顺铂对膀胱癌细胞自噬和凋亡的作用及其相关性

Effects of cisplatin on autophagy and apoptosis and their correlation in bladder cancer cells

目的研究顺铂(DDP)能否激活人膀胱癌T24细胞自噬以及其中可能的机制,并且探索自噬和凋亡之间相关性。方法用MTT法检测不同浓度的顺铂(0、10、20和40μg/m L)对T24细胞的增殖作用。透射电镜观察细胞内自噬小体;用Western blot技术检测细胞内LC3-Ⅱ、P62蛋白和细胞外信号调节蛋白激酶1/2(ERK1/2)和磷酸化ERK1/2蛋白的表达量。研究自噬对于DDP处理后T24细胞增殖和凋亡的影响。结果 DDP可显著抑制T24细胞的增殖(P<0.05),并呈浓度依赖性,半数抑制浓度(IC50)为(30.3±2.4)μg/m L。DDP可以诱导T24细胞发生自噬,细胞自噬体数目明显增加;明显上调LC3-Ⅱ蛋白的表达(P<0.05),下调P62蛋白的表达(P<0.05)。DDP能上调ERK1/2的磷酸化(P<0.05);ERK1/2通路抑制剂U0126可以抑制DDP诱导的自噬,表现为LC3-Ⅱ蛋白表达明显下降(P<0.05)。自噬抑制剂渥曼青霉素(WTM)抑制自噬后明显加强DDP对T24细胞的增殖抑制和凋亡促进作用(P<0.05);在DDP和WTM联合处理组,细胞凋亡相关蛋白多聚ADP-核糖聚合酶1(PARP 1)裂解增强,cleaved-caspase-3增多(P<0.05)。结论自噬对T24细胞具有保护作用,可抑制DDP诱导的细胞凋亡,自噬活化的机制可能与ERK信号通路的激活有关。

Objective To identify whether cisplatin can induce autophagy of bladder cancer T24 cells and the possible mechanism,and to observe the relationship between outophagy and apoptosis. Methods MTT assay was applied to investigate the effects of various concentration of cisplatin（ 0,10,20 and 40 μg / m L） on T24 survival. TEM was performed to detect the autophagosome formation. Western blot assay was used to analyze the expression changes of LC3-Ⅱ,P62 and extracellular signal-regulated kinase（ ERK1 /2） and p-ERK at the protein level. The effects of autophagy on the survival and apoptosis of bladder cancer cells were investigated. Results DDP observably inhibited proliferation of bladder cancer cells in a dose-dependent manner（ P〈0. 05）,the 50 % inhibiting concentration（ IC50） was（ 30. 3 ± 2. 4） μg /m L; DDP induced autophagy of bladdercancer cells,observably increased autophagosome induced by DDP; up-regulated expression levels of LC3-Ⅱproteins（ P〈0. 05）,down-regulated expression of P62 proteins（ P〈0. 05）; DDP increased the protein level of p-ERK（ P〈0. 05）; The inhibitor of ERK pathway U0126 inhibited DDP-induced autophagy,as evidenced by decrease in the expression of LC3-Ⅱ proteins（ P〈0. 05）. After inhibition of autophagy by WTM in DDP-treated cells,cell viability was obviously decreased and apoptosis was increased（ P〈0. 05）; DDP combined with WTM observably enhanced cleavage of poly ADP-ribose polymerase 1（ PARP-1） and cleaved-caspase-3 which is apoptosis related proteins（ P〈0. 05）. Conclusions Autophagy can protect T24 cells against ciplatin-induced apoptosis,the possible mechanism of autophagy is the ERK signaling pathway is activated.