Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells
Inhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cells
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DOI：
10.3892/mmr.2018.8686
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发表时间：
2018-05-01
影响因子：
3.4
通讯作者：
Yi, Huihuang
中科院分区：
文献类型：
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作者：
Chen, Jianhua;Zhang, Lemeng;Yi, Huihuang
最近的研究表明，自噬参与肿瘤的发生，并参与获得性化疗耐药。本研究旨在探讨顺铂诱导A549人肺癌细胞自噬的功能及其机制。通过LC 3B-I/II转化、LC 3B斑点和自噬体形成来测量自噬。通过caspase-3活性、caspase-3裂解和LDH释放来测量凋亡细胞死亡。采用逆转录-定量聚合酶链反应（RT-PCR）和蛋白质印迹法（Western blot）检测自噬相关蛋白的转录和表达水平。用Beclin 1和Atg 5 siRNA转染细胞，观察顺铂诱导细胞自噬的作用。结果表明，顺铂诱导A549细胞中的凋亡细胞死亡并触发自噬反应，如通过增加的微管相关蛋白1轻链3（LC 3B）-I/II转化、增加的LC 3B斑点和自噬体形成所示。顺铂诱导的自噬反应的机制进行了研究。顺铂通过上调自噬蛋白（Atg）5和Beclin 1的mRNA和蛋白表达水平诱导自噬，而丝氨酸/苏氨酸蛋白激酶ULK 1、Atg 3、Atg 7、Atg 12和螯合体-1的mRNA和蛋白表达水平没有显著上调。此外，敲低Atg 5和Beclin 1的小干扰RNA转染损害顺铂诱导的自噬反应的激活，增加半胱天冬酶-3切割和抑制细胞活力。这些发现表明，通过抑制Atg 5和Beclin 1破坏自噬可能促进顺铂诱导的A549人肺癌细胞凋亡。总之，本研究表明，靶向自噬可能在未来用于治疗肺癌。
Recent studies have indicated that autophagy contributes to tumorigenesis and participates in acquired chemotherapeutic resistance. The present study aimed to determine the function and underlying mechanism of cisplatin-induced autophagy in A549 human lung cancer cells. Autophagy was measured by LC3B-I/II conversion, LC3B puncta and autophagosomes formation. Apoptotic cell death was measured by caspase-3 activity, caspase-3 cleavage and LDH release. The transcriptional and expressional level of autophagy related proteins were measured by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Beclin 1 and Atg 5 siRNA transfection was used to explore the function of cisplatin-induced autophagy. The results demonstrated that cisplatin induces apoptotic cell death in A549 cells and triggers an autophagic response, as indicated by increased microtubule-associated protein 1 light chain 3 (LC3B)-I/II conversion, increased LC3B puncta and autophagosome formation. Mechanisms underlying cisplatin-induced autophagic responses were also investigated. Cisplatin induced autophagy by upregulating the mRNA and protein expression levels of autophagy protein (Atg)5 and Beclin 1, whereas the mRNA and protein expression levels of serine/threonine-protein kinase ULK1, Atg3, Atg7, Atg12, and sequestosome-1 were not markedly upregulated. In addition, knockdown of Atg5 and Beclin 1 by small interfering RNA transfection impaired cisplatin-induced activation of autophagic responses, increased caspase-3 cleavage and inhibited cell viability. These findings suggested that disruption of autophagy via the inhibition of Atg5 and Beclin 1 may promote cisplatin-induced apoptotic cell death in A549 human lung cancer cells. In conclusion, the present study demonstrated that targeting autophagy may be used in the future for the treatment of lung cancer.