The Effect Of PI3K Inhibitor PI-103 On The Proliferation Of Diffuse Large B Cell Lymphoma

Lymphoma is a group of malignant tumors originating in lymph nodes and extra-nodal lymphoid tissue,which can be classified into Hodgkin’s lymphoma(HL)and non-Hodgkin’s lymphoma(NHL)according to pathological.Diffuse large B-cell lymphoma(DLBCL)is the most common type of non-Hodgkin’s lymphoma and is the most common lymphatic malignancy in adults.Although 60%of patients with DLBCL are remission with the R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisone),Clinical studies has shown that one-third of DLBCL patients still have the potential for relapse.Also,there are some refractory patients who are not sensitive to this treatment.The phosphatidylinositol 3kinase(PI3K)pathway is highly and frequently activated in tumors and effects tumor cell growth,proliferation,survival,motility,invasion and angiogenesis through the production of phospholipids,phosphatidylinositol-3,4,5-trisphosphate.The PI3K/Akt/mTOR signaling pathway play a major role in the proliferation and survival of a variety of malignancies,including DLBCL.Therefore,PI3K is expected to be a potential therapeutic target in DLBCL,and inhibition of PI3K activity could be an effective therapeutic approach for DLBCL.ObjectiveIn this study,we used OCI-LY10,OCI-LY7,HBL-1 and SC-1 cell to investigate the effect of PI3K inhibitor PI-103 on the proliferation of human DLBCL cell lines.Highthroughput sequencing and bioinformatics were also used to explore the molecular mechanism of PI-103 and to provide a basis for clinical application.Methods(1)Trypan blue exclusion assay was used to detect the effects of PI-103 on the proliferation of human diffuse large B cell lymphoma cell lines in vitro.(2)The CCK-8 assay was used to detect the effect of PI-103 drug concentrations on the proliferation of human diffuse large B cell lymphoma cell lines,and detect the half maximal inhibitory concentration(IC50)on human DLBCL cell lines.(3)The subcutaneous tumor was used to detect the effect of PI-103 on the proliferation of human DLBCL cells in vivo.(4)Propidium Iodide staining and flow cytometry were used to detect the cell cycle changes of diffuse large B cell lymphoma cell lines treated with PI-103 and DMSO control treatment.(5)The cell cycle of DLBCL cell lines was detected by EdU assay and flow cytometry with PI-103 and DMSO control treatment.(6)The apoptosis of DLBCL cell lines at PI-103 and DMSO control treatment were detected by Annexin V and flow cytometric.(7)RNA-seq and bioinformatics was used to detect the effect of PI-103 on the expression of related genes and signaling pathways.(8)Real-time qPCR analyses were performed to verify the changes of PTPN6 at the transcriptional level in human diffuse large B cell lymphoma cell lines treated with PI-103.(9)WB were used to examine the changes of PTPN6 at the translational level in human diffuse large B cell lymphoma cell lines treated with PI-103.(10)WB were used to examine the changes of AKT phosphorylation in human diffuse large B cell lymphoma cell lines treated with PI-103.(11)PTPN6 was cloned into MSCV PIG vector,PTPN6 overexpression plasmid was constructed,and human DLBCL cell line OCI-LY10 was retrovirally infected to overexpress PTPN6,and the effect of PI-103 on the cycle of the overexpressed cell line was also examined.Results(1)The results of viable cell count assay and CCK-8 assay showed that PI-103 inhibited the proliferation of human DLBCL cell lines in a dose-and time-dependent,and the number of viable cells decreased significantly with the increase of drug concentration and treatment time.(2)Subcutaneous tumor showed that PI-103 could inhibit the proliferation of DLBCL cells in vivo.(3)Cell cycle assay showed that PI-103 could block human DLBCL cell lines in G1 phase,and with the increase of drug concentration the block of G1 phase was more obvious.(4)Apoptosis assay showed that PI-103 did not increase the proportion of apoptotic cells in human DLBCL cell lines.(5)The results of bioinformatic analyses showed that the expression of genes was significantly up-or down-regulated in OCI-LY10 cell after PI-103 treatment,and PTPN6 being the most significantly down-regulated gene.GSEA analysis suggested that PI-103 may be directly regulate cell cycle.(6)Real-time qPCR showed that PI-103 inhibited the expression of PTPN6 at the transcriptional level in human DLBCL cell lines.(7)WB showed that PI-103 could inhibit the expression of PTPN6 at the translational level in human DLBCL cell lines,also inhibit the phosphorylation of AKT.(8)Overexpression of PTPN6 can rescue the effect of PI-103 on the proliferation of human DLBCL cell lines.Conclusions(1)PI-103 inhibits cell proliferation in vitro by blocking diffuse large B cell lymphoma cell lines in the G1 phase.(2)PI-103 significantly inhibits the proliferation of human diffuse large B cell lymphoma cell lines by targeting PI3K,PTPN6,and phosphorylation of AKT.