Unstable cellular differentiation in adenosquamous cell carcinoma.

For the determination of whether two or more stem lines with fixed of stable differentiation are present in a growing mixed adenosquamous cell carcinoma, two different mixed tumors, derived from transformed F344 rat tracheal epithelium, were dissociated and ten single cells were isolated from each tumor. Each of these single cells was allowed to grow 27 population doublings to form a clonal population. All clones were then inoculated into animals for the assessment of in vivo differentiation. All cell inocula produced carcinomas within 7-70 days. Of the ten single cell clones isolated from the first tumor, seven clones produced mixed adenosquamous carcinomas and three produced adenocarcinomas in which no squamous cell component was found. From the second tumor, eight clones produced mixed tumors and two produced squamous cell carcinomas in which no adenocarcinoma component was found. The mixed tumors derived form clones contained widely varying proportions of adeno and squamous components. Recloning of the cloned lines yielded populations that produced mixed adenosquamous cell carcinomas upon inoculation in vivo. Comparison of tumor phenotypes from clones of early and late passages further confirmed the instability of differentiation in these neoplastic epithelial cells. These studies strongly suggest that the mixture of differentiated cell types in adenosquamous cell carcinomas is not the result of a mixture of stem cells with one fixed phenotype within the tumor, but rather is the result of an instability of differentiation; the neoplastic cells can express both types of differentiation.

Effects of dietary N-(4-hydroxyphenyl)retinamide on N-nitrosomethylbenzylamine metabolism and esophageal tumorigenesis in the Fischer 344 rat.

BACKGROUND: 9-cis-Retinoic acid (9-cis-RA) and N-(4-hydroxyphenyl)retinamide (4-HPR) are effective chemopreventive agents against epithelial tumors in the oral cavity, breast, and prostate. We tested the inhibitory activity of these retinoids against N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. METHODS: Male Fischer 344 rats were randomly assigned to receive diets either lacking or containing 9-cis-RA or 4-HPR for 1 week before tumor initiation with NMBA and then for the duration of the study. NMBA metabolism, O(6)-methylguanine adduct formation, and cytochrome P450 messenger RNA (mRNA) expression in the esophagi of the rats were studied to investigate the mechanisms by which dietary 4-HPR affects tumorigenesis. All statistical tests were two-sided. RESULTS: Dietary 4-HPR resulted in a dose-dependent and statistically significant enhancement (P<.05) of tumorigenesis in response to NMBA. In two different tumor bioassays, the mean tumor multiplicity for rats fed the highest concentration of dietary 4-HPR (0.8 g/kg diet) was increased by 5.9 tumors (95% confidence interval [CI] = 1.7 to 10.1 tumors) and 6.7 tumors (95% CI = 5.6 to 7.8 tumors) compared with the mean tumor multiplicity for rats that received the control diet lacking 4-HPR. Animals fed diets containing 9-cis-RA displayed no statistically significant increase in tumorigenesis. Compared with animals fed a diet lacking 4-HPR, animals fed 4-HPR had increased NMBA metabolism in esophageal explant cultures and had higher levels of O(6)-methylguanine DNA adducts and CYP2A3 mRNA in their esophagi. CONCLUSIONS: Dietary 4-HPR enhances tumorigenesis in response to NMBA in the rat esophagus by increasing tumor initiation events. Dietary 4-HPR may exert paradoxical effects at some sites, such as the aerodigestive tract, by modulating the bioactivation of carcinogens in target tissues.

Establishment of epithelial cell lines following exposure of cultured tracheal epithelium to 12-O-tetradecanoyl-phorbol-13-acetate.

In vitro exposure of rat tracheal epithelium to the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate results in a marked increase in growth capacity. The growth changes are manifested in an increased rate of cell division and growth in primary cultures and in the establishment of permanent epithelial cell lines. Such changes did not occur in control cultures. Fourteen of the cell lines have been inoculated into immunosuppressed recipients, and all are nontumorigenic.

Natural history of intraepithelial neoplasia in humans with implications for cancer chemoprevention strategy.

