抑制自噬增敏天花粉蛋白诱导HeLa细胞凋亡的作用机制研究

Mechanism of autophagy inhibition for sensitizing HeLa cell apoptosis induced by trichosanthin

探讨天花粉蛋白(trichosanthin,TCS)诱导宫颈癌HeLa细胞发生细胞自噬和凋亡的作用和机制。两步层析法制备TCS,MTT法检测TCS抑制HeLa细胞的增殖情况;透射电镜下观察TCS处理HeLa细胞后自噬囊泡和凋亡小体的形成;激光共聚焦显微镜下观察绿色荧光蛋白(green fluorescent protein,GFP)-自噬微管相关蛋白轻链β3(microtubule-associated protein 1 light chain beta 3,LC3B)融合蛋白定位的改变;Western blot检测细胞自噬和凋亡相关蛋白表达的变化情况。研究发现制备的TCS纯度高,且具有生物学活性。TCS能明显抑制宫颈癌HeLa细胞的增殖,且呈浓度和时间依赖性。透射电镜下观察TCS作用12 h后有明显的自噬囊泡形成,48 h后有典型的凋亡小体形成。激光共聚焦显微镜下观察到LC3B蛋白在胞浆中由弥散分布转向斑点状聚集分布。Western blot结果显示,TCS处理HeLa细胞12 h后自噬活化蛋白LC3BⅡ表达开始升高,且随药物浓度的增加而增加。TCS作用24 h后凋亡相关蛋白多腺苷二磷酸多聚酶(poly ADP-ribose polymerase,PARP)开始出现活性剪切带,TCS与3-甲基腺嘌呤(3-methyladenine,3-MA)联用后进一步促进PARP的剪切活化。结果表明,TCS能明显抑制宫颈癌HeLa细胞的生长,诱导HeLa细胞发生自噬和凋亡,且TCS诱导的HeLa细胞自噬早于凋亡,抑制自噬能够增敏天花粉蛋白诱导的HeLa细胞凋亡,表明TCS诱导的细胞自噬是一种保护性细胞反应,与自噬抑制剂联用可增强TCS的抗宫颈癌作用。

This research investigated the effect and mechanism of trichosanthin(TCS)in inducing autophagy and apoptosis of HeLa cells in cervical cancer.Two-step chromatography was used to prepare TCS.MTT assay was used to detect the inhibition effect of TCS on the proliferation of HeLa cells.The formation of autophagic vesicles and apoptotic bodies in HeLa cells treated with TCS was observed under transmission electron microscopy,and green fluorescent protein(GFP)-microtubule-associated protein 1 light chain beta 3(LC3B)fusion protein location was observed under laser confocal microscopy.The expression of autophagy and apoptosis-related proteins was detected by Western blot.It was found that the prepared TCS had high purity and biological activity.TCS could inhibit the proliferation of HeLa cells significantly in a concentration-dependent and time-dependent manner.Obvious autophagic vesicles were observed after HeLa cells were treated with TCS for 12 h,and typical apoptotic bodies were formed after 48 h.Under laser confocal microscopy,LC3B protein was observed to shift from a diffuse distribution to a spotted aggregation distribution in the cytoplasm.Western blot results showed that the expression of autophagy-activating protein LC3BⅡbegan to increase in HeLa cells after being treated with TCS for 12 h and increased with the increase in drug concentration.Apoptosis-related protein poly ADP-ribose polymerase(PARP)began to activate as cleaved PARP after being treated with TCS for 24 h.The combination of TCS and 3-methyladenine(3-MA)further promoted the activation of cleaved PARP.The results showed that TCS could significantly inhibit the growth of HeLa cells in cervical cancer.Moreover,TCS induced autophagy in HeLa cells earlier than apoptosis,and inhibiting autophagy could sensitize apoptosis of HeLa cells induced by TCS.This indicated that autophagy induced by TCS was a protective cell response,and TCS combined with autophagy inhibitors could enhance the anti-cervical cancer effect of TCS.