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San-Zhong-Kui-Jian-Tang, a traditional Chinese medicine prescription, inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis. 

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Breast cancer is one of the most common malignancies in women, and is the leading cause of death worldwide for women between the ages of 40 and 55 years in the world.1) This pathology is currently controlled by surgery and radio-therapy, and is frequently supported by adjuvant chemo- or hormonotherapies.2)However, breast cancer is highly

resist-ant to chemotherapy, and there is still no effective cure for patients with advanced stages of the disease, especially in cases of hormone-independent cancer.2)Effective

chemopre-ventive treatment for breast cancer would have an important impact on breast cancer morbidity and mortality. Apoptosis has been characterized as a fundamental cellular activity to maintain the physiological balance of the organism.3) It is

also involved in immune defense machinery and plays a nec-essary role as a protective mechanism against carcinogenesis by eliminating damaged cells or abnormal excess cells prolif-erated owing to various chemical agents’ induction.4,5)

Emerging evidence has demonstrated that the anticancer ac-tivities of certain chemotherapeutic agents are involved in the induction of apoptosis, which is regarded as the preferred way to manage cancer.4,6)

Zhong-Kui-Jian-Tang (SZKJT; Japanese name: San-shu-kaigen-to), a traditional Chinese medicine prescription, has been used for treating patients with various cancers. In this study, we determined the antiproliferative activity of SZKJT, and examined its effect on cell cycle distribution and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-231. Furthermore, to establish the anticancer mechanism of SZKJT, we assayed the levels of p53, p21/WAF1, Fas/APO-1 receptor, Fas ligand, Bcl-2 family protein and caspases activity, which are strongly associated with the signal transduction pathway of apoptosis and affect

the chemosensitivity of tumor cells to anticancer agents.7)

MATERIALS AND METHODS

Chemicals and Reagents Fetal calf serum (FCS), nonessential amino acids, sodium pyruvate, insulin, Dul-becco’s modified Eagle’s medium (DMEM) and RPMI 1640 were obtained from GIBCO BRL (Gaithersburg, MD, U.S.A.). Dimethyl sulfoxide (DMSO), ribonuclease (RNase) and propidium iodide were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Nucleosome ELISA, WAF1 ELISA, Fas Ligand, Fas/APO-1 ELISA, and caspase-9, cas-pase-8 activity assay kits were purchased from Calbiochem (Cambridge, MA, U.S.A.). The antibodies to b-actin, cy-clinD1, cyclinD2, cyclinB, Bax, Bak, Bcl-2, and Bcl-XLwere

obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

Plant Materials It was prepared as a lyophilized-dry powder of hot water extracts from 17 species of medical herbs consisting of Coptis chinensis FRANCH (5.6 g), Cimi-cifuga heracleifolia KOMAR (8.6 g), Scutellaria baicalensis GEORGI(22.9 g), Gentiana scabra BUNGE. (14.3 g), Trichosan-thes cucumeroides (SER.) MAXIM. (14.3 g), Phellodendron amurense RUPR. (22.9 g), Anemarrhena asphodeloides BUNGE. (14.3 g), Platycodon grandiflour (JACQ.) (14.3 g), Laminaria japonica ARESCH. (14.3 g), Bupleurum chinese DC. (14.3 g), Glycyrrhiza uralensis FISCH. (8.6 g), Sparga-nium stoloniferum BUCCH. (8.6 g), Curcuma aeruginosa ROXB (8.6 g), Forsythia suspense (THUMB.) VAHL(8.6 g), Pueraria lobata OHWI(8.6 g), Paeonia lactiflora PALL. (5.6 g) and An-gelica sinensis (OLIV.) DIELS. (5.6 g). They were purchased from a local herb store. The authenticity of the plant species

San-Zhong-Kui-Jian-Tang, a Traditional Chinese Medicine Prescription,

Inhibits the Proliferation of Human Breast Cancer Cell by Blocking Cell

Cycle Progression and Inducing Apoptosis

Ya-Ling HSU,aMing-Hong YEN,bPo-Lin KUO,cChien-Yu CHO,dYu-Ting HUANG,c

Chien-Jung TSENG,dJu-Ping LEE,cand Chun-Ching LIN*, b

aDepartment of Pharmacy, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan; No. 60, Erh-Jen Road, Sec. 1,

