Beta-carotene interaction with NNK in the AJ-mouse model: effects on cell proliferation, tumor formation and retinoic acid responsive genes.

We studied the influence of beta-carotene on the tobacco smoke carcinogen 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor development in the A/J-mouse model. The normally low beta-carotene absorption was facilitated with a diet enriched in fat and bile salt, resulting in plasma and lung tissue levels similar to humans. beta-Carotene enhanced NNK-induced early bronchial cell proliferation, however, this effect was not predictive for later tumor development. Tumor multiplicity was not significantly affected by beta-carotene, neither in carcinogen-initiated nor in uninitiated mice, and regardless of dose and time point of supplementation during tumor development. RARbeta isoform and CYP26 gene expression levels analyzed by quantitative RT-PCR were weakly, but significantly, inversely correlated and showed evidence for altered retinoid signaling and catabolism in the lungs of NNK-initiated, beta-carotene supplemented mice. However, this interaction did not translate into enhanced tumor multiplicity. These results indicate that impaired retinoid signaling is not likely a key factor in lung tumorigenesis in this mouse model.

Beta-carotene and apocarotenals promote retinoid signaling in BEAS-2B human bronchioepithelial cells.

High dose beta-carotene supplementation of smokers was associated with increased lung cancer risk in two intervention trials. It was proposed that generation of apocarotenals in smoke-exposed lungs impaired retinoic acid (RA) signaling, leading to squamous metaplasia and cell proliferation. To test this, we compared RA target gene regulation by retinoids, apocarotenals or beta-carotene by transcriptomics in BEAS-2B cells cultured to promote squamous differentiation. Retinoids, beta-carotene as well as apocarotenals induced known RA target genes. Retinoids upregulated involucrin, indicating that retinoids did not rescue BEAS-2B cells from squamous differentiation. Muc5AC, a marker for mucous differentiation, was transiently induced. beta-Carotene and apocarotenals less strongly induced involucrin and did not induce muc5AC. In summary, apocarotenals or beta-carotene upregulated RA target genes suggesting promotion, not inhibition, of RA signaling in BEAS-2B cells. Furthermore, apocarotenals and beta-carotene regulated gene expression independently of RA signaling. Squamous differentiation is not unequivocally linked to RA deficiency in BEAS-2B cells.

beta-carotene-induced changes in RARbeta isoform mRNA expression patterns do not influence lung adenoma multiplicity in the NNK-initiated A/J mouse model.

A number of epidemiological studies have reported associations of beta-carotene plasma levels or intake with decreased lung cancer risk. However, intervention studies in smokers reported increased lung tumor rates after high long-term beta-carotene supplementation. For insight into these conflicting results, we studied the influence of beta-carotene on tobacco smoke carcinogen-induced lung cancer development in the A/J-mouse using 4-(N-Methyl-N-nitro samino)-1-(3-pyridyl)-1-butanone (NNK) as the initiator and lung adenoma multiplicity as the functional endpoint. Gene regulation of the putative tumor suppressor RARbeta in mouse lung was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for its relevance in predicting the endpoint of lung cancer. A/J-mice achieved plasma beta-carotene levels of up to 3 micromol/L within 4 wk and up to 6 micromol/L after 6 mo of supplementation on a diet modified to enhance beta-carotene absorption. Despite high lung beta-carotene concentrations of up to 6 micromol/kg, tumor multiplicity was not significantly affected by the beta-carotene treatment, either in carcinogen-initiated or non-initiated mice, and was unrelated to beta-carotene dose and the time point of treatment during cancer formation. Tumor multiplicity did not correlate with beta-carotene plasma levels in NNK-treated animals. All RARbeta isoforms were significantly suppressed in the lungs of NNK- and NNK plus high dose beta-carotene-treated animals. However, the number of tumors per mouse did not correlate with the RARbeta-isoform expression levels. beta-carotene alone after 3 mo of supplementation mildly but significantly increased levels of RARbeta1, beta2, and beta4. This increase persisted for 6 mo for RARbeta2 and beta4. In summary, we found no effect of beta-carotene on tumor formation in the NNK-initiated A/J-mouse lung cancer model with respect to dose or time point of treatment. beta-Carotene-induced changes in RARbeta isoform gene expression levels were not predictive for the number of lung tumors but were indicative of intact beta-carotene metabolism and persistent sensitivity to retinoic acid in the mice. Down-regulation of RARbeta in NNK-induced adenoma-bearing lungs was similar to that observed in human lung cancer and further confirms the A/J-mouse as a valuable model for lung carcinogenesis.