Neuropilin-1 (NRP-1) is a nontyrosine kinase coreceptor for semaphorin 3A and the vascular endothelial growth factor involved in tumor angiogenesis, growth, and metastasis and is regarded as a promising target for cancer therapy. In the present study, we investigated the effects of an anti-NRP-1 monoclonal antibody (mAb) that we generated for MCF7 breast cancer cellular adhesion studies. MTT, colony formation, and adhesion assays showed that our anti-NRP-1 mAb dose-dependently inhibited MCF7 proliferation and fibronectin adhesion, leading to a rounded cellular morphology. Further, rhodamine phalloidin stain revealed that fibronectin-dependent formation of actin stress fibers was inhibited by anti-NRP-1 mAb. Immunoprecipitation and western blot showed that anti-NRP-1 mAb treatment inhibited the formation of NRP-1-α5β1 integrin complexes and suppressed the phosphorylation of focal adhesion kinase and p130cas in MCF7 cells. These findings contribute to further understanding the NRP-1 function in cell adhesion and tumor metastasis. Moreover, our anti-NRP-1 mAb is a prospective drug candidate for tumor treatment.
Our previous work indicates the lymphatic network and perivascular spaces or tissues might be involved in the facial intradermal brain-targeted delivery of Evans blue (EB). In this article, we presented the detailed involvement of both, and the linkage between lymphatic network and perivascular spaces or tissues. The
in-vivo
imaging, the trigeminal transection and immunohistochemistry were used.
In-vivo
imaging indicated intradermal injection in the mystacial pad (i.d.) delivered EB into the brain at 2-, 6- and 24 h, while intranasal injection (i.n.) delivered EB into the rostral head and intravenous injection (i.v.) diffused EB weakly into the brain. Trigeminal perineurial and epineurial EB occurred along the perivascular spaces or tissues and along brain vessels. EB diffused into the lymphatic vessels and submandibular lymph nodes. Moreover, perineurial and epineurial EB co-located or overlaid with Lyve1 immuno-reactivity and VEGF antibody, and lymphatic network connected with perivascular spaces or tissues, suggesting lymphatic system-perivascular spaces might involve in the EB delivery with i.d. The trigeminal transection reduced the trigeminal epineurial and perineurial EB and brain EB along vessels. EB diffused in the fasciculus and the perineurium, blood and lymphatic vessels in the mystacial pad, mystacial EB overlaid VEGF or Lyve1 antibody. In summary, the dermal-trigeminal-brain perivascular spaces or tissues and the linkage to the lymphatic network mediated the intradermal brain-targeted delivery.
We investigated the inhibitory effects of anti-Neuropilin-1 monoclonal antibody (NRP-1 MAb) on adhesion of glioma cells to Fibronectin (FN) and the associated mechanisms. The effects of NRP-1 MAb on adhesion of U251 glioma cells and formation of stress fibers were studied using adhesion assays and confocal fluorescence microscopy. After treatment of U251 glioma cells with NRP-1 MAb, changes of related proteins adhering to FN were detected by co-immunoprecipitation and Western blot. Adhesion assays showed that NRP-1 MAb played an inhibitory role in adhesion of U251 glioma cells. Confocal fluorescence microscopy, co-immunoprecipitation and Western blot showed that NRP-1 MAb can inhibit the formation of stress fibers adhering to U251 cells. NRP-1 MAb can also affect the formation of Integrin 5 1-NRP-1 complex, and also reduce the level of phosphorylation of FAK and p130cas. In conclusion, NRP-1 MAb has the inhibitory effects on adhesion of glioma cells to FN. This effect is related to the inhibition of NRP-1 MAb on the formation of Integrin 5 1-NRP-1 complex, which leads to the inhibition of FAK/p130cas signaling pathway.
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