盐酸吡柔比星通过上调ATG4B增强HeLa细胞保护性自噬

Pirarubicin induces cytoprotective autophagy in HeLa cells through up-regulating autophapy related 4B

目的探讨盐酸吡柔比星增强宫颈癌He La细胞自噬水平的可能机制。方法采用CCK-8检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对He La细胞增殖的影响;台盼蓝染色实验检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对He La细胞死亡的影响;GFP-LC3质粒转染He La细胞后观察盐酸吡柔比星对自噬小体形成的影响;Western blot检测细胞中自噬标志分子Ⅱ型微管相关蛋白轻链3(microtuble-associated protein light chain 3-Ⅱ,LC3-Ⅱ)、自噬相关分子自噬相关蛋白4B(autophapy related 4B,ATG4B)的表达情况。CCK-8实验及台盼蓝染色实验观察在有或者无自噬抑制剂氯喹作用下以及shRNA下调He La细胞中的ATG4B后盐酸吡柔比星对He La细胞的杀伤效应的影响。结果盐酸吡柔比星能剂量依赖地抑制He La细胞的增殖并促进其死亡(P<0.05);盐酸吡柔比星处理能显著增加He La细胞中自噬小体的数量,并显著上调自噬标志分子LC3-II、自噬关键酶ATG4B的表达;自噬抑制剂氯喹可显著增强盐酸吡柔比星的杀细胞效应(P<0.05),shRNA干扰ATG4B可抑制盐酸吡柔比星对He La细胞中自噬的增强作用(P<0.05),并增强盐酸吡柔比星的杀细胞效应(P<0.05)。结论盐酸吡柔比星通过上调ATG4B而增强的自噬对He La细胞起保护作用。

Objective To investigate the mechanism and significance of pirarubicin in enhancing the autophagy of He La cells. Methods Trypan blue exclusion assay and CCK-8 assay were used to detect cell mortality and proliferation in He La cells treated with different doses of pirarubicin alone or combined with chloroquine（ autophagy inhibitor）. The effect of pirarubicin on autophagosome formation in He La cells transfected with GFP-LC3 plasmid was observed with fluorescence microscopy. Western blotting was used to determine the protein levels of autophagy markers microtuble-associated protein light chain 3-Ⅱ（ LC3-Ⅱ）,and autophapy related 4B（ ATG4B）. After ATG4 B was down-regulated with shRNA in He La cells,the effect of pirarubicin on killing He La cells in the presence of chloroquine was observed. Results Pirarubicin inhibited cell proliferation and induced cell death in a dose-dependent manner in He La cells（ P〈0. 05）.Pirarubicin induced the formation of LC3-GFP puncta in He La cells,and increased the expression of LC3 Ⅱand ATG4 B markedly. Chloroquine enhanced pirarubicin-triggered cell death in He La cells（ P〈0. 05）.Knockdown of ATG4 B via shRNA suppressed pirarubicin-induced autophagy and increased pirarubicin-induced cell death in He La cells. Conclusion Pirarubicin induces cytoprotective autophagy in He La cells through upregulating ATG4 B.