Studies Of A Monoclonal Antibody Targeting Neuropilin-1on Growth Inhibition Of Skin Cancer Cells And Its Mechanisms

Background and PurposeIn the abundant sunshine state, the morbidity rate of skin cancer is extremely high, its etiology is complex and pathogenesis is different. But traditional radiotherapy and chemotherapy are used in a restrict range for the disadvantages of low effects and large side effects. Molecular targeted drugs with high tumor-targeting ability and low side effects provide a new strategy for cancers. Recent studies show that Neuropilin-1(NRP-1) is a new target for cancer therapy. NRP-1is a multifunctional receptor and a single-spanning transmembrane glycoprotein. It is involved in vascular generation and activation and participated in the malignant phenotype of tumor through independent signal pathway as co-receptor of semaphorin3(Sema3), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and so on. Many studies proved that NPR-1was highly expressed in skin cancer and was tightly related to the initial and progress, but mechanism among them remains unclear. This paper aims to detect effect of anti-NRP-1mAb produced by our laboratory on the growth of skin cancer cell lines and explore the mechanism, which lay a foundation for the development of a new anti-tumor strategy targeting NRP-1.MethodsNRP-1mAb was produced from ascites of Balb/c mice and purified by a column with rprotein A. SDS-PAGE detected the concentration and purity of the antibody. Indirect ELISA was used to identify the specialty and titers of the NRP-1mAb respectively. The expressions of NRP-1in skin cancer cells A431, B16F10and their corresponding tissue were detected by flow cytometry, Western blotting, immunofluorescence and immunohistochemistry. MTT was used to evaluate the inhibitory effect of NRP-1mAb on proliferation. Cell morphology was observed by microscopy. Nuclear staining and flow cytometry were used to observe cell apoptosis. Western blotting detected the expression of Caspase-3, Bax, Bcl-2, PI3K (85), p-Akt, p-ERK1/2.ResultsThe purity of NRP-1mAb processed by rProteinA is as high as96.5%and indirect ELISA showed that the NRP-1mAb got the titer of1×10-6. Western blotting showed the bands of39KDa,100KDa and130KDa respectively, indicating the NRP-1mAb could bind to both recombinant NRP-1blb2and full-length NRP-1in cancer cells. Flow cytometry showed that skin cancer cell lines positively expressed NPR-1. Western blotting and immunofluorescence staining indicated that the NRP-1was localized on the membrane of skin cancer cell lines and immunocytochemistry showed that NRP-1highly expressed in the skin cancer tissues. MTT and colony forming assay showed that NRP-1mAb have inhibitory effect on the skin cancer cell lines in dose-dependent method and time-dependent method. Nuclear Hoechst staining and flow cytometry showed that NRP-1mAb led to apoptosis of skin cancer cell lines in dose-dependent method. Western blotting showed upregulation of the expression of Caspase-3and Bax, along with the downregulation of Bcl-2, PI3K (85), p-Akt and p-ERKl/2expression after treatment of NRP-1mAb on skin cancer cell linesConclusions1. We successfully produce NRP-1mAb with high purity, high titer and stable titration.2. NRP-1mAb can bind to the NRP-1expressed in the skin cancer cell lines.3. NRP-1mAb have inhibitory effect on the skin cancer cell lines in dose-dependent method and time-dependent method.4. The growth inhibitory effect of NRP-1mAb on the skin cancer cell lines is related to the down regulation of PI3K/Akt and MEK/ERK signaling pathway.