Jen-Te, Tainan 717, Taiwan: bFaculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University; No. 100,

Shin-Chuan 1’st Road, Kaohsiung 807, Taiwan: cDepartment of Biotechnology, Chia-Nan University of Pharmacy and Science;

No. 60, Erh-Jen Road, Sec. 1, Jen-Te, Tainan 717, Taiwan: and dGraduate Institute of Natural Products, Kaohsiung

Medical University; No. 100, Shin-Chuan 1’st Road, Kaohsiung 807, Taiwan. Received July 17, 2006; accepted September 19, 2006

San-Zhong-Kui-Jian-Tang (SZKJT; Japanese name: Sanshu-kaigen-to), a traditional Chinese medicine pre-scription, has been used for treating patients with various cancers. This study first investigates the anticancer ef-fect of SZKJT in two human breast cancer cell lines, MCF-7 and MDA-MB-231. SZKJT exhibited efef-fective cell growth inhibition by inducing cancer cells to undergo G0/G1 phase arrest and apoptosis. Blockade of cell cycle was associated with increased p21/WAF1 levels, and reduced amounts of cyclinD1, cyclinD2 in a p53-independ-ent manner. SZKJT treatmp53-independ-ent triggered the mitochondrial apoptotic pathway indicated by changing Bax/Bcl-2 ratios, cytochrome c release and caspase-9 activation, but did not act on Fas/Fas ligand pathways and the activa-tion of caspase-8. Further investigaactiva-tion revealed that SZKJT’s inhibiactiva-tion of cell growth effect was also evident in a nude mice model. Taken together, our study suggests that the induction of p21/WAF1 and activity of the mito-chondrial apoptotic system may participate in the antiproliferative activity of SZKJY in human breast cancer cells.

Key words San-Zhong-Kui-Jian-Tang; breast cancer; cell cycle; apoptosis; p21; mitochondrial

© 2006 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: aalin@ms24.hinet.net

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was confirmed by Doctor MH Yen of Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan. Each recipe (200 g) was decocted three times with 1 l boiling distilled water for 2 h. The decoction was filtered, collected, concentrated, and lyophilized. The average yield obtained for SZKJT was 20.7%.

Cell Cultures Breast cancer cell lines MCF-7 (ATCC HTB-22), MDA-MD-231 (ATCC HTB-26), and IMR-90 (ATCC CCL-186) normal lung fibroblast cells and BNL CL.2 (ATCC TIB-73) normal murine liver cells were ob-tained from the American Type Cell Culture Collection (Manassas, VA, U.S.A.). Normal mammary epithelial cell H184B5F5/M10 was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). MCF-7, IMR-90, H184B5F5/M10, and BNL CL.2 cells were cultured in DMEM with nonessential amino acids, 0.1 mMsodium pyru-vate, 10mg/ml insulin, and 10% FCS. The MDA-MB-231 cells were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY, U.S.A.) supplemented with 10% FCS and 1% penicillin–streptomycin solution (Life Technologies, Inc.).

Cell Proliferation Assay (XTT) Inhibition of cell pro-liferation by SZKJT was measured by XTT (sodium 3 -[1- (phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) assay. Briefly, cells were plated in 96-well culture plates (1104cells/well). After 24 h

incubation, the cells were treated with SZKJT (0, 100, 200, 300mg/ml) for 48 h. Fifty microliters of XTT test solution, which was prepared by mixing 5 ml of XTT-labeling reagent with 100ml of electron coupling reagent, was then added to each well. After 4 h incubation, absorbance was measured on an ELISA reader (Multiskan EX, Labsystems) at a test wave-length of 492 nm and a reference wavewave-length of 690 nm. Data were calculated as the percentage of inhibition by the follow-ing formula: inhibition %[100(ODt/ODs)100]%. ODt and ODs indicated the optical density of the test substances and the solvent control, respectively. The concentration of 50% cellular cytotoxicity of cancer cells (IC50) of test

sub-stances was calculated based on 48 h absorbance values.

