Nitrosamine-Induced Lung Carcinogenesis and Ca2+/Calmodulin Antagonists

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[CANCER RESEARCH (SUPPL.) 52. 2723s-2726s. May I. 1992) Nitrosamine-induced Lung Carcinogenesis and Ca2+/Calmodulin Antagonists

Hildegard M. SchÃ¼ller2

Experimental Oncology Laboratory, Department ofPathobiology, I'nirersily of Tennessee, Knoxville, Tennessee 37901

Abstract exposure. When the treatment is continued, only Clara cells give rise to tumors (peripheral adenomas and adenocarcinomas) This review summarizes recent data which implicate cell membrane (8, 9), while the neuroendocrine cells undergo terminal differ receptors and their associated signal transduction pathways as molecular targets of tobacco-related lung carcinogenesis as well as therapy of such entiation into squamous cells without progressing into tumors cancers. (9, 11). On the other hand, treatment of hamsters exposed to It is shown that the two nitrosamines /V-nitrosodiethylamine and 4- continuous hyperoxia with an identical dosing regimen of DEN (methylnitrosamino)-l-(3-pyridyl)-l-butanone bind to nicotinic elioliti- or NNK selectively stimulates peptide secretion by PNE cells ergic receptors in hamster lung. Binding of the nitrosamines as well as during the acute phase (12), while chronic exposure results in nicotine to this receptor stimulates proliferation of human lung carcinoid the development of neuroendocrine lung tumors (9, 13). cells in vitro. These data suggest chronic stimulation of nicotinic receptors The fact that the nitrosamines stimulated secretion by Clara by nicotine and nitrosamines in smokers as one of the molecular events cells and PNE cells during the initial phases of these experi responsible for stimulation of neuroendocrine cell proliferation and ulti ments led us to investigate the role of signal transduction mately the development of lung tumors with neuroendocrine pathways which regulate secretion for cancer initiation by these differentiation. On the other hand, a selective antiproliferative effect of the dihydro- chemical carcinogens. Subsequently, we have explored if signal pyridine derivative B859-35 on neuroendocrine lung tumor cells in vivo transduction pathways involved in the regulation of cell prolif eration can be exploited for the development of a target-ori and in vitro suggests the potential use of such agents as cancer therapeu tics. The demonstrated inhibition of Ca2+/calmodulin and protein kinase ented therapy of neuroendocrine lung tumors. C by B859-35 as reported in other in vitro systems suggests interference with such elements of signal transduction pathways as the molecular Nicotinic Cholinergic Receptor of PNE Cells and mechanism of the observed antiproliferative effects. Nitrosamines

Introduction Under physiological conditions, the secretion of calcitonin and mammalian bombesin by PNE cells is stimulated by the yV-Nitrosamines and their precursors are common environ neurotransmitter acetylcholine via high-affinity ligand binding mental contaminants, and they are formed in the saliva, stom to cholinergic receptors in the cell membrane (14). Exposure to ach, and gut from nitrate and amines (1, 2). Tobacco products tobacco smoke or nicotine has been shown to stimulate calci contain significant levels of nitrosamines and are believed to tonin secretion in humans and hamsters (14-16). This effect contribute substantially to the high lung cancer risk of smokers was inhibited by specific antagonists of nicotinic cholinergic (3). It is generally believed that nitrosamines are metabolically receptors (14-16), an expression of pharmacological properties activated in the host organism to generate intermediates which of the nicotinic subtype of cholinergic receptors. Although the react with DNA (4). The formation of the DNA-adduct O6- signal transduction pathway and molecular biology of nicotinic methylguanine from the tobacco-specific NNK' has recently cholinergic receptors have been extensively investigated, all been shown to correlate with the occurrence of mutations in such studies have been conducted on muscle or neuron receptors codons 12 and 61 of the K-ras oncogene (5). Mutations of this (17, 18). Since signal transduction pathways and molecular particular type have been identified in many chemically induced biology of nicotinic cholinergic receptors in muscles and neu and spontaneous tumors and are believed to be a key event in rons are slightly different (18), it is conceivable that this recep the molecular mechanisms leading to the development of many tor expresses yet another variation of functional aspects when cancer types. In view of the many different cell types in the located in PNE cells. Discussions and interpretations on the mammalian body capable of metabolizing nitrosamines, it is, mechanisms of action of this receptor in PNE cells and tumor however, difficult to understand why some members of this cells with neuroendocrine differentiation may therefore change class of chemical carcinogens selectively cause cancers in the in the future as more data become available. Nevertheless there liver while others selectively affect other organs, such as the are a number of functional similarities among the reported respiratory tract or pancreas (6). subtypes of nicotinic receptors which are likely to apply to this DEN and the nicotine-derived nitrosamine NNK are both receptor in PNE cells as well. Binding of an agonist to the potent lung carcinogens in Syrian golden hamsters when s.c. receptor results in the opening of an ion channel with high administered (6, 7). However, there is a pronounced difference affinity for Ca2+ via membrane depolarization. Agonist/recep in the acute and chronic cellular responses when these two tor binding results in a momentary desensitization of the recep nitrosamines are administered under ambient air conditions as tor which is mediated by phosphorylation of the receptor via opposed to a simultaneous continuous exposure to hyperoxia. cyclic AMP-dependent protein kinase (19), protein kinase C Under ambient air conditions, DEN and NNK both stimulate (20), and tyrosine-specific-protein kinase (21). Prolonged ex secretion and hyperplasia of pulmonary Clara cells (8, 9) and posure to agonists results in permanent desensitization of the neuroendocrine cells (9, 10) during the first few weeks of receptor accompanied by channel closing and an increase of ' Presented at the NCI Workshop "Investigational Strategies for Detection receptor affinity by two orders of magnitude (17). It has been and Intervention in Early Lung Cancer," April 21-24, 1991, Annapolis, MD. shown in neuroendocrine cells of the adrenal medulla and in 2To whom requests for reprints should be addressed. 3The abbreviations used are: NNK, 4-(methylnitrosamino)-l-(.1-pyridyl)-l- pheochromocytoma cells (which are derived from these cells) butanone; DEN, /V-nitrosodiethylamine; SCLC, small cell lung cancer; PNE. that stimulation of the nicotinic receptor by agonist binding pulmonary neuroendocrine. causes the nonspecific secretion of the entire contents of neu- 2723s

