The Role And Mechanism Of Pimozide On Reversing Lenalidomide Resistance In Multiple Myeloma

Background:Multiple myeloma(Multiplemyeloma,MM)is a malignant plasma cell disease originating from the bone marrow,which has not been completely cured at present.Most patients relapse or progress because of drug resistance(Drug Resistance,DR).Bone marrow microenvironment(bone marrow microenvironment BMME)plays a key role in drug resistance of MM.In BM the soluble cytokines IL-6 activates JAK/STAT3 pathway.Then the abnormal activation of STAT3 can promote the expression of related genes involved in proliferation,angiogenesis,invasion and migration,regulation of redox homeostasis and inducible drug resistance,which also is the important mechanism of lenalidomide resistance of MM cells.In previous studies,our team has confirmed that the stromal cell culture supernatant(SCCS)can induce MM cells to be resistant to lenalidomide by activating the downstream JAK/STAT3 pathway of IL-6,which subsequently up-regulated the expression of xCT and increased the activity of peroxidase,thus caused the decrease of intracellular ROS.Pimozide,a novel inhibitor of STAT3,has been reported to be resistant to a variety of tumors,but the antitumor effect of Pimozide in MM cells and its effect on lenalidomide resistance have not been studied.Objective:1.To explore the anti-tumor effect of Pimozide on MM cells;2.To explore the effect of Pimozide on MM cells which has been resistant to lenalidomide and its mechanism.Methods:1.MM cell lines were pretreated with IL-6/SCCS for 6 hours to reduce the sensitivity to lenalidomide and cell count kit(CCK8)was used to detect the proliferation of cells.2.Apoptosis rate of MM cell lines(lenalidomide sensitive/resistant)treated with Pimozide、lenalidomide or both was detected by AnnexinV/PI staining and flow cytometry.3.Intracellular reactive oxygen species probe DCFH-DA was loaded into MM cell lines(lenalidomide sensitive/resistant)treated with Pimozide、lenalidomide or both,and the intracellular ROS level was detected by flow cytometry.4.Western blot was used to detect the expression of related proteins in MM cell lines(lenalidomide sensitive/resistant)treated with Pimozide、lenalidomide or both:STAT3,p-STAT3,xCT,apoptosis protein PARP,Bax,anti-apoptosis protein Bcl-2.Results:1.IL-6 is the main cytokine in SCCS.SCCS/IL-6 can induce MM cell lines(H929,MM.1S)to be resistant to lenalidomide.2.Pimozide inhibited cell proliferation viability and promoted apoptosis of MM cell lines MM.1S and H929 in a time-and dose-dependent manner.Pimozide combined with lenalidomide had obvious synergistic effect,which could attenuate the inhibition of cell proliferation viability and the increase of intracellular ROS level induced by lenalidomide in MM cell lines3 After pretreatment ofSCCS,Pimozide combined with lenalidomide increased the level of ROS,decreased the expression of anti-apoptotic protein Bcl-2,increased the expression of pro-apoptotic protein PARP and Bax,and inhibited the expression of p-STAT3 and xCT in MM cell linesConclusion:1.Lenalidomide resistance of MM cell lines is related to the activation of JAK/STAT3 pathway mediated by IL-6 or SCCS and the decrease level of downstream intracellular ROS.2.Pimozide can directly act on MM cells that inhibits JAK/STAT3/xCT pathway,increases the level of intracellular ROS and induces cell apoptosis.3.The resistance of MM cell lines to lenalidomide mediated by IL-6 or SCCS was reverse under the treat of pimozide combined with lenalidomide,that further strengthenes the inhibition of JAK/STAT3/xCT pathway,and further increases the level of intracellular ROS and the rate of apoptosis.Statistical analysis:All experiments were repeated at least 3 times and the data for each group were described as mean±standard deviation.Data analysis was performed using SPSS 25.0 software.The measurement data of the two groups were compared by independent sample t-test,and the mean of multiple groups were compared by one-way ANOVA.Levene tested the homogeneity of the variance.If the variance is uniform,the LSD method is used for multiple comparisons.If the variance is not uniform the approximate F test is used for multiple comparisons.P<0.05 is determined to be statistically significant.