The Mechanism Of PP2A/c-MYC Signaling Pathway Affecting Drug Resistance In Diffuse Large B-cell Lymphoma

Objective: 1.To explore the expression of PP2A-56α and c-MYC in the parent and drug-resistant cells of diffuse large B-cell lymphoma(DLBCL);2.To investigate the effect of PP2A-B56α activator(DT-061)on the cell viability and apoptosis of rituximab resistant DLBCL cells;3.To study the effect of DT-061 on AKT/GSK-3β/c-MYC signaling pathway in DLBCL cells resistant to rituximab.Methods: 1.Two DLBCL cell lines(OCI-LY8 and OCI-LY18)resistant to rituximab,which were successfully constructed by using the "concentration gradient dosing method" in the previous project,were tested for drug resistance by CCK-8 法 and flow cytometry.2.DT-061 was treated with concentration gradient(1,5,10,15,20 μg/ml),respectively,and cytotoxicity test was used to detect the CDC effect of rituximab on drug-resistant DLBCL cells at 24 h,48h,72 h,respectively.3.Semi-inhibited DT-061 was co-cultured with rituximab resistant strains of DLBCL,and the cell viability and apoptosis rate of resistant cells were measured 48 h later by CCK-8 法 and flow cytometry.4.Western Blot was used to detect the expression of PP2 A and c-MYC proteins in DLBCL drugresistant cells and parent cells,and then semi-inhibited concentration of DT-061 was cocultured with DLBCL drug-resistant cells for 48 h,and cell proteins were extracted.The expression levels of PP2A-B56α,AKT,GSK-3β and c-MYC protein were detected by Western Blot and other methods,so as to study the effect of DT-061 on AKT/GSK-3β/cMYC signaling pathway and its antagonistic effect on Rituximab resistance and its molecular mechanism.Results: 1.After rituximab was treated with different concentrations(0,2,10,50,100 μg/ml),the cell viability of parent and drug-resistant cells of DLBCL decreased more significantly than that of drug-resistant cells,and the decreasing trend was concentrationdependent.The apoptosis rate of cells increased significantly,and the trend was also rituximab concentration dependent,and the results were statistically significant compared with the control group(P<0.05).2.DT-061 was treated with different concentrations(1,5,10,15,20 μg/ml)for 24 h,48 h and 72 h,and cell viability decreased in concentrationdependent and time-dependent manner,and the results were statistically significant(P<0.05).3.In the treatment of DLBCL drug-resistant cells with semi-inhibitory concentration of DT-061,the cell viability of drug-resistant cells decreased significantly,and the cell apoptosis rate increased significantly.4.Western Blot was used to detect the expressions of PP2A-B56α and c-MYC in DLBCL parent cells and drug-resistant cells respectively.Compared with the parent cells,the expression of PP2A-B56α in drugresistant cells was significantly decreased,while the expression of c-MYC was significantly increased.5.The protein expressions of PP2A-B56α,AKT,GSK-3β and c-MYC were detected by Western Blot after treatment with semi-inhibited concentration of DT-061.Compared with parental cells,the expression of PP2A-B56α and GSK-3βwere significantly increased,the expression of AKT and c-MYC were significantly reduced in the dosing group,the difference had statistical significance(P < 0.05).Conclusion: 1,The expression of PP2A-56α decreased obviously in Rituximab resistant DLBCL cell lines,while the expression of c-MYC rise significantly.2.DT-061 can reduce the cell viability and induce apoptosis of DLBCL cell lines resistant to Rituximab.3.By activating PP2A-B56α,DT-061 can decrease the expression of AKT protein in drug-resistant cells,enhance the activity of GSK-3β protein,accelerate the degradation of c-MYC protein and resist the resistance of rituximab,thus affecting the development process of rituximab resistant DLBCL.