The Investigation Of New Proteasome Inhibitors In Multiple Myeloma

MM（Multiple Myeloma）is a malignant tumor originated from B cells,its cytological characteristics is malignant lesions of plasmacyte of B cells terminal differentiated stage cells.The incidence of multiple myeloma is the second highest in malignant tumors of the blood system,only to non-Hodgkin’s lymphoma,and the incidence are increasing year by year in recent years.The disease is characterized by a large number of abnormal plasmacyte infiltration in peripheral blood and bone marrow and non-functional monoclonal immunoglobulin was producted.Because there are a large number of abnormal plasmacyte infiltration in the bone marrow of the patients,the hematopoietic function of the three lines of bone marrow is suppressed,and the patients have pathological osteolytic changes,pathological fracture,spinal enlargement,organ infiltration,anemia infection and other systemic symptoms.A large number of monoclonal immunoglobulin in peripheral blood often lead to hyperviscosity,and a large number of abnormal immunoglobulin light chain is excreted through the kidney,making the renal tubule in a state of high metabolism for a long time,resulting in renal tubular sclerosis.Therefore,late patients often have renal failure,and the quality of life of patients with the disease is poor.At present,the research on the pathogenesis of multiple myeloma is mainly focused on the NF-κB signaling pathway,which mediates the expression of adhesion molecules and IL-6 growth dependence of multiple myeloma cells.With the deepening of basic research,more and more inhibitors have been used in clinical treatment of tumors,one of the target pathways is ubiquitin-proteasome degradation system.The main physiological function of the system is to mediate the ubiquitin of cell endogenous proteins,which are recognized by proteasomes and degrade substrate proteins.At present,proteasome inhibitor Velcade（Bortezomib）,which has been approved by FDA for clinical treatment of multiple myeloma,is effective in the treatment of multiple myeloma,but the current situation of clinical treatment is not optimistic.42%of the patients have developed drug resistance.Moreover,the main side effect of the drug was neutropenia,which further aggravated the infection of the patients.Therefore,it is urgent to find a kind of therapeutic drug with strong effect,quick effect and little side effect.The chemical small molecule,named of Y1,W3,W4 are new type of proteasome inhibitors and a boric acid derivative.We will continue to explore the mechanism of three new proteasome inhibitors in multiple myeloma cell line U266 and mouse models.Objectives:To screen a new proteasome inhibitor against the growth inhibition of multiple myeloma cell line U266,and compare the effect of new proteasome inhibitor W3with Velcade in U266 cells and tumors in mice to explore the mechanism of its role in multiple myeloma cells.Methods:（1）Inhibitory effect of new proteasome inhibitor on multiple myeloma cell line U266 was screened by CCK8.U266 cells were treated with different concentrations of Velcade at the concentration of 2 n M,4 n M,8 n M,10 n M,Y1 at the concentration of 4 n M,10n M,20 n M,40 n M,80 n M,W3 at the concentration of 1 n M,2 n M,4 n M,8 n M,10 n M,W4 at the concentration of 2 n M,4 n M,8 n M,10 n M,20 n M for 48 h,and their IC₅₀ concentrations were determined by CCK8.W3 was the new proteasome inhibitor with the strongest inhibitory effect on the growth of U266 cells.（2）The difference of late apoptosis induced by new proteasome inhibitor w3 and Velcade were detected by Hoechst33342 staining and Flow Cytometry.（3）The effect of new proteasome inhibitor W3 and Velcade on U266 cell cycle arrest was detected by Flow Cytometry.（4）The effect of new proteasome inhibitor W3 and Velcade on the enhancement of endogenous protein polyubiquitin in U266 cells was detected by western-blotting technique.The effect of inhibiting the expression of p65 in U266 cells and nude mice was detected by Western-Blotting.（5）The inhibitory effect of new proteasome inhibitor W3 and Velcade on tumor growth in vivo was detected by nude mice tumorigenesis test.Results:（1）New proteasome inhibitors Y1,W3 and W4 could inhibit the growth of multiple myeloma cells U266 cells.The inhibitory effect of W3 on the growth of U266 cells was the strongest.（2）The effect of new proteasome inhibitor W3 on inducing the late apoptosis of U266 cells was stronger than that of Velcade.（3）The cell cycle of U266 cells was arrest by new proteasome inhibitor W3.and the effect was stronger than that of Velcade.（4）The effect of W3,a new proteasome inhibitor,on enhancing the ubiquitin level of endogenous protein in U266 cells was stronger than that of Velcade.The inhibitory effect of p65 on the expression in U266 cells and mouse tumor tissues was stronger than that of Velcade.（5）New proteasome inhibitor W3 on tumor growth in mice,and the inhibitory effect was earlier than that of Velcade,and the function time was earlier than that of Velcade.Conclusions:（1）W3,a new proteasome inhibitor,could significantly inhibit the growth of U266,H1299 and Hela cells,promote apoptosis and induce cell cycle arrest.The effect of W3,a new proteasome inhibitor,was better than that of Velcade.（2）W3 could enhance the level of intracellular proteins polyubiquitin,and decrease the level of free ubiquitin in cells,W3 could inhibit the expression of p65 protein in U266 cells.The effect was stronger than that of Velcade.（3）W3 could inhibite the growth of tumor in mice,the function time was earlier than that of velcade,and significantly inhibited the expression of p65 in mice tumor tissues,which of W3 was better than that of Velcade.