Intraepithelial neoplasia is of critical importance to the cancer chemoprevention field because it is a target condition for which drugs must be sought that will prevent its development or stop its progression. The term "dysplasia" refers to the morphological alterations that characterize intraepithelial neoplasia and according to many authors consists of seven basic morphological changes that occur in the majority of human epithelia, as well as in the epithelium of mouse skin papillomas induced by 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate: increased nuclear size; altered nuclear shape; increased nuclear stain uptake; nuclear pleomorphism (increased variation in nuclear size, shape, and stain uptake); increased mitoses; abnormal mitoses; and disordered or absent maturation. Clonal evolution appears to begin early in the neoplastic process during intraepithelial neoplasia. Aneuploidy has been found during intraepithelial neoplasia in many human epithelia, and, in association with other forms of genetic instability, may provide the increase in genetically variant cells required for clonal evolution to occur. It is postulated that two major factors affecting the rate of progression of intraepithelial neoplasia are the cellular mutation rate, which is enhanced by environmental carcinogens, and the cellular proliferation rate, which is enhanced by agents that include sex hormones, inducers of chronic inflammation, and irritant chemicals which stimulate reactive hyperproliferation. A preferred chemoprevention strategy should consist of the development of drugs and drug combinations which will block mutagenic carcinogens or prevent epithelial hyperproliferation or its causes. Two examples of the induction of regression of intraepithelial neoplasia by chemopreventive drugs are the regression of oral leukoplakia produced by beta-carotene and the regression of colorectal polyps in patients with familial polyposis produced by sulindac. It is evident that there is a strong need for more research on the induction of regression of intraepithelial neoplasia with chemopreventive agents. There is also a critical need to identify and develop biomarkers that correlate with the appearance and regression of intraepithelial neoplasia.

Enhanced induction of the anchorage-independent phenotype in initiated rat tracheal epithelial cell cultures by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The purpose of the studies reported here was to compare the response of noninitiated and initiated primary rat tracheal epithelial (RTE) cell cultures to the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The endpoints measured were number of cells per culture, colony-forming efficiency, subculturability, and colony formation in soft agarose. Primary RTE cell cultures were exposed on Day 1 to either 0.2% dimethyl sulfoxide, or to 0.1 micrograms per ml of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG). Thereafter, the same cultures were exposed twice weekly from Days 6 to 30 to either 0.2% dimethyl sulfoxide or to TPA (10 pg/ml). Sequential exposure to MNNG and TPA did not increase the number of viable cells per culture beyond that seen in MNNG-exposed cultures. Determination of the frequency of colony-forming cells 10 days after the end of the initiation-promotion treatment (Day 40 of culture) revealed a marked enhancement in colony-forming efficiency of treated cultures compared to dimethyl sulfoxide-exposed control cultures. However, sequential exposure to MNNG and TPA had an additive or slightly more than additive effect on the colony-forming efficiency of RTE cells exposed to MNNG or TPA only. Treatment of the primary cultures with MNNG alone or TPA alone increased the subculturability of RTE cells to a similar extent. The sequential exposure to MNNG followed by TPA appeared to have an additive effect on the frequency of subculturability. The most pronounced effect of the sequential MNNG-TPA exposure as compared to single-agent exposure was a marked enhancement of the anchorage-independent (ag+) phenotype. Of the cultures treated with MNNG followed by TPA, over 50% were ag+ at 60 days. In contrast, of the cultures treated either with MNNG alone or with TPA alone, only 3% were ag+ on Day 60. (All control cultures were ag-.) Colony-forming efficiency in soft agarose also increased disproportionately between 60 and 120 days in initiated-promoted cultures. These experiments indicate that the major effect of the tumor promoter TPA on initiated RTE cell cultures is to enhance the appearance of the late ag+-phenotype.

Screening of potential chemopreventive agents using biochemical markers of carcinogenesis.

