| Background: Multiple myeloma(MM)is the second most common hematological malignancy and is characterized by the infiltration of clonal malignant plasma cells in the bone marrow.Although the development of new drugs and treatment regimens in the past decade has improved the survival of patients,multiple myeloma is still incurable,and the vast majority of patients will inevitably relapse and progress,and eventually die due to refractory treatment.Therefore,the development and exploration of new drugs and treatment regimens are of great significance for the treatment of patients with multiple myeloma.Objective: DCZ5731 is a novel salicylanilide derivative obtained by collaboration with the Shanghai Institute of Materia Medica,Chinese Academy of Sciences.In this study,we will explore the effect of DCZ5731 against multiple myeloma and its mechanism of action.Methods: The effect of DCZ5731 on the viability of MM cells was detected by Cell Counting Kit-8(CCK-8).5-ethynyl-2’-deoxyuridine(Ed U)kit and soft agar colony formation assay were used to detect the effect of DCZ5731 on the proliferation of MM cells.By Ficoll-Hypaque density gradient centrifugation and CD138 immunomagnetic bead enrichment,peripheral blood mononuclear cells(PBMCs)from normal people and CD138-positive primary MM cells from clinical MM patients were extracted.The effects of DCZ5731 on normal PBMCs and primary MM cells were investigated by detecting cell viability.The effect of DCZ5731 on MM cell apoptosis was detected by Annexin V/PI double staining and TUNEL/DAPI staining analysis.Western blot was used to detect the activation of caspase-mediated apoptosis pathway after DCZ5731 acted on MM;Z-VAD-FMK,a broad-spectrum caspase inhibitor,was used to further explore whether DCZ5731-induced MM cell apoptosis depended on caspase-mediated apoptosis pathway.The effects of DCZ5731 on MM cell cycle were detected by PI staining and flow cytometry;the effects of DCZ5731 on cycle-related proteins were detected by western blot.The protein that DCZ5731 acted on was predicted by Pharm Mapper,and the effect of DCZ5731 on HSP90 was studied by molecular structure docking and cellular thermal shift assay.The effect of DCZ5731 on the complex of HSP90 and chaperone Cdc37 was detected by Co-IP assay.Molecular signaling pathways affected by DCZ5731 in MM cells were analyzed by transcriptome sequencing.Western blot was used to detect the effect of DCZ5731 on HSP90 client proteins and downstream signaling pathways in MM cells.The effect of DCZ5731 on heat shock response was detected by western blot and immunofluorescence.By inducing the differentiation of bone marrow mononuclear cells,we explored the effect of DCZ5731 on osteoclast differentiation.By co-culturing MM cells with cytokines and bone marrow stromal cells,we investigated whether DCZ5731 can overcome the protective effect of bone marrow microenvironment on MM cells;western blot was used to detect whether DCZ5731 inhibited Akt and ERK activation in the bone marrow microenvironment.A subcutaneous MM xenograft tumor model in nude mice was established to study the anti-multiple myeloma effect of DCZ5731 in vivo.The safety of DCZ5731 in vivo was studied by observing the body weight changes of nude mice and detecting the biochemical indexes of liver and kidney function in the serum of nude mice.The effects of DCZ5731 on cell proliferation,apoptosis,and Akt and ERK signaling were detected by immunohistochemical staining of tumor tissue.The effects of DCZ5731 and bortezomib alone or in combination on MM cell lines and primary MM cells were detected by CCK-8 and flow cytometry.Calcu Syn software was used to calculate the combination index(CI)to study the combined effect of the two drugs.In nude mice,we compared the effects of DCZ5731 and bortezomib alone or in combination in inhibiting tumor growth.Meanwhile,immunohistochemical staining of tumor tissue was used to preliminarily explore the combined mechanism of DCZ5731 and bortezomib in vivo.Results: DCZ5731 was able to inhibit the activity and proliferation of MM cells in a concentration-and time-dependent manner and also played a role in CD138-positive primary MM cells from clinical MM patients,while it had no significant effect on normal PBMCs.DCZ5731 can induce MM cell apoptosis through caspase-mediated intrinsic and extrinsic apoptotic pathways,and this apoptotic effect can be blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK.DCZ5731 triggered G0/G1 arrest in MM cells by downregulating the protein expression levels of CDK4,CDK6,and Cyclin D1.DCZ5731 can act on HSP90 to disrupt the interaction between HSP90 and Cdc37 and inhibit the formation of HSP90-Cdc37 complex.DCZ5731 didn’t induce the up-regulation of heat shock factor HSF-1 and heat shock proteins HSP70 and HSP27 in MM cells which would result in heat shock side effects.DCZ5731 can induce the down-regulation of HSP90 client protein levels and inhibit JAK2/STAT3 and MEK/ERK signaling pathways in MM cells.DCZ5731 can inhibit the differentiation of osteoclasts and overcome the protective effect of the bone marrow microenvironment on MM cells.DCZ5731 also inhibited the phosphorylation of Akt and ERK in MM cells in the bone marrow microenvironment.DCZ5731 inhibited the growth of MM xenograft tumors in nude mice,but had no effect on the body weight and liver and kidney functions of nude mice,further indicating the safety of DCZ5731 in vivo.The results of immunohistochemistry on tumor tissue showed that DCZ5731 inhibited the expression of p-Akt and p-ERK in vivo,down-regulated the proliferation marker Ki67,activated caspase 3,and up-regulated the apoptosis marker TUNEL-positive cells.Finally,DCZ5731 combined with bortezomib can exert a synergistic effect on MM cells and inhibit tumor growth more effectively in vivo.Conclusion: DCZ5731 exhibited potent anti-multiple myeloma effects in vitro and in vivo,which were associated with the inhibition of cell proliferation,induction of G0/G1 phase arrest,and apoptosis.DCZ5731 was well tolerated in vivo,with no significant effect on normal peripheral blood mononuclear cells.Mechanistically,DCZ5731 could act on HSP90,disrupt the HSP90-Cdc37 interaction,affect the expression of HSP90 client proteins,and inhibit the JAK2/STAT3 and MEK/ERK signaling pathways in MM cells,thus blocking the protective effect of the bone marrow microenvironment on MM cells.In addition,the combination of DCZ5731 and the clinical first-line anti-myeloma drug bortezomib can exert a synergistic effect.Our study suggests the potential clinical application of DCZ5731 in the treatment of multiple myeloma,providing a preliminary research basis and new ideas for the combination of DCZ5731 and bortezomib in myeloma patients. |
