OBJECTIVE To explore the effect of pentagalloylglucose(PGG) on the apoptosis of ovarian cancer cells and the mechanism underlying the induced caspase-dependent apoptosis pathways in ovarian cancer HO-8910 cells. METHODS HO-8910 cells were cultured with PGG 10, 20,40 and 80 μmol·L-1 for 48, 72 or 96 h. The cell survival rate was detected by MTT assay. The morphological alteration of nucleus of HO-8910 cells was observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis ratio was determined by Annexin V -FITC/PI double-staining combined with flow cytometry assay. Western blotting was employed to detect the pro-caspases or active caspases( c-caspases). The mRNA expression of apoptosis regulator genes Bcl-2, Bcl-XL, CIAP-1, CIAP-2, survivin, NIAP, XIAP and cycle regulator gene cyclin D1 was visualized by RT-PCR. RESULTS PGG inhibited the growth in vitro of HO-8910 cells in a time- and concentration-dependent manner during 48, 72 and 96 h treatment (P＜0. 05) ; the correlation coefficient was 0. 93 , 0.95 and 0. 86, respectively. PGG treatment induced nuclear, chromatin pycnosis of HO-8910 cells and the morphological alteration of nuclei in apoptosis. The ratio of apoptosis in HO-8910 cells rose with lengthened treatment and increased doses of PGG. PGG 20-80 μmol·L-1 promoted the cleavage of caspases 3, 7 and 9 and the related PARP substrate in HO-8910 cells. However, PGG 20 - 80 μmol·L-1 inhibited the protein expression of the death receptor FAS and the cleavage of caspase 8 at 24 and 36 h, but down-regulated the mRNA expression of cyclin D1, Bcl-2, Bcl-XL and NIAP genes vat 12 h. It had on effect on Bax, CIAP-2 and XIAP mRNA, but up-regulated the mRNA expression of CIAP-1. The effect of PGG on the surviving mRNA was not concentration-dependent. CONCLUSION PGG might induce caspase 9-dependent endogenous caspase pathways by inhibiting the expression of apoptosis inhibitor genes, and trigger HO-8910 cell apoptosis. It' s likely that PGG has repressed the caspase 8-dependent apoptosis pathway in HO-8910 cells.%目的 探讨五没食子酰基葡萄糖(PGG)诱导卵巢癌H0-8910细胞凋亡的作用及诱导胱天蛋白酶凋亡途径的机制.方法 PGG 10,20,40和80μmol·L-1处理H0-8910细胞48,72和96 h后,MTT法检测细胞存活率；Hoechst 33258染色观察HO-8910细胞核形态改变,AnnexinV -FITC/PI双染流式细胞术检测细胞凋亡率；Western印迹法检测细胞内胱天蛋白酶酶原及活性形式；RT-PCR检测凋亡调控基因Bax、Bcl-2、Bcl-XL、凋亡抑制因子1(CIAP-I)、CIAP-2、存活蛋白、神经元凋亡抑制蛋白(NIAP)、X连锁凋亡抑制蛋白(XIAP)和细胞周期蛋白DI mRNA表达.结果 PGG 10～80 μmol·L-1分别作用48,72和96 h,随浓度的增加,细胞存活率明显降低,r分别为0.93,0.95和0.86(P＜0.05).PGG 40 μmol·L-1使HO-8910细胞的细胞核染色质固缩,出现凋亡形态学改变,早期凋亡率从正常对照组的(0.6±0.1)％分别增加到(3.4±1.1)％,(9.8±3.7)％和(19±4.5)％,对晚期凋亡率影响不明显.PGG 20～80 μmol·L-1使HO-8910细胞内胱天蛋白酶3,胱天蛋白酶7和胱天蛋白酶9及其底物多聚腺苷二磷酸核糖聚合酶(PARP)的剪切水平增加,PGG20～80 μmol·L,均抑制死亡受体FAS的蛋白表达水平并使胱天蛋白酶8总剪切水平降低.PGG 20～80 μmol·L-1抑制HO-8910细胞中细胞周期蛋白D1,Bcl-2,Bcl-XL和NIAP mRNA的表达,上调CIAP-1 mRNA 的表达,对基因Bax,CIAP-2和XIAP mRNA表达影响不明显；PGG 20 μmol·L-1抑制存活蛋白基因mRNA的表达,但是增加处理浓度却上调存活蛋白基因mRNA的表达.结论 PGG可能通过抑制凋亡抑制基因Bcl-2和Bcl-XL的表达从而诱导HO-8910细胞内胱天蛋白酶9依赖的内源性凋亡途径,并诱导细胞凋亡.
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