Ninety potential chemopreventive agents were screened using 6 chemoprevention-associated biochemical end points. These compounds were tested using rodent (tracheal epithelial or liver) cells and human cells [neonatal foreskin fibroblasts, bronchial epithelial cells, or human leukemic cells (HL-60)]. The effects measured were: (a) inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tyrosine kinase activity in HL-60 cells; (b) inhibition of TPA-induced ornithine decarboxylase (ODC) activity in rat tracheal epithelial cells; (c) inhibition of poly(ADP-ribose)polymerase in propane sultone-treated primary human fibroblasts; (d) inhibition of benzo[a]pyrene(B[a]P)-DNA binding in human bronchial epithelial cells; (e) induction of reduced glutathione in Buffalo rat liver cells; and (f) inhibition of TPA-induced free radical formation in primary human fibroblasts or HL-60 cells. Fifty compounds were highly effective in inhibiting TPA-induced tyrosine kinase activity. This assay identified compounds from a wide variety of chemical classes as effective inhibitors, including all the vitamins, retinoic acid analogues, protein kinase C inhibitors, and chemicals belonging to the amino acid category. Fifty-two chemicals were classified as highly positive compounds when examined for their ability to inhibit TPA-induced ODC activity. These agents showed a dose-dependent inhibition or inhibition at all doses. Retinoids, in general, exhibited strong inhibition of ODC activity. A category of compounds showing dose-dependent inhibition were the sulfur compounds, especially the thiols and thiones. Among the natural products, terpenes were strong inhibitors of ODC. Forty-seven compounds were classified as strong inhibitors of poly(ADP-ribose)polymerase. In the carcinogen-DNA binding inhibition assay, 21 compounds were identified as strong inhibitors, which include phenolic compounds as well as sulfur compounds. Vitamins and their analogues were also good inhibitors. Testing for induced glutathione yielded 19 compounds that were good inducers. Sulfur-containing compounds and most of the phenolic compounds were also inducers of glutathione. Twenty compounds were highly positive for inhibition of TPA-induced free radical formation. A significant number of phenolic and sulfur compounds were again strong oxygen radical scavengers. Some antiinflammatory agents were also identified as free radical inhibitors. In general, retinoids were quite active in all the assays. Eight compounds were positive in all of the six assays; these were vitamin C (ascorbic acid), bismuththiol, esculetin, etoperidone, folic acid, hydrocortisone, indole-3-carbinol, and tocopherol succinate. Agents that were positive in these assays may inhibit the carcinogenesis process by similar mechanisms in humans and are identified as candidates for development as chemopreventive agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of chemopreventive agents in different mechanistic classes using a rat tracheal epithelial cell culture transformation assay.

The rat tracheal epithelial (RTE) cell focus inhibition assay was used to identify potential chemopreventive agents. Ninety-nine agents were evaluated for their ability to inhibit benzo[a]pyrene-induced transformation of RTE cells. Freshly isolated RTE cells were exposed to benzo[a]pyrene alone or in combination with a chemopreventive agent. After 30 days in culture, transformed foci were scored and inhibition was quantitated. In these studies, foci formation was inhibited mainly by agents which modulate the initiation of carcinogenesis by altering drug-metabolizing enzymes, inhibiting the binding of benzo[a]pyrene to DNA, enhancing detoxification of activated carcinogens, or by inducing epithelial cell differentiation. Such agents include antioxidants, free radical scavengers, glutathione S-transferase enhancers, vitamins, retinoids, and sulfhydryl compounds. Agents which inhibit ornithine decarboxylase and arachidonic acid metabolism were not as effective. The RTE assay provides important data for agent selection prior to whole animal-screening assays in the development of chemoprevention drugs.

Inhibition of transformation in cultured rat tracheal epithelial cells by potential chemopreventive agents.

Twenty-eight compounds were screened for chemopreventive activity by using a rat tracheal epithelial cell transformation inhibition assay. In this new assay, chemicals were tested for their ability to inhibit the formation of transformed rat tracheal epithelial cell colonies which arise following exposure to the carcinogen benzo(a)pyrene. The 15 positive compounds were N-acetylcysteine, bismuththiol, calcium glucarate, (+/-) catechin, diallyl disulfide, glycaric acid, D-glucaro-1,4-lactone, N-(4-hydroxyphenyl)retinamide, D-limonene, mesna, retinoic acid, rutin, quercetin, silymarin, and taurine. In examining the nature of compounds that inhibited rat tracheal epithelial cell transformation, several possible chemopreventive mechanisms appeared to be predominant: compounds that were positive (a) increased glutathione levels or enhanced conjugation; (b) increased cytochrome P-450 activity; (c) displayed nucleophilic activity; or (d) induced differentiation. Thirteen compounds were negative in the rat tracheal epithelial transformation inhibition assay: crocetin, difluoromethylornithine, ellagic acid, esculetin, enoxalone, ibuprofen, levamisole, nordihydroguaiaretic acid, L-2-oxothiazolidine-4-carboxylate, piroxicam, sodium butyrate, D-alpha-tocopherol acetate, and polyethylene glycol 400. It was evident from these results that this assay would not detect compounds that were (a) anti-promoting in nature; (b) glutathione inhibitors; (c) differentiation inhibitors; (d) O6-methylguanine inhibitors; (e) organ specific; or (f) inactive. The rat tracheal epithelial cell transformation inhibition assay appeared to identify chemopreventive compounds that act at early stages of the carcinogenic process.

