The Anti-lymphoma Activity And Mechanisms Of Novel Synthetic 4-chlorobenzoyl Berbamine In C-Myc Positive Diffuse Large B Cell Lymphoma Cells

Background.B cell lymphoma is a heterogeneous group of lymphoproliferative disorders originating in B lymphocytes.In U.S,DLBCL occupied 32.5%in all non-Hodgkin lymphoma,is a most common histological subtype of lymphoma.Introduction of multiple new treatment strategy including rituximab and hematopoietic stem cell transplantation have greatly improved response rate and survival of DLBCL patients,however,30-40%of patients are refractory/relapsed.In DLBCL,over-expression of c-Myc can be observed in a group of DLBCL cases using immunohistochemistry always associated with constant failures of standard chemotherapeutic regimens and poor outcomes.Currently,c-Myc is still identified as"undrugable" oncoprotein.It is difficult to target partially attribute to its short half-life and rapid metabolism in cancer cells,another reason is special structure called helix-loop helix topology lacking domains that binding to drug.Berbamine,which is isolated from the Chinese traditional medicine Berberis amurensis,has been largely used to elevate the number of neutrophils and improve immune function in China for decades.Several studies have demonstrated that Berbamine has anti-tumor activity against different types of cancers,including myeloid and T cell leukemia,T cell lymphoma,multiple myeloma,melamona and hepatocellular cancer.However,the higher therapeutic doses of Berbamine limit its clinical application,the novel derivative 4-chlorobenzoyl Berbamine(CBBM)was synthesized for efficacy improving from natural Berbamine.In this study,we first investigated the clinical traits and overall survivial of c-Myc positive DLBCL.Sencond,to demonstrate the anti-lymphoma activity of CBBM,we carried out a panel of DLB CL cells after CBBM treatment and access its anti-lymphoma activity;Last,to explore its potential mechanisms of anti-lymphoma,we investigated the c-Myc-and its associated protein after CBBM treatment,and the atuocrine IL 10 and its downstream JAK2/STAT3 pathway.Part I DLBCL with c-Myc expression is associated with a poor prognosis:a 116 DLBCL patients clinical analysisObjectiveThe purpose of this study was to investigate the clinical traits,therapeutic response,prognostic values of c-Myc expression in DLBCL results from immunohistochemistry.Methods116 newly diagnosed DLBCL patients treated in the department of Hematology,Second affiliated hospital and SRRS hospital,Zhejiang university School of medicine were analyzed retrospectively.Immunohistochemistry was performed on archived paraffin-embedded tissue using c-Myc,BCL2,CD 10,BCL6,MUM1,Ki-67.Analysis was carried out to identy the prognosis significance and clinical traits such as sex,age,clinical stage,IPI score,LDH,GCB/non-GCB subtype,Ki-67 index,centre nervous system involvement,GI tract involvement and the treatment therapeutic response and overall survival of c-Myc positive lymphoma.Results1.In all 116 newly diagnosed DLBCL,c-Myc protein expression were detected in 40.52%(47/116)of patients,and c-Myc protein high expression(>80%)were detected in 6.90%(8/116)of patients,c-Myc and BCL2 double expression were detected in 34.48%(40/116)of patients.2.C-Myc protein expression was associated with inferior overall,the median survival time was 23 months,and c-Myc negetive group did not reach.The similar result was found in c-Myc and BCL2 double expression DLBCL(p =0.005,p =0.004,respectively).3.Compared with c-Myc negative expression,c-Myc protein expression was significantly associated with high IPI score(?60 years old group)(p =0.033),high Ki-67 index(p =0.002),central nervous system involvement(p =0.003)and low complete response rate after six cycle of chemotherapy(p =0.028).ConclusionsC-Myc protein expression detected by immunohistochemistry defined a subset of DLBCLs was strongly associated with aggressive clinical course,poor response to chemotherapy and poor outcome in newly diagnosed patients.Part ? Novel synthetic 4-chlorobenzoyl Berbamine inhibits the proliferation of diffuse large B cell lymphoma cells and its potential mechanismObjectiveThe aims of this part is to investigate the potent anti-lymphoma activity of CBBM as compared with Berbamine,and to access the potential anti-lymphoma activity CBBM and explore possible molecular mechanism of DLBCL inhibition caused by CBBM.Methods1.To evaluate proliferative inhibition of CBBM and its parent natural compound Berbamine,we treated a panel of DLBCL cell lines at 24h,48h,72h at various concentretions by MTT assay.To access the toxicity of