Cell Cycle Analysis To determine cell cycle distribution analysis, 5105 cells were plated in 60 mm dishes and treated with SZKJT (100, 200mg/ml) for 12 h. After treat-ment, the cells were collected by trypsinization, fixed in 70% ethanol, washed in phosphate-buffered saline (PBS), resus-pended in 1 ml of PBS containing 1 mg/ml RNase and 50mg/ml propidium iodide, incubated in the dark for 30 min at room temperature, and analyzed by EPICS flow cytometer. The data were analyzed using Multicycle software (Phoenix Flow Systems, San Diego, CA, U.S.A.).

Measurement of Apoptosis Quantitative assessment of apoptosis was analyzed by an Annexin V assay kit (BD Bio-sciences PharMingen, San Jose, CA, U.S.A.). Briefly, cells grown in 10 cm Petri dishes were harvested with trypsin and washed in PBS. Cells were then resuspended in binding buffer (10 mmol/l HEPES/NaOH (pH 7.4), 140 mmol/l NaCl, 2.5 mmol/l CaCl2) and stained with Annexin V-FITC and PI

at room temperature for 15 min in the dark. Cells were then analyzed in an EPICS flow cytometer (Coulter Electronics) within 1 h after staining. Data from 10000 cells were col-lected for each data file. Apoptotic cells were defined as An-nexin V-positive, PI-negative cells.

Quantitative assessment of apoptotic cells was also as-sessed by the terminal deoxynucleotidyl transferase-medi-ated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNA-strand breaks during apopto-sis by using BD ApoAlert™ DNA Fragmentation Assay Kit. Briefly, cells were incubated with 100 and 200mg/ml SZKJT for the indicated times. The cells were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 min at 37 °C. The stained cells were then analyzed with an EPICS flow cy-tometer.

Assaying the Levels of p53, p21/WAF1, Fas/APO-1 and Fas Ligand (mFasL and sFasL) p53 pan ELISA, WAF1 ELISA, Fas/APO-1 ELISA and Fas Ligand ELISA kits were used to detect p53, p21/WAF1, Fas/APO-1 receptor and FasL. Briefly, cells were treated with 0, 100, and 200mg/ml SZKJT for the indicated times. The samples of cell lysate were placed in 96 well (1106 per well) microtiter plates

coated with monoclonal detective antibodies, and were incu-bated for 1 h (Fas/APO-1), 2 h (p53 or p21/WAF1) or 3 h (Fas ligand) at room temperature. It was necessary to determine the soluble Fas ligand in cell culture supernatant by using Fas Ligand ELISA kit. After removing unbound material by washing with washing buffer (50 mMTris, 200 mMNaCl, and 0.2% Tween 20), horseradish peroxidase conjugated strepta-vidin was added to bind to the antibodies. Horseradish perox-idase catalyzed the conversion of a chromogenic substrate (tetramethylbenzidine) to a colored solution, with color in-tensity proportional to the amount of protein present in the sample. The absorbance of each well was measured at 450 nm, and concentrations of p53, p21/WAF1, Fas/APO-1 and Fas L were determined by interpolating from standard curves obtained with known concentrations of standard pro-teins.

Assay for Caspase-8 and -9 Activities The assay is based on the ability of the active enzyme to cleave the chro-mophore from the enzyme substrate: Ac-IETD-pNA (Ac-Ile-Glu-Thr-Asp-pNA) for caspase-8, and LEHD-pNA (Ac-Leu-Glu-His-Asp-pNA) for caspase-9. Cell lysates were incu-bated with peptide substrate in assay buffer (100 mM NaCl, 50 mMHEPES, 10 mMdithiothreitol, 1 mMEDTA, 10% glyc-erol, 0.1% CHAPS, pH 7.4) for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm. Results are repre-sented as the percentage of change of activity compared to the untreated control.

