The Effect Of TLR4 Inhibitor TAK-242 On Proliferation And Apoptosis Of Human Multiple Myeloma Cells

ObjectiveTo investigate the effect of Toll-like receptor 4（TLR4）specific inhibitor TAK-242 on the proliferation and apoptosis of human multiple myeloma cell line RPMI8226,and to explore its possible mechanism of action.MethodsThe human multiple myeloma cell line RPMI8226 with logarithmic growth was divided into group A,group B,group C and control group（CON）.Adding TAK-242 with final concentrations of 20,40 and 80μmol/L to group A,group B and group C(selection of concentration refer to reference^[1]),and adding the same amount of RPMI 1640 culture solution into the control group.The cell proliferation inhibition rate was detected by CCK-8 method;the apoptosis rate was detected by Annexin-V/PI;the expression levels of Toll-like receptor 4（TLR4）mRNA and myeloid differentiation factor 88（MyD88）mRNA were detected by RT-PCR;the protein expression levels of Myeloid differentiation factor 88（MyD88）,nuclear factor-κB in the nucleus（NF-κB）P65,nuclear factorκB inhibitory proteinα（IκBα）,cysteine aspartate protease-3（caspase-3）,cell cycle protein D1（Cyclin D1）were detected by Western blot.Results1.The results of CCK-8 showed that compared with the control group,in the same action time,the proliferation rate of RPMI8226 cells decreased gradually with the increase of inhibitor TAK-242 concentration,and the difference was statistically significant（P<0.05）.At the concentration of inhibitor 40μmol/L,the proliferative activity of RPMI8226 cells decreased gradually with the prolongation of inhibitor TAK-242,which was significantly lower than that of the control group,and the difference was statistically significant（P<0.05）.TAK-242has inhabiting effect on the proliferation of RPMI8226 cells.2.The results of flow cytometry showed that the apoptosis rate of RPMI8226 cells treated with different concentrations of inhibitor TAK-242increased gradually with the increase of inhibitor concentration.Compared with the control group,the difference was statistically significant（P<0.01）.TAK-242can induce apoptosis in RPMI8226 cells.3.The results of RT-PCR showed that the relative expression of MyD88mRNA and NF-κB mRNA decreased compared with the control group after RPMI8226 cells was treated with different concentrations of inhibitor for 24h,and the difference was statistically significant（P<0.05）.TLR4/MyD88 signaling pathway plays an important regulatory role on NF-κB in human multiple myeloma cells.4.The results of Western Blot showed that the expression level of NF-κB P65 protein and Cyclin D1 protein decreased compared with the control group after RPMI8226 cells was treated with different concentrations of inhibitor for24h,and the difference was statistically significant（P<0.05）.The expression level of IκBαprotein and caspase-3 protein increased,and the difference was statistically significant（P<0.05）,which further proved that TAK-242 inhibited the proliferation of RPMI8226 cells and induced RPMI8226 cells apoptosis.ConclusionToll-like receptor 4 specific inhibitor TAK-242 could inhibit the proliferation and promote the apoptosis of human multiple myeloma cell line RPMI8226,which may be related with the inhibition of NF-κB signaling pathway function,down-regulation of NF-κB P65 and Cyclin D1 protein and up-regulation of IκBα,caspase-3 protein.