CBBM,we treated two cases of normal hematopoietic cells by MTT assay.2.To reveal the celluler mechanism of anti-lymphoma of CBBM,we next evaluated whether CBBM mediated anti-lymphoma activity is related to apoptosis and cell cycle arresting by Flow cytometry.3.To verify the mechanism of apoptosis and autophagy caused by CBBM,we investigated Caspase 3,Cleaved-Caspase 3,PARP,Cleaved-PARP,LC3B expression in CBBM treated OCI-Ly3 cells by Western blot.4.To determine if CBBM treatment affect the expression of c-Myc proteins in DLBCL cells by Western blot;To reveal the mechanisms by which CBBM reduces c-Myc protein in OCI-Ly3 cells,we examined c-Myc mRNA expression levels by qPCR and MG132,a proteasome inhibitor,or/and CBBM co-culture with OCI-Ly3 by Western blot.5.To explore the expression of CaMKIIy in DLBCL,we examined CaMKIIy expression in vitro in a panel of DLBCL lines,showed the relationship between IC50 values of CBBM and the grey level of CaMKIIy/GAPDH.6.To investigate the IL10 mRNA and protein expression level of five DLBCL cell lines by qRCP and ELISA.To determine if CBBM treatment affect the level of autocrine IL10 by ELISA.7.To reveal the mechanisms of IL 10 inhibition,we examined JAK2/STAT3 pathway and BCL2 expression after CBBM treatment by Western blot.8.To vertified the if the downregulation of JAK2/STAT3 pathway caused by CBBM induced IL10 inhibition,we investigated the change of JAK2/STAT3 signaling using recombinant human IL10 co-treatment with CBBM by Western blot.9.To investigate the expression of PD-L1,we carried out PD-L1 expression after CBBM treatment at various concentretions.To find out if the inhibition of PD-L1 associated with PD-L1 protein degradation,we examined the PD-L1 expression after CBBM and/or MG132 treatment by Western blot.Results1.The derivative CBBM showed 1.39±0.04,2.56±0.04,3.89±0.07,1.82±0.05,3.73±0.06 ?mol/L IC50 values in OCI-Ly3,OCI-Ly10,U2932,SU-DHL 16 and Pfeiffer cells,exhibited 8.78±0.25-,9.24±0.33-,4.57±0.017-,9.64±0.53-,and 7.01 ±0.031-fold increase in anti-tumor activity compared to natural Berbamine.The survival rates of CBBM for two normal hematopoietic cell samples at 10 ?mol/L were 87.93±4.70%and 93.15±1.50%,respectively.Compared to OCI-Ly3,the selective index of CBBM was 7.20±0.22.2.Apoptosis of OCI-Ly3 cells were increased obviously after drug treatment in a dose-dependent manner.The proportion of G0/G1 phase cells were increased significantly arresting by CBBM.3.Cleaved-Caspase 3,Cleaved-PARP,and LC-3B were increased after CBBM treatment.4.C-Myc mRNA and protein level in OCI-Ly3 cells both reduced after exposure to CBBM.Further studies showed that CBBM treatment led to proteasome-dependent degradation of c-Myc protein in OCI-Ly3.5.We carried out correlation analysis between the IC50 values of CBBM and the grey level of.CaMKIIy/GAPDH in the five cell lines tested,the results indicated that CBBM-mediated cell inhibition was positively correlated with expression levels of CaMKII?,R2=0.8624.Western Blot demonstrated that both CaMKII? and its phosphorylation were obviously inhibited by CBBM.6.CBBM down-regulates autocrine IL10 protein in OCI-Ly3:cells in time-depentent and dose-dependent manners.7.CBBM reduced levels of phospho-JAK2,phospho-STAT3 at 24h after treatment in OCI-Ly3 cells,whereas total JAK2,STAT3 remained unchanged.BCL2 was obviously inhibited by CBBM subsequencely.8.Recombinatant human IL10 stimulation experiment showed elevated IL10 level led to the increased expression of JAK2/STAT3 signaling and c-Myc,CBBM had inhibitory effect on activated JAK2/STAT3 signaling,c-Myc and BCL2 caused by IL10.9.PD-L1 protein,a c-Myc downstream molecular of adaptive immune checkpoint,CBBM treatment greatly reduced PD-L1 protein expression of OCI-Ly3 cells,and could not rescued by MG132.Conclusions1.CBBM potently induced apoptosis of DLBCL cells and exhibited more anti-tumor activity compared to natural Berbamine.Meanwhile,CBBM inhibit the proliferation of DLBCL cells selectively with sparing normal blood cell.2.CBBM inhibite the proliferation of DLBCL line OCI-Ly3 cells through G0/G1 phase arrest and induced apoptosis.And the inhibition effect of proliferation by CBBM through activating apoptosis and autophagy related protein in OCI-Ly3 cells.3.CBBM treatment led to proteasome-dependent degradation of c-Myc protein in OCI-Ly3 whereas slight decreased c-Myc mRNA.CaMKIIy,a key positive up-regulator of c-Myc,were inhibited by CBBM.4.CBBM could downregulate JAK2/STAT3 signaling,c-Myc and BCL2 protein though inhibiting autocrine IL10 expression in OCI-Ly3.5.PD-L1 expression was downregulated simultaneously in DLBCL cells.