Immunoblot Assay Cells were treated with 200mg/ml SZKJT for specified intervals of time. Mitochondrial and cy-toplasmic fractions were separated using Cytochrome c Re-leasing Apoptosis Assay Kit (BioVision, California, U.S.A.). For immunoblotting, the cells were lysed on ice for 40 min in a solution containing 50 mM Tris, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 2 mM Na3VO4, 2 mM EGTA, 12 mM

b-glycerolphosphate, 10 mM NaF, 16mg/ml benzamidine hy-drochloride, 10mg/ml phenanthroline, 10 mg/ml aprotinin, 10mg/ml leupeptin, 10 mg/ml pepstatin, and 1 mM phenyl-methylsulfonyl fluoride. The cell lysate was centrifuged at 14000g for 15 min, and the supernatant fraction was col-lected for immunoblotting. Equivalent amounts of protein were resolved by SDS-PAGE (10—12%) and transferred to PVDF membranes. After blocking for 1 h in 5% nonfat dry

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milk in Tris-buffered saline, the membrane was incubated with the desired primary antibody for 1—16 h. The mem-brane was then treated with appropriate peroxidase-conju-gated secondary antibody, and the immunoreactive proteins were detected using an enhanced chemiluminescence kit (Amersham, U.S.A.) according to the manufacturer’s instruc-tions.

In Vivo Tumor Xenograft Study Female nude mice [6

weeks old; BALB/cA-nu (nu/nu)] were purchased from Na-tional Science Council Animal Center (Taipei, Taiwan) and maintained in pathogen-free conditions. MDA-MB-231 cells were injected subcutaneously into the flanks of nude mice (5106cells in 200ml). Tumors were allowed to develop for ca. 30 d until they reached ca. 75 mm3, when treatment was

initiated. Twenty mice were randomly divided into two groups. SZKJT was dissolved in sterilized distilled water, and a nude mouse received 1000 mg/kg of SZKJT in 0.5 ml of water through a naso-gastric tube (twice a day). The con-trol group was treated with an equal volume of normal saline. After transplantation, tumor size was measured using calipers and tumor volume was estimated according to the formula: tumor volume (mm3)LW2/2, where L is the

length and W is the width.

Statistical Analysis Data were expressed as means

S.D. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences ( p<0.05) between the means of control and SZKJT-treated cells were analyzed by Dunnett’s test.

RESULTS

Effect of SZKJT on Cell Proliferation in MCF-7 and MDA-MB-231 Cell Lines As shown in Fig. 1A, SZKJT inhibited cell growth in two human breast cancer cell lines in a concentration-dependent manner, with MCF-7 being more sensitive to SZKJT-induced cell growth inhibition than MDA-MB-231. The IC50values of SZKJT were 103.2mg/ml

for MCF-7 and 116.2mg/ml MDA-MB-231. Interestingly, the proliferation inhibitory effect of SZKJT on H184B5F5/ M10 normal mammary epithelial cells, IMR-90 normal lung fibroblast cells, and BNL CL.2 normal murine liver cells was not significant at the same concentrations as on tumor cells (Fig. 1B).

Effect of SZKJT on Cell Cycle Distribution in MCF-7 and MDA-MB-231 Cell Lines To examine the mechanism responsible for SZKJT-mediated cell growth inhibition, cell cycle distribution was evaluated using flow cytometric analy-sis. The results showed that treating cells with SZKJT caused a significant inhibition of cell cycle progression in both MCF-7 and MDA-MB-231 cells at 12 h and 24 h (Figs. 2A, B), resulting in a clear increase of the percentage of cells in

Fig. 1. Effect of SZKJT on the Proliferation Inhibition in MCF-7 and MDA-MB-231 Cells

The effect of SZKJT in breast cancer cells (A) and various normal cells (B). Cells were seeded into 96-well plates (104

cells/well) and allowed to adhere overnight. The next day, the cells were incubated with vehicle (0.1% DMSO) and different concentra-tions of SZKJT for 48 h. Cell proliferation was determined by XTT assay. Results are expressed as percent cell proliferation relative to the proliferation of control. Each value is the meanS.D. of three determinations.

