丹皮酚通过内质网应激和线粒体损伤途径促进卵巢癌细胞凋亡

Paeonol Promotes Apoptosis of Ovarian Cancer Cells through Endoplasmic Reticulum Stress and Mitochondrial damage Pathways

【目的】探讨丹皮酚对卵巢癌细胞凋亡的影响及其机制。【方法】用不同剂量(0、1、2、3、4、5、6、7、8 mmol/L)丹皮酚处理人正常卵巢表面上皮细胞HOSEpiC和人类卵巢癌细胞SKOV3。应用细胞计数试剂盒8(CCK8)测定丹皮酚对HOSEpiC和SKOV3细胞的毒副作用,选择低剂量丹皮酚(1、2、5 mmol/L)进行后续实验。用丹皮酚(1、2、5 mmol/L)处理SKOV3细胞,溴脱氧尿嘧啶核苷(BrdU)染色法观察SKOV3细胞的增殖能力,蛋白免疫印迹(Western Blot)法检测SKOV3细胞Ki67、增殖细胞核抗原(PCNA)、Bcl-2、Bax、葡萄糖调控蛋白78(GRP78)、CCAAT增强子结合蛋白(C/EBP)同源(CHOP)、cleaved Caspase-12、cleaved Caspase-3、细胞色素c(Cyto-c)、细胞凋亡诱导因子(AIF)、细胞色素c氧化酶4(COX IV)、cleaved Caspase-9的表达,流式细胞术检测细胞SKOV3凋亡和Ca2+水平。【结果】丹皮酚(2、5 mmol/L)对正常细胞无害,可剂量依赖性地降低SKOV3细胞存活率。丹皮酚(2、5 mmol/L)抑制SKOV3细胞增殖,促进SKOV3细胞凋亡。与空白对照组比较,丹皮酚(2、5 mmol/L)均升高内质网应激相关的凋亡通路蛋白GRP78、CHOP、cleaved Caspase-12、cleaved Caspase-3表达水平及Ca2+含量(均P<0.05)。与空白对照组比较,丹皮酚2、5 mmol/L均降低线粒体损伤相关的凋亡通路Cyto-c、AIF(线粒体内)水平,升高Cyto-c、AIF(胞质)的表达水平,升高cleaved Caspase-9表达水平,下调Bcl-2及上调Bax表达水平(均P<0.05),且呈剂量依赖性。【结论】丹皮酚剂量依赖性地调控SKOV3细胞中内质网应激和线粒体损伤相关的凋亡通路蛋白,可促进卵巢癌细胞凋亡。

Objective To explore the effects of paeonol on apoptosis of ovarian cancer cells and its mechanism.Methods Human normal ovarian surface epithelial cells HOSEpiC and human ovarian cancer cells SKOV3 were treated with different doses(0,1,2,3,4,5,6,7,and 8 mmol/L)of paeonol. The toxic side effects of paeonol on HOSEpiC and SKOV3 cells were observed by Cell count kit 8(CCK8),so 1,2 and 5 mmol/L of paeonol were selected for the next tests. After SKOV3 cells were treated with paeonol(1,2 and 5 mmol/L),the proliferation activity of SKOV3 cells was observed by BrdU staining,expression levels of Ki67,proliferating cell nuclear antigen(PCNA), Bcl-2, Bax, glucose-regulated protein-78(GRP78), CCAAT/enhancerbinding protein homologous protein(CHOP), cleaved Caspase-12, cleaved Caspase-3, Cytochrome c(Cyto-c),apoptosis induce factor(AIF),Cytochrome c oxidase IV(COX IV),cleaved Caspase-9 were detected by Western blotting assay. The apoptosis and Ca2+ levels were detected by flow cytometry. Results Paeonol of 2,5 mmol/L was harmless to normal cells,but decreased the survival rate of SKOV3 cells in a dose-dependent manner. Paeonol of 2, 5 mmol/L inhibited the proliferation activity of SKOV3 cells, and promoted apoptosis of SKOV3 cells.Compared with the blank control group, paeonol of 2, 5 mmol/L increased the endoplasmic reticulum stressrelated apoptosis pathway proteins such as GRP78,CHOP,cleaved Caspase-12,cleaved Caspase-3 expression levels and Ca2+ content(all P<0.05). Compared with the blank control group,paeonol of 2,5 mmol/L decreased the mitochondrial injury-related apoptosis pathway proteins such as Cyto-c,AIF(in mitochondria)expression levels,increased Cyto-c,AIF(in cytoplasm)expression levels,increased cleaved Caspase-9 expression level,downregulated Bcl-2 and up-regulated Bax expression level(all P<0.05). Conclusion Paeonol promoted ovarian cancer cell apoptosis by regulating and controlling the apoptosis pathways associated with endoplasmic reticulum stress and mitochondrial injury in SKOV3 cells in dose-dependent manner.