The objective of this study was to elucidate the role and mechanism of
nitric oxide (
NO) synthase (NOS) in modulating the growth of the Caco-2 human colon
carcinoma cell line. The two novel observations reported here are, first, that NG-hydroxy-
L-arginine (NOHA) inhibits Caco-2
tumor cell proliferation, likely by inhibiting
arginase activity, and, second, that NO causes cytostasis by mechanisms that might involve inhibition of
ornithine decarboxylase (ODC) activity. Both
arginase and ODC are
enzymes involved in the conversion of
arginine to
polyamines required for cell proliferation. Cell growth was monitored by cell count, cell
protein analysis, and
DNA synthesis. NOHA (1-30 microM) and NO in the form of
DETA/NO (1-30 microM) inhibited cell proliferation by 30-85%. The
cytostatic effect of NOHA was prevented by addition of excess
ornithine,
putrescine,
spermidine, or
spermine to cell cultures, whereas the
cytostatic effect of NO (
DETA/NO) and
alpha-difluoromethylornithine (ODC inhibitor) was unaffected by
ornithine but was prevented by
putrescine,
spermidine, or
spermine. The
cytostatic effect of NOHA appeared to be independent of its conversion to NO, and the effect of NO appeared to be independent of cGMP. NOHA inhibited
urea production by Caco-2 cells and inhibited
arginase catalytic activity (85% at 3 microM), whereas NO (
DEA/NO and SNAP) inhibited ODC activity (>/=60% at 30 microM) without affecting
arginase activity. Coculture of Caco-2 cells with
lipopolysaccharide/
cytokine-activated rat aortic endothelial cells markedly slowed Caco-2 cell proliferation, and this was blocked by NOS inhibitors. These observations that NOHA and NO may inhibit sequential steps in the
arginine-polyamine pathway suggest a novel
biological role for NOS in the inhibition of cell proliferation of certain
tumor cells and possibly other cell types.