Grape seed proanthocyanidins inhibit the migration of human lung cancer cells through the sequential inhibition of nitric oxide synthase, guanylate cyclase and mitogen-activated protein kinase

2119

Lung cancer is the leading cause of cancer related deaths in both menand women worldwide. Therefore, it is desirable to develop newer and more effective chemopreventive agents and strategies that can reduce the risk of lung cancer development. Grape seed proanthocyanidins (GSPs) have been shown to exert anticancer effects in various tumor models. Chronic induction of nitric oxide (NO), an inorganic free radical gas, has been shown to promote tumor development and metastasis by multiple mechanisms. As NO has been shown to stimulate tumor cell migration, a major event in metastatic cascade, our present study examined the effect of GSPs on the signaling mechanisms underlying NO-mediated migration using metastatic-specific non-small cell human lung cancer cells, A549, as an in vitro model. Using an in vitro transwell migration assay, we found that treatment of A549 cells with GSPs (0, 20, 40, 60 and 80 μg/ml) for 24 h resulted in a dose-dependent inhibition (26-87%) of migration of A549 cells. The migration capacity of cells was reduced in the presence of N^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), and restored in the additional presence of excess L-arginine (NOS substrate), confirming the role of endogenous NO as a promoter of migration. GSPs significantly suppressed the elevated levels of endogenous NO in A549 cells in a dose-dependent manner (11-56%), and also blocked the migration promoting capacity of L-arginine. Treatment with guanylate cyclase (GC) inhibitor 1-H-[1,2,4]oxadiaxolo[4,3-a]quinolalin-1-one (ODQ) reduced the migration of A549 cells whereas additional presence of 8-bromoguanosine 3’5’-cyclic monophosphate (8-Br cGMP), an analogue of cGMP, restored the migration of these cells, suggesting the role of GC in migration of A549 cells. GSPs significantly reduced the elevated level of cGMP in A549 cells (10-62%) and also checked the migration restoring activity of 8-Br cGMP. The mitogen-activated protein kinase kinase (MAPKK) inhibitor, UO126, inhibited the migration of A549 cells in a dose-dependent manner, indicating the role of MAPKK in the migration. In addition, UO126 and ODQ inhibited the migration restoring effects of L-arginine in L-NAME-treated cells, suggesting the involvement of cGMP and MAPK pathways in NO-mediated migration. Treatment of GSPs also down-regulated the phosphorylation levels of ERK1/2 in A549 cells. Together these results indicate sequential inhibition of NO, GC and MAPK pathways by GSPs in mediating signals for A549 human lung cancer cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human lung cancer progression, the present results are relevant for the development of GSPs as a potential chemopreventive agent for lung cancer prevention.