Lung cancer promotion by beta-carotene and tobacco smoke: relationship to suppression of retinoic acid receptor-beta and increased activator protein-1?

A considerable number of epidemiologic studies conducted in the 1970s and 1980s indicated that an inverse relationship exists between estimated intakes of b-carotene and the risk of developing various types of cancer, especially lung cancer. These studies have led to the suggestion that dietary b-carotene can reduce human cancer rates (1,2). Subsequent observational cohort and nested case–control studies based on measurement of carotenoids in blood and tissues showed consistent inverse association between blood b-carotene and risk of lung cancer (1). Initially, it was thought that the conversion of b-carotene to retinol (1,3) is the mechanism of its cancer preventive effects. However, the inability to demonstrate a consistent negative association between plasma retinol levels and cancer risk has led to the proposal that b-carotene itself might exert chemopreventive effects. Several mechanisms have been proposed for such effects: 1) action as an antioxidant, 2) induction of cytochrome P450 xenobiotic detoxifying enzymes, 3) enhancement of gapjunctional communication, and 4) metabolism to retinoic acid that could exert biologic effects by activating nuclear retinoic acid receptors (RARs) (1,4,5). This metabolism occurred through either central cleavage to retinal and subsequent oxidation to retinol and retinoic acid or excentric cleavage to form b-apocarotenoic acids and subsequently retinoic acids (4,5). Several large-scale intervention studies were then designed to test the ability of b-carotene alone or combined with a-tocopherol or retinyl palmitate to prevent lung cancer (6–10). Unexpectedly, these studies failed to demonstrate prevention of lung cancer. Furthermore, both the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study conducted in Finland (6,7) and the b-Carotene and Retinol Efficacy Trial study conducted in the United States (8,9) demonstrated a higher incidence of lung cancer in current smokers, alcohol drinkers, and individuals exposed to asbestos who received b-carotene. In contrast, exsmokers not exposed to asbestos showed no increased risk upon supplementation with b-carotene. The mechanism by which b-carotene increased lung cancer risk in heavy smokers and asbestos workers is not clear. It has been suggested that the relatively high partial oxygen pressure in the lung combined with reactive oxygen species derived from tobacco smoke or induced by asbestos is conducive for b-carotene auto-oxidation and that the oxidative metabolites can act as propagators of free-radical formation in smokers’ lungs (11–13). The report by Wang et al. (14) in this issue of the Journal provides a possible explanation for the enhancing effect of b-carotene supplementation on lung carcinogenesis in smokers, based on findings in an animal model. Using the ferret (Mustela putorius furo), Wang et al. have determined the effects of supplementation with high-dose b-carotene and exposure to tobacco smoke on lung tissue. The authors observed an increase in squamous metaplasia and in the level of a marker of cell proliferation (proliferating-cell nuclear antigen [PCNA]) in b-carotene-supplemented animals, and this increase was enhanced further by tobacco smoke. The concentrations of b-carotene and retinoic acid and the levels of RARb in lung tissue were lower in animals supplemented with high-dose b-carotene, exposed to tobacco smoke, or both compared with control animals. Furthermore, ferrets on combined treatment showed a severalfold increase in the expression of c-Jun and c-Fos proteins. Often induced by mitogenic stimuli and tumor-promoting agents, these proteins can form a heterodimer that binds an activator protein-1 (AP-1) site on DNA and enhances gene transcription (15). Wang et al. (14) concluded that the enhanced lung tumorigenesis after high-dose supplementation with b-carotene and exposure to tobacco smoke is the result of RARb suppression and overexpression of c-jun and c-fos genes. The presumed sequence of events is as follows: An increase in b-carotene in the presence of tobacco smoke in the ferret’s lung results in an increase in P450 enzymes. This effect enhances the formation of b-carotene oxidative metabolites such as b-apo-carotenals, which further increase the level of P450 enzymes and may abrogate retinoid signaling. Consequently, b-carotene and retinoic acid levels decrease further. The decrease in retinoic acid may lead to a decrease in the expression of RARb and an increase in the expression of c-fos and c-jun. The decrease in RARb may in turn facilitate the development of squamous metaplasia, and the increase in AP-1 may lead to hyperproliferation and an increase in PCNA. Wang et al. (14) propose that these changes may eventually enhance lung tumorigenesis. Overall, the above events represent a plausible mechanism; however, some of the biochemical and molecular changes that occur in ferret lung tissue and their relationship to the development of squamous metaplasia, PCNA increase, and lung tumorigenesis deserve further comments. Wang et al. (14) used western blotting to analyze the levels of PCNA, RARs, c-Jun, and c-Fos. It would have been more informative had they used immunohistochemical techniques to determine the expression of these proteins in consecutive tissue sections to determine whether the diverse changes occur in the same or different cells. For example, are RARb decreased and c-Jun and c-Fos increased in the same cells? Do these changes occur in squamous metaplasia lesions only or throughout the epithelium? Wang et al. (14) suggested that b-carotene oxidation metabolites cause diminished retinoid signaling by down-regulating RARb. It is not clear how this would happen in view of the report that b-apo148-carotenoic acid can transactivate the response element of the RARb gene (5), which could result in an increase in RARb