Effect Of MG132 On Cell Proliferation, Apoptosis And Cks1,P27 Expression Of Human Hepatocarcinoma BEL-7402 Cell Line

Background:The pathogenesis of malignant tumors is complicated.More and more study results are related to the disorder of cell cycle regulation mechanism and the disruption of cell differentiation process.It is very important for the researchers to reveal the pathogenesis and treatment of hepatic carcinoma for reducing the mortality and improving the quality of patients'life.Therefore,this study designs human hepatocarcinoma BEL-7402 Cell Line as the research object to look for a safe and effective anti-tumor drug,and detect the variety of Cks1,P27 protein from protein and gene level in proteasome inhibitor,and detect the mechanism of proteasome inhibitor inhibiting the BEL-7402 cell proliferation and apoptosis.Objective:To investigate whether MG132(Z-Leu-Leu-Leu-CHO) reduced the growth of the human hepatocarcinoma BEL-7402 Cell Line cells through induction of apoptosis in vitro.To observe the effect of MG132 on the expression of Cks1 and P27.Methods:A human hepatocarcinoma BEL-7402 Cell Line cells were cultured in vitro and MG132 was divided into 1,2.5,5.0,10.0,20.0umol/L at concentration,and treated with 5.0umol/L MG132 for different hours.Cells in logarithmic growth phase were selected and examined.(1)Cell inhibition rate was calculated with MTT assay. (2)The morphological features of BEL-7402 cell were observed by inverted microscope.Staining of BEL-7402 Cells with Hoechst 33258 to detect the nuclear changes by fluorescence microscope.(3) Apoptosis rate with Annexin V/PI and cell cycle was detected staining by flow cytometry.(4)The expression of Cks1,P27 mRNA were detected by FQ-RT-PCR.(5) the expression of Cks1,P27 were detected by immunohistochemistry method and western blotting analyse.Results:(1) After treated with MG132,the growth of BEL-7402 cells were inhibited obviously.MG132 inhibited the growth of BEL-7402 cells in a time and dose dependent manner.(2)BEL-7402 cells became round,small,cell shrinkage,membrane blebbing and so on under inverted microscope.Hoechst 33258 staining detect the apoptotic features of nuclear condensation and fragmentation.(3) The quantification of the apoptosis effect by flow cytometric analysis showed that it was in a correspondence with the concentration and hours in cell viability induced by MG132. The percentage of G2/M phase of BEL-7402 cells increased significantly compared with the control guoup.(4) In contrast to control guoup,the Cks1 and P27 mRNA expression level have no significant change.(5) In contrast to control guoup,the Cks1 and P27 protein expression level increased significantly.Conclusions:Proteasome inhibitor MG132 effectively inhibited the human hepatocarcinoma BEL-7402 Cell Line cells proliferation and blocked the cell cyclin at G2/M phase and promoted apoptosis in a time and dose dependent manner.It has no difference in the Cks1 and P27 mRNA expression level compared with the control group,while in the Cks1 and P27 protein expression level increased significantly,which maybe related with the ubiquitin-proteasome pathway.MG132 inhibited cell proliferation and promote apoptosis may be associated with increasing Cks1,P27 protein expression to be further confirmed.