Anti-apoptotic/cytoprotective effects of the nitric oxide/cGMP/protein kinase G signaling pathway in SV40-immortalized endothelial cells: implications for tumor angiogenesis

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Previously, our lab has shown that low-level nitric oxide (NO), via activation of the cGMP/protein kinase G (PKG) signaling pathway, plays a key role in preventing spontaneous apoptosis and protecting against toxin-induced apoptosis in a variety of mammalian cells, including neural cells, pancreatic islets and various tumor cells (e.g. human ovarian cancer cells). In contrast, hyper-stimulation of cGMP/PKG by high-level NO causes induction of apoptosis in certain cells (e.g. endothelial cells, pancreatic islets and colon cancer cells). Thus, NO has a dichotomous role in regulating apoptosis that depends on the level of NO exposure. Although high-level activation of cGMP/PKG causes apoptosis in endothelial cells, the role of low-level/basal cGMP/PKG activation in regulating endothelial apoptosis has not been elucidated. The present study used SV40-immortalized endothelial cells (SVECs) as a model of tumor angiogenesis and tested the pro-apoptotic/anti-apoptotic effects of S-nitroso-N-acetylpenicillamine (SNAP, NO donor) over a large concentration range. ODQ, inhibitor of NO-activated cGMP synthesis, was used to determine the role of endogenous NO-induced cGMP/PKG activation in regulating apoptosis. Levels of apoptosis were quantified by Cell Death Detection ELISA. Low-level NO, generated by SNAP (5, 10 and 50 μM), significantly (P<0.001) lowered spontaneous apoptosis, whereas high-level NO, generated by SNAP (500 and 1000 μM), induced apoptosis. Blocking cGMP synthesis with ODQ (40 μM) significantly (P<0.01) increased apoptosis, suggesting that low-level/basal activation of cGMP/PKG by endogenous NO is needed to prevent spontaneous apoptosis in SVECs. When cells were pre-incubated with 8-Br-cGMP (cell-permeable direct activator of PKG), apoptotic effects of both ODQ and high-level NO were completely prevented, suggesting PKG plays a key role in preventing both spontaneous apoptosis and high-level/toxic-NO-induced apoptosis. The data suggest that basal activation of low-level-NO/cGMP/PKG plays an essential role in protecting rapidly-growing endothelial cells against induction of apoptosis. Because many tumor cells over-express inducible NO synthase (iNOS), resulting in NO release within the tumor, this NO could promote endothelial cell survival via activation of cGMP/PKG pathway, promoting tumor angiogenesis.