Fig. 2. The Distribution of Cell Cycle in SZKJT Treated MCF-7 (A) and MDA-MB-231 Cells (B)

Cells were treated with vehicle (0.1% DMSO), 100 and 200mg/ml SZKJT for 12 and 24 h. The distribution of cell cycle was assessed by flow cytometry. Each value is the meanS.D. of three determinations. The asterisk indicates a significant difference be-tween control and SZKJT-treated cells as analyzed by Dunnett’s test, p<0.05.

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the G0/G1 phase when compared with the control.

SZKJT Induced Apoptosis in Both MCF-7 and MDA-MB-231 A quantitative evaluation of apoptosis was sought using an Annexin V-FITC dye to detect the translocation of phosphatidylserine from the inner (cytoplasmic) leaflet of the plasma membrane to the outer (cell surface). Compared with vehicle-treated cells, 200mg/ml SZKJT induced 48.6% and 40.6% of apoptotic cells in MCF-7 and MDA-MB-231 at 48 h, respectively (Figs. 3A, B). Additionally, a quantitative evaluation was sought using TUNEL to detect the amount of DNA fragmentation. Compared with vehicle-treated cells, 200mg/ml SZKJT induced 50.6% and 42.6% of cytoplasmic

oligonucleosome in MCF-7 and MDA-MB-231 at 48 h, re-spectively (Figs. 3C, D). The proapoptotic effect of SZKJT was also observed in a dose-dependent manner after 48 h treatment (Fig. 3E).

Effect of SZKJT on Cell Cycle-Related Molecules We next examined the effect of SZKJT on cell cycle-regulatory molecules, including p53, p21/WAF1, cyclinD1, cyclinD2, and cyclinB. Previous reports have indicated that MCF-7 cells have a normal tumor suppression gene, p53, whereas in MDA-MB-231 cells the major protein of the p53 gene has mutated and is accompanied by the absence of p53 func-tion.8,9)As shown in Fig. 4A, SZKJT failed to affect the ex-Fig. 3. The Induction of Apoptosis of MCF-7 and MDA-MB231 by SZKJT Treatment

The quantitation of apoptosis in MCF-7 (A) and MDA-MB-231 (B) cells by Annexin V-FITC/PI staining. The analysis of apoptosis in MCF-7 (C) and MDA-MB-231 (D) cells by using TUNEL method. (E) The effect of SZKJT on apoptosis in a dose-dependent manner. Cells were treated with vehicle alone (0.1% DMSO) and various concentration of SZKJT for the indicated time. The phosphatidylserine translocation and cytoplasmic oligonucleosome of SZKJT treated cells was estimated by Annexin V assay. Each value is the meanS.D. of three determinations. The asterisk indicates a significant difference between control and SZKJT-treated cells as analyzed by Dunnett’s test, p<0.05.

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pression of p53 at any of the examined time points in MCF-7 cells, but increased the expression of p21/WAF1 in both MCF-7 and MDA-MB-231 cells (Figs. 4B, C). Furthermore, SZKJT treatment of the cells resulted in a time-dependent decrease in the protein expression of cyclinD1, cyclinD2 in both MCF-7 and MDA-MB-231 cells (Fig. 4D).

Fas/Fas Ligand Is Not Involved in SZKJT-Mediated Apoptosis To establish the sequence of events occurring during SZKJT-induced apoptosis, we measured some of the molecular activity of the death receptor apoptotic pathway, including Fas/APO-1 receptor and its two ligands, mFas lig-and lig-and sFas liglig-and. However, treatment of either of these two cell lines with 100 or 200mg/ml SZKJT failed to affect the levels of these proteins at any of the examined time points, including Fas/APO-1, mFas ligand and sFas ligand (data not shown). In addition, SZKJT also failed to affect the activation of caspase-8 in both MCF-7 and MDA-MB-231 cells (data not shown).

