Enhanced Effect Of Resveratrof On Sensitivity Of Laryngeal Carcinoma Hep-2Cells To Cisplatin And Its Mechanism

Objective:To study the effect of co-treatment with cisplatin and resveratrol or each drugseparately on cell proliferation and apoptosis of human laryngeal carcinoma Hep-2cells,and this effect relationship between the changes of Sirt1protein and mRNA. In order toexplore the enhanced effect of resveratrol on the sensitivity of human laryngealcarcinoma Hep-2cells to cisplatin and its mechanism.Methods:（1） Hep-2cells were randomly divided into blank control group and differentdoses（3,6,12,24μmol L-1）of cisplatin（DDP） groups and the cells were treated for6,12,24,48h.The suitable concentration of DDP(6μmol L-1) and culture time(24h) werechoosen. Hep-2cells were randomly divided into blank control group and different doses（10,30,50,70μmol L-1）of resveratrol（Res） groups and the cells were treated for6,12,24,48h.The suitable concentration of Res(50μmol L-1) and culture time(24h) werechoosen.（2）Hep-2cells treated under the suitable condition were randomly divided intoblank control group, DDP group, Res group, sirtinol group, sirtinol+Res group, DDP+Res group, DDP+sirtinol group and DDP+Res+sirtinol group. The survival rates ofHep-2cells in various groups were detected by CCK-8assay;the expressions levels ofSirt1protein in the Hep-2cells various groups in various groups were detected withimmunofluorescence method; the apoptotic rates of Hep-2cells in various groups weredetermined by AnnexinV-FITC Kits.The levels of Sirt1mRNA in Hep-2cells in variousgroups were detected with RT-PCR.Results：CCK-8assay results showed that the survival rate of Hep-2cells in DDP（6μmol L-1） and Res（50μmol L-1） for24hours group were declined significantly(P<0.01）,compared with blank control group；the survival rate of Hep-2cells in Res group and DDP group were declined significantly （P<0.01）, compared with blank controlgroup；compared with DDP group，significant decline cell survival rate of Hep-2cells inDDP+Res group（P<0.01）；compared with DDP+Res+sirtinol group，the survival rateof Hep-2cells in DDP+Res group were decreased (P<0.05）. The immunofluorescenceresults showed that the mean fluorescence intensity of Sirt1protein of the cells in DDP＋Res group was increased significantly with DDP group（P<0.01）.Compared with DDP+Res+sirtinol group,the mean fluorescence intensity of Sirt1protein of the cells in DDP+Res group was increased significantly （P<0.01）. The Annexin V-FITC Kit results showedthe apoptosis rate of DDP＋Res group higher than those in DDP group and DDP＋Res＋sirtinol group（P<0.01）.RT-PCR results showed The levels of Sirt1mRNA in DDP group,Res group and DDP+Res group were increased significantly compared with blank controlgroup (P＜0.01); compared with DDP+Res group,the levels of Sirt1mRNA in DDPgroup and Res group were decreased significantly (P＜0.01). The level of Sirt1mRNA inHep-2cells in DDP＋Res group is about0.94times higher than DDP group, about1.82times higher than Res group.Conclusion:(1)Cisplatin and resveratrol can induce Hep-2cells to apoptsis.Cisplatin andresveratrol inhibits Hep-2cell growth in a dose-and time-dependent manner.(2)Cisplatinand resveratrol induce Sirt1expression and enhance the level of Sirt1mRNA in Hep-2cell.(3)Resveratrol can improve the sensitivity of Hep-2cells to cisplatin,and itsmechanism may be related to increasing the expression of Sirt1protein.