As2O3联合γ分泌酶抑制剂对HepG2细胞凋亡的诱导作用及其机制

目的:探讨As₂O₃通过Notch信号通路对HepG₂细胞凋亡的影响,阐明其作用机制。方法:体外培养的HepG₂细胞分为空白组、As₂O₃组、MW167组和联合组;采用不同浓度As₂O₃和MW167处理HepG₂细胞24、48和72 h,MTT法检测细胞增殖抑制率。根据MTT结果选取无细胞毒剂量As₂O₃和MW167(As₂O₃ 0.25 mg·L^-1、MW167 10 μmol·L^-1)分组干预HepG₂细胞48 h;流式细胞术检测细胞凋亡率;RT-PCR和Western blotting法检测Notch-1、Bcl-2和Caspase-3 mRNA和蛋白表达水平。结果:不同浓度As₂O₃和MW167处理HepG₂细胞均可抑制细胞增殖,呈剂量效应关系;无细胞毒剂量As₂O₃和MW167处理不同分组HepG₂细胞48 h后,与空白组比较,As₂O₃组、MW167组和联合组细胞凋亡率升高(P<0.05),其中以联合组升高最为明显;与空白组比较,As₂O₃组、MW167组和联合组细胞中Bcl-2 、Notch-1 mRNA和蛋白表达水平降低(P<0.05),其中以联合组降低最为明显;与空白组比较,As₂O₃组、MW167组和联合组细胞中Caspase-3 mRNA和蛋白表达水平升高(P<0.05),其中以联合组升高最为明显。结论:As₂O₃可诱导肝癌HepG₂细胞凋亡,其作用机制可能与通过Notch信号通路下调细胞中Bcl-2 、Notch-1基因和蛋白表达水平以及上调细胞中Caspase-3基因和蛋白表达水平有关联。

Induction of As2O3 in combination with γ-secretase inhibitor on apoptosis of HepG2 and its mechanism

Objective To discuss the effect of As₂O₃ on the apoptosis of HepG₂ cells via Notch signal pathway, and to clarify its mechanism. Methods The HepG₂ cells cultured in vitro were divided into blank group, As₂O₃ group, MW167 group and combination group.The HepG₂ cells were treated with As₂O₃ and MW167 at different concentrations for 24, 48 and 72 h, and the inhibitory rates of proliferations of the HepG₂ cells were detected by MTT method.The HepG₂ cells in various groups were intervened by MW167 and As₂O₃ at non-cytotoxic doses (As₂O₃ 0.25 mg·L^-1, MW167 10 μmol·L^-1) based on The Results of MTT for 48 h, then the apoptotic rate was tested by flow cytometry;RT-PCR and Western blotting Methods were employed to detect the expressions of Notch-1, Bcl-2 and Caspase-3 mRNA and protein. Results The HepG₂ cells treated with both As₂O₃ and MW167 at different concentrations could inhibit the cell proliferation and showed a dose-effect relationship.For the HepG₂ cells in different groups treated with MW167 group and As₂O₃ group at non-cytotoxic doses for 48 h, compared with blank group, the apoptotic rates in As₂O₃ group, MW167 group and combination group were increased(P<0.05), especially in combination group; the expression levels of Bcl-2, Notch-1 mRNA and protein in As₂O₃ group, MW167 group and combination group were lower than those in blank group(P<0.05), especially in combination group;compared with blank group, the expressions levels of Caspase-3 mRNA and protein in As₂O₃ group, MW167 group and combination group were increased(P<0.05), especially in combination group. Conclusion As₂O₃ can induce the apoptosis of hepatocelular carcinoma HepG₂ cells and the mechanism may be related to the down-regulation of Bcl-2 and Notch-1 and up-regulation of Caspase-3 mRNA and protein through Notch signal pathway.