Knock-down ATG5 gene inhibits autophagyand enhances celastrol-induced apoptosis in human lung cancer cell H1299 / 基础医学与临床

ABSTRACT

Objective To establish the lung cancer cell strain with low ATG5 expression and to detect the effect of celastrol on lung cancer cell apoptosis after downregulation of autophagy.Methods H1299 was infected by lentivirus-mediated ATG5 shRNA.RT-qPCR and Western blot assays were applied to confirm the effect of ATG5 knock down.Autophagy was measured by Western blot and RFP-LC3 transfection.Cell apoptosis of ATG5 normal expression group and of ATG5 low expression group of H1299 cells was detected by FACS.Finally, Western blot was used to detect the expression of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3.Results The expression of ATG mRNA and protein significantly decreased after ATG5 knockdown in H1299 cells (P<0.05).The autophagy marker of LC3-Ⅱ level was downregulated and P62 expression was upregulated after inhibition of ATG5, and the RFP-LC3 puncta reduced significantly after ATG5 knockdown (P<0.05).Compared with control group,the apoptosis rate in ATG5 downregulation group increased significantly after celastrol treatment (P<0.01).Pro-apoptotic proteins of Bax and cleaved caspase-3 levels were upregulated and anti-apoptotic protein of Bcl-2 level decreased after ATG5 inhibition (P<0.05).ConclusionsThe effect of celastrol-induced apoptosis of lung cancer cells was enhanced after downregulation of autophagy, demonstrating inhibition autophay may be a new target of lung cancer treatment.