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NG-hydroxy-L-arginine and nitric oxide inhibit Caco-2 tumor cell proliferation by distinct mechanisms
1Department of Molecular and Medical Pharmacology, University of California Los Angeles School of Medicine, Los Angeles, California 90095-1735, USA.
Abstract:
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.
Insights
Nitric oxide (NO) and NG-hydroxy-L-arginine (NOHA) inhibit colon cancer cell growth by targeting key enzymes in the arginine-polyamine pathway. NOHA inhibits arginase, while NO inhibits ornithine decarboxylase (ODC), offering novel therapeutic targets.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nitric oxide (NO) synthase (NOS) plays a role in cell growth modulation.
- Arginase and ornithine decarboxylase (ODC) are critical enzymes in polyamine synthesis, essential for cell proliferation.
Purpose of the Study:
- To investigate the mechanism by which NOS modulates the growth of Caco-2 human colon carcinoma cells.
- To elucidate the specific roles of NG-hydroxy-L-arginine (NOHA) and NO in Caco-2 cell proliferation.
Main Methods:
- Cell proliferation was assessed using cell counts, protein analysis, and DNA synthesis.
- Enzyme activity assays were performed for arginase and ODC.
- Experiments involved the use of NOHA, DETA/NO, alpha-difluoromethylornithine, and cocultures with activated endothelial cells.
Main Results:
- NOHA inhibited Caco-2 cell proliferation by inhibiting arginase activity, with effects reversed by ornithine or polyamines.
- NO inhibited Caco-2 cell proliferation by inhibiting ODC activity, with effects reversed by polyamines but not ornithine.
- Coculture with activated endothelial cells slowed proliferation, an effect blocked by NOS inhibitors.
Conclusions:
- NOHA and NO inhibit sequential steps in the arginine-polyamine pathway, impacting Caco-2 cell proliferation.
- These findings suggest a novel role for NOS in suppressing tumor cell growth.
- Targeting arginase and ODC presents potential therapeutic strategies for colon cancer.