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. INDUCTION AND THERAPY OF N1TROSAMINE-INDUCED LUNG CANCER roendocrine storage granules by exocytosis (22). Thus opioids, ( ;r+/< alinoduliii Antagonists and Neuroendocrine Lung catecholamines, and neuropeptides were simultaneously se Cancer creted in this system (22). Ca2+/calmodulin antagonists have been suggested as thera Structural similarities of DEN with acetylcholine and of peutics for neuroendocrine lung cancer, because the signal NNK with nicotine along with the observed stimulation of PNE transduction pathways of many growth factors for this tumor cell secretion by these nitrosamines in the hamster led us to category are Ca2+/calmodulin dependent (31). In particular, investigate their potential interaction with the nicotinic cholin- mitogenic action of the neuropeptides gastrin-releasing peptide ergic receptor which regulates the secretory activity of this cell (synonym, mammalian bombesin), bradykinin, cholecystokinin, type under physiological conditions (14-16). Radioreceptor galanin, neurotensin, and vasopressin involves Ca2+-dependent assays with 'H-labeled (Â£)-(â€”)-nicotinein cell membrane frac signaling events (32). However, the use of conventional Ca2+- tions (23) from normal hamster lung homogenates revealed antagonists such as verapamil as cancer therapeutics is prohib measurable and saturable binding under steady-state conditions ited by their pronounced cardiovascular effects. (24). However, when an identical assay was conducted on cell Recently, a novel type of Ca2+/calmodulin antagonists has membrane fractions from hamsters which had been preexposed been developed from the dihydropyridine, niguldipine, by sep for 4 wk to hyperoxia (resulting in PNE cell hyperplasia), the aration of the racemic mixture into two stereoisomers (33). While the Ca2+ channel-mediated cardiovascular effects rest levels of specific nicotine binding were substantially increased primarily with the (H-)-enantiomer, the (â€”)-enantiomer (B859- (24). Saturation was reached in both cases at a concentration 35) has a 40-fold lower affinity to C&2+channels, therefore of 20 nM 'H-labeled (S)-(-)-nicotine, suggesting that the ob affecting the cardiovascular system only at relatively high dose served increase in specific binding was due to an increase in levels (33). On the other hand, B859-35 is a potent inhibitor receptor number rather than changes in the sensitivity of recep of calmodulin (34) and protein kinase C (35) and is thus likely tors. Scatchard analysis confirmed that binding was to a single to inhibit cell proliferation stimulated by pathways involving class of binding sites (24). Competition experiments with non- these regulatory components. We therefore tested the antipro- radioactive DEN and NNK resulted in complete displacement liferative effects of B859-35 in the neuroendocrine, lung tumor of 3H-labeled (5)-(~)-nicotine from the receptor at equimolar cell line NCI-H727. Exposure of cells for 48 h to B859-35 dose levels (24). Even 20 times lower concentrations of the caused a dose-dependent inhibition of cell proliferation, which nitrosamines caused significant displacement of nicotine. Ac was highly significant until the end of the assay (216 h after cordingly, the affinity of DEN and NNK to the nicotinic recep seeding of cells or 168 h after termination of exposure to B859- tor is significantly higher than that of nicotine itself. 35) (Fig. 1). Concentrations as low as 1 pM were still highly Having thus established that DEN and NNK do in fact bind effective in inhibiting cell growth in these assays. In contrast, to nicotinic cholinergic receptors in the lungs, we then asked verapamil inhibited cell proliferation under identical exposure the question: Does such binding result in a stimulation of PNE conditions only at significantly higher concentrations (10 to cell proliferation? In the absence of an in vitro system of normal 100 /Â¿M),andthis effect was tapering off during the last part of PNE cells, we studied the effects of DEN and NNK on the the observation period (144 to 216 h after seeding) (Ref. 36; growth kinetics of the human cell line NCI-H727 which is Fig. 1). In order to explore if B859-35 also has an antiproliferative derived from a lung carcinoid. This cell line is very well differ effect on neuroendocrine lung tumors in vivo we conducted a entiated, grows as a monolayer, and expresses several neuroen bioassay experiment in hamsters. The animals were simulta docrine markers as well as functional bombesin and epidermal neously exposed to hyperoxia and DEN as previously described growth factor receptors (25-28). Contrary to human lung can (13) to induce neuroendocrine lung tumors. Nitrosamine injec- cer lines of other histolÃ³gica! types, NCI-H727 only grows in tissue culture when maintained at relatively high CO2 levels (8 to 10%), thus mimicking the well-established dependence of Control normal PNE cells in vivo on abnormal pulmonary oxygen levels Verapamil for proliferation (reviewed in Ref. 27). Exposure of the cells for 48 h to various concentrations of acetylcholine, nicotine, DEN, or NNK resulted in a dose-dependent stimulation of cell prolif eration in all cases (26, 29). This effect was inhibited by preex- posure to hexamethonium or pentolineum, which are specific 'S antagonists of nicotinic cholinergic receptors. Antagonists of other receptor types, such as muscarinic cholinergic or adrener- I gic receptors, did not inhibit the mitogenic effects of DEN, NNK, nicotine, or acetylcholine. Our data thus confirm the hypothesis that the two nitrosamines stimulate proliferation of 24 72 144 cells with PNE cell differentiation via direct interaction with Hours Fig. 1. Antiproliferative effects of B859-35 and verapamil in the human lung the nicotinic cholinergic receptor. A mechanism such as this is carcinoid cell line NCI-H727. Cells were seeded at a concentration of 5 x IO4 likely to contribute significantly to the well-documented prev cells/ml and allowed to attach for 24 h. B859-35 or verapamil was then added to alence of lung cancer with neuroendocrine differentiation in the culture medium at the concentration indicated. After 48 h of incubation, the cells were washed, and fresh medium without drug was added. Cells were counted smokers (30). Moreover, the mitogenic effects of nicotine re manually at the intervals indicated after trypan blue dye exclusion (Neubauer vealed in line NCI-H727 strongly suggest that nicotine itself chamber). Under these conditions. B859-35 demonstrated a pronounced and sustained antiproliferative effect at a 1 pM concentration. Inhibition of cell aids in the selective propagation of a neuroendocrine lung proliferation by verapamil required significantly higher dose levels and was of tumor type. shorter duration. 2724s

