Lemeng Zhang, Jianhua Chen, Lemeng Zhang 

Thoracic medicine department, hunan cancer hospital

Objective:Recent studies indicate that autophagy is adouble-edged sword in tumor genesis and plays an important role in theresistance of cancer cells to chemotherapy. We hypothesize that autophagy protects against cisplatin induced apoptotic cell death in A549 human lung cancer cells. Method:Autophagy activity was measured by Western blotting, immunofluoresence staining, and transmission electron microscopy. Apoptoticcell death was measured by caspase-3 activity, flowcytometry, and MTT and LDH release. Cisplatin induced autophagy related gene activation was measured by QPCR and WB. Result: Cisplatin leads to apoptotic cell death in A549 cells in a dose- and time- dependent manner; meanwhile, it triggers autophagy response as demonstrated by increased conversion of LC3BI to LC3BII, increased LC3B punctate and autophagosomes formation. We further explore the relationship between cisplatin induced apoptotic cell death and autophagy. Pharmacologic inhibitionof autophagy by 3-MA and CQ impaired the elimination of damaged mitochondria, increased caspase-3 activity and thus decreased A549 cell viability. Furthermore, we found that cisplatin induced autophagy with alternating significantly thetranscription and expression of Beclin 1, Atg5, while the up regulation of Atg1/Ulk1, Atg3, Atg7, Atg12, LC3B, and SQSTM1/p62 were not significant. And down regulating Atg5 and Beclin 1 by siRNA, impaired cisplatin induced autophagic activation, increased accumulation of damaged mitochondrial in cytoplasm and increased caspase-3activity and thus decreased A549 cell viability. It demonstrates thatdisruption of autophagy may sensitize A549 cells to cisplatin induced apoptoticcell death. Conclusion: Suppression of autophagy, both by pharmacologic interventions and by knockdown autophagy related genes atg5 and beclin-1, sensitizes A549 tumor cells to cisplatin induced apoptotic cell death. These findingssuggest a rationale to modulate autophagy in anti-lung cancer therapy.

Key Words: autophagy  apoptosis  cisplatin