The BET Inhibitor Nitroxoline Activates Protective Autophagy And Synergizes With Bortezomib To Induce Apoptosis Of Multiple Myeloma Cells

[Background and Objectives]Multiple myeloma(MM)is an incurable hematological malignancy characterized by overproduction monoclonal immunoglobulins within the bone marrow.The survival outcome of MM patients can range from several months to many decades due to the high heterogeneity of MM.In spite of the rapid advancement of novel agents in MM treatment disease recurrence or progression is inevitable.Therefore,it is imperative to develop agents with novel mechanisms of actions and explore the effective combination therapy for MM treatment.As the mechanism of epigenomes and regulation of gene transcription in tumorigenesis have been more and more clearly elucidated,epigenetics-targeted drugs are widely used in the treatment of various cancers,especially hematological malignancies.BRD4 protein functions as transcriptional regulator.In recent years,a large number of studies have demonstrated that BRD4 expression and its abnormal function are closely related to the occurrence of various malignant tumors.Therefore,the small molecular inhibitors designed and developed for BRD4 have been preliminarily confirmed to have strong anti-tumor activity by targeting BRD4.Nitroxoline,a selective BET inhibitor,has been confirmed to have anti-tumor activity in MLL leukemia.However,its role in MM remains unclear.Therefore,we further explored the role of BET inhibitor nitroxoline and its underlying molecular mechanisms in MM.[Methods]1.Transmission electron microscopy,qRT-PCR,western blotting assays and other assay were used to detect the effects of BET inhibitor nitroxoline on key autophagy-related genes in multiple myeloma cell lines(RPMI8226,H929).2.Autophagy inhibitors 3-MA,BAF and HCQ alone or combined with BET inhibitor nitroxoline to treat RPMI8226 and H929 cells,and then examine the effects of nitroxoline on autophagy vesicles and autophagy-related proteins of MM cells by transmission electron microscopy and western blotting assays.3.The effects of nitroxoline alone and combined with autophagy inhibitors on the proliferation and apoptosis of RPMI8226 and H929 cells were performed by MTT,flow cytometry and western blotting assays.4.qRT-PCR and western blotting were used to detect the effects of BET inhibitor nitroxoline on the expression of histone methylation tags(EZH2,H3K27me3)and histone acetylation tags(SIRT1,H4K16ac,hMOF)in RPMI8226 and H929 cells.5.The effects of histone H3K27 methylation and H4K16 acetylation on nitroxoline-induced autophagy were detected by chromatin immunoprecipitation(CHIP-qPCR).6.RPMI8226 and H929 cells were treated with different concentrations of nitroxoline and/or bortezomib,and then MTT assay was used to detect the effects of nitroxoline and/or bortezomib on the proliferation of RPMI8226 and H929 cells,and the CompuSyn1.0 software was used to evaluate the effect of the combination.7.Flow cytometry was used to detect the effects of nitroxoline and bortezomib on the cell cycle and apoptosis of RPMI8226 and H929 cells.8.Western blotting was used to detect the effect of nitroxoline and bortezomib on the expression of mitochondrial apoptosis-related proteins and cycle arrest-related proteins.9.To illustrate the effect of nitroxoline and bortezomib on the growth of subcutaneous transplanted tumors in a nude mouse animal model of myeloma cells.HE staining was used to observe the pathological changes of tumor tissue.Immunohistochemical was used to detect the expression of Ki-67.TUNEL assay was utilized to determine the apoptosis of xenograft tumor cells.[Results]1.The BET inhibitor nitroxoline can inhibit the cell proliferation and induce the apoptosis in RPMI8226 and H929 cells.2.BET inhibitor nitroxoline can promote autophagy by up-regulating the expression of LC3B-II,Atg5 and Atg 7 and down-regulating the expression of P62 at the mRNA and protein levels in RPMI8226 and H929 cells.3.Compared with the nitroxoline treatment alone group,the combination group of nitroxoline and autophagy inhibitor significantly suppressed the proliferation and induced apoptosis in RPMI8226 and H929 cells.4.Autophagy inhibitors enhance nitroxoline-induced apoptosis of RPMI8226 and H929 cells by up-regulating the expression of apoptosis-related protein cleaved caspase3.5.In RPMI8226 and H929 cells,nitroxoline down-regulated the expression of EZH2 and hMOF at the mRNA level,and up-regulated the expression of SIRT1 in a concentration-dependent manner.6.In RPMI8226 and H929 cells,nitroxoline down-regulated the expression of EZH2,hMOF,H3K27me3,H4K16ac,and up-regulated the expression of SIRT1 in a dose-dependent manner.7.Compared with the untreated control group,the recruitment of H4K16ac and H3K27me3 in the LC3B promoter region was reduced,accompanied by increased binding of RNA polymerase Ⅱ in the corresponding promoter region,which in turn activated the expression of LC3B gene in RPMI8226 and H929 cells.8.The BET inhibitor nitroxoline can enhance the apoptosis induced by bortezomib in RPMI8226 and H929 cells by activating the mitochondrial-dependent apoptosis pathway.9.In vivo experiments further confirmed that the combined drug group significantly inhibited tumor growth compared with the bortezomib or nitroxoline alone.TUNEL and HE staining indicated that tumor cell apoptosis was significantly increased in the combination group.[Conclusion]Based on the above research evidence,our results support the following three main conclusions:1.Nitroxoline,a new BET inhibitor,can induce protective autophagy in myeloma cells.2.The BET inhibitor nitroxoline can reduce the recruitment of H3K27me3 and H4K16ac in the promoter region of the LC3B gene,and increase RNA polymerase Ⅱ,thereby activating the expression of autophagy genes in myeloma cells.3.The BET inhibitor nitroxoline synergistically sensitizes the anti-myeloma effect of the proteasome inhibitor bortezomib through the mitochondrial apoptosis pathway.In summary,myeloma cell lines RPMI8226 and H929 were used as the research object in this study.We firstly discovered that nitroxoline,a novel BET inhibitor,can activate autophagy to exert anti-myeloma effects.We also found that the expression of SIRT1,EZH2,hMOF,H3K27me3,H4K16ac in epigenetics is closely related to the transcriptional regulation of autophagy-related genes.In addition,we also confirmed that nitroxoline activates protective autophagy and synergizes with bortezomib to induce apoptosis of multiple myeloma cells.We also further demonstrated its clinical significance in vivo experiments.Through the above research,we evaluate that the new BET inhibitor nitroxoline can be used as a new agent for anti-myeloma treatment,thus providing a theoretical basis for the combinative treatment strategies.