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. INDUCTION AND THERAPY OF NITROSAMINE-INDUCED LUNG CANCER lions were then discontinued, and the animals were returned to The mitogenic effect of nitrosamines via binding to nicotinic ambient air conditions and given B859-35 by stomach tube receptors does not exclude additional molecular events com every day excluding weekends for 20 wk, while a positive control monly associated with carcinogenesis, such as metabolic acti group was maintained under identical conditions but without vation and DNA interaction. In light of the fact that agonists receiving B859-35. In analogy to the in vitro studies with the bound to a cell membrane receptor are usually internalized and human carcinoid-derived cell line (see above), B859-35 dem enzymatically degraded, such high-affinity uptake will result in onstrated a pronounced antiproliferative effect on neuroendo- substantially higher nitrosamine concentrations in normal and crine lung tumors in this system (37). In contrast, Clara cell- neoplastic PNE cells than in other cell types which are lacking derived peripheral adenomas induced in the same animal spe this mechanism. As we have previously shown with the carci cies under ambient air conditions by DEN progressed further noid-derived cell line NCI-H727 and DEN, neuroendocrine when treated with an identical dosing regimen of B859-35 (37). lung cells are well able to metabolize nitrosamines (28). In conjunction with the data obtained with cell line NCI- Whether the frequently reported changes in oncogene expres H727 (see above), these data suggest that B859-35 may be sion are the result of such DNA/metabolite interactions or an effective in the treatment of neuroendocrine lung cancer. In adaptive response to continuous receptor stimulation and sub order to arrive at a realistic assessment of these data, we have sequent receptor desensitization remains yet to be established. to consider that line NCI-H727 and the neuroendocrine ham The antiproliferative effect of the dihydropyridine derivative ster lung tumors are atypical carcinoids, a well-differentiated B859-35 and the phenylalkylamine verapamil on lung tumor and only moderately malignant tumor type. A potential effect of B859-35 on SCLC, a highly malignant tumor type of com cells with neuroendocrine differentiation in the hamster model and in the human carcinoid cell line NCI-H727 is unrelated to paratively poor differentiation, is difficult to predict at this time. Clinical trials in human cancer patients which are cur the discussed functional aspects of the nicotinic cholinergic receptors. Although we have shown that B859-35 inhibits the rently in progress at the Thompson Cancer Survival Center, mitogenic effects of nicotinic agonists in line NCI-H727 by Knoxville, TN (clinical investigator. Dr. J. J. Costanzi) and the blocking this receptor-mediated pathway,4 the experiments dis University of Colorado Health Sciences Center (clinical inves tigator. Dr. P. A. Bunn, Jr.), as well as at the Clinical Center, cussed in this review are dealing with an antiproliferative effect Philips University, Marburg, Germany (clinical investigator, on cells not stimulated by nicotinic agonists. As is evident from Dr. K. Havemann), will have to show if the high expectations the relatively high concentrations of verapamil required to yield in this novel type of antitumor agent are justified. a significant antiproliferative effect in line NCI-H727, blocking of Ca2* channels alone results in significantly less inhibition of Conclusions proliferation than the simultaneous inhibition of calmodulin and protein kinase C by the dihydropyridine, B859-35. Cal Our data suggest that the nicotinic cholinergic receptor in modulin and protein kinase C interface with a large number of normal and neoplastic lung cells with