Mechanism Of Inhibition In Hela Cell Proliferation Response To Betulinic Acid

Betulinic acid(BA)is a naturally occurring pentacyclic triterpene that exhibits a variety of biological activities including potent antitumor properties,which showed a cell specific property.Previous study suggested BA could inhibit cervical cancer cell line,but the molecular mechanism is still not known,in present study,HeLa cells were detected in proliferation and apoptosis by BA treatment,especially to investigate the underlying mechanisms about the involved signaling pathways,which may provide some experimental data for further treatment about cervical cancer.1.Using different concentrations of BA treated HeLa cells in different treatment time,suggested that BA inhibited the cell proliferation and induced apoptosis of HeLa cells in a dose-and time-dependent manner.Since this result verified the inhibition of cell proliferation,the IC50 calculated by MTT measure at 48 h treatment was 30.42 ± 2.39 μmol/L.To comply with a standard that cell apoptosis should be induced but enough living cells should also be maintained for protein extraction and proteomic study,30 μmol/L,48 h treatment condition was chosen to treat HeLa cells.2.We implemented a 2-DE technique to globally search for differentially expressed proteins in HeLa cells affected by BA,36 proteins were chosen for more than 1.5-fold expression differences between the control group and treatment group,30 of them were increased and 6 proteins were decreased.Identified proteins were categorized into metabolic process,biological process,protein localization and other biological process.According to their molecular function,the modulated proteins were classified into catalytic activity,redox activity,translated regulation and more.The protein interaction network could not offer specific signaling pathway related to cell proliferation and cell apoptosis,but the expression level of 14-3-3β and 14-3-3ε was decreased by BA treatment,and the mRNA expression change was consistent with proteomic result.14-3-3 proteins could point us more based on previous study suggested 14-3-3 protein family was involved in PI3K/Akt signaling pathway and mitochondrial apoptotic pathway.3.Since BA(30 μmol/L)was able to inhibit HeLa cell proliferation and induce cell apoptosis significantly after 12 h,we prioritized the proteins expression level before 12 h,and the time point order of modulated proteins was:PI3K(p110a)1 h,Phospho-Akt(Ser473)2 h,Phospho-Akt(Thr308)2 h,p2lwaf1/Cip1 2 h,p27Kip 3 h,Bad 3 h,caspase-9 6 h.Meanwhile,the dose-dependent experiment also indicated BA definitely inhibited PI3K/Akt signaling pathway,further infected downstearm pathway like cell cycle and mitochondrial pathway.4.HeLa cell cycle was arrested at GO/G1 phase by BA treatment,which response to activation of p27Kip,p21Wafl/Cipl expression.This result illustrated BA inhibited the cell proliferation since be effective on cell cycle.5.The changement of mitochondrial membrane potential significantly decreased at 1 h in BA treated-HeLa cells.Meanwhile,mitochondrial Bcl-2 family proteins Bcl-xl,Bad and caspase-9 were analyzed by western blot.As the ratio of Bcl-xl/Bad was significantly reduced and the expression of caspase-9 increased,indicated that mitochondrial pathway was involved in BA-treated HeLa cells.6.The level of ROS production detected by flow cytometry was significantly increased after 0.5 h treatment of HeLa cells with BA and shown in a dose-and time-dependent manner.GSH could prevent inhibition of PI3K/Akt by BA and also influenced the expression of cell cycle related proteins and mitochondrial apoptotic proteins,further block the apoptotic rate with BA treatment.Therefore,this part suggested ROS was the key factor for BA-inhibited cell proliferation and induction of cell apoptotisis.