| Objective:Celastrol has been reported to have multiple effects,such as anti-inflammation,suppression of tumor angiogenesis,inhibition of tumor growth,inhibition of tumor metastasis.In order to investigate the effect of tripterine on Ezrin phosphorylation in inhibiting hepatocellular carcinoma cell migration.Ezrin phosphorylation level in cell lysates was detected to investigate the migration of liver cancer.Ezrin expression and its phosphorylation effect before and after treatment with tripterine were detected in liver cancer cells to explore the important mechanism of tripterine on the migration of liver cancer cells.In this experiment,we used celastrol to study the possible mechanism by which Ezrin phosphorylation inhibits tumor migration.Two representative liver cancer cell lines,including MHCC97H and HepG2,were selected in our experiments.However,It is unclear whether the two cell lines have similar expression levels of Thr567 phosphorylation under the stimulation of celastrol.Therefore,the role and mechanism of Ezrin in the invasion and migration of HCC cells were clarified,and further theoretical basis was provided for elucidation of the mechanism of tumor metastasis.The aim of this study was to elucidate the changes of Ezrin and its phosphorylation during the migration and invasion of hepatocellular carcinomacells before and after treatment with tripterine and the mechanism by which tripterine inhibits HCC metastasis.Methods:Western blot was used to detect the protein marker of cell proliferation to investigate the effect of different concentrations and time of tripterine on the phosphorylation level of Ezrin in HepG2 and MHCC97H cells of liver cancer.The levels of Ezrin Thr567 in cell lysate were detected by immunofluorescence and western blot.The effects of different concentrations and time of tripterine on the phosphorylation of HepG2 and MHCC97H cells were investigated.Cell scratch test and live cell tracking test were used to detect the migration of tumor cells,and the effects of different concentrations and time of tripterine treatment on the migration of HepG2 and MHCC97H cells were investigated.Cell migration was observed by constructing Ezrin Thr567 mutant,and the MHCC97H migration of HCC cells transfected with Ezrin Thr567A and Ezrin Thr567D was observed by live cell tracking assay.The interaction between ROCK-Ezrin and celastrol was investigated by IP experiment.Results:(1)The effects of tripterine on Ezrin phosphorylation in hepatocellular carcinoma cells were studied by western blotting and immunofluorescence assays.The western blotting experiment indicated that,Ezrin pT567 has been influenced by celastrol,and the influence of Ezrin pT567 has the tendency of concentration dependence.(2)Western blot assay of the effect of tripterine on the phosphorylation of Ezrin Thr567 in hepatocellular carcinoma cells showed that Ezrin pT567 in both MHCC97H and HepG2 cells were significantly lower than those in the control group when treated by 1 μM celastrol.(3)According to the analysis of the scratch test results of MHCC97H and HepG2 cell lines,the migration rate of cells within 24 h was significantly decreased by 1 μM celastrol.(4)Based on the results of living cell tracking experiment,by the treatment of 1μM celastrol for 6 h,the movement speed of a single cell was slowed down,and the range of motion was restricted.(5)After transfection of Ezrin WT,Ezrin T567A and Ezrin T567D,MHCC97H cell of Ezrin T567A was significantly inhibited.(6)The results of IP assay showed that the expression of ROCK1 was significantly decreased after celastrol treatment.Conclusion:(1)Celastrol can inhibit tumor metastasis by reducing the phosphorylation level of Ezrin pT567.The optimal treatment concentration is 1μM and the optimal time is 6h.(2)Our results suggest that the mechanism by which celastrol inhibits liver cancer metastasis is achieved by blocking Rho kinase-mediated phosphorylation of Ezrin. |
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