Progress in cancer chemoprevention: perspectives on agent selection and short-term clinical intervention trials.

The basic cancer-related chemical and biological sciences, pathology, and epidemiology have contributed to the understanding that antimutagenesis and antiproliferation are the important general mechanisms of chemoprevention and to the development of antimutagenic and anti-proliferative agents as potential chemopreventive drugs. These disciplines have also provided the biochemical and histopathological bases for identifying intermediate biomarkers that can be used as surrogate end points for cancer incidence in clinical chemoprevention trials and for selecting cohorts for these trials. Particularly important as histological biomarkers of cancer are the cytonuclear morphological and densitometric changes that define intraepithelial neoplasia (IEN). IEN changes are on the causal pathway to cancer. They may serve as target lesions in Phase II chemoprevention trials and as standards against which other earlier cellular and molecular biomarkers can be evaluated. Strategies for the clinical evaluation of chemopreventive agents have been defined for seven targets--colorectal, prostate, lung, breast, bladder, oral, and cervical cancers. Cohorts have been identified for short-term Phase II trials that investigate the effects of chemopreventive agents on IEN and on earlier biomarkers. Patients with adenomas serve as a cohort for trials in colon. One cohort for Phase II trials in prostate is patients with early stage cancers scheduled for prostatectomy; another is patients with prostatic intraepithelial neoplasia (without prostatic carcinoma). Patients treated for lung cancer are at high risk for bronchial dysplasia and second cancers; such patients are a cohort for Phase II trials in lung cancer. Presurgical breast cancer patients and patients with ductal or lobular carcinoma in situ are cohorts for studies in breast. Patients with superficial bladder cancers (Ta/T1 with or without carcinoma in situ) are cohorts for studies of chemoprevention in bladder, and patients with dysplastic oral leukoplakia are evaluated for chemoprevention of oral cancers. Cervical intraepithelial neoplasia is a prototype IEN, and patients with cervical intraepithelial neoplasia are a cohort for studies of cervical cancer.

Chemopreventive efficacy of sulindac sulfone against colon cancer depends on time of administration during carcinogenic process.

Epidemiological and model studies with laboratory animals have provided evidence that nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer. Sulindac, a nonsteroidal anti-inflammatory drug, has been shown to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats when administered continuously before, during, and after carcinogen treatment (initiation and postinitiation periods) or when given continuously beginning 14 weeks after carcinogen administration (promotion/ progression stage). The present study was designed to investigate the chemopreventive efficacy of sulindac sulfone (exisulind), the sulfone metabolite of sulindac, when administered during the promotion/progression stage of colon carcinogenesis in comparison to the effect during the initiation and postinitiation periods. We have also studied the modulating effect of exisulind on colonic tumor apoptosis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0%, 0.06%, and 0.12% exisulind. At 7 weeks of age, groups of animals were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 weeks). Animals intended for the promotion/progression study and receiving 0% exisulind were switched to an experimental diet containing 0.12% exisulind at 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, 50 weeks after the second AOM injection. Colon tumors were evaluated histopathologically for tumor type. Administration of 0.06% and 0.12% exisulind during the initiation and postinitiation periods significantly inhibited the incidence and multiplicity of invasive and/or noninvasive adenocarcinomas of the colon. The inhibition of colon tumorigenesis by exisulind was associated with a significant retardation of body weight gain shortly after sulfone administration and increased apoptosis in the colon tumors. In contrast, administration of the higher dose (0.12%) of exisulind during the promotion/progression stage had only minimal effects on colon tumorigenesis and apoptosis in the colon tumors, suggesting that early administration, but not late administration, may be required for chemopreventive efficacy of this drug.

Cytokeratin, lectin, and acidic mucin modulation in differentiating colonic epithelial cells of mice after feeding Western-style diets.

