Vol. 33 No Journal of Jinan University Medicine Edition Apr HepG2 5-FU. HepG2. HepG2 Western blotting. HepG2 MTT

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1 33 2 Vol. 33 No Journal of Jinan University Medicine Edition Apr HepG2 5-FU HepG2 5-FU MTT HepG2 Hoechst HepG2 Western blotting Caspase-3 Caspase FU DDP HepG2 MTT HepG2 HepG2 48 h IC μmol /L Caspase-3 Caspase μmol /L 5-FU DDP HepG2 48 h HepG HepG2 HepG2 5-FU DDP HepG2 R A Silibinin induces apoptosis in human hepatoma HepG2 cells and enhances its chemo-sensitivity CHEN Jing-hong 1 ZHANG Peng 1 CHEN Qing 1 LIU Ping-li 1 WANG Li 2 JIANG Jian-wei 1 1. Department of Biochemistry Medical College Jinan University Guangzhou China 2. Department of Laboratory Panyu Center Hospital Guangzhou China Abstract Aim To investigate the inhibitory effect of Silibinin and its ability of enhancing chemo-sensitivity to human hepatoma HepG2 cells lines. Methods The effects of Silibinin on the proliferation of HepG2 cells were evaluated by the MTT assays and colony formation assays. The cellular morphology of HepG2 cells was observed using fluorescence microscopy after Hoechst staining. Western blotting was used to detect the expression of caspase-3 and -9 protein levels. HepG2 cells were treated with Silibinin in combination with different concentrations of 5-fluorouracil 5-FU or cisplatin DDP cell proliferatory inhibition rates were detected by MTT method. Results Silibinin inhibited the proliferation of HepG2 cell lines in a dose dependent manner P < The IC 50 of the Silibinin on human hepatoma HepG2 cells for 48 hours was μmol / L. Cell clone formation inhibition assay demonstrated that B Tel @ qq. com

2 2 HepG2 5-FU 139 cell clones decreased with the increase of Silibinin's concentrations. Hoechst staining showed the occurrence of nuclear fragmentation and chromosomal condensation cell detachment of HepG2 cells treated with Silibinin. Western-blotting assay showed that Caspase-3 and Caspase-9 protein expression levels decreased gradually as the concentration of Silibinin increased. MTT assay indicated that the chemo-therapeutic effects of 5-FU and DDP on HepG2 cells were enhanced independently when they were combined with Silibinin 150 μmol /L and the sensitivities increased were enhanced about and times respectively. Conclusion Silibinin inhibited the proliferation by inducing apoptotic of human hepatoma HepG2 cells and enhanced the chemo-sensitivity to 5-FU and DDP. Key words Silibinin HepG2 cell lines apoptosis sensitivities HepG2 3 SB SB DMSO RPMI-1640 DMSO silymarin SM 0. 1% silibinin SB HepG2 10% RPMI % CO % 0. 25% 2 MTT HepG /ml100 μl 96 1 I 2 2DMSO μmol /L % CO 2 48 h 10 μl MTT 4 h μl DMSO ΔA = 570 nm 1. 1 C 25 H 22 O 10 H 2 O % = 1 - / RPMI % BLISS Gibco Hochest33258 IC 50 Trypsin Sigma dimethyl sulfoxide DMSO Sigma Dimethylthiazol-2-yl -2 5-diphenyltetrazolium bromide -3- glyceraldehyde-3-phosphate dehydrogenase GAPDH -3 cysteinglaspartatespecificprotease-3 Caspase- MTT cysteinglaspartatespecificprotease-9 Caspase-9 HepG /ml 12 4 Hoechst Cell Signaling Technology 5-5-FU DDP 3 HepG2 15% RPMI DMSO μmol /L 37 5% CO 2 7 d 15 min 2 1 2DMSO μmol /L 48 h PBS 200 μl

3 ml V V = min 10 mg /L Hoechst min 5 6 PBS 2 15 min BCA 15% PVDF 110 ma 150 min 5% TBS-T 2 h GAPDH Caspase-3 Caspase DMSO h ECL X DMSO 6 HepG2 P < μmol / /ml100 μl 96 L HepG % CO μmol /L h 40% ~ 50% μmol /L 150 μmol /L 48 h MTT IC 50 = IC 50 / IC 50 Q Q = Ea + b / Ea + Eb - Ea Eb Ea Eb Ea + b Q Q < Q < Q SPSS ± x 珋 ± s P < HepG HepG2 HepG2 7 d 1 2 DMSO 3 20 μmol /L 4 30 μmol /L 5 40 μmol 6 50 μmol /L. 2 HepG Hoechest HepG2 HepG2 48 h HepG2 48 h 150 μmol /L DMSO DMSO 3 P < IC μmol /L

