Study On The Mechanism Of BCL6 Down-regulation By Disulfiram/Copper On Diffuse Large B-cell Lymphoma

Objective To study the changes of BCL6 and BCL6 related pathway proteins after using disulfiram(DSF)/Copper(Cu)on human diffuse large B-cell lymphoma(DLBCL)cell line and primary cells,explore the molecular mechanism of DSF/Cu inhibiting DLBCL by down regulating BCL6 signal pathway.At the same time,analyzed the circulating follicular helper T cells(Tfh)and their subtypes in DLBCL patients and analyze the changes of Tfh after using DSF/Cu in order to provide experimental basis for the clinical treatment of DLBCL.Methods 1.The biological characteristics of BCL6 and its expression in DLBCL patients,and relationship with DLBCL patients and patients’ survival were analyzed by using online tools of The Cancer Genome Atlas(TCG A),Go and KEGG were analyzed by David online website.Using TIMER2.0 online database to analyze the correlation between BCL6 and immune infiltrating cells.2.The effect of DSF/Cu on the apoptosis of primary DLBCL cells were detected by Annexin-V PI staining.3.The effect of DSF/Cu on the mitochondrial membrane potential primary DLBCL cells were detected by JC-1 method.4.Western blotting was used to detect the expression of related protein of BCL6 in DLBCL cell lines and primary DLBCL cells after DSF/Cu treatment.5.Detection of Tfh and its subtypes in peripheral blood of DLBCL patients by flow cytometry.6.Detection of the effect of DSF/Cu on Tfh in DLBCL patients by flow cytometry.Results 1.Compared with normal control,the expression level of BCL6 gene was obviously higher in female than male patients.The expression of BCL6 in White patients was higher than that in Asia patients.2.The prognosis of DLBCL patients with high expression of BCL6 gene is better than that of low expression of BCL6 gene.3.GO/KEGG gene enrichment analysis showed that BCL6 related genes could be enriched in MAPK pathway.4.The results of immune infiltration showed that BCL6 was associated with a variety of immune cells.5.DSF/Cu induced apoptosis of primary DLBCL cells.6.DSF/Cu induced a decreased in mitochondrial membrane potential in primary DLBCL cells.7.After treatment of DSF/Cu,BCL6 was down-regulated,p53 was up-regulated in OCI-LY7 and OCI-LY10 cell lines and down-regulated in U2932 cell line.And BCL6 was down-regulated,BCL2 was up-regulated,BCL-XL was down-regulated,BAX remained unchanged and p53 was up-regulated or down-regulated in primary cells.8.BCL6 related proteins AIP and LITAF in DLBCL cell line and primary cells were down-regulated by DSF/Cu.9.p38 was down-regulated or remained unchanged,JNK and ERK were down-regulated in DLBCL cell lines and primary cells by DSF/Cu.10.In DLBCL patients,the frequency of circulating CD4+CXCR5+Tfh increased,Tfhl subtype decreased,Tfh2 and Tfh17 subtypes increased,CD4+CXCR5-Tph decreased,Tph1 subtype decreased,and Tph17 subtype increased;Circulating CD4+PD-1+T cells were up-regulated in patients with DLBCL,and PD-1 was highly expressed in Tfh,Tfh subtypes,Tph and Tph subtypes.11,CD4+CXCR5+Tfh,CD4+PD-1+T cells,CD4+CXCR5+PD-1+T cells,CD4+CXCR5-PD-1+T cells and CD4+CXCR5+BCL6+T cells significantly decreased,while CD4+CXCR5-Tph increased in DLBCL patients after DSF/Cu treatment.Conclusion 1.DSF/Cu induced apoptosis and decreased mitochondrial membrane potential of primary DLBCL cells.2.Down-regulation of BCL6 related ROS-MAPK signaling pathway may be one of the antitumor mechanisms of DSF/Cu.3.In DLBCL patients,the frequency of circulating CD4+CXCR5+Tfh increased,Tfh2 and Tfh17 subtypes increased,CD4+CXCR5-Tph decreased,Tph17 subtype increased,PD-1 was highly expressed in Tfh,Tfh subtypes,Tph and Tph subtypes.4.DSF/Cu may inhibit the expression of transcription factor BCL6 leading to inhibiting the differentiation of Tfh cells,and then intervene the growth process of B cells.