neuroendocrine differ different signal transduction pathways and Ca2+-dependent in- entiation plays an important role in the mechanisms of tumor tracellular reactions involved in the regulation of cell prolifer initiation and progression of smoking-related lung cancer. The ation (39, 40). Their inhibition is thus likely to inhibit prolif pronounced mitogenic effect of nicotine and tobacco-related eration in many different cell types. The selective effect of nitrosamines on normal and carcinoid-derived neuroendocrine B859-35 on tumor cells with neuroendocrine differentiation lung cells is mediated by binding of these amines to this receptor observed in our in vitro and in vivo studies may in part be type. Multiple exposures to these three nicotinic receptor ago explained by the dependence of many neuropeptides which are nists over 20 to 30 yr thus provide neuroendocrine tumor cells autocrine growth factors for these cells on Ca2+/calmodulin. with a growth advantage over other cell types, a fact clearly Moreover, selective uptake by dihydropyridine receptors may reflected in the prevalence of SCLC in smokers. On the other further potentiate the effects of B859-35 in neuroendocrine hand, such chronic exposure to receptor agonists will ultimately lead to receptor desensitization. In the case of a Ca:+ channel- tumor cells by yielding a relatively high intracellular concentra operated cell membrane receptor, such as the nicotinic cholin tion of the drug. Although our findings to date support the development of ergic receptor, this results in a higher receptor affinity to its inhibitors of second messengers such as Ca2+ channels, protein ligands which is reflected by receptor saturation at substantially lower agonist concentrations (17). At the same time, the closing kinase C, or calmodulin as cancer therapeutics, it is possible of Ca2+ channels characteristic for desensitized receptors of this that cancer cells such as SCLC are less susceptible to their category (17) blocks the int race) lular transduction chain nor actions than the relatively well-differentiated carcinoid cells mally initiated by binding of an agonist to the receptor. This, used in our studies. Moreover, it is conceivable that, during in turn, results in an inhibition of cellular responses such as progression to a more malignant state under continuous expo secretion of peptides or cell proliferation regulated by the sure to multiple carcinogenic signals from the environment, receptor. The data presented by J. D. Minna in this conference multiple adaptive changes occur in these cells, thus providing and elsewhere (38) on functional aspects of nicotinic cholinergic them with their well-documented superiority over normal cells. receptors in SCLC lines are a classic example for desensitized Signal transduction pathways which regulate the proliferation receptors; saturation of the receptor in such highly malignant of normal cells in response to external stimuli may thus become tumor cells, which are the end stage of decades of exposures to replaced by new ones. Further studies are needed to elucidate nicotine and nitrosamines by smoking, is reached at signifi the molecular changes in such signal transduction pathways cantly lower concentrations of nicotine than in the normal PNE under continuous exposure to environmental agents, such as cells of our hamster experiments. Moreover, because signal transduction via these receptors is blocked by closed Ca2+ the nitrosamines and nicotine, and during progression into a highly malignant state. channels, nicotine fails to stimulate cell proliferation in these cells. * H. M. SchÃ¼llcr,unpublished observations. 2725s

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2726s

Hildegard M. Schüller

Cancer Res 1992;52:2723s-2726s.

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