4 2 HepG2 5-FU 141 HepG2 48h Hoechst DMSO μmol /L Western blotting Caspase-3 Caspase-9 Caspase n μg ml Caspase-3 Caspase FU 9 4 Caspase-3 Caspase-9 Caspase HepG2 1 2 DMSO 3 80 μmol /L ± μmol /L μmol /L ± Western blotting HepG2 Caspase-3 Caspase HepG2 5-FU IC μg /ml 5-FU 10 μg /ml 20 μg /ml 150 μmol /L IC μg / ml IC μg /ml μg ρ 5-FU / 5-FU HepG2 x 珔 ± s /% Q 5-FU + 5-FU ± ± ± ± ± ± ± ± FU P < HepG2 x 珔 ± s ρ μg ml - 1 / n /% Q ± ± ± ± ± ± ± ± P < silibinin SB Survivin 2 μg /ml 150μmol /L G1 G2 /M IC μg /ml G TNF NF-k B TNF-a

5 MTT 5-FU HepG2 5-FU 150 μmol /L 5-FU HepG2 5-FU HepG2 200 μmol /L HepG HepG2 IC Q 5-FU μmol /L Li 9 Caki μmol /L 48 h 200 μmol /L 78. 9% 90% Ge 10 HepG2 200 μmol /L BxPC - 3 HepG2 5-FU 50% 400 μmol /L 48 h 20% 5-FU LO-2 5-FU C Caspase-9 Caspase silymarin from experimental pharmacology to clinical medicine J. Indian J Med Res Hoechst J. Western blotting Caspase-9 Caspase DEEP G AGARWAL R. Antimetastatic efficacy of silibi- HepG2 Survivin Caspase Singh 11 Survivin Survivin Survivin 1 PRADHAN S GIRISH C. Hepatoprotective herbal drug nin molecular mechanisms and therapeutic potential a- gainst cancer J. Cancer and Metastasis Reviews ZI XIAOLI ZHANG JIANCHUN AGARWAL R et al. Survivin p53 Silibinin up-regulates insulin-like growth factor- binding Tyagi 12 RT4 protein 3 expression and inhibits proliferation of androgenindependent prostate cancer cells J. Cancer Res 200 μmol /L h Survivin survivin ZHAO JIFU LAHIRI-CHATTERJEE SHARMA Y et mrna Survivin Survivin 13 al. Inhibitory effect of a flavonoid antioxidant silymarin on benzoyl peroxide-induced tumor promotion oxidative Survivin SMMC stress and inflammatory responses in SENCAR mouse skin FU J. Carcinogenesis Survivin 6 HOGAN F S KRISHNEGOWDA N K MIKHAILOVA DDP Survivin M et al. Flavonoid silibinin inhibits proliferation and DDP 15 Survivin promotes cell-cycle arrest of human colon cancer J. SKOV3 Surg Res

6 2 HepG2 5-FU DEEP G SINGH R P AGARWAL C et al. Silymarin and silibinin cause G1 and G2- M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells A comparison of flavanone silibinin with flavanolignan mixture silymarin J. Oncogene MATEEN S TYAGI A AGARWAL C et al. Silibinin inhibits human nonsmall cell lung cancer cell growth through cell-cycle arrest by modulating expression and function of key cell-cycle regulators J. Mol Carcinog LI LEI GAO YE ZHANG LINLIN et al. Silibinin inhibits cell growth and induces apoptosis by caspase activation down-regulating survivin and blocking EGFR -ERK activation in renal cell carcinoma J. Cancer Lett GE YAKUN ZHANG Y CHEN YUNPENG et al. Silibinin causes apoptosis and cell cycle arrest in some human pancreatic cancer Cells J. Int J Mol Sci SINGH P TYAGI A SHARMA G et al. Oral silibinin inhibits in vivo human bladder tumor xenograft growth involving down-regulation of surviving J. Clinical Cancer Research TYAGI K AGARWAL C SINGH R et al. Silibinin down-regulates survivin protein and mrna expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells J. Biochem Biophys Res Commun J Survivin J survivin SKOV3 J 櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆 CA