Correspondence to: Reuben Lotan, Ph.D., Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
A considerable number of epidemiologic studies conducted in the 1970s and 1980s indicated that an inverse relationship exists between estimated intakes of ß-carotene and the risk of developing various types of cancer, especially lung cancer. These studies have led to the suggestion that dietary ß-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 ß-carotene and risk of lung cancer (1). Initially, it was thought that the conversion of ß-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 ß-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 gap-junctional 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 ß-apocarotenoic acids and subsequently retinoic acids (4,5).
Several large-scale intervention studies were then designed to test the
ability of ß-carotene alone or combined with -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
ß-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 ß-carotene. In contrast, ex-smokers not exposed to
asbestos showed no increased risk upon supplementation with ß-carotene.
The mechanism by which ß-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 ß-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 ß-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 ß-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 ß-carotene-supplemented animals, and this increase was enhanced further by tobacco smoke. The concentrations of ß-carotene and retinoic acid and the levels of RARß in lung tissue were lower in animals supplemented with high-dose ß-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 ß-carotene and exposure to tobacco smoke is the result of RARß suppression and overexpression of c-jun and c-fos genes. The presumed sequence of events is as follows: An increase in ß-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 ß-carotene oxidative metabolites such as ß-apo-carotenals, which further increase the level of P450 enzymes and may abrogate retinoid signaling. Consequently, ß-carotene and retinoic acid levels decrease further. The decrease in retinoic acid may lead to a decrease in the expression of RARß and an increase in the expression of c-fos and c-jun. The decrease in RARß 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 RARß 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 ß-carotene oxidation metabolites cause diminished retinoid signaling by down-regulating RARß. It is not clear how this would happen in view of the report that ß-apo-14'-carotenoic acid can transactivate the response element of the RARß gene (5), which could result in an increase in RARß levels rather than in a decrease. It is more likely that the decrease in retinoic acid concentration in the lung results in suppression of RARß expression, which depends on the presence of retinoic acid. Moreover, a relationship has been found between retinoid levels and RARß expression in oral premalignant lesions in vivo (16).
The mechanism underlying the development of squamous metaplasia in ferret lung remains elusive. It cannot be explained by the decrease in lung concentration of retinoic acid alone because ferrets exposed to tobacco smoke alone showed a decrease in lung retinoic acid concentration but no squamous metaplasia. Likewise, the data do not provide strong support for a relationship between the increase in AP-1 and squamous metaplasia because squamous metaplasia increased in ferrets supplemented with ß-carotene alone even though c-Jun levels did not change by more than 20%.
The decreased expression of RARß observed in the ferret lung tissue is very interesting because it extends and is compatible with other studies suggesting a tumor suppressor role for RARß (17) and a report on the loss of RARß expression in about 50% of non-small-cell lung cancers in vivo (18). The increase in c-Fos and c-Jun proteins in ferrets supplemented with ß-carotene and exposed to tobacco smoke can be explained on the basis of a decrease in retinoic acid concentrations. This decrease could relieve antagonism of AP-1 transcriptional activity (19) and thereby increase the expression of c-jun and c-fos genes, which contain AP-1 sites in their promoter regions (15,19). If the increase in c-Fos and c-Jun proteins is accompanied by an increase in AP-1 transcriptional activity, it could lead to enhanced cell proliferation and increased PCNA. It is noteworthy that the major effects on PCNA, c-Jun, and c-Fos proteins were observed in ferrets that received high-dose ß-carotene and were exposed to tobacco smoke, whereas each treatment alone had only a small effect on the expression of these proteins. Wang et al. (14) suggest that the mechanism of the synergistic effects of the combined treatment is the formation of ß-carotene oxidative metabolites. However, it remains to be shown that such metabolites can enhance cell proliferation and suppress RARß expression.
The ferret absorbs carotenoids in intact form, transports them to various tissues, and metabolizes ß-carotene in a manner similar to that in humans. Thus, the choice of this animal model is appropriate from some perspectives. However, there are several findings in the ferret study that are not characteristic of the response of human lung tissue to either tobacco smoke or ß-carotene. Specifically, chronic smoking in humans often leads to the development of squamous metaplasia in vivo (20), yet no squamous metaplasia was observed in the lungs of ferrets exposed to tobacco smoke only. Of note is the difference between the effect of tobacco smoke on mouse lung, where it caused the development of lung tumors (21), and in the ferret, where it failed to even cause squamous metaplasia. It is not clear whether the duration of exposure of the ferrets to smoke or the amount of harmful tobacco smoke constituents was insufficient or whether the ferret is less sensitive than mice and humans to tobacco smoke.
Surprisingly, squamous metaplasia was observed in lung tissue of all ferrets supplemented with ß-carotene, whether or not they were exposed to tobacco smoke. This finding suggests that ß-carotene was more detrimental to ferret lung tissue than was tobacco smoke. Although it is not known whether squamous metaplasia occurs in lungs of human nonsmokers supplemented with high-dose ß-carotene, it has been reported that supplementation with ß-carotene for 14 weeks did not affect metaplasia scores in the sputum of 75 smokers (22). Furthermore, administration of ß-carotene with retinol on alternate days to asbestos workers in Texas had no effect on the frequency of cytologic abnormalities (e.g., moderate atypia or worse) in sputum samples (23).
In conclusion, the mechanism of enhanced lung cancer risk among smokers given high-dose ß-carotene is of great importance because of the general interest in the potential of ß-carotene as a chemopreventive agent and the disturbingly conflicting results of the observational studies and the intervention studies discussed above. For obvious reasons, it is not likely that another clinical trial will be conducted in smokers receiving high-dose ß-carotene supplements just to collect bronchial specimens to assess the mechanism of the enhanced tumorigenesis. Therefore, the use of an animal model like the ferret model is the best surrogate approach. The possibility that oxidative metabolites of ß-carotene may abrogate retinoid signaling and eventually enhance carcinogenesis is intriguing. However, the end point of this study was not lung cancer development but rather the appearance of squamous metaplasia and an increase in PCNA in ferret lung tissue. It is not clear that these changes are related directly to carcinogenesis. In humans, such changes are often reversible (e.g., upon smoking cessation), and there is no general agreement that they are premalignant lesions. Therefore, extrapolation of the findings with the ferret to human lung carcinogenesis must be done with caution because causality has not been established directly for high-dose ß-carotene, tobacco smoke, and lung cancer development.
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