The Investigation Of Effect Of Apatinib On The Diffuse Large B-cell Lymphoma Cells And Its Molecular Mechanism

BackgroundDiffuse large B-cell lymphoma(DLBCL)is the most common type of non-Hodgkin lymphoma and accounts for 30%to 40%of new diagnoses.DLBCL is an aggressive lymphoma that is heterogeneous clinically,morphologically,biologically and cytogenetically.In recent years,the introduction of antilymphoma monoclnoe antibodies,rituximab,in combination with chemotherapy(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisolone,R-CHOP)has significantly improved the survival outcomes of the patients with DLBCL.While,nearly up to one-third patients suffered from relapsed and refractory disease after R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisolone)treatment,and limited patients got benefits from the salvage strategy of autologous stem cell transplantation(ASCT),indicating new agents are required to be tested in this group of people.Nowadays,the targeted drugs are the focus of the research in the malignant tumor.Angiogenesis plays an important role in tumor development,progression,metastasis,and selective inhibitors of angiogenesis have become a strategy for anti-tumor therapy.At present,more and more targeted angiogenic drugs are used clinically in tumors.Apatinib is an inhibitor of vascular endothelial growth factor receptor-2(VEGFR-2)tyrosine kinase,which is an inhibitor of targeted blood vessels.Its main mechanism is competitive binding to the intracellular tyrosine ATP binding site,Highly selective inhibition of VEGFR2 tyrosine kinase activity,blocking the signal transduction of VEGFR2 binding to endothelial growth factor(VEGF),which effectively inhibit tumor angiogenesis.After the binding of the VEGF and VEGFR2,they can activate a variety of signaling pathways,including PI3K/Akt,Ras pathway.Ras pathway is closely related to the vast majority of human tumors.Apatinib can inhibit the activation of VEGF/VEGFR2,and inhibit its activation of Ras signaling pathway and play an anti-tumor effect.ObjectiveApatinib,a small-molecule vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor,has an anti-tumour effect on kinds of tumor.We investigate the effect of apatinib on the proliferation and apoptosis of Diffuse large B-cell lymphoma(DLBCL)cell line,including oci-lyl,SU-DHL-4(S4)(the subtypes of GCB)and oci-ly3、SU-DHL-2(S2)(the subtypes of ABC).Furthermore,we will explore the relationship between the role of apatinib in the killing of diffuse large B-cell lymphoma and the Ras signaling pathway.Method1.Using the cell counting kit-8(CCK-8)to detect the proliferation inhibition rate after treatment with various concentration(2.5、5、10、20、40μmol/L)of apatinib for 48h on DLBCL cells,and applied graph prism5 to count the corresponding IC50 values.2.Using the Annexin-V/PI double staining assay flow cytometric analysis the percentage of apoptotic cells in DLBCL after treatment with various concentration(0、10、20、30、40μmol/L)of apatinib for 48h.3.Western blotting analysis of the expression level of proteins of pVEGFR2 and the Ras signaling pathway(Ras、c-Raf、pMEK1/2 and pErK1/2),after treatment with apatinib for 48 h on oci-lyl and SU-DHL-2 cells.4.The statistical analyses were performed with the statistical software SPSS 19.0.Because of the small sample,non-parametric test Mann-Whithey U test was used to compare the two groups of independent samples;non-parametric test Kruskal-Wallis H test was used to compare the difference of rates in different groups.A value of P<0.05 was accepted as an indication of statistical significance.Results represent the mean± SEM of at least three independent experiments.All tests were bilateral test.Results1.The cell counting kit-8 cytotoxicity experiment results indicated that various concentration apatinib could significantly inhibit the GCB-DLBCL cells(oci-ly1 and SU-DHL-4)proliferation with dose-dependent mode:for 48h,the IC50 are 19.30±0.07 and 18.15±0.15,and the differences had statistical significance compared with control group(χ2=16.460,P=0.006 and χ2=16.248,P=0.006).AS well as,the cell counting kit-8 cytotoxicity experiment results indicated that various concentration apatinib could significantly inhibit the ABC-DLBCL cells(oci-ly3 and SU-DHL-2)proliferation in a dose-dependent mode for 48h:the IC50 are 15.29±0.13 and 12.34±0.27,and the differences had statistical significance compared with control group(x2=16.460,P=0.006 and χ2=16.648,P=0.005).The proliferation inhibition rate of ABC-type cell oci-ly3 was significantly higher than that of two GCB-type cell lines oci-lyl and SU-DHL-4(P =0.047 and P=0.047).Similarly,the proliferation inhibition rate of SU-DHL-2 was higher than that of two GCB cell lines oci-ly1 and SU-DHL-4(S4)(P =0.047 And P =0.047).2.The results of Annexin V/PI showed that various concentration of apatinib induced significantly apoptosis on GCB-DLBCL cells(oci-lyl and SU-DHL-4)in a dose-dependent mode,and the differences had statistical significance compared with control group(χ2=12.767,P=0.012 and χ2=13.233,P=0.010).The results of Annexin V/PI showed that various concentration of apatinib induced significantly apoptosis on ABC-DLBCLcells(oci-ly3 and SU-DHL-2)in a dose-dependent mode,and the differences had statistical significance compared with control group(χ2=13.033,P=0.011 and χ2=12.933,P=0.012).The apoptosis rate of ABC-type cell line oci-ly3 was not significantly different from that of two GCB cell lines oci-ly1 and SU-DHL-4(S4)by Mann-Whitney U analysis(P = 0.275 and P=0.06).Similarly,the apoptosis rate of another ABC-type cell line SU-DHL-2 was not significantly different from that of GCB cell lines oci-ly1 and SU-DHL-4(S4)(P = 0.275 and PP=0.06).3.Western blotting results indicated decrease the expression of the pVEGFR2,and inhibited the Ras(Ras,c-Raf,pMEK1/2,pErk1/2)pathway in oci-ly1 and SU-DHL-2 cells treated with apatinib for 48h.After the relative grayscale analysis of the protiens,the expression of pVEGFR2,Ras,c-Raf,pMEK1/2,pErk1/2 protein in the apatinib group(20,40μmol/L)has the statistically significant(all χ2 = 7.2,all P =0.027)with the control group(0μmol/L).Conclusions1.Apatinib can inhibit the proliferation and induce the apoptosis of DLBCL cells(oci-ly1,SU-DHL-4,oci-ly3,SU-DHL-2),and apatinib was more effective in the proliferation inhibition of ABC-type DLBCL cell lines.2.Apatinib selectively binds to and inhibits VEGFR2 which may inhibit VEGF/VEGFR2 signaling pathway and then inhibited the downstream-Ras pathway,thereby playing a killing effect on DLBCL cells.