Andre Castonguay

INHIBITION OF COX-2 AND INDUCTION OF APOPTOSIS: Two Determinants of Nonsteroidal Anti-Inflammatory Drugs' Chemopreventive Efficacies in Mouse Lung Tumorigenesis

Recent studies suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit lung tumorigenesis under conditions that are immunosuppressive. We hypothesized that this inhibition of mouse lung tumorigenesis requires induction of apoptosis and inhibition of COX(cyclooxygenase)-1, COX-2, and the incidence of K-ras mutation. The NSAIDs used in this study include acetylsalicylic acid (ASA) that is anti-inflammatory with COX-1and COX-2inhibition and N-[2-(cyclohexyloxy)4-nitrophenyl]-methanesulfonamide (NS398) that is a specific COX-2inhibitor. Wehave previously demonstrated that ASA (147 and 294 mg/kg diet) and NS398 (7 mg/kg diet) inhibited lung tumorigenesis by 31%, 44%, and 34%, respectively, in 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-treated A/Jmice. No difference in the incidence and types of K-ras mutations was found between the lung tumors treated with NNK and those treated with NNK/ASA and NNK/NS398. In NNK-treated mice, ASA (294 mg/kg diet) or NS398 significantly increased the apoptotic index, from 0.07 to 0.30 or to 0.33, respectively. ASA (294 mg/kg diet) and NS398 also inhibited the expression of COX-2. Finally, modulation of gene expression by NS398 and ASA (294 mg/kg diet) was determined using Atlas cDNA expression arrays. Expression of cyclin B2 was decreased and expression of Fas-L and BAD were increased in lung tissues treated with both NS398 and ASA. Treatment with NS398 also increased expression of p 57kip2 and myosin. These genes modulated by NSAIDs may play a role in mediating the observed chemopreventive effects of the NSAIDs in the mouse lung. Our results demonstrate that lung tumor prevention with NSAIDs involve both the induction of apoptosis and the inhibition of COX-2 expression.Recent studies suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit lung tumorigenesis under conditions that are immunosuppressive. We hypothesized that this inhibition of mouse lung tumorigenesis requires induction of apoptosis and inhibition of COX (cyclooxygenase)-1, COX-2, and the incidence of K-ras mutation. The NSAIDs used in this study include acetylsalicylic acid (ASA) that is anti-inflammatory with COX-1 and COX-2 inhibition and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398) that is a specific COX-2 inhibitor. We have previously demonstrated that ASA (147 and 294 mg/kg diet) and NS398 (7 mg/kg diet) inhibited lung tumorigenesis by 31%, 44%, and 34%, respectively, in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-treated A/J mice. No difference in the incidence and types of K-ras mutations was found between the lung tumors treated with NNK and those treated with NNK/ASA and NNK/NS398. In NNK-treated mice, ASA (394 mg/kg diet) or NS398 significantly increased the apoptotic index, from 0.07 to 0.30 or to 0.33, respectively. ASA (294 mg/kg diet) and NS398 also inhibited the expression of COX-2. Finally, modulation of gene expression by NS398 and ASA (294 mg/kg diet) was determined using Atlas cDNA expression arrays. Expression of cyclin B2 was decreased and expression of Fas-L and BAD were increased in lung tissues treated with both NS398 and ASA. Treatment with NS398 also increased expression of p57kip2 and myosin. These genes modulated by NSAIDs may play a role in mediating the observed chemopreventive effects of the NSAIDs in the mouse lung. Our results demonstrate that lung tumor prevention with NSAIDs involve both the induction of apoptosis and the inhibition of COX-2 expression.

Inhibition of Lung Tumorigenesis by Nsaids: A Working Hypothesis

A 7-week treatment with the tobacco carcinogen NNK induced 8-10 lung adenomas per A/J mouse. NNK suppressed humoral and cellular immune responses and increased plasma PGE2 and LTB4 levels. This protocol is particularly suitable for testing NSAIDs and lipoxygenase inhibitors as cancer preventive agents. Sulindac and ASA inhibited lung tumorigenesis by 52 and 60%, respectively, attenuated the suppressive effect of NNK, and lowered the plasma PGE2 to basal levels. In contrast, naproxen neither inhibited lung tumorigenesis nor increased NNK-suppressed NK cell cytotoxicity. NSAIDs and lipoxygenase inhibitors had additive preventive efficacies against NNK-induced lung tumorigenesis. However, sulindac was not effective in preventing lung tumorigenesis induced by B[a]P, which lacks immunosuppressive activity. These results and those published by other investigators lead to the following hypothesis: Reactive intermediates derived from NNK interfere with the stimulation of the complex NF-kappa B/I kappa B. NF-kappa B is involved in the regulation of immune and inflammatory responses. The authors propose that NNK-derived intermediates induce the expression of COX-2 and lipoxygenase involved in NNK activation. This hypothesis provides a rationale for the lack of efficacy of naproxen to prevent tumorigenesis, to attenuate NNK-induced synthesis of PGE2, and to increase NK cell cytotoxicity. According to this hypothesis, PGE2 synthesis and induction of apoptosis contribute to varying degrees to the mechanism of cancer prevention.

