Differential induction of heme oxygenase-1 against nicotine-induced cytotoxicity via the PI3K, MAPK, and NF-kappa B pathways in immortalized and malignant human oral keratinocytes.

BACKGROUND: Heme oxygenase-1 (HO-1) exhibits cytoprotective effects in many different cell types and is induced by nicotine exposure in human gingival fibroblasts. However, the role of HO-1 in cancer cells exposed to nicotine has not previously been described. METHODS: We investigated the effects of nicotine on HO-1 protein expression and cell viability in immortalized (IHOK) and malignant (HN12) human oral keratinocyte cells using the 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay and Western blotting. We also examined the involvement of the phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and nuclear factor-kappaB (NF-kappaB) signaling pathways in nicotine-induced cytotoxicity and HO-1 levels in IHOK and HN12 cells. RESULTS: Nicotine-induced HO-1 production and had cytotoxic effects on cells in both a concentration- and time-dependent manner. Nicotine-induced cytotoxicity and accumulation of HO-1 were greater in IHOK cells than in HN12 cells. Molecular inhibitors of the ERK, p38 MAP kinase, PI3 K, and NF-kappaB signaling pathways blocked the cytotoxic effects and induction of HO-1 expression by nicotine. Treatment with antioxidants (bilirubin, N-acetylcysteine) protected cells against nicotine-induced cytotoxicity and blocked the upregulation of HO-1, the effects of which were more pronounced in IHOK cells than in HN12 cells. CONCLUSIONS: Collectively, these results suggest that HO-1 plays a principal role in the protective response to nicotine in oral cancer and immortalized keratinocytes. Moreover, the addition of exogenous antioxidants may help to protect oral epithelial cells as chemopreventive agents against nicotine-induced oxidative stress.

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes.

BACKGROUND: Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. METHODS: We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. RESULTS: Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. CONCLUSIONS: We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.