FIG. 8A) because the HAs-triggered apoptosis was
mediated by caspase-8/-3 cascade, and Bid was a proapoptotic Bcl-2
family protein that was activated by caspase-8 in response to
Fas/TNF-R1 death receptor signals. Activated Bid (named as tBid) is
translocated into the mitochondria and induces cytochrome c
release, which in turn activates downstream caspases (Yin, 2000,
Cell Res. 10: 161). Thus, it was wondered whether HAs could trigger
Bid activation and mitochondrial cytochrome c release. Bid/tBid
levels were tested by using a polyclonal anti-Bid antibody that
detects both the 23-kDa intact form and the 15-kDa truncated form
of the active Bid. The cellular level of the cleaved fragment of
p15 active form of Bid increased to 3.86-fold after exposed to HAs
(3 mg/ml) for 360 min. In contrast, the level of full-length Bid
(p23) decreased (FIG. 8A). The effect of on Bid/tBid was in a
time-dependent manner (FIG. 8B). It was next examined whether
mitochondrial cytochrome c was released into cytosol using Western
blot analysis. Mitochondrial fractions were purified as described
previously, and cytochrome oxidase subunit IV (COX 4) levels were
used as a control protein to show equal amounts of protein loads
(Marchenko et al., 2000, J Biol. Chem. 275: 16202). As shown in
FIG. 8C, HAs treatment caused an accumulation of cytochrome c in
the cytosolic fraction, compared to that of control cells,
accompanied with a reduction in the mitochondrial fraction in a
concentration-dependent manner. After 12 h of treatment, a decrease
in cytochrome c in the mitochondria (about 0.63-fold) and an
accumulation in the cytosol (about 2.10-fold) were clearly observed
(FIG. 8D). Taken together, these results indicated that the cell
death caused by HAs were dependent on Bid activation and cytochrome
c release in HL-60 cells.
Example 8
Effects of SB203580 in the HAs-Induced Apoptosis
[0055] The effects of specific inhibitor of p38, SB203580 on
repressing HAs-mediated apoptosis were illustrated in FIGS. 4 and
5. It was supposed that this inhibitor might also suppress the
downstream effectors of p38, c-Jun, Fas, and FasL, which are
involved in the HAs-induced apoptosis. As shown in FIG. 9, HL-60
cells treated with HAs (3 mg/ml) alone showed significantly
elevated levels of c-Jun, Fas, and FasL (lane 2) compared with lane
1. These pretreatment of HL-60 cells with SB203580 (50 AM) was
down-regulated by the expressions of these proteins (lane 3). These
results again emphasize an important role for p38 activation
involved in the HAs-induced apoptosis pathway.
Example 9
HAs-Induced Apoptosis Signaling Pathway Model
[0056] The invention was schematically presented in FIG. 10. HAs
induced apoptosis via activating p38 MAP kinase that subsequently
phosphorylates target protein c-jun and transduced the signal to
further activate the apoptotic protein cascades that contained
Fas-mediated signaling (Fas/caspase-8/tBid signaling module). As an
outcome to the events, cytochrome c released from the mitochondria,
leading to the cleavage of caspase-3.
Example 10
Animal Model
[0057] The male 49-day-old S-D rats were divided into different
groups including negative control, positive control and
experimental groups. The positive control rats were intravenous
injected with 35 mg/kg NMU (N-Nitroso-N-Methylurea), and the
experimental rats were intravenous injected with 35 mg/kg NMU and
the HAs (0.1% and 0.2%, respectively). Every group contained 12
rats, totally 48 rats in this experiment. These rats were
intravenous injected 1 time per 2 weeks, total 6 times. After
injecting 6 times, the rats were observed another 150 days and the
body weight and the blood analysis were recorded.
[0058] The pathological analysis of the rat liver and spleen were
shown in FIGS. 11, 12 and 13. The blood analysis was shown in FIG.
14 and the body weight was shown in FIG. 15.
[0059] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0060] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The cell lines, embryos, animals, and processes and methods for
producing them are representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Modifications therein and other uses will occur to those
skilled in the art. These modifications are encompassed within the
spirit of the invention and are defined by the scope of the
claims.
[0061] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0062] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0063] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations, which are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
[0064] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
6 1 21 DNA Artificial primer 1 caagtgactg acatcaactc c 21 2 22 DNA
Artificial primer 2 ctattttggc ttcattgaca cc 22 3 23 DNA Artificial
primer 3 ggattgggcc tggggatgtt tca 23 4 24 DNA Artificial primer 4
ttgtggctca ggggcaggtt gttg 24 5 24 DNA Artificial primer 5
cggagtcaac ggatttggtc gtat 24 6 25 DNA Artificial primer 6
agccttctcc atggttggtg aagac 25
* * * * *