The Effects And Mechanisms Of Apoptosis In Lung Adenocarcinoma Cells Induced By Combination Of Resveratrol And SB203580

ObjectiveThis artical is designed to assess whether SB203580enhances the resveratrol-inducedapoptosis in human lung adenocarcinoma A549cells, and explore its specificmolecular mechanisms.Methods1. Establishing a stable model of apoptosis: A549cells was treated with differentconcentrations of SB203580(0,1and10μM) and/or resveratrol (0,50,75,100and125μM), and then cell viability was assessed by CCK-8assay.2. Estimating the forms of cell death: FCM analysis of apoptosis with Annexin/PIdouble staining method or with PI staining was to determine the fashion by whichSB203580sensitized the RV-induced cell death.3. FCM method was used to examine the effect of SB203580on the RV-induced lossof mitochondrial membrane potential.4. Immunofluorescence was used to assess the differences between the activation ofBax and Bak in combination treatment with SB203580and resveratrol andtreatment with resveratrol alone.5. Fluorogenic substrates were utilized to measure the activation of caspases incombination treatment with SB203580and resveratrol and treatment withresveratrol alone.6. Detecting the release of AIF and Smac from mitochondria by confocalfluorescence imaging.7. Detecting the expression level of FasL in extrinsic apoptotic pathway.Results1. Different concentrations of resveratrol induced a dose-dependent cytotoxicity;treatment with SB203580alone did not affect the cells viability, but combinationtreatment with SB203580and SB203580significantly enhanced the resveratrol-induced cytotoxicity, and the statistically significant concentrations75μM resveratrol and10μM SB203580were chose for the subsequentexperiments.2. A variety of apoptotic detecting methods had confirmed that the fashion by whichSB203580enhanced the resveratrol-induced cell death was apoptosis.3. SB203580pretreatment promoted the resveratrol-induced loss of mitochondrialmembrane potential.4. SB203580pretreatment increased the resveratrol-induced activation of Bax,however, it is opposist that activation of Bak decreased.5. SB203580increased the resveratrol-induced activation of caspase-9slightly andcaspase-3potently.6. SB203580contributed to the caspase-8activation which was not activate whentreated with resveratrol alone.7. AIF and Smac did not release from the mitochondria in the late stage ofresveratrol-induced apoptosis enhanced by SB203580.8. SB203580increased the resveratrol-induced cleavage of FasL.Conclusion1. SB203580enhanced the resveratrol-induced apoptosis in A549cells;2. SB203580potently enhances the RV-induced apoptosis via initiating extrinsic andenhancing intrinsic apoptosis pathways.