| Objective:c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis.Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival and the USP5/c-Maf axis could be a potential target for myeloma therapy.As a concept of principle,the present study established a USP5/c-Maf-based luciferase system that was used to screen a FDA-approved drug library to find novel anti-MM agent and to clarify their mechanisms.Methods:(1)HEK293T cells were transfected with USP5,c-Maf and MARE.luci plasmids to construct a screening system.A mid-throughput screen was performed based on this system to search USP5/c-Maf inhibitors from the FDA-approved drug library.(2)LP1 cells were treated with the potential inhibitors from the screen,and c-Maf and PARP were detected by WB.(3)RT-PCR assay was performed to detect the mRNA level of USP5 and c-Maf.(4)LP1 and RPMI-8226 cells were treated with mebendazole(MBZ).The interaction between USP5 and c-Maf was measured by immunoprecipitation and WB.(5)LP1 and RPMI-8226 cells were treated with MBZ and c-Maf ubiquitination level was detected by immunoprecipitation and WB.(6)Luciferase assay and RT-PCR were performed to evaluate whether MBZ can influence the transcriptional activity of c-Maf.(7)WB and flow cytometer were performed to examine whether MM cell apoptosis induced by MBZ.(8)Western blot was carried out to evaluate whether MBZ enhances WP1130 and daunorubicin in MM cell apoptosis.(9)The human myeloma xenograft models were established in nude mice with LP1 and RPMI-8226 cells,respectively,to examine the effects of MBZ on MM tumor growth in vivo.At the same time,body weight was measured and blood samples were analyzed to evaluate the toxicity of MBZ in vivo.Results:(1)The Mid-throughput screen found about 30 compounds that significantly reduced the luciferase activity of c-Maf.(2)WB and RT-PCR analyses showed that only mebendazole(MBZ)induced MM cell apoptosis and promoted c-Maf protein degradation without affecting its mRNA level.(3)MBZ significantly reduced the protein level of USP5 and c-Maf,and downregulated the expression of USP5 at the mRNA level,but it didn't affect the mRNA level of c-Maf.(4)Co-IP results showed that MBZ significantly inhibited the interaction between USP5 and c-Maf in both HEK293T cells and MM cells.(5)The degradation of c-Maf induced by MBZ can be reversed by the proteasomal inhibitor MG132.MBZ can significantly increase the ubiquitination levels of c-Maf,thereby inducing its degradation.(6)MBZ inhibited the luciferase activity of c-Maf and also downregulated the mRNA levels of CCND2,ITGB7 and ARK5,three typical downstream target genes of c-Maf.(7)WB analysis showed that MBZ can induce PARP and caspase-3 cleavage in c-Maf expressed MM cell lines(LP1,RPMI-8226).Further flow cytometry analysis showed that MBZ could induce marked apoptosis in LP1 and RPMI-8226 but not in U266 cells that lack c-Maf.(8)Compared to the single-drug treatment groups,MBZ promoted the cleavage of PARP and deceased more c-Maf protein when combined with WP1130 or DNR.(9)MBZ delayed MM tumor growth in xenograft mice but displayed no effects on mice body weights.In addition,analyses of blood biochemistry showed that there were no significant changes in liver and renal functions.These results indicated that MBZ displayed potent anti-myeloma activity without overt toxicity.The WB results shown that MBZ can decrease the protein level of USP5 and c-Maf and induce MM cell apoptosis in vivo.Conclusions:MBZ elicits great anti-MM activity in vivo and in vitro with low toxicity.MBZ significantly inhibits USP5 and promotes c-Maf degradation through the ubiquitin-proteasome pathway,thus inducing the MM cell apoptosis.Taken together,this study identifies MBZ as a USP5/c-Maf inhibitor that could be developed as a novel anti-myeloma agent. |
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