The Effect Of A DJ-1 Inhibitor STK793590 On The Proliferation And Apoptosis Of Multiple Myeloma Cells:An In Vitro Study

Objectives:1.To determine whether a DJ-1 inhibitor STK793590 can affect the proliferation and apoptosis of multiple myeloma(MM)cells in vitro.2.To explore the underlying mechanisms,if the above effect can be observed.3.To determine whether STK793590 can sensitize MM cells to bortezomib(Bz).Methods:1.RPMI8226 cells were retrospectively treated with increasing concentrations of STK793590(12.5μM-100μM)and the control(DMSO)for indicated time periods(24h、48h、72h),and then cell proliferation was evaluated with CCK-F method;Edu cell proliferation analysis:RPMI8226 cells were treated by STK793590(50μM)for 48h,and the changes of cell fluorescence intensity were recorded.2.RPMI8226 cells were treated with increasing concentrations of STK793590(25μM,50μM,100μM)and DMSO for 48h,then flow cytometry(FCM)was used to detect the distribution of cell cycle after propidium iodide(PI)incubation,and the expression of cell cycle-related proteins CDK1 and Cdc25c was detected by Western-blot(WB).3.Soft agar clony formation assay:STK793590(50μM)and DMSO were respectively used to treat RPMI8226 cells for two weeks,and the clone formation was supervised.4.RPMI8226 cells were treated with increasing concentrations of STK793590(25μM-100μM)and DMSO for 48h,and 50μM STK793590 was used to treat RPMI8226 cells for indicated time periods(24h、48h、72h),the cell apoptosis was detected with Annexin V/PI double-staining method by FCM;Meanwhile,the apoptosis-related protein Bax and Bcl-2 were also detected by WB.5.Preliminary study of the underlying mechanisms:DCFH-DA was used to incubate RPMI8226 cells after being exposed to STK793590 and DMSO,and the intracellular reactive oxygen species(ROS)was detected by FCM.The angiogenesis-related cytokines including the basic fibroblast growth factor(bFGF)and vascular endothelial growth factor-A(VEGF-A)were detected,and the PTEN/PI3K/Akt signaling pathway-related proteins were also evaluated by WB.6.The CCK-F method was used to detect the proliferation inhibition,and FCM was used to detect cell cycle and apoptosis changes when RPMI8226 cells were treated with bortezomib/STK793590 alone or the combination of both.Results:1.The effect of STK793590 on the proliferation,cell cycle,clone formation and apoptosis of RPMI8226 cells:1.1 CCK-F experiment:STK793590 can significantly inhibit the proliferation of RPMI8226 cells in the time and concentration-dependent manner(P<0.001);Edu cell proliferation assay showed that the fluorescence intensity of RPMI8226 cells treated with STK793590(50μM)for 48h was significantly lower than DMSO(P=0.0148,P<0.05).1.2 The cell cycle progression of RPMI8226 cells was blocked in the G2/M phase after varied concentrations of STK793590 treatment except for 25μM(P<0.01);As the concentrations of STK793590 increased,the expression of cyclin CDK1 and Cdc25c was also significantly reduced,indicating that STK793590 can result in G2/M phase arrest.1.3 Clone formation of RPMI8226 cells treated with STK793590 was significantly reduced compared with the cells treated with DMSO.1.4 After the treatment of STK793590 on RPMI8226 cells,the rate of apoptotic cells increased with the rising concentrations and time periods(P<0.05,P<0.01),and the expression of pro-apoptotic protein Bax increased,while the anti-apoptotic protein Bcl-2 reduced significantly,which finally leads to an increased ratio of Bax/Bcl-2.2.Preliminary mechanism research2.1 The intracellular changes of ROS in RPMI8226 cells treated with STK793590 were detected by fluorescent probe loaded with DCFH-DA.With the increasing concentrations of STK793590,the intracellular ROS level of RPMI8226 cells was not obviously changed.2.2 With the increasing concentrations of STK793590,the expression of bFGF protein in RPMI8226 cells was reduced,while the VEGF-A protein did not change significantly;In addition,the expression of PTEN protein increased significantly,while the phosphorylated protein Akt(p-Akt)protein expression decreased,but the total AKT(total-AKT)protein expression did not change significantly.3.Compared with Bz or STK793590 treatment alone,the inhibition of cell proliferation was more obviously observed after the treatment of Bz combined with STK793590(P<0.01),and the proportion of apoptotic cells was also significantly higher in the combination group(P<0.01).Conclusions:1.STK793590 can inhibit the proliferation of myeloma RPMI8226 cells in vitro,cause cell cycle G2/M phase arrest and promote cell apoptosis.2.The inhibitory effect of STK793590 on RPMI8226 cells maybe through the influence of bFGF expression and PTEN/PI3K/Akt signaling pathway,but further verification is needed.3.STK793590 can sensitize myeloma cells to bortezomib treatment in vitro,deserving a further exploration of its anti-tumor potential.