Several studies have recently reported the development of colonic epithelial cell hyperproliferation in rodents following the ingestion of Western-style diets. In this study, additional measurements related to differentiation and maturation of the colonic epithelial cells were made after feeding this type of diet. Two Western-style diets high in fat and phosphate content and low in calcium and vitamin D were fed to C57BL/6J mice for 12, 24, and 52 weeks. Diet A contained American Blend fat as a source of lipids, diet B contained corn oil, and control diet C was a standard AIN-76A semisynthetic diet which is lower in fat content and higher in calcium and vitamin D. Colonic epithelial cells were studied for three biomarkers: cytokeratin catalogue no. 18 (clone LE64) expression, soybean agglutinin carbohydrate lectin binding, and acidic mucins including sialo- and sulfomucins. Feeding of diets A and B revealed that colonic epithelial cells had increased expression of cytokeratin catalogue 18 and SBA carbohydrate lectin binding compared to controls (P = 0.0001 for diet A versus C and diet B versus C). Significant differences were found between diets B and C (P = 0.0001) and diets A and C (P = 0.0001) in total acidic mucins and in the ratio of sialomucin:sulfomucin (P = 0.0001). These findings demonstrate that both functional and structural modifications occurred in colonic epithelial cells under these dietary conditions, and further defined this rodent model for preclinical evaluation of nutritional and chemopreventive interventions.

Chemoprevention of rat prostate carcinogenesis by 9-cis-retinoic acid.

A chemoprevention study was conducted to evaluate the activity of 9-cis-retinoic acid (9-cis-RA) as an inhibitor of prostate carcinogenesis in male Wistar-Unilever (HsdCpb:Wu) rats. After pretreatment with a sequential regimen of cyproterone acetate (50 mg/kg/day for 21 days) and testosterone propionate (100 mg/kg/day for 3 days), groups of 40 rats received a single i.v. injection of N-methyl-N-nitrosourea (MNU; 30 mg/kg body weight). Beginning 2 weeks after carcinogen administration, rats received chronic exposure to testosterone administered in s.c. implanted silastic capsules. The study was terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. Continuous dietary administration of 9-cis-RA at 100 mg/kg diet or 50 mg/kg diet beginning 1 week before MNU administration reduced cancer incidence in the dorsolateral + anterior prostate from 65% in dietary controls to 18 and 20%, respectively (P < 0.001 for both comparisons). Similarly, these dose levels of 9-cis-RA reduced the incidence of cancer in all accessory sex glands from 79% in dietary controls to 48 and 33% (P < 0.01 for both comparisons), respectively. Chronic dietary administration of 9-cis-RA induced no gross or organ-specific toxicity in any animal and did not suppress group mean body weight gain. The potent anticarcinogenic activity of 9-cis-RA in the rat prostate, when considered with its apparent lack of toxicity in rodents, suggests that this and other ligands for the retinoid X receptor merit consideration for evaluation in clinical prostate cancer chemoprevention trials.

Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression stages of colon cancer.

Curcumin, derived from the rhizome of Curcuma longa L. and having both antioxidant and anti-inflammatory properties, inhibits chemically induced carcinogenesis in the skin, forestomach, and colon when it is administered during initiation and/or postinitiation stages. This study was designed to investigate the chemopreventive action of curcumin when it is administered (late in the premalignant stage) during the promotion/progression stage of colon carcinogenesis in male F344 rats. We also studied the modulating effect of this agent on apoptosis in the tumors. At 5 weeks of age, groups of male F344 rats were fed a control diet containing no curcumin and an experimental AIN-76A diet with 0.2% synthetically derived curcumin (purity, 99.9%). At 7 and 8 weeks of age, rats intended for carcinogen treatment were given s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight per week. Animals destined for the promotion/progression study received the AIN-76A control diet for 14 weeks after the second AOM treatment and were then switched to diets containing 0.2 and 0.6% curcumin. Premalignant lesions in the colon would have developed by week 14 following AOM treatment. They continued to receive their respective diets until 52 weeks after carcinogen treatment and were then sacrificed. The results confirmed our earlier study in that administration of 0.2% curcumin during both the initiation and postinitiation periods significantly inhibited colon tumorigenesis. In addition, administration of 0.2% and of 0.6% of the synthetic curcumin in the diet during the promotion/progression stage significantly suppressed the incidence and multiplicity of noninvasive adenocarcinomas and also strongly inhibited the multiplicity of invasive adenocarcinomas of the colon. The inhibition of adenocarcinomas of the colon was, in fact, dose dependent. Administration of curcumin to the rats during the initiation and postinitiation stages and throughout the promotion/progression stage increased apoptosis in the colon tumors as compared to colon tumors in the groups receiving AOM and the control diet. Thus, chemopreventive activity of curcumin is observed when it is administered prior to, during, and after carcinogen treatment as well as when it is given only during the promotion/progression phase (starting late in premalignant stage) of colon carcinogenesis.

