Studies On Netrin-1 Expression, Function And Mechanisms During The Process Of Vascular Mimicry And Angiogenesis

Objective:Netrin-1 which is on axonal growth and guidance and Matrix metalloproteinase-2 (MMP-2) play very important role in cancer cell proliferation, survival and angiogenesis. MMP-2 activity is critical in the formation of vasculogenic-like networks. While the relationship between Netrin-1 pathway and formation of vasculogenic mimicry (VM) remains unclear. In this study, we will explore relationship between Netrin-1 and MMP-2 to investigate the role of Netrin-1 in the formation of VM in lung cancer.Methods:53 resected tissues were collected including 45 patients with lung cancer and 9 patients with lung benign disease during from May,2008 to Decem,2009 in the Union Hospital and Xiang fan Center Hospital, tongji Medical College, Huazhong University of Science and Technology. Periodic acid schiffreaction(PAS) histochemistry and immunohistochemistry double staining was used to observe the occurrence of VM in lung tissues. Immunohistochemistry was used to detect the expression of Netrin-1 and MMP-2 in pathological tissue sections. The correlation of Netrin-1 and VM in lung tissues was analyzed statistically.Results:Netrin-1 was expressed in 23 cases (51.1%), MMP-2 in 27 cases (60%) in lung cancer tissue sections. Netrin-1 was expressed in 1 case (11.1%) and MMP-2 in 2 cases (22.2%) in lung benign disease tissue sections. The expression of Netrin-1 and MMP-2 is higher in lung cancer than that in lung benign disease. VM existed in 6 cases (13.3%) with lung cancer, while the expression of Netrin-1 and MMP-2 was positive. The occurrence of VM was 0% in those cases in lung cancer which the expression of Netrin-1 was negative.Conclusion:VM was existed in lung cancer. Within these lung cancers, Netrin-1 and MMP-2 were highly expressed.Netrin-1 might play an important part in the formation of VM by MMP-2 signal pathway in lung cancer. The advanced research is needed to investigate the precise mechanism of the formation of VM in lung cancer.Objective:Our studies suggested that Netrin-1 might play an important role in the formation of VM by MMP-2 signals pathway in lung cancer A549 cells. The precise molecular mechanism of Netrin-1 on VM in lung cancer remains unclear. In the present study, we studied the effect of Netrin-1 on the formation of vasculogenic mimicry channels of lung cancer cells in vitro.Methods:The migration and invasion activity of A549 cells was detected by Transwell chamber assay. The effect of Netrin-1 on the tube formation of A549 was observed by the tube formation assay. Western blot assay and RT-PCR was used to investigate the expression of DCC?MMP-2 and 9 in A549cells after treated with Netrin-1.Results:Netrin-1 increased the migration and invasion activity of A549 cells. After treated with Netrin-1 at 24h, the expression of DCC?MMP-2 and 9 was up-regulated by western blot assay and RT-PCR. The effect of Netrin-1 on the tube formation of A549 was increased by the tube formation assay.Conclusion:A549 cells can form cords and vascular networks when cultured on 3-D collagen matrices. Netrin-1 can enhance the ability to form vascular networks of A549 cells through increasing cell proliferation, the activity of migration and invasion, the expression of DCC and MMP-2/9 were up-regulated to augment the ability of tube formation in lung cancer cells A549.Objective:Vascular endothelial growth factor (VEGF) is among the most important angiogenic factors. Endostar is a novel recombinant human endostatin as an angiogenesis inhibitor. In part I, our results showed that Netrin-1 might play an important role in the formation of VM in lung cancer. The precise molecular mechanism of Netrin-1 with other vascular growth factors in lung cancer cells remains unclear. Our present study will explore the relationship between Endostar and Netrin-1 expression in lung cancer cells. We also studied the effect of Netrin-1 on the level of VEGF in lung cancer cells.Methods:The cytotoxic effect and the invasion of Endostar on A549 cells were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fluorescence-immunocytochemistry and Western blot assay was used to detect the expression of Netrin-1 after treated with Endostar. Enzyme-linked immunosorbent assay(ELISA) was used to investigate the expression of VEGF of A549 cells after treated with Netrin-1.Results:Endostar inhibited proliferation and invasion activity of A549 cells in concentration-dependent manner. The IC50 of Endostar to A549 cell was20.7±5.6?mol/L at 24h,32.9±6.3?mol/L at 48h and 56.3±4.6?mol/L at 72h. After treated with Endostar with the dose of IC50 at 24h, the expression of Netrin-1 was down-regulated by western blot assay. Endostar inhibited the migration activity of A549 cells. After treated with Netrin-1 at 24h, the expression of VEGF was up-regulated by ELISA.Conclusion:Our findings indicate that Netrin-1 facilitates angiogenesis through up-regulating expression of VEGF after treated with Netrin-1 in lung cancer A549 cells. Endostar might inhibit cell proliferation, migration, invasion through down-regulating the expression of Netrin-1 in lung cancer cells A549.