Studies Of Monoclonal Antibody Targeting Neuropilin-2 On Anti-tumor Activity And Its Mechanism Of Colon Cancer Cells

Research background and purposeColon cancer is one of the common malignant tumors in China,the incidence in recent years is getting higher and higher.And the existing treatment has not been able to improve the poor prognosis of colon cancer.Therefore,the development of new therapeutic drugs and treatment programs are of great significance.At present,molecular targeted therapy is a hot topic in clinical research.Colon cancer commonly used targeted drugs are bevacizumab,cetuximab,etc.,mainly for epidermal growth factor receptor(EGFR),vascular endothelial growth factor(VEGF)and other targets,clinical treatment is often treated with chemotherapy combined with targeted drug therapy.Neuropilin-2(NRP-2)is highly expressed in colon cancer cells.Its ligands and receptors,such as VEGF and VEGFR,Semaphorin and Plexins,TGF-?and TGFR,HGF and c-met,are involved in the conduction of multiple signaling pathways,and play an important role in the formation of tumor blood vessels,regulate cell proliferation and apoptosis,death,migration,invasion and other biological reactions.Therefore,NRP-2 may become a new target for tumor therapy.The aim of this study is to prepare anti-NRP2-b1b2 fragment monoclonal antibody by hybridoma technique,and then study the anti-tumor effect of the antibody on colon cancer and its mechanism,which provided a new idea for the targeted therapy of colon cancer.MethodsThe laboratory had constructed 3 hybridoma cell lines C3?E4?E8 that secreted monoclonal antibodies against NRP2-b1b2.After in vitro expansion,the cells were injected into the abdominal cavity of Balb / c mice,and then ascites containing NRP2-b1b2 monoclonal antibody was obtained.The antibody was purified by rProtein A affinity column,then dialyzed into dialysis bag and lyophilized on freeze dryer.Then,the purity and concentration of the antibody were analyzed by SDS-PAGE electrophoresis.The activity of the antibody was identified by indirect ELISA.The binding of NRP2-b1b2 monoclonal antibody to NRP2 protein in colon cancer cell line and colon cancer tissue was detected by flow cytometry,Western blotting,immunofluorescence and immunohistochemical staining.CCK8 assay,apoptosis assay and Transwell assay were used to detect the effect of NRP2 antibody on the biological behavior of colon cancer cells.The expression of Bcl-2 / Bax / Caspase-3,PI3 K / Akt,MEK / ERK and other signal pathways were analyzed by Western blotting.The anti-tumor mechanism of NRP2 antibody was also analyzed.ResultsThe results show that the prepared NRP2 mAb has high purity and good affinity,the concentration is about 1mg / mL,the titer is about 1 × 10-6.Western blotting showed that the total protein of four colon cancer cells had a specific band at 34 KDa and 130 KDa,respectively,which indicated that NRP2 mAb had high specificity,and that four colon cancer cells had NRP2,in which colon cancer cell SW480 was expressed highest.Flow cytometry and immunofluorescence assay showed that NRP2 antigen was positive in colon cancer cells and was mainly expressed in cell membrane.Immunohistochemical results showed that the expression of NRP2 in colon cancer was up-regulated compared with adjacent tissues.At the same time,different pathological grades and different tumor staging colon cancer tissue staining results are different.CCK8 experiment,apoptosis test,Transwell experiments show that NRP2 mAb can significantly inhibit colon cancer SW480 cell proliferation,migration,invasion,and promote cell apoptosis,and showed a certain concentration-dependent and time-dependent.Western blotting showed that the expression of Caspase-3 and Bax was up-regulated and the expression of Bcl-2,PI3 K,p-ERK and p-AKT was down-regulated after NRP2 mAb was detected in colon cancer cells,indicating that the anti-tumor mechanism of NRP2 mAb might be related to MEK / ERK,Bcl-2 / Bax / Caspase-3,PI3 K / Akt signaling pathway.Conclusions1.The NRP2 mAb prepared in this experiment has high purity and good affinity.2.NRP2 mAb can well bind specifically to colon cancer cells and NRP2 proteins in colon cancer tissues.3.NRP2 mAb significantly inhibited colon cancer SW480 cell proliferation,migration,invasion,promoted cell apoptosis.4.The mechanism of NRP2 mAb on the proliferation and apoptosis of colon cancer cells was related to MEK / ERK,Bcl-2 / Bax / Caspase-3 and PI3 K / Akt signaling pathway.