SiPGK1 Enhanced Sensitivity Of Melanoma Cells To Vemurafenib And The Possible Mechanisms

Objective:To explore whether targeting phosphoglycerate kinase 1(PGK1)can enhance the sensitivity of BRAFV600 E mutation melanoma cells to vemurafenib and the possible mechanisms.Methods:1)Melanoma cell lines of A375 m,A375mR,1205 LU,UACC903,C8161-C9 were devided into 2 groups(si-non-target,siNT and siPGK1),cells in siPGK1 group were transfected with Small interfering RNA technology(siRNA),cells in siNT group were transfected with si-non-target RNA.2)Survival ability of melanoma cell lines’ were measured with MTT assay.Then the cell viability were measured after treated with different concentration of vemurafenib.Cells viability were also detected after transfection of siNT and siPGK1 and treated with different concentration of vemurafenib.3)Western blot technology was used to determine the expression of PGK1 protein,the PGK1 silencing results,and the expression of apoptosis index of PARP in 5 melanoma cell lines.4)Used conlongenic assay technology to detect the colony formation,colony shape and proliferation ability of melanoma cell lines’.Then the cells in the siNT group and siPGK1 group were treated with different concentration of vemurafenib,and the colony numbers were cauculated as colony formation indexes.5)Cells were treated with different concentration of vemurafenib in siNT group and siPGK1 group and Flow cytometry(FCM)was used to detect the apoptosis level and the different stages of apoptosis.Results:1)PGK1 was highly expressed in BRAF mutated melanoma cell lines than in wild type melanoma cell lines.The expression of PGK1 protein in A375 m,A375mR,1205 LU and UACC903 cells were 2.26 fold(P<0.01),1.69 fold(P<0.05),1.20 fold,2.04 fold(P<0.01)higher respectively than in C8161-C9 wild type cells.2)Silencing of PGK1 expression increases the efficacy of vemurafenib in melanoma cells,as evidenced by greater killing in the tumor cells subjected to combined treatment of vemurafenib with siPGK1.MTT Assay showed there is no significant inhibition effect on wide type melanoma cell line(C8161-C9).Low concentration of vemurafenib can inhibit A375 mR proliferation and survival ability,while showed drug resistance with high concentration of vemurafenib(>10μM).Other 3 BRAF mutated melanoma cell lines showed high sensitivity to vemurafenib,especially A375 m,the inhibite rate up to 78%(P<0.05).3)Enhanced sensitivity of melanoma cells to vemurafenibis by si PGK1 was associated with activation of apoptotic cell death pathway.A375 mR and UACC903 showed high activation of apoptotic cell death pathway,while C8161-C9 showed the lowest activation of apoptotic.4)Early apoptosis plays more important roles in 1205LU、UACC903、C8161-C9 cell lines than in A375m、A375mR cell lines.Before knock down of PGK1,A375 m and A375 mR cells had a feature of late apoptosis.Conclusion:Targeting siPGK1 may enhanced the sensibility of melanoma cells to vemurafenib,and therefore lower the viability and proliferation of melanoma cells.And this enhanced sensitivity is related to the activation of apoptosis.