Ethanol promotes chemically induced oral cancer in mice through activation of the 5-lipoxygenase pathway of arachidonic acid metabolism

Abstract

Alcohol drinking is a known risk factor for oral cancer in humans. However, previous animal studies on the promoting effect of ethanol on oral carcinogenesis were inconclusive. It is necessary to develop an animal model with which the molecular mechanism of ethanol-related oral carcinogenesis may be elucidated to develop effective prevention strategies. In this study, mice were first treated with 4-nitroquinoline-1-oxide (4NQO, 100 μg/mL in drinking water) for 8 weeks and then given water or ethanol (8%) as the sole drink for another 16 weeks. During the experiment, 8% ethanol was well tolerated by mice. The incidence of squamous cell carcinoma (SCC) increased from 20% (8/41) to 43% (17/40; P < 0.05). Expression of 5-lipoxygenase (5-Lox) and cyclooxygenase 2 (Cox-2) was increased in dysplasia and SCC of 4NQO-treated tongues and further enhanced by ethanol. Using this mouse model, we further showed that fewer cancers were induced in Alox5(-/-) mice, as were cell proliferation, inflammation, and angiogenesis in the tongue, as compared with Alox5(+/+) mice. Interestingly, Cox-2 expression was induced by ethanol in knockout mice, whereas 5-Lox and leukotriene A4 hydrolase (LTA4H) expression and leukotriene B4 (LTB4) biosynthesis were dramatically reduced. Moreover, ethanol enhanced expression and nuclear localization of 5-Lox and stimulated LTB4 biosynthesis in human tongue SCC cells (SCC-15 and SCC-4) in vitro. In conclusion, this study clearly showed that ethanol promoted 4NQO-induced oral carcinogenesis, at least in part, through further activation of the 5-Lox pathway of arachidonic acid metabolism.

Figure 1

Overexpression of 5-Lox and Cox-2 in oral lesions induced by 4NQO/ethanol. (A, D) Normal epithelium; (B, E) Dysplasia; (C, F) SCC. Expression of 5-Lox and Cox-2 in the epithelial cells oral tissues was semi-quantified after immunohistochemical staining (G), and confirmed by Western blotting with frozen tissue samples (H). Protein samples of 3 representative tongues (one from each group) were shown. * Statistically different from Group 1B (p<0.05); ** Statistically different from dysplasia and SCC (p<0.01).

Figure 2

Cell proliferation, inflammation, and angiogenesis in 4NQO/ethanol-treated mouse tongue of Alox5^+/+ and Alox5^−/− mice. (A) Cell proliferation (BrdU-labeling index). ** Statistically different from the remaining groups (p<0.01). Values were expressed as mean ± SD. (B) Inflammation (number of infiltrating mast cells per mm²). ** Statistically different from Group 2C (p<0.01). * Statistically different from Group 2E (p<0.05). (C) Microvessel density (number of CD31-positive microvessels per mm²). ** Statistically different from Group 2C (p<0.01). * Statistically different from Group 2E (p<0.05).

Figure 3

5-Lox and Cox-2 expression and LTB4 biosynthesis in 4NQO/ethanol-treated mouse tongue of Alox5^+/+ and Alox5^−/− mice. (A) Overexpression of 5-Lox and Cox-2 in mouse tongue induced by ethanol as determined by immunohistochemical staining. ** Statistically different from Group 2C (p<0.01); * Statistically different from Group 2E (p<0.05), based on ANOVA test. (B) Overexpression of 5-Lox, LTA4H and Cox-2 in mouse tongue induced by ethanol as determined by Western blotting. Protein samples of representative tongues (2 from Group 2A, and 3 each from other groups) were shown. (C) Increased LTB4 biosynthesis in mouse tongue induced by ethanol as determined by enzyme immunoassay. * Statistically different from Group 2C (p<0.01).

Figure 4

Effect of ethanol on cell growth, 5-Lox expression, cellular localization of 5-Lox, and LTB4 biosynthesis, in SCC-15 and SCC-4 cells. (A) Dose-dependent effect of ethanol on cell proliferation of SCC-15 and SCC-4 cells as determined by MTT assay. (B) Increased expression of 5-Lox in SCC-15 and SCC-4 after ethanol treatment (250mM) for 6h, 12h and 24h as determined by Western blotting. (C) Increased nuclear localization of 5-Lox in SCC-15 cells after ethanol treatment (250mM) as determined by immunofluoresent staining with DAPI counterstaining. (D) Increased LTB4 biosynthesis in SCC-15 and SCC-4 cells after ethanol treatment (25–500mM) for 24 hr as determined by enzyme immunoassay. * Significantly different from control (p<0.05) ** Significantly different from control (p<0.01)