Study On The Drug-resistance Mechanism And Molecular Targeted Therapy Of Non-GCB Diffuse Large B Cell Lymphoma

PART Ⅰ PRDMl/Blimpl is involved in the doxorubicin resistance of non-GCB DLBCLObjective:To investigate drug-resistance mechanisms in non-germinal center B cell(non-GCB)diffuse large B cell lymphoma(DLBCL).Methods:Doxorubicin(DOX)resistant OCI-Ly3 cells were generated through long-term incubation of cells in the medium with gradually increasing concentrations of DOX.Expression of genes related to drug metabolism were determined using a Functional Gene Grouping PCR array.Drug resistant proteins were identified using bioinformatics,and molecular association networks were subsequently generated.The association and mechanism of key genes was determined using a dual-luciferase reporter assay system and CHIP.Expression of drug resistant genes and target genes was then measured using Western blotting and immunohistochemistry.The correlation between gene expressions was analyzed using Spearman’s rank correlation coefficient.Results:Using a PCR array,MDR1 was identified as the key gene that regulates DOX resistance in OCI-Ly3/DOX-A100,a non-GCB DLBCL cell line.The dual-luciferase reporter assay system demonstrated that the transcription of MDR1 could be inhibited by PRDM1.CHIP results showed that PRDM1 had the ability to bind to the promoter region(-1132 to-996)of MDR1 gene.In OCI-Ly3/DOX cells,the activity of NF-κB and the expression of PRDM1 decreased with an increase in drug resistant index,while the expression of MDR1 increased with enhanced drug-resistance.OCI-Ly3/DOX-A100 cells were infected with lenti-virus particles contain PRDM1 gene.The results showed that the MDR1 expression level of the cells decreased rapidly accompany with over-expression of PRDM1 and the ability of drug-resistance decreased,and the sensitivity of the OCI-Ly3/DOX-A100 cells to doxorubicin increased;Xenograft model of OCI-Ly3/DOX-A100 cells treatment experiment confirmed that the growth of transplanted tumor could be significantly inhibited with treatment of zosuquidar combined with doxorubicin in nude mice,compared with treatment of doxorubicin or zosuquidar alone.P<0.01 vs control;Immunohistochemical analysis revealed that relative MDR1 expression was higher than that of PRDM1 in human DLBCL tissue samples.A negative correlation between MDR1 and PRDM1 was found.Conclusion:OCI-Ly3/DOX cell line of non-GCB DDLBCL was successfully induced and obtained,and the chemoresistance index was about 40 times more than that of the parental cell OCI-Ly3.PRDM1 is a negative regulator of MDR1;PRDM1 can bind to the promoter of MDR1 and inhibit the expression of MDR1.MDR1 is an important molecule involved in the drug-resistance of OCI-Ly3/DOX cells;Chemoresistance of OCI-Ly3/DOX cell can be reversed by MDR1 inhibitor Zosuquidar(ZSQ);Chemoresistance of OCI-Ly3/DOX cell can be reversed when the expression of PRDM1 restored.Weakening of the NF-kappa B signal can down regulate the expression of PRDM1,and remove the transcriptional repression of MDR1,triggering the expression of MDR1 transcription.In clinical samples,the expression of PRDM1 was negatively correlated with the expression of MDR1.PRAT ⅡCombination of Oridonin and PI3K/mTOR inhibitor NVP-BEZ235 in non-GCB type of DLBCLObjective:To investigate the antitumor effect and its molecular mechanisms in non-germinal center B cell(non-GCB)diffuse large B cell lymphoma(DLBCL)with combination of Oridonin and PI3K/mTOR inhibitor.Methods:CCK-8 assay was used to observe the influence of Oridonin and NVP-BEZ235 alone or combined on cell proliferation,and to assess the whether the two drugs have synergistic effect.Flow Cytometry was performed to exam the apoptosis and cell cycle.The intrinsic apoptosis pathway,AKT/mTOR pathway,NF-κB pathway and the marker of DNA damage yH2AX were detected by Western blot.The changes of intracelluar reactive oxygen species(ROS)was also detected by the flow cytometry analysis.The combined treatment inducing tumor regression in vivo was performed in nude mice of SU-DHL-2 xenograft mouse model.Results:The combination of Oridonin and PI3K/mTOR inhibitor NVP-BEZ235 was more effective for inhibiting cell proliferation compared with single-agent therapy.Oridonin combined with NVP-BEZ235 remarkably increased apoptosis in tumor cells but the synergistic effect cannot obviously increased cell cycle arrest.Combination treatment downregulated the AKT/mTOR and NF-κB signaling pathway and caused reactive oxygen species(ROS)-mediated DNA damage response.ROS inhibitor NAC reversed apoptosis and decreased the phosphorylation levels of histone H2AX.Mice treated with Oridonin/NVP-B EZ235 represented obvious tumor growth regression and prolonged overall survival.Conclusions:Our findings demonstrated the synergistic antitumor effect of Oridonin and NVP-BEZ235 in non-GCB type of DLBCL,the underlying mechanism may be multifunctional,involving apoptosis,AKT/mTOR and NF-κB inactivation and ROS-mediated DNA damage response.Cotreatment was also effective in vivo.These data pave the way for potential treatment of non-GCB type of DLBCL with combination of Oridonin and NVP-BEZ235.