Impact And Mechanism Of Melatonin On Diffuse Large B-cell Lymphoma Cells' Sensitivity To Epirubicin

Background and Objective:Diffuse large B-cell lymphoma(DLBCL)is the most common type of B-cell non-Hodgkin's lymphoma.As a malignant invasive tumor with obvious heterogeneity,the patient covers all ages and has extensive clinical manifestations.The R-CHOP regimen consisting of anthracyclines is a first-line treatment for DLBCL.Epirubicin is widely used as a third-generation anthracycline chemotherapy for the treatment of DLBCL.Although the clinical treatment of DLBCL is significant,it can be cured even in advanced stages,but as many as one-third of patients still relapse or become resistant during treatment.These patients often have poor clinical prognosis and high mortality.Multidrug resistance is considered to be the main cause of reduced sensitivity to chemotherapy in patients with DLBCL.The expression of P-glycoprotein(P-gp)is one of the main factors determining multidrug resistance.Studies have shown that melatonin can affect the expression of P-gp,and has the possibility of increasing the sensitivity of tumors to chemotherapy drugs.The purpose of this study was to investigate whether melatonin has a sensitizing effect on epirubicin in DLBCL and its specific molecular mechanism.Methods:1.Using CCK8 assay to detect the cell viability of SUDHL-10 and SUDHL-6 cell lines under different treatments(melatonin alone,epirubicin alone,and combination of melatonin and epirubicin).To reflect the effect of combined melatonin on the proliferation of DLBCL cell lines;2.To detect the apoptosis of SUDHL-6 cell line under different drug treatment groups by AO/EB assay.Western blotting and cytochrome c release experiments were used to further reveal the molecular mechanism of by which melatonin was combined to promote apoptosis of DLBCL cell lines;3.Western blotting and immunofluorescence experiments were used to detect the expression of P-gp of SUDH-10 and SUDHL-6 cell lines under different drug treatment groups.At the same time,rhodamine-123 accumulation test and epirubicin accumulation test were used to detect the function of P-gp;4.Under different concentration gradients of epirubicin,expression of NF-?B P65 and P-gp in SUDHL-10 and SUDHL-6 cell lines was detected by western blotting.After higher concentrations of epirubicin to induce high expression of P-gp in DLBCL cell lines,the specific inhibitors of NF-?B pathway were used to reflect the relationship between P-gp and the NF-?B pathway in the DLBCL cell line;5.Western blotting assay was used to detect the expression of NF-?B pathway after melatonin treatment;Pulldown assay was used to detect the combination between NF-?B P65 and ABCB1 gene promoter region.Results:1.Comparing with epirubicin alone,the combination of melatonin and epirubicin inhibited cell proliferation more significantly,and the IC50 value of epirubicin decreased significantly.In particular,when the two drugs were used in the SUDHL-6 cell line,the IC50 value of epirubicin decreased from 156.388 ?g/L to69.827 ?g/L;2.Combination of melatonin and epirubicin increases the expression of pro-apoptotic proteins,decreases the expression of apoptosis-inhibiting protein(BCL-2),and promotes cytochrome C release from mitochondria to cytoplasm;3.Comparing with the control group,epirubicin alone can increase P-gp expression.After epirubicin combined with melatonin,the tendency of P-gp increase is inhibited.Combing with melatonin treatment can inhibit the function of P-gp and increase the accumulation of rhodamine 123 dye or epirubicin in cells;4.With the increasing drug concentration of epirubicin,the expression of P-gp and NF-?B P65 in DLBCL cell line also showed an increasing trend.After QNZ blocked the NF-?B pathway,the expression of P-gp was also inhibited;5.When treated with epirubicin alone,the NF-?B P65 in the nucleus of SUDHL-10 and SUDHL-6 increased,and the binding to the promoter region of ABCB1 gene also increased.This increased trend is inhibited after melatonin is combined with epirubicin.Conclusions:1.Melatonin can enhance the inhibitory effect of epirubicin on the proliferation and apoptosis of DLBCL cell lines;2.Melatonin can enhance the sensitivity of DLBCL cell lines to epirubicin by affecting NF-?B pathway and the expression of P-gp.These findings not only provide melanin as a potential adjuvant for chemotherapy,but also provide a new perspective for the treatment of refractory diffuse large B-cell lymphoma.