SZKJT Induces the Execution of Apoptosis through Activation of the Mitochondrial Pathway To investigate the mitochondrial apoptotic events involved in SZKJT-in-duced apoptosis, we first analyzed the changes in the levels of pro-apoptotic protein Bax and Bak, and anti-apoptotic protein Bcl-2. Western blot analysis showed that treatment of MCF-7 and MDA-MB-231 cells with SZKJT increased Bax and Bak protein levels (Fig. 5A). In contrast, SZKJT de-creased Bcl-2, which led to an increase in the proapoptotic/ antiapoptotic Bcl-2 ratio (Fig. 5A).

Cytosolic extracts were prepared under conditions to pre-serve the mitochondria, and cytosolic cytochrome c protein levels were measured by immunoblotting analysis. Figure 5B shows that the cytosolic fraction from untreated MCF-7 and MDA-MB-231 cells contained no detectable amounts of cy-tochrome c, whereas it did become detectable after 12 h of 200mg/ml SZKJT treatment in both MCF-7 and MDA-MB-231 cells.

Hallmarks of the apoptotic process include the activation of cysteine proteases, which represent both initiators and ex-ecutors of cell death. Upstream caspase-9 activities increased significantly as shown by the observation that treatment with SZKJT increased caspase-9 activity in both MCF-7 and MDA-MB-231 cells. This is consistent with the release of cytochrome c into the cytosol (Figs. 5C, D).

To verify the relation of apoptosis and cytotoxicity in SZKJT-treated breast cancer cells, we used pan-caspase in-hibitor to block caspase activity in both cell lines and deter-mine whether the cell proliferation inhibition was changed after SZKJT treatment. Blocked of caspases activation re-sulted in a completely decreased in SZKJT-mediated prolifer-ation inhibition in both cell lines (Fig. 5E), suggesting that SZKJT inhibits cell proliferation inhibition by apoptosis in-duction.

SZKJT Inhibits Tumor Growth in Nude Mice To de-termine whether SZKJT inhibits tumor growth in vivo, equal numbers of MDA-MB-231 cells were injected subcuta-neously into both flanks of the nude mice. Tumor growth in-hibition was most evident in mice treated with SZKJT at 1000 mg/kg, (twice day) where ca. 40% reductions in tumor size were observed, in contrast with mice treated with the ve-hicle (Figs. 6A, B). No sign of toxicity, as judged by parallel monitoring body weight, was observed in SZKJT-treated Fig. 4. Effects of SZKJT on the Expressions of Cell Cycle-Related

Pro-teins

The level of p53 in MCF-7 cells (A). The amount of p21/WAF1 in MCF-7 (B) and MDA-MB-231 (C) cells. The expressions of cyclinD1, cyclinD2, and cyclinB in SZKJT-treated cells (D). Cells were treated with vehicle (0.1% DMSO), 100 and 200mg/ml SZKJT for the indicated time. The level of p53 and p21/WAF1 protein was measured by p53 pan and WAF1 ELISA kit. The amount of cyclinD1, cyclinD2, and cyclinB was assessed by Western blot assay. Each value is the meanS.D. of three de-terminations. The asterisk indicates a significant difference between control and SZKJT-treated cells as analyzed by Dunnett’s test, p<0.05.

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mice.

DISCUSSION

Breast cancer is the most common neoplasm in human in both developed and developing countries.10,11) In our study, we have found that SZKJT effectively inhibits tumor cell growth in vitro, concomitant with induction of cell cycle ar-rest and apoptosis, and inhibits tumor cell growth in nude mice. Furthermore, because SZKJT do not exhibit any sig-nificant toxicity on various normal cells, this suggests that SZKJT possesses selectivity between normal and cancer cells. This selection of SZKJT to cancer cells may be related to the different genomic stability between cancer and normal cells.12)

Eukaryotic cell cycle progression involves sequential acti-vation of Cdks, whose actiacti-vation is dependent upon their as-sociation with cyclins.13)A complex formed by the associa-Fig. 5. SZKJT Induced Apoptosis through the Initiation of the