Apoptosis, cell replication, and Western-style diet-induced tumorigenesis in mouse colon.

In this study, feeding Western-style diets (WDs) to mice for a duration of two years without any chemical carcinogen led to the development of gross colonic lesions that were histologically classified as dysplastic crypts and focal hyperplasias with or without atypical nuclei. To better understand early biological events contributing to the development of colonic neoplasia, grossly normal colonic mucosa was investigated; mitotic and apoptotic colonic epithelial cells, atypical mitosis, and atypical nuclei were studied. A significant and transient increase of mitotic activity in the basal and intermediate portions of the colonic crypts was seen in young mice after feeding them the WDs. This was accompanied by diffuse activation of apoptosis of the colonic epithelial cells. In the middle of the rodents' life span, after administration of both the WDs and control diet, the rodents developed a marked depletion of apoptotic epithelial cells in the mid-region of the colonic crypts; this was followed by the expansion of an epithelial cell population containing atypical nuclei, and the emergence of the gross lesions noted above. With this sequence of events, prolonged feeding of WDs to mice produced single-crypt dysplastic lesions and focal hyperplasias indicative of tumorigenesis.

Exceptional chemopreventive activity of low-dose dehydroepiandrosterone in the rat mammary gland.

To determine if the chemopreventive activity of dehydroepiandrosterone (DHEA) in the rat mammary gland can be dissociated from its toxicity, two studies were conducted in which low doses of DHEA were administered alone and in combination with other agents to rats treated with N-methyl-N-nitrosourea. Beginning 1 week prior to administration of 35 mg N-methyl-N-nitrosourea per kg body weight, groups of 20 female Sprague-Dawley rates were fed AIN-76A diet supplemented with DHEA alone (800 or 400 mg/kg diet), DHEA + tamoxifen (80 or 40 microgram/kg diet), DHEA + carbenoxolone (3500 or 1750 mg/kg diet), or DHEA + tamoxifen + carbenoxolone. When administered alone at either 800 or 400 mg/kg diet, DHEA reduced mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence from >70% in dietary controls to 0%; mammary cancer incidence in all DHEA combination regimens was also < or = 5%. The dose levels of DHEA used induced no toxicity or alteration in body weight gain. These results indicate that dietary supplementation with low doses of DHEA has chemopreventive efficacy greater than or equal to that of endocrine ablation. This protection may be mediated by the induction of differentiation in the mammary parenchyma.

Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice.

This investigation is part of a continuing effort to develop effective chemoprevention for carcinogenesis of the lung. The present study explores the use of aerosol administrations for this purpose. The agent selected for initial study was the synthetic glucocorticoid budesonide. This selection was based on previous work in which budesonide added to the diet was found to inhibit pulmonary adenoma formation in female A/J mice. However, high dose levels were required, i.e., of the order of 300 microg/kg, of body weight [L. W. Wattenberg and R. D. Estensen, Carcinogenesis (Lond.), 18: 2015-2017, 1997]. For aerosol administration of budesonide, a nose-only technique has been developed that entails nebulization of the compound dissolved in ethanol and subsequent stripping off of the solvent (less than 3 microl ethanol/liter of air remaining at the site of inhalation). The budesonide particles produced by the apparatus had a mass median aerodynamic diameter of less than 1 microm. An experiment has been carried out in which the inhibitory effects of aerosolized budesonide, given for 1 min six times a week, were studied. Concentrations of budesonide of 26, 81, and 148 microg/liter of air (calculated doses of 23, 72, and 126 microg/kg of body weight) were used. The aerosols were started 1 week after three oral administrations of benzo(a)pyrene (2 mg/20 g of body weight) to female A/J mice. All three doses of budesonide resulted in more than 80% inhibition of pulmonary tumor formation compared to the aerosol control and 90% or greater compared to mice not exposed to aerosol. The difference in inhibition is due to the aerosol procedure itself, which produces a reduction in tumor formation. A decrease in splenic weight (evidence of a systemic effect) occurred at all doses of budesonide. To the best of our knowledge, this is the first published effort at the use of aerosol administration to prevent neoplasia of the respiratory tract. The results of the present study show that administration of a potential chemopreventive agent by aerosol at a low dose can inhibit the occurrence of pulmonary carcinogenesis in female A/J mice.

