Nox1 downstream of 12-lipoxygenase controls cell proliferation but not cell spreading of colon cancer cells

The catalytic subunit of the NADPH oxidase complex, Nox1 (homologue of gp91phox/Nox2), expressed mainly in intestinal epithelial and vascular smooth muscle cells, functions in innate immune defense and cell proliferation. The molecular mechanisms underlying these functions, however, are not completely understood. We measured Nox1-dependent O-2(-) production during cell spreading on Collagen IV (Coll IV) in colon carcinoma cell lines. Knocking down Nox1 by shRNA, we showed that Nox1-dependent O-2(-) production is activated during cell spreading after 4 hr of adhesion on Collagen IV. Nox1 activation during cell spreading relies on Rac1 activation and arachidonic metabolism. Our results showed that manoalide (a secreted phospholipase A2 inhibitor) and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (a 12-lipoxygenase inhibitor) inhibit O-2(-) production, cell spreading and cell proliferation in these colonic epithelial cells. 12-Lipoxygenase inhibition of ROS production and cell spreading can be reversed by adding 12-HETE, a 12-lipoxygenase product, supporting the specific effect observed with cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. In contrast, Nox1 shRNA and DPI (NADPH oxidase inhibitor) weakly affect cell spreading while inhibiting O-2(-) production and cell proliferation. These results suggest that the 12-lipoxygenase pathway is upstream of Nox1 activation and controls cell spreading and proliferation, while Nox1 specifically affects cell proliferation. (c) 2007 Wiley-Liss, Inc.