Mitochon-drial Pathway

(A) The effect of SZKJT in Bcl-2 family proteins. (B) The release of cytochrome c in MCF-7 and MDA-MB-231 cells. The activation of caspase-9 in MCF-7 (C) and MDA-MB-231 (D) cells. (E) The effect of pan-caspase inhibitor on SZKJT-mediated cell proliferation inhibition. For (A) and (B), cells were treated with 200mg/ml SZKJT for the indicated time. The extraction of cytoplasm and mitochondria were separated from cell pellet by lysis buffer and centrifugation. Western blotting analysis assessed the protein expressions. For (C) to (D), the activity of caspase-9 was assessed by cas-pase-9 activity assay kit. For (E), cells were treated z-VAD-FMK (20mM) for 1 h, and then incubated with SZKJT (200mg/ml) for 48 h. The cell proliferation was assessed by XTT assay. Each value is the meanS.D. of three determinations. The asterisk indi-cates a significant difference between control and SZKJT-treated cells as analyzed by Dunnett’s test, p<0.05.

Fig. 6. SZKJT Inhibits Growth in Nude Mice

(A) Representative tumor-possessing nude mice and tumors from the control and SZKJT-treated groups. (B) Mean of tumor volume measured at the indicated number of days after implant. Animals bearing pre-established tumors (n15 per group) were dosed daily for 40 d with p.o. of SZKJT (1000 mg/kg, twice a day) or vehicle. During the 40-d treatment, tumor volumes were estimated using measurements taken from ex-ternal calipers (mm3

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tion of Cdc4/6 and cyclin D1/D2 plays a major role at entry into S phase.14,15)Cell cycle progression is also regulated by

the relative balance between the cellular concentration of cy-clin-dependent kinase inhibitors (CKIs), such as members of the cyclin-dependent kinase-interacting protein/cyclin-de-pendent kinase inhibitory protein (CIP/KIP) and inhibitor of dependent kinase (INK) families, and that of cyclin-CDK complexes. The Cip/Kip family, including p21/WAF1, and p27/KIP, bind to cyclin-CDK complexes and prevent ki-nase activation and subsequently blocks the progression of cell cycle at G0/G1 or G2/M phase.15,16) In our result, we

found that SZKJT treatment not only causes a significant in-crease in the expression of p21 both in MCF-7 and MDA-MB-231 cells, but also decreases the expressions of cy-clinD1, and cyclinD2. Thus, it is reasonable to postulate that SZKJT treatment may cause cell cycle arrest by regulating the expressions of G0/G1 regulating proteins.

Two major distinct apoptotic pathways have been de-scribed for mammalian cells. One involves caspase-8, which is recruited by the adapter molecule Fas/APO-1 associated death domain protein to death receptor upon Fas ligand bind-ing.3,17)We did not observe any alteration of either

Fas/APO-1 or Fas ligand (mFas ligand and sFas ligand) expression or caspase-8 activation in SZKJT-treated MCF-7 and MDA-MB-231 cells. On the other hand, SZKJT treatment resulted in a significant increase of Bax and Bak expression, and de-creased the amount of Bcl-2, suggesting that changes in the ratio of proapoptotic and antiapoptotic Bcl-2 family proteins might contribute to the apoptosis-promotion activity of SZKJT. These regulatory effects of SZKJT on the Bcl-2 fam-ily are correlated with the release of cytochrome c from the mitochondria into the cytoplasm and the activation of cas-pase-9.

In this study, we concluded that the molecular mechanisms during SZKJT-mediated growth inhibition in MCF-7 and MDA-MB-231 cells involved the (1) induction of apoptosis, (2) blockade of cell cycle progression by regulating cell cycle-related factor, (3) trigger of mitochondrial pathway, (4)

modulation of Bcl-2 family protein, and (5) inhibition of can-cer growth in nude mice. We demonstrated that SZKJT may be a promising chemotherapy agent for treating breast can-cer.

Acknowledgements This study was supported by Com-mittee on Chinese Medicine and Pharmacy of Taiwan (CCMP94-RD-045).

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17) Chopin V., Slomianny C., Hondermarck H., Le Bourhis X., Exp. Cell

數據

Fig. 2. The Distribution of Cell Cycle in SZKJT Treated MCF-7 (A) and MDA-MB-231 Cells (B)
Fig. 6. SZKJT Inhibits Growth in Nude Mice

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