The Effect Of P38MAPK On Crotoxin-induced Apoptosis And Autophagy In Human Lung Cancer SK-MES-1Cells

Objective: Crotoxin (CrTX) is the main toxin of South American rattlesnake(Crotalus durissus terrificus) venom, including a weakly toxic basic Phospholipase A2(CB) and a non-enzymatic, non-toxic acidic component (crotapotin, CA). CrTX is ahetero-dimeric β-neurotoxin. The classic biological activities of CrTX includeneurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. Numerous studies inrecent years have shown that CrTX also has immunomodulatory, anti-inflammatory,anti-microbial, anti-tumor and analgesic actions. CrTX exhibits antitumor activity invivo and in vitro, but the molecular mechanism has not yet been clearly explained. Herewe investigate the anti-tumor effect and molecular mechanism of CrTX in human lungcancer SK-MES-1cells.Methods: Firstly the cytotoxicity of CrTX on SK-MES-1cells was determined byMTT method and colony formation determined by colony formation assay. The cellcycle changes were investigated using flow cytometry, and the level of PCNA wasdetected with Western blot analysis. Apoptosis was investigated with Hoechst33258staining, the ratio of apoptotic cells was measured with Annexin V-FITC/PI doublestaining, and the level of Cleaved Caspase-3was detected by Western blot analysis.Autophagy activation was monitored by changes of protein levels of LC3, p62andBeclin1. The protein levels of P-p38and p38were evaluated by Western blotting. Theeffects of the p38MAPK inhibitor SB203580on CrTX-induced apoptosis andautophagy in SK-MES-1cells were determined with flow cytometry, AnnexinV-FITC/PI double staining and Western blotting analysis.Results: The proliferation of SK-MES-1cells was significantly inhibited by CrTXin a dose-and time-dependent manner, and the IC50was25.13μg/ml for72h. CrTXsuppressed colony formation of human lung cancer SK-MES-1cells. CrTX induced S phase arrest, and PCNA downregulation. CrTX increased the apoptosis rate from4.65%±1.73%(control) to12.44%±0.45%(50μg/ml)(p <0.01), and increased expressionof Cleaved Caspase-3. The protein levels of LC3and Beclin1were increased but p62decreased. CrTX activated p38MAPK pathway. Pretreatment of cells with SB203580(aspecific inhibitor of p38MAPK, SB) increased cell viability. SB203580pretreatmenthad no influence to CrTX-induced S phase arrest, but reduced the rate of apoptosis andthe level of Cleaved Caspase-3, compared with CrTX treatment alone. The expressionof LC3and Beclin1was decreased, and p62was increased by SB pretreatment.Conclusions: CrTX inhibits the proliferation and induces S phase arrest, apoptosisand autophagy of SK-MES-1cells. The p38MAPK signal transduction pathway isactivated by CrTX and mediates CrTX-induced apoptosis and autophagy.