| Objective: To clarify the expression of PKM2 in breast cancer cells MCF-7 and SKBR3 and human normal cells MCF-10 A treated 4-OHT,to explore the relationship between PKM2 and MST1 and functions of PKM2 in mediating MST1-mediated MCF-7 and SKBR3 cells apoptosis.Methods: 1.MCF-7 and SKBR3 cells were treated with 4-OHT,cells apoptosis were detected by flow cytometry analysis of apoptosis assay,the expression of PKM2 and MST1 were measured by western blot assay.The tamoxifen-resistant breast cancer cell line(MCF-7R)was established by increasing tamoxifen concentration in a stepwise manner.CCK-8 was used to measure the proliferation of cells.The protein expression of PKM2 and MST1 were determined by Western blot.2.Co-IP and immunofluorescence experiments were performed to clarify the relationship between PKM2 and MST1.3.shRNA for PKM2 gene lentiviral interference vectors were infected into MCF-7R cells,the cells were treated with 4-OHT subsequently,Western blot was employed to measure the effect of interference and the expression of Cleaved-Caspase3 as well as MST1.Caryoplasm protein separation experiment and immunofluorescence staining assay were used to detect the cell translocation of MST1.4.Flag-MST1 were transient transfected into breast cancer cells treated with 1?M 4-OHT,the cells were transfected with pCDNA3.1-Flag-PKM2 plasmids or PKM2 shRNA,western blot was used to measure the expression of Cleaved-Caspase3,flow cytometry analysis of apoptosis assay was used to detected cells apoptosis.Results: 1.The apoptosis of MCF-7 and SKBR3 cells were triggered by 4-OHT in a dose-dependent manner,however,TAM had no effect on apoptosis in MCF-10 A at the indicated concentrations.Western blot results showed the levels of PKM2 were significantly decreased after 4-OHT treatment in a dose-dependent manner;however,Cleaved-Caspase3 and Cleaved-MST1 proteins were markedly increased.The expression of PKM2 was markly increased in MCF-7R cells.2.Co-IP results showed that PKM2 interacts with MST1,and the N-terminal kinase domain of MST1(cl-MST1)mediates its interaction with PKM2,note that the interaction of MST1 with PKM2 was decreased by treatment of the cells with 4-OHT.The immunofluorescence staining analysis showed that PKM2 and MST1 are co-localized mainly in the cytoplasm,either examined in MCF-7 cells or SKBR3 cells.3.Western-blot showed the protein levels of PKM2 declined obviously,and the expression of cl-Caspase3 and cl-MST1 were significantly increase in PKM2-silenced MCF-7 and PKM2-silenced SKBR3 cells.Subcellular fractionation revealed that the majority of the cl-MST1 was located in the nuclear fraction in PKM2-silenced groups,the immunofluorescence staining showed the amount of MST1 in nuclei was increased in PKM2-knockdown cells consistent with that of MST1 cleavage.4.Western blot showed that overexpression of PKM2 significantly decreased the activity of Caspase3 in MST1 overexpression groups,additionally,PKM2-depletion significantly increased the activity of Caspase3 in MCF-7 and SKBR3 cells.In the MST1 transfected cells,flow cytometry analysis showed that overexpression of MST1 alone effectively induced apoptosis in MCF-7 cells;however,the apoptosis rate was markedly reduced by co-transfection of PKM2 with MST1,In addition,the MST1-induced apoptosis rate was significantly increased in MCF-7 cells which were silenced the endogenous expression of PKM2 by PKM2-shRNA compared with transfection of MST1 alone.Conclusion: 1.4-OHT restrained the expression of PKM2 and TAM-induced apoptosis is associated with Caspase-dependent MST1 cleavage.2.PKM2 interacts with MST1,and cl-MST1 mediates its interaction with PKM2.3.Downregulation of PKM2 upregulates the level of cl-MST1 and promotes its nuclei translocation.4.PKM2 inhibits MST1-mediated apoptosis,and PKM2 is required for MST1-mediated apoptosis. |
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