Effect Of V-ATPase Inhibition On The Cell Behavior Of Human Hepatocellular Carcinoma Cells HepG2and Its Mechanisms

Objective:In a previous study, we found that there was a higher vacuolar ATPase (V-ATPase) expression in human hepatocellular carcinoma (HCC) tissues and the inhibition of V-ATPase markedly retarded the growth of HCC in nude mice model, indicating that V-ATPase may play an important role in the development and progress of HCC. In this study, we made an investigation on the effect of V-ATPase inhibition on the proliferation and apoptosis of HCC cells and underlying mechanisms, which aimed to provide new evidence for pH adjustment mechanism of tumor cell as a target for anticancer strategies and to provide experimental evidence for the clinical application of V-ATPase inhibitor, PI-230.Methods:The study was performed in human hepatocellular carcinoma (HCC) cells HepG2cells and human normal liver cell lines. The cells were synchronized. The proliferation of cells was evaluated with a fine MTT assay, cell colony formation experiments, and cell viability analyzer cell counting. Cellular DNA cycle was examined with flow cytometry. AnnexinV-FITC/PI in the cells was analyzed by using flow cytometry to detect early apoptosis rate and the late apoptosis rate was examined by using Tunel method. The expression of caspase-3protein was analyzed by western blot. Mitochondrial membrane potential was measured by using flow cytometry.Resμlts:(1) Compared with the control group,80μM PI-230, a V-ATPase inhibitor, incubating for24hours, had no significant effect on the survival of HepG2and LO2cells (p>0.05), but160μM,320μM, and640μM of PI-230concentration-dependently decreased the survival of cells (p<0.05).(2) Compared with controls,10μM PI-230, incubating for14days, significantly reduced the number and rate of colony formation in HepG2cells (P<0.05), and the effect of PI-230was concentration-dependent (10-120μM). But, both10μM and20μM PI-230had no significant effect on the number and rate of colony formation in LO2cells (p>0.05). However, PI-230(40μM-120μM) concentrationdepend- ently reduced the number and rate of colony formation in LO2cells.(3) The numbers of HepG2and LO2cells by cell counting were significantly decreased after PI-230(40,80, and120μM) incubating for1day,2days,3days,4days,5days, and7days, time-and concentration-dependently, compared with the control group. And PI-230also concentration-dependently affected the cell doubling time.(4) Compared with control,40μM PI-230for48h had no significant effect on inhibition rate of HepG2, and80,120,160and200μM of PI-230had significant concentration-dependent effect (p<0.05). For LO2cells,40and80μM PI-230had no significant effects, and120μM,160μM the,200μM had significant effect (p<0.05).(5) Compared with control, after120μM,160μM, and200μM PI-230for48h, the percentage of cells in G0/G1phase and PI values were significantly higher in HepG2cells (p<0.05), while the LO2cells did not change significantly (p>0.05).(6) Compared with control,80μM PI-230for24h had no significant effect on early apoptosis rate of HepG2cells,(p>0.05), but120μM,160μM, and200μM PI-230significantly increase the apoptosis (p<0.05), without concentration-dependently. And for LO2cells, only200μM PI-230had significant effect on the early apoptosis.(7) Compared with the control,120,160, and200μM PI-230for48h, increased significantly the late apoptosis rate of HepG2cells.(8)200μM PI-230for48h induced the increase of cleaved caspase-3protein expression in HepG2cells, not LO2cells, compared with control (p-<0.05).(9) Compared with control,160and200μM PI-230for48h induced the changes of high mitochondrial potential membrane potential and low mitochondrial membrane potential in both HepG2cells and LO2cells (p<0.05).Conclμsion:(1) The V-ATPase inhibitor (PI-230) can inhibit the proliferation of HCC cells and induce the apoptosis of HCC cells, so it may be a potential anticancer drug.(2) The role of (PI-230) is a cell cycle specific toxicity, and it mediates cell apoptosis mainly through mitochondrial membrane potential and caspase pathway (3) The toxicity of v-ATPase inhibitor PI-230is cell selective and sensitive.