Study of induced nitric oxide enzymeregulation mechanism in melanoma cells and its effect on lymphatic endothelial cell proliferation

Objective To research induced nitric oxide enzyme (iNOS) signal regulation mechanism in B16 melanoma cells, and the effect on lymphatic endothelial cell (LEC) proliferation. Methods In the iNOS signal regulation experiment, the cultivation of B16 cells were divided into 4 groups: blank control group, Vascular endothelial growth factor-C (VEGF-C) group, VEGF-C+ iNOS antagonist aminoguanidine (Ag) group, VEGF-C+ VEGF receptors (VEGFR-3) antagonist MAZ-51 group. We used the real-time quantitative polymerase chain reaction, Western blot test and nitrate reduction method to detect the level of iNOS mRNA, protein and nitric oxide (NO) in B16 cellsfor each grouprespectively after co-cultured 48 h. In LEC proliferation study, B16 cellswere co-cultured with LEC. The sampleswere divided into 6 groups. Group A (LEC); GroupB (LEC+ VEGF-C); Group C (LEC+ B16); Group D (LEC+ B16+ VEGF-C); Group E (LEC+ B16+ VEGF-C+ Ag); Group F (LEC+ B16+ VEGF-C+ MAZ-51). EDU method was used to detect LEC proliferation. Results In iNOS signal regulation experiment: as compared to blank control group, the iNOS mRNA and protein expression level in B16 cells were significantly improved in group VEGF-C (1.10±0.03 vs. 0.69±0.02, P=0.008). While the group with Ag or MAZ-51, the level of iNOS mRNA and protein expression was not only lower than VEGF-C group (0.35±0.03 vs. 1.10±0.03, P=0.005; 0.42±0.02 vs. 1.10±0.03, P=0.004), but also lower than the blank control group (0.35±0.03 vs. 0.69±0.02, P=0.028; 0.42±0.02 vs. 0.69±0.02, P=0.025). The optical density of NO in VEGF-C group was 0.25±0.03, which was significantly higher than the blank control group (0.19±0.03, P=0.008), and significantly higher than the group with Ag (0.07±0.02, P=0.017) or MAZ-51 (0.07±0.02, P=0.015). The LEC proliferation experiment showed: The proliferative rates were (0.45±0.02)%, (0.47±0.03)%, (0.48±0.05)%, (0.51±0.02)%, (0.24±0.01)%, and (0.28±0.03)% respectively. Compared with the blank control group (A), the level of LEC proliferation in group B, C, and D were all significantly improved(P=0.029, P=0.024, P=0.009). In Group E andF were obviously lowered (P=0.006, P=0.005). Conclusion To inhibit the VEGF-C/VEGFR-3 signaling pathways in B16 cells can reduce the expression of iNOS, and further reducing the proliferation of LEC. Key words: Melanoma; Vascular endothelial growth factor-C; Vascular endothelial growth factor receptors-3; Induced nitric oxide enzyme; Lymphatic endothelial cell