Institute for Biological Research, Belgrade, Serbia.
J Cell Mol Med. 2009 Sep;13(9B):3644-54. doi: 10.1111/j.1582-4934.2009.00663.x.
The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.
自噬在顺铂抗癌作用中的作用通过人 U251 神经胶质瘤、大鼠 C6 神经胶质瘤和小鼠 L929 纤维肉瘤细胞系进行了研究。顺铂处理后，肿瘤细胞中观察到自噬的剂量和时间依赖性诱导，如自噬诱导蛋白 beclin-1 的上调以及随后吖啶橙染色的酸性自噬小泡的出现。电镜也证实了顺铂处理细胞中自噬体的存在。用溶酶体抑制剂巴弗洛霉素 A1 和氯喹，或磷脂酰肌醇 3-激酶抑制剂渥曼青霉素抑制自噬，明显增强了顺铂引发的氧化应激和半胱天冬酶激活，导致 DNA 片段化和凋亡细胞死亡增加。自噬的保护作用的机制显然涉及干扰顺铂诱导的 Bcl-2 家族蛋白的调节，因为自噬的抑制增强了顺铂介导的促凋亡 Bax 的上调和抗凋亡 Bcl-2 的下调。顺铂处理细胞中的自噬诱导之前是腺苷单磷酸激活蛋白激酶 (AMPK) 的激活和哺乳动物靶标雷帕霉素 (mTOR) 介导的 p70S6 激酶磷酸化的同时下调。小干扰 RNA (siRNA) 介导的 AMPK 沉默降低了顺铂触发自噬的能力，而 mTOR siRNA 的转染足以在肿瘤细胞中触发自噬。最后，siRNA 介导的 AMPK 下调和 AMPK 抑制剂化合物 C 增加了顺铂诱导的肿瘤细胞死亡，而 mTOR siRNA 和 AMPK 激活剂二甲双胍保护肿瘤细胞免受顺铂的影响。总之，这些数据表明，顺铂触发的 AMPK 激活和随后的 mTOR 活性抑制可以诱导自噬反应，从而保护肿瘤细胞免受顺铂介导的凋亡死亡。
