The Research Of The Effect And Mechanism Alkaline Microenvironment And V-ATPase Inhibitor KM91104 On Hepatocellular Carcinoma

Objective: In order to understand the biological effects and mechanism of the tumor microenvironment p H on hepatocellular carcinoma cells;to determine the biological effects and mechanism of V-ATPase inhibitor KM91104 on hepatocellular carcinoma cells;to explore the action mechanism of hepatocellular carcinoma microenvironment p H and the relationship with V-ATPase.Methods: The hepatocyte LO-2 cells and hepatocellular carcinoma cells SMC7721 and HCCLM3 cells were regarded as object of study,cell culturing with different extracellular microenvironment p H and different concentrations of KM91104 were regarded as the experimental group,and with original culture medium as the control group;CCK8 method was used to detect the survival rate of hepatocellular carcinoma cells in experimental group and control group under different p H microenvironment and different concentrations of KM91104,then selected the appropriate p H and KM91104 concentration forthe next experiment.The changes of hepatocellular carcinoma cells migration were examined by scratch test in this study,and the intracellular H+ levels were measured by BCECF-AM fluorescent probe in experiment group and control group,then the changes of fluorescence intensity were detected by flow cytometry;The cells were stained with PI and Annexin V-FITC / PI staining and cell cycle and apoptosis were detected by flow cytometry.Finally,the total RNA in each group was extracted by one step TRIzol method,and the expressions V-ATPase subunits V1C2,V0D2,caspase-3 and cyclin B1 were detected by RT-PCR.Results: 1.When the extracellular microenvironment p H was greater than 8.2,the proliferation of LO-2 cells and hepatocellular carcinoma cells were significantly inhibited and survival rate of which decreased with time.In the extracellular alkaline environment,the migration of SMC7721 cells was inhibited,but the migration of HCCLM3 cells was enhanced,and the extracellular acidic microenvironment could promote the migration of hepatocarcinoma cells;the role of V-ATPase was significantly inhibited leaded to intracellular H + concentration significantly higher than control group and the apoptosis of hepatocellular carcinoma cells also increased when the extracellular microenvironment p H was 8.4;the ratio of G1 phase cells in hepatocellular carcinoma cells increased,HCCLM3 cell cycle extension,SMC7721 cycle shortened when the extracellular microenvironment changed,and the expression levels of V-ATPase subunits V1G1 and V0D2 in hepatocellular carcinoma cells were significantly increased.2.V-ATPase inhibitor KM91104 significantly inhibited the growth of HCC cells,and the inhibitory effect was enhanced with increasing concentration;KM91104 significantly inhibited the migration of SMC7721 but promoted themigration of HCCLM3 cells.KM91104 could inhibit the role of V-ATPase liver cells so that the H+ concentration increase,KM91104 could also promote the apoptosis of hepatocellular carcinoma cells and lead to the cycle of liver cancer cells significantly changed,S phase cell ratio increased,G2 phase cell ratio decreased;V-ATPase subunits V1 G,V0D2 expression level were significantly increased after KM91104 on liver cancer cells.Conclusion: 1.Alkaline microenvironment could inhibit the growth of hepatocellular carcinoma cells and promote the apoptosis of HCC cells in a certain extent.2.V-ATPase inhibitor KM91104 could inhibit the growth of hepatocellular carcinoma cells and promote the apoptosis of hepatocarcinoma cells through inhibiting the function of V-ATPase.3.The mechanism of alkaline microenvironment in hepatocellular carcinoma may be consistent with the action mechanism of V-ATPase inhibitor,which is caused by inhibiting the function of V-ATPase.