Secoemestrin C抗肝癌作用机制研究

The anti-cancer mechanism of secoemestrin C in liver cancer cells

目的 通过验证secoemestrin C(Sec C)对肝癌细胞增殖的影响,观察细胞内脂质过氧化水平,检测内质网应激(ERS)相关信号通路变化,进而揭示SecC在抑制肝癌增殖过程中的作用机制,为针对肝癌的药物研发提供新的思路。方法 四甲基偶氮唑蓝(MTT)实验和平板克隆法测定Sec C对Hep G2和BEL7404细胞增殖的影响;流式细胞术测定SecC对肝癌细胞脂质过氧化和凋亡的影响;Westernblot法探究SecC对肝癌细胞内质网应激和细胞凋亡相关蛋白表达的影响。结果 SecC以剂量依赖性方式抑制Hep G2和BEL7404细胞增殖,IC50分别为1.556和3.489μmol/L;平板克隆实验结果显示,1μmol/L Sec C处理7 d后,肝癌细胞的克隆数目和大小显著降低,表明SecC显著抑制肝癌细胞增殖且呈现浓度依赖。流式结果显示,与对照相比,Sec C处理后,Bodipy的阳性率增加且呈现浓度依赖,表明SecC促进肝癌细胞内脂质过氧化,且流式结果显示SecC以剂量依赖性的方式诱导肝癌细胞凋亡。Western blot结果表明,SecC会引起内质网应激相关蛋白免疫球蛋白结合蛋白、1型内质网转膜蛋白激酶、活化转录因子4和磷酸化的真核生物起始因子2表达增多,其作用呈现浓度依赖性;同时Western blot结果显示,凋亡相关蛋白多聚腺嘌呤二核苷酸核糖聚合酶的表达显著降低,以及凋亡剪切产物聚腺苷二磷酸核糖聚合酶、活化的含半胱氨酸的天冬氨酸蛋白水解酶3和活化的含半胱氨酸的天冬氨酸蛋白水解酶7的表达显著增多,其作用呈现浓度依赖性。结论 Sec C能够显著抑制肝癌细胞的增殖,其作用机制可能通过诱导细胞内质网应激导致细胞凋亡。

Objective We aimed to verify the effect of secoemestrin C(Sec C)on the proliferation of hepatocellular carcinoma cells,measure intracellular lipid peroxidation level,and detect the changes of endoplasmic reticulum stress(ERS)related signaling pathways.Furthermore,the mechanism of Sec C in inhibiting the proliferation of liver cancer was investigated.Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay and plate cloning were used to determine the effects of Sec C on the proliferation of HepG2 and BEL7404 cells.Effects of Sec C on lipid peroxidation and apoptosis of hepatocellular carcinoma cells were determined by flow cytometry.Effects of Sec C on the expression of endoplasmic reticulum stress and apoptosis-related proteins in hepatocellular carcinoma cells were investigated by Western blot.Results Sec C inhibited HepG2 and BEL7404 cell proliferation in a dose-dependent manner with IC50 of 1.556 and 3.489μmol/L,respectively.The results of plate cloning experiment showed that the numbers and size of liver cancer cell clones were significantly decreased after treatment with 1μmol/L Sec C for 7 days.Therefore,the results indicated that Sec C significantly inhibited the proliferation of liver cancer cells in a dose-dependent manner.The flow results showed that,compared with the control group,the positive rate of bodipy after Sec C treatment was increased in a dose-dependent pattern,indicating that Sec C promoted lipid peroxidation in hepatocellular carcinoma cells;Also the flow results showed that Sec C induced apoptosis of hepatocellular carcinoma cells in a dose-dependent manner.Western blot showed that ER stress-related proteins binding immunoglobulin protein(BIP),ER type-1 inositolrequiring enzyme 1(IRE1α),activating transcription factor 4(ATF4),and phosphorylated eukaryotic initiation factor 2(p-eIF2)expression levels were increased by Sec C treatment concentration-dependently.Meanwhile,Western blot results showed that the expression levels of apoptosis-related protein poly-ADP-ribose polymerase(PARP)were significantly decreased.The expression levels of polyadenosine diphosphate ribose polymerase(cleaved-PARP),activated cysteine-containing aspartate proteolytic enzyme 3(cleaved-caspase3)and activated cysteine-containing aspartate proteolytic enzyme 7(cleaved-caspase7)were significantly increased by Sec C treatment concentration-dependently.Conclusion Sec C significantly inhibits the proliferation of hepatocellular carcinoma cells through inducing endoplasmic reticulum stress and cell apoptosis.