NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)

NADPH oxidase/dual‐oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa‐B (NFκB)‐mediated inflammation and inflammation‐associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo4,5‐b]pyridine (PhIP). In the PhIP‐induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB‐p50 and NFκB‐p65 were all highly overexpressed compared with their levels in adjacent normal‐looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco‐2 cells there was a strong apoptotic response, with increased levels of cleaved caspase‐3, ‐6, ‐7 and poly(ADP‐ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide‐induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP‐induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.