Chemoprevention of colon carcinogenesis by dietary perillyl alcohol.

Epidemiological studies suggest that consumption of diets containing fruits and vegetables, major sources of phytochemicals and micronutrients, may reduce the risk of developing cancer of the colon. Several phytochemicals and micronutrients present in fruits and vegetables are known to exert cancer-chemopreventive effects in several organs, including the colon. Monoterpenes such as d-limonene and perillyl alcohol derived from orange peels and lavender, respectively, have been shown to possess chemopreventive properties against mammary, liver, and/or lung carcinogenesis. The present study was designed to investigate the efficacy of dietary 40 and 80% maximum tolerated dose (MTD) levels of perillyl alcohol on azoxymethane (AOM)-induced colon carcinogenesis. The effect of this agent on the process of apoptosis in colon tumors was also investigated. Prior to the efficacy study, the MTD of perillyl alcohol was determined in male F344 rats in a 6-week subchronic toxicity study and found to be a 2.5-g/kg diet when added to the AIN-76A diet. At 5 weeks of age, groups of male F344 rats were fed control (AIN-76A) diet or diets containing 1 and 2 g perillyl alcohol/kg diet, representing 40 and 80% MTD levels, respectively. At 7 weeks of age, all animals except those in the vehicle-treated groups were given two weekly s.c. injections of AOM (15 mg/kg body weight/week). All animals were continued on their respective dietary regimen for 52 weeks after AOM treatment and then sacrificed. Colon tumors were evaluated histopathologically using routine procedures. Perillyl alcohol at the 1-g/kg level significantly inhibited the incidence (percentage of animals with tumors) and multiplicity (tumors/ animals) of invasive adenocarcinomas of the colon, whereas perillyl alcohol at 2 g/kg diet inhibited the incidence of total adenocarcinomas of the colon and small intestine as compared to the control diet. Our studies also indicate that the colon tumors of animals fed perillyl alcohol exhibited increased apoptosis as compared to those fed the control diet. These results demonstrate the potential chemopreventive activity of perillyl alcohol against colon carcinogenesis. The chemopreventive activity of perillyl alcohol is mediated through the tumor cell loss by apoptosis.

Chemoprevention of rat prostate carcinogenesis by early and delayed administration of dehydroepiandrosterone.

Two in vivo bioassays were conducted to evaluate the efficacy of dehydroepiandrosterone (DHEA) as an inhibitor of prostate carcinogenesis in rats. Prostate adenocarcinomas were induced in male Wistar-Unilever rats by a sequential regimen of cyproterone acetate and testosterone propionate, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU) and chronic androgen stimulation. In the first experiment, DHEA (1000 or 2000 mg/kg diet) was administered continuously to rats beginning 1 week before MNU exposure. In the second experiment, continuous administration of DHEA (2000 mg/kg diet) was begun either 1 week before, 20 weeks after, or 40 weeks after MNU exposure. Controls received basal diet without added DHEA. Studies were terminated at 13 months after MNU administration, and prostate cancer incidence was determined by histopathological evaluation of step sections of accessory sex glands. In the first study, continuous dietary administration of DHEA beginning 1 week before MNU resulted in a dose-related inhibition of prostate cancer induction. In the second experiment, comparable reductions in prostate cancer incidence were observed in groups exposed to DHEA beginning 1 week before, 20 weeks after, and 40 weeks after carcinogen exposure. These data demonstrate that nontoxic doses of DHEA confer significant protection against prostate carcinogenesis in rats. The efficacy of delayed administration of DHEA suggests that the compound confers protection against later stages of prostate cancer induction and can suppress the progression of existing preneoplastic lesions to invasive disease.

Influence of N-methyl-N-nitrosourea, testosterone, and N-(4-hydroxyphenyl)-all-trans-retinamide on prostate cancer induction in Wistar-Unilever rats.

