| Neuropilin-2(NRP2)is a multifunctional receptor that plays an important role in the development of nervous,cardiovascular and immune system etc.NRP2 is low or not expressed in normal adult tissues,but highly expressed in various tumor cell membranes,such as colon cancer,breast cancer and glioma.NRP2 can co-receptor with vascular endothelial growth factor receptor,participate in neovascularization and lymphatic angiogenesis,and participates in the regulation of biological functions such as proliferation,migration and invasion of cancer cells.Because NRP2 is highly expressed on various tumor cell membranes and is involved in the regulation of biological characteristics of cancer cells,NRP2 has been considered as a good biomarker and molecular targeted therapeutic target for cancer cells.Using cell fusion technology to prepare functional antibodies against NRP2 and develop targeted therapy for NRP2 is a novel strategy.Different colon cancer cells express different levels of NRP2 protein,and the regulatory effects of NRP2 on the biological activity of different cancer cells and the molecular mechanisms involved are also different.Up to now,the effect of anti-NRP2 antibody on the biological activity of different colon cancer cell lines and its molecular mechanism has not been clarified.The aim of this study is to detect the anti-neoplastic activity of anti-NRP2 blb2 monoclonal antibody(Anti-NRP2 mAB)prepared in our laboratory against colon cancer cell lines and to study its molecular mechanism,so as to provide basic parameters and research route for the development of anti-NRP2 antibody drugs targeting NRP2.METHODS:Antibodies were produced from ascites of mice,purified by protein A column,identified by SDS-PAGE electrophoresis,detected byELISA,identified by WB and immunohistochemical staining.Changes of cell morphology induced by antibody were observed by optical microscopy.The effect of antibody on the viability of different colon cancer cell lines was detected by CCK8 assay.Colony forming assay was used to observe the inhibitory effect of antibody on the growth of colon cancer cells.Flow cytometry and Annexin V-PI staining were used to observe the apoptosis induced by antibody.Migration test and Transwell invasion test were used to detect the effect of antibody on the migration and invasion of colon cancer cells.CRISPER technique was used to establish nrp2 knockdowned colon cancer cell line.JC-1 staining was used to detect the effect of antibody on mitochondrial membrane potential.Western blot was used to detect apoptotic signal molecular and EMT related protein levels.ROS production was detected by H2-DCFDA probe.Cytochrome C and COX IV distribution and EMT key molecule expression were detected by cellular immunofluorescence assay.SW480 and SW620 xenotransplantation mice models were used to evaluate the inhibiton of antibodies on tumor growth.RESULTS:SDS-PAGE electrophoresis results showed that Anti-NRP2 mAB purified by protein A column had high purity and few impurity proteins.The titer of antibody activity determined by ELISA was over 1:105.Anti-NRP2 mAB showed good specificity by immunohistochemical staining.Immunohistochemical staining analysis of 75 normal and 103 samples of colon cancer showed that NRP2 was highly expressed in most of colon cancer,but not in normal colonic tissues.Microscopic observation showed that anti-NRP2 mAB could cause significant changes in growth morphology of SW480,SW620,HT29 and LoVo cell lines.In Anti-NRP2 mAB treated group,colon cancer cells became rounded,exfoliated and died.CCK8 results showed that Anti-NRP2 mAB significantly inhibited the growth of SW480,SW620,HT29 and LoVo cell lines in a dose-and time-dependent manner.Colony formation results showed that Anti-NRP2 mAB inhibited colony formation in low-dose group,while in 200 μg/ml high-dose group,SW480 and SW620 colon cancer cells completely inhibited colony formation.Flow cytometry and Annexin V-PI staining showed that Anti NRP2 mAB induce significantly apoptosis in SW480 and SW620 cells.Migration and Trans well invasion experiments showed that Anti-NRP2 mAB could significantly inhibit the migration and invasion of SW480 and SW620 cells.Wound healing assay showed that anti-NRP2 mAB significantly inhibited SW620 migration but knocking down Nrp2 did not inhibit SW620 migration.JC-1 staining assay showed that anti-NRP2 mAB could decrease the mitochondrial membrane potential of SW480 and SW620 cells.H2-DCFDA probe assay showed that anti NRP2 mAB could increase ROS production in SW480 and SW620 cells.Immunofluorescence and Western blot showed that anti-NRP2 mAB could increase the distribution of cytochrome C in the cytoplasm of SW480 and SW620 cells.Western blot results showed that Anti-NRP2 mAB could up-regulate the expression of caspase-3/9 and Bax protein,but the level of Caspase-8 protein did not change significantly.Anti-NRP2 mAB also down-regulated the expression of Bcl-2 protein in SW620 cell line.The results of EMT biomarker test showed that Anti-NRP2 mAB up-regulated the expression of E-cadherin and down-regulated the expression of vimentin and N-cadherin in SW620 cell line,similar to the result of knocking down Nrp2 in SW620 cell line.However in SW480 cell line,the results showed that the expression of N-cadherin and vimentin was significantly up-regulated after Anti-NRP2 mAB treatment,while the expression of E-cadherin was slightly up-regulated.It was observed that Anti-NRP 2 mAB inhibit significantly the growth of SW480 and SW620 xenotransplantation mice in a dose-dependent manner.Conclusion:our results suggest that Anti-NRP2 mAB can significantly inhibit the proliferation,migration,invasion and EMT of colon cancer cell lines SW480 and SW620;the inhibition of Anti-NRP2 mAB on the proliferation of colon cancer cells may be partly related to its induction of apoptosis mediated by caspase-9/cytochrome c;In SW620 cell line,the EMT inhibition of Anti-NRP2 mAB was related to the up-regulation of E-cadherin and down-regulation of vimentin and N-cadherin expression.In SW480 cell line,the EMT inhibition of Anti-NRP2 mAB was mainly related to the up-regulation of E-cadherin expression by Anti NRP2 mAB.Anti NRP2 mAB can inhibit the growth of transplanted tumors of colon cancer cell lines SW480 and SW620 in vivo,which indicates that Anti-NRP2 mAB is a promising candidate therapeutic antibody. |
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