Mechanisms Of Doxorubicin-induced Cardiotoxicity Mediated By Transcription Factor E2F1

Objective: To investigate mechanisms of doxorubicin(DOX)-induced cardiotoxicity mediated by transcription factor E2F1 and the antagonistic effects of resveratrol(RSV)on DOX-related myocardial cytotoxicity.Methods: Part 1.The starved rat cardiac cell line(H9c2)was treated with DOX(1μmol/L)for 24 hours.The autophagy was observed by GFP-LC3 assay using fluorescence microscope,while the apoptosis was detected by flow cytometry double-stained with Annexin V and propidium iodide(PI).The protein expressions of E2F1,AMPKα2,cleaved caspase-3,m TORC1,LC3-II/I were analyzed by Western blot.H9c2 cells transfected with or without small interfering RNA molecules(si E2F1,sim TORC1,si AMPKα2)were divided into four groups: control group,DOX group,si RNA(si E2F1,sim TORC1,si AMPKα2)group and DOX+si RNA group.The final concentration of DOX was 1 μmol/L.Then the autophagy and apoptosis were analyzed.Part 2.The 24-hour starved H9c2 cells were divided into four groups: control group,DOX group,RSV group and DOX+RSV group.The cells were treated with DOX and/or RSV.The final concentration of DOX and RSV were 1 μmol/L and 20 μmol/L,respectively.The autophagy was observed by GFP-LC3 assay using fluorescence microscope,while the apoptosis was detected by flow cytometry double-stained with Annexin V/PI.The protein expressions of E2F1,AMPKα2,cleaved caspase-3,m TORC1,LC3-II/I were analyzed by Western blot.The expression of E2F1 in H9c2 cell was up-regulated through transfected with p EGFP-C1-E2F1.Then cells were divided into control group,RSV group,E2F1 group and RSV+E2F1 group.The autophagy and apoptosis were analyzed.Results: Part 1.DOX resulted in inhibited autophagy and upregulated apoptosis in myocardial cells,accompanied by increased expression of E2F1,AMPK2,cleaved caspase-3,and m TORC1(P<0.001),and decreased expression of LC3-II/LC3-I(P<0.01).The transfection of si E2F1,sim TORC1 and si AMPKα2 would significantly inhibit the expression of E2F1,m TORC1 and AMPKα2.The increased apoptosis and the inhibited autophagy triggered by DOX were partially reversed by inhibiting E2F1 and m TORC1.Meanwhile,the suppressed E2F1 partially reversed the up-regulated AMPKα2,cleared caspase-3,m TORC1(P<0.001)and down-regulated LC3-II/LC3-I(P<0.001)induced by DOX.The abrogated m TORC1 partially reversed the up-regulation of cleaved caspase-3(P<0.001)and the down-regulation of LC3-II/LC3-I(P<0.001)induced by DOX,while the expression of E2F1 did not change.The repressed AMPKα2 did not reverse the decreased autophagy,but reduced apoptosis induced by DOX.And the expression of cleaved caspase-3 decreased(P<0.001).Part 2.RSV treatment partially reversed the increased apoptosis and inhibited autophagy induced by DOX.In RSV+DOX group,the expression of E2F1,AMPKα2,cleaved caspase-3 and m TORC1 significantly decreased compared with DOX group(P<0.001),meanwhile,the expression of LC3-II/LC3-I increased(P<0.001).With the increased expression of E2F1,the autophagy of H9c2 cells was reduced.The apoptosis and the expression of m TORC1,AMPKα2,and cleaved caspase-3 were significantly increased(P<0.001),and the expression of LC3-II/LC3-I was decreased(P<0.01).Compared with RSV group,the expression of m TORC1,AMPKα2,and cleaved caspase-3and the apoptosis in RSV+E2F1 group were up-regulated(P<0.001),however,the autophagy in RSV+E2F1 group was inhibited.Conclusion: E2F1 might play a very crucial role in DOX-induced cardiotoxicity which correlated with the regulation of E2F1/AMPKα2 and E2F1/m TORC1 pathway.Meanwihle,RSV could attenuate DOX-induced cardiotoxicity by inhibiting E2F1.