The classic action of retinoids is thought to involve the activation of nuclear retinoid receptors (26
). Our data show, for the first time, that β-carotene significantly promoted and accelerated the development of small airway-derived PAC induced by NNK in vivo by mechanisms that involved non-classical cellular signaling via p-ERK1/2 and p-CREB. The observed four-fold increase in the incidence of nasal cavity tumors may have been caused by similar mechanisms, even though regulatory signal transduction in this type of cancer has not been studied to date. Having harvested serum and lung tissues for HPLC analysis of retinoids two hours after the last β-carotene injection, we were able to identify ATRA, 9-cis-RA and RP as the predominating retinol metabolites in serum and lungs. These findings are in accord with reports that have described non-genomic signaling induced by β-carotene, retinol or its metabolites in human PAC cells of small airway epithelial phenotype, in immortalized normal human small airway epithelial cells and in human bronchial epithelial cells (18
). While signaling via cAMP PKA CREB and PKA-dependent transactivation of ERK1/2 downstream of the EGFR stimulated the proliferation of small airway-derived PAC cells and its normal cells of origin, the same signaling cascade inhibited the proliferation of bronchial epithelial cells or small cell lung cancer cells (18
). In conjunction with our current findings, these data suggest that the non classical signaling cascade stimulated by β-carotene and its metabolites modulates the regulation of lung cells with different outcomes for different types of cells. Small airway epithelia and the PACs derived from them are stimulated in their growth whereas large airway epithelia and SCLC derived from them are inhibited. Since PAC was the predominating lung cancer type in the CARET trial (28
), the stimulation of non-genomic signaling in the current study may therefore justify the hypothesis that increased cAMP signaling may have contributed to the unfortunate outcome of this and similar clinical trials.
Immunoassays showed a significant increase in intracellular cAMP in blood cells from hamsters treated with NNK that was further increased by β-carotene treatment. These findings suggest that hyperactive cAMP signaling in response to NNK and β-carotene is a systemic event that can be detected by a simple blood test. This interpretation is in accord with the reported increase in lung cancer deaths in the CARET trial (4
). In analogy to the assessment of cardiovascular function by analysis of cAMP in blood cells (22
), it may hence be possible to identify individuals with elevated systemic cAMP who would respond with lung cancer promotion to β-carotene or retinoid treatment. On the other hand, individuals with below normal cAMP levels in blood cells may benefit from the selective cancer preventive effects of these agents.
It has been shown that vitamin A deficiency alone or in combination with benzo (a)-pyrene caused squamous metaplasia, a precursor of squamous cell carcinoma, in the epithelium of the trachea and stem bronchi in hamsters in vivo and in organ culture (17
). Treatments with β-carotene, retinol or other retinoids reversed or reduced this response (30
). The hamster trachea and stem bronchi are coated by a pseudostratified respiratory epithelium found in large airways (trachea, stem bronchi, lobar bronchi, segmental bronchi) of humans (32
) and comprised of basal cells, mucous cells and ciliated cells. In accord with the cited data in hamsters, recent studies with immortalized human large airway epithelial cells have shown a significant retinoid-induced inhibition of cell proliferation involving a cAMP-dependent inhibition of ERK1/2 phosphorylation (19
). Dietary β-carotene administered to A/J mice enhanced bronchial epithelial cell proliferation in NNK-treated animals but did not promote the NNK-induced PACs (20
). The mouse lung is a model for the human lung periphery, with all intrapulmonary airways being coated by the simple respiratory epithelium comprised of nonciliated Clara cells and sparse ciliated cells that is restricted to small airways (bronchioles) in humans (32
). The stimulation of bronchial epithelial cell proliferation in mice is thus equivalent to the stimulation of human small airway epithelial cells in vitro by β-carotene and retinoids (18
). However, spontaneous and NNK-induced lung tumors in the mouse are derived from alveolar type II cells (33
) and not from small airway epithelia as most human PACs (5
) or the PACs induced in the hamster by NNK (7
). In turn, human PAC cell lines with features of alveolar type II cells are not stimulated in their growth by agents that increase intracellular cAMP (34
). The observed β-carotene induced proliferation of bronchial epithelial cells in mice did therefore not promote the development of PAC.
Collectively, our data in NNK-induced small airway-derived PAC and the in vitro responses of human PAC cells of this phenotype (18
) suggest that β-carotene and retinoids promote the development of this PAC type.