The influence of chemical carcinogen, hormonal stimulation, and chronic dietary administration of the synthetic retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), on the induction of prostate cancer in male Wistar-Unilever rats was determined. Three different tumor induction regimens were used: (a) a single i.v. dose of 50 mg of N-methyl-N-nitrosourea (MNU) per kg body weight, followed by chronic androgen stimulation via s.c. implantation of two silastic capsules containing 40 mg testosterone each; (b) a single i.v. dose of 50 mg of MNU per kg body weight (no testosterone treatment); and (c) chronic androgen stimulation with implanted testosterone capsules (no MNU treatment). In a fourth series of animals, the incidence of spontaneous prostate tumors was determined in groups of rats receiving neither carcinogen nor hormone stimulation. Within each series, parallel groups of animals were fed a control (vehicle-supplemented) diet or control diet supplemented with 4-HPR beginning 1 day after carcinogen administration; retinoid administration was continuous until termination of the study at 450 days. The incidence of accessory sex gland cancer in rats treated sequentially with MNU + testosterone was >60%, in comparison with cancer incidences of <20% in rats receiving MNU only and <5% in rats treated with testosterone only. No spontaneous accessory sex gland tumors were observed in rats receiving no carcinogen and no testosterone. Tumor induction in the accessory sex glands by MNU + testosterone was relatively specific for the prostate: the incidence of carcinoma of the dorsolateral/anterior prostate was more than 5-fold greater than the incidence of cancer present only in the seminal vesicle. 4-HPR conferred no protection against cancer induction in the prostate by any regimen of MNU and/or testosterone. These results demonstrate the importance of both carcinogen exposure and hormone stimulation on the induction of neoplasia in the prostate of Wistar-Unilever rats.

Celecoxib inhibits N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats.

Epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) may have a role in the prevention of human cancers. A number of preclinical studies have also suggested that inhibition of cyclooxygenase (COX) with NSAIDs has an anticancer effect in animal models of colon, urinary bladder, skin, and breast. In these studies, we evaluated the COX-2 inhibitor celecoxib in two rodent models of urinary bladder cancer. Male B6D2F1 mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) developed transitional and squamous cell urinary bladder cancers, many of which grew rapidly and caused substantial morbidity that required sacrifice of the mice. Groups of mice received various daily doses of celecoxib in the diet (1250, 500, or 200 mg/kg of diet) beginning 7 days before the initiation of 12 weekly doses of OH-BBN. Mice were checked weekly for the presence of palpable urinary bladder masses. The study was terminated at 8 months following the initial treatment with OH-BBN. The percentage of mice with large palpable bladder lesions, which necessitated sacrifice of the mice, was 40% in the OH-BBN control group. In contrast, only 10% of all celecoxib-treated mice required sacrifice before the scheduled termination of the experiment, implying that all three doses of celecoxib inhibited the formation of large palpable lesions. Celecoxib did not significantly alter the incidence of preneoplastic bladder lesions, but did dose-dependently decrease the total number of urinary bladder cancers/mouse, palpable plus microscopic, by 77, 57, and 43% at dosages of 1250, 500, and 200 mg of celecoxib/kg of diet, respectively. In the second model, female Fischer-344 rats were administered OH-BBN twice/week for a period of 8 weeks. After 8 months, all rats developed preneoplastic lesions, whereas roughly 60% of the rats developed relatively small urinary bladder cancers. Rats were treated continually with celecoxib in the diet (500 or 1000 mg/kg of diet) beginning either 1 week prior to the initial OH-BBN treatment or beginning 1 week following the last OH-BBN treatment. Neither celecoxib treatment regimen significantly altered the number of preneoplastic lesions. Whereas celecoxib treatment initiated prior to OH-BBN administration decreased cancer incidence roughly 65%, celecoxib treatment initiated beginning 1 week after the last dose of OH-BBN profoundly decreased cancer incidence (>95%). Celecoxib did not alter the body weights of the mice or rats, or cause other signs of toxicity at any of the doses studied. Taken together these results demonstrate that: (a) celecoxib effectively inhibits tumor growth and enhances survival in the mouse model of urinary bladder cancer; and (b) celecoxib profoundly inhibits development of urinary bladder cancers in the rat model even when administered following the last dose of OH-BBN. Clinical trials will be necessary to determine whether COX-2 inhibitors will provide a clinical benefit in human bladder cancer.