On the formation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone during smoking☆

Tobacco-specific N-nitrosamines (TSNA) are the most abundant carcinogens identified in tobacco and its smoke. Reducing their levels in tobacco products and especially in cigarette smoke is, therefore, a primary goal towards minimizing the carcinogenic burden of the tobacco consumer. This study delineates the mechanisms of formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most powerful of the carcinogenic TSNA during cigarette smoking. It demonstrates, by means of radiolabeled tracer compounds that 6.9-11.0% of the NNK formed in tobacco during the curing process transfers into the mainstream smoke. This constitutes 26-37% of the NNK present in the smoke. Addition of [methyl-14C]-nicotine to cigarettes, prior to smoking, led to the finding that 0.001% of nicotine in the cigarette column appears in the smoke as NNK. Thus, 63-74% of NNK in smoke is formed during smoking. NNK yield in the smoke was independent of nitrate content of the tobacco. These data serve to devise methods of reducing TSNA in smoke.

Tobacco specific nitrosamines: Carcinogenicity, metabolism, and possible role in human cancer

(1983). Tobacco specific nitrosamines: Carcinogenicity, metabolism, and possible role in human cancer. Journal of Environmental Science and Health. Part C: Environmental Carcinogenesis Reviews: Vol. 1, No. 1, pp. 1-54.

Strain A/J Mouse Lung Adenoma Growth Patterns Vary When Induced by Different Carcinogens*

The histogenesis of mouse lung adenomas is currently being investigated in several laboratories. Based upon studies of a limited number of carcinogens in different mouse strains, some investigators suggest that all lung adenomas in mice are derived from alveolar type II cells, whereas others suggest a Clara cell origin for a majority of the tumors. This report differs from previous investigations in that 12 different carcinogens were evaluated for the types of lung tumor growth patterns they induced in a single mouse strain (strain A mice). The carcinogens aflatoxin B, (AFB,), benzo(a)pyrene (BP), 1,2-dimethylhydrazine (DMH), 3-meth-ylcholanthrene (MCA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N-nitrosomethylurea (MNU) induced tumors with a predominately solid/alveolar growth pattern, whereas N-nitrosodiethylamine (NDEA) induced predominantly papillary tumors. Most of the other carcinogens induced a higher proportion of lung tumors with the solid/alveolar growth pattern than with the papillary growth pattern; however, ratios between the 2 growth patterns varied. If, as suggested by others, solid tumors are derived from alveolar type II cells and papillary tumors from Clara cells, then carcinogens may differ with respect to their ability to transform one cell type or the other.

Lung Tumorigenicity of NNK Given Orally to A/J Mice: Its Application to Chemopreventive Efficacy Studies

The ability of five chemopreventive agents to inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors in A/J mice was determined. The carcinogen was administered in the drinking water during 7 weeks (at doses of 9.2 to 3.1 mg/mouse). Three chemopreventive agents: (dose, g/kg diet) ellagic acid (4.0), 2(3)-BHA (5.0), and sulindac (0.13) inhibited the multiplicity of lung adenomas by 52, 88, and 52%, respectively, when compared to NNK controls. beta-Carotene + retinol (2.14 + 0.009), in combination, and selenium (0.0022) were ineffective. NNK was absorbed more rapidly from the duodenum than from the stomach and was metabolized in both tissues. The activation of NNK by alpha-carbon hydroxylation and its deactivation by pyridine N-oxidation was more extensive in the duodenum than in the stomach. Carbonyl reduction of NNK was 10 times higher in the duodenum. Liver microsomes were more active than lung microsomes in the alpha-carbon hydroxylation of NNK, suggesting that some liver isozymes of cytochrome P-450 have a high affinity for NNK. Pyridine N-oxidation was five times more extensive in lung microsomes than in liver microsomes. Collectively, these results demonstrate that NNK given orally to A/J mice provides a suitable model from which to assess the relative activity and mechanisms of action of chemopreventive agents in pulmonary carcinogenesis.

Nasal Cavity Rhabdomyosarcoma (NCR) in Rats Treated with Tobacco‐Specific N‐Nitrosamines (TSNA)a

Four hundred male and female rats have been treated with N N N (”-nitrosonornicotine) and NNK [4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone], in trioctanoine, injected S.C. three times a week for 12 weeks. All spontaneously dead or sacrificed animals were autopsied and the gross lesions examined microscopically. After removing the skin, the heads were decalcified and the nasal cavity separated from the rest of the head by a cross-cut posterior to the eyeglobes. The specimen obtained was further divided in four equidistant parts by cross (frontal) sections. Microscopic examination was performed after paraffin embedding and H and E and PTAH staining.