HepG hotmail. com. HepG2 Bid Bcl-2. HepG2 G0 /G1 Bid Bcl-2. HepG2. HepG2

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1 588 HepG huan_chaos@ hotmail. com HepG2 MTT PI HepG2 Western Blot HepG2 Bid Bcl-2 HepG2 HepG2 G0 /G Bid Bcl-2 Bax Bid Bcl-2 Bax HepG2 HepG2 R A DOI /j. issn Matrine Induces Apoptosis of HepG2 Cells via Mitochondrial Apoptotic Pathway ZHOU Huan XU Ming-ying 2 LEI Yu 2 LIANG Kai GUO Qing HU Tian-hui 2 Department of Pharmacy Affiliated Tumor Hospital of Guangxi Medical University Nanning China 2 Cancer Research Center School of Medicine Xiamen University Xiamen 3602 China Abstract Objective To explore the mechanism of matrine inducing apoptosis of HepG2 cells via mitochondrial apoptotic pathway. Methods MTT assay PI staining flow cytometry were used to evaluate the anti-proliferation cell cycle regulation and inducing apoptosis effects of matrine on HepG2 cells. Western Blot assay was used to detect the expression levels of anti-apoptotic protein Bid and Bcl-2 in HepG2 cells. Results Matrine could inhibit the proliferation and induce the apoptosis of HepG2 cells in a time- and dose-dependent manner and the cell cycle of HepG2 was arrested in G0/G phase. Apoptosis of HepG2 cells induced by matrine was associated with collapse of mitochondrial membrane potential which made the Bid and Bcl-2 expression levels down-regulated but the Bax expression level up-regulated. Conclusion Matrine induces the apoptosis of human hepatoma cell line HepG2 via the mitochondrial apoptotic pathway by regulating the expression levels of Bid Bcl-2 and Bax proteins. Key words Matrine HepG2 Mitochondrial apoptotic pathway / HepG J ~ 973 ~ thu@ xmu. edu. cn

2 h. 0 mg /ml h EDTA. HepG2 Annexin V-FITC /PI Bid Bcl-2 Bcl-xl Bax Santa Cruz Biotech DMEM 500 μl Binding buffer Gibco PAA 5 μl Annexin V-FITC 5 μl PI MTT Sigma Chemical Annexin-FITC 5 min h 488 nm 50 mg /ml g 50 ml ddh 2 O h HepG2 DMEM mg /ml 24 h 37 5% CO 2 DMEM 0 μg /ml 0% 00 U /ml 00 U /ml min 37 80% ~ 90% PBS 60 min 0. 5 ml EDTA 0. 25%. 7 Western Blot 4x Loading buffer 00 5 min. 3 MTT 0 5 /ml 96 marker 4 24 h TBS-T 0 min % 60 min 2. 0 mg /ml h 20 μl ECL 3 min 5 mg /ml MTT 4 h MTT 50 μl DMSO 0 min x ± s 570 nm P < OD 630 nm 2. 4 PI 2. MTT HepG2 24 h HepG mg /ml 24 h P < EDTA HepG2 70% h - DNase RNase HepG2 x ± s % min 5 μl PI 30 min 488 nm. 5 Annexin V-FITC /PI 24 h mg /ml. 8 GraphPad Prism 5. 0 mol /L 2 h 24 h 48 h ± ± * ± * ± 2. 6 * ± * ± * ± * ± * ± 3. 5 * ± * ± * ± 4. 0 * ± 5. 6 * ± * ± * * P < 0. 05

590 2. 2 HepG2 G0 /G 0. 25 0. 5. 0 mol /L HepG2 G0 /G 7. 97 ± 2. 54 % 74. 487 ± 0. 64 % 77. 223 ± 0. 4 % 66. 772 ± 0. 65 % G0 /G F = 45. 579 P = 0. 000 HepG2 G0 /G P < 0.

3 HepG2 G0 /G mol /L HepG2 G0 /G ± % ± % ± 0. 4 % ± % G0 /G F = P = HepG2 G0 /G P < A HepG2 Western Blot G0 /G p27 p27 2B HepG HepG mol /l 24 h HepG ±. 8 % ± % ± % ±. 33 % ± % ± 5. 6 % F = P < HepG2. 67 ± % P < F = P < B P < A. 0 mol /l h HepG ± % ± % 3 HepG HepG ± ± ± mg /L ± HepG F = P < mol /l P < 0. 05

4 HepG HepG2 HepG2 HepG2 p27 Western Blot G S p27 Bid Bcl-2 3 Bax HepG2 G0 /G p27 PARP Bcl-xl AIF 5 5 HepG2 HepG2 ψm 3 HepG2 MMP MMP Bcl-2 MMP BxPC PANC- SMMC-772 Bcl-2 Bcl-XL Bax Bak 8 7 Bik Bad Bid Akt ERK /2 9 BH3 0 c-myc p53 Bcl-2 7 AIF - 2 Western Blot HepG2 24 h p27 p27 G0 /G ψm MMP ψm ATP 4-5 Rhodamine 23 HepG2 - DNA 8 G0 /G S HepG2 G0 /G Bid

5 592. P53 HBV J Liang CZ Zhang JK Shi Z et al. Matrine induces caspasedependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas /FasL and downregulation of Bcl-2 J. Cancer Chemother Pharmacol Luo SX Deng WY Wang XF et al. Molecular mechanism of indirubin-3'-monoxime and Matrine in the reversal of paclitaxel resistance in NCI-H520 /TAX25 cell line J. Chin Med J Engl Zhao B Li B Bai S et al. Effects of matrine on proliferation and apoptosis of cultured retinoblastoma cells J. Graefes Arch Clin Exp Ophthalmol Liu T Song Y Chen H et al. Matrine inhibits proliferation and induces apoptosis of pancreatic cancer cells in vitro and in vivo J. Biol Pharm Bull Zhang Y Zhang H Yu P et al. Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration J. Cytotechnology J Bcl-2 Bax 9 Zhang S Zhang Y Zhuang Y et al. Matrine induces apoptosis in human acute myeloid leukemia cells via the mitochon- Bcl-xL AIF AIF drial pathway and Akt inactivation J. PLoS One e C6 TRADD J. HepG HepG2 Müller M Strand S Hug H et al. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 APO- /Fas receptor / ligand system and involves activation of wild-type p53 J. J Clin Invest Nagao T Kondo F Sato T et al. Immunohistochemical detection of aberrant p53 expression in hepatocellular carcinoma correlation with cell proliferative activity indices including mitotic index and MIB- immunostaining J. Hum pathol Polyak K Kato JY Solomon MJ et al. p27kip a cyclin- 2. CDK inhibitor links transforming growth factor-beta and J contact inhibition to cell cycle arrest J. Genes Dev Sareen D Darjatmoko SR Albert DM et al. Mitochondria calcium and calpain are key mediator of resveratrol-induced apoptosis in breast cancer J. Mol Pharmaeol Kroemer G Zamzai N Susin SA. Mitochondria control of apoptosis J. Inununol Today Shangary S Johnson DE. Peptiedes derived BH3 domains of Bcl-2 family members a comparative analysis of inhibition of Bcl-2 Bcl-x L and Bax oligomerizatio induction of cytochrome c release and activation of cell death J. Biochemistry De Falco M De Luca L Acanfora F et al. Alteration of the Bcl-2 Bax ratio in the placenata as pregnancy proceeds J. Histochem J Joza N Pospisilik JA Hangen E et al. AIF not just an apoptosis-inducing factor J. Ann NY Acad Sci ~ 206

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