Combination chemoprevention of rat mammary carcinogenesis by indomethacin and butylated hydroxytoluene

Indomethacin, a nonsteroidal antiinflammatory agent which inhibits prostaglandin biosynthesis, is an effective inhibitor of mammary carcinogenesis in rats. However, the activity of indomethacin as a chemopreventive agent is limited by toxicity. The present studies were conducted to determine if the toxic and anticarcinogenic effects of indomethacin can be modified by the phenolic antioxidant, butylated hydroxytoluene (BHT). Simultaneous administration of BHT resulted in a dose-related inhibition of indomethacin toxicity in female Sprague-Dawley rats, and increased the tolerable indomethacin dose from 50 to 150 mg/kg diet. When BHT (5000 mg/kg diet) and indomethacin (50 mg/kg diet) were administered in combination, no increased inhibition of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis was observed above that attained by administration of BHT alone or indomethacin alone at those doses. However, when the indomethacin dose was increased to 100 mg/kg diet, an enhanced inhibition of carcinogenesis was attained when BHT and indomethacin were administered from 2 weeks prior to until 1 week after 7,12-dimethylbenz(a)anthracene administration. These data indicate that "combination chemoprevention" regimens can be utilized to reduce the toxicity of anticarcinogenic drugs. However, the BHT-indomethacin interaction appears to involve a functional or dispositional antagonism which limits the anticarcinogenic efficacy of increasing indomethacin dose level.     

Lactobacilli, anticarcinogenic activities and human intestinal microflora.

Lactobacilli belong to the normal oropharyngeal and intestinal microflora in humans. These microorganisms contribute to the stabilization of the microflora and maintain the colonization resistance against pathogens. Lactobacilli have been used as dietary supplements in order to prevent gastrointestinal disturbances. Claims have been made that certain strains of lactobacilli possibly exert anticarcinogenic activities. The activity of bacterial enzymes, implicated in colon carcinogenesis may be elevated by a high meat, Western-type diet. Supplements of Lactobacillus acidophilus decreased these levels in both rats and humans. Colon cancer patients given L. acidophilus fermented milk showed a significant increase both in numbers of intestinal lactobacilli and dietary calcium intake, while decreasing trends in levels of both soluble faecal bile acids and faecal bacterial enzymes, two risk makers for colon cancer, were observed. In vitro studies have revealed that lactobacilli and other lactic acid bacteria have the ability to absorb cooked food mutagens. Recent studies in humans have shown that intake of L. acidophilus significantly reduced the mutagen excretion after consumption of fried meat. Several mechanisms by which lactobacilli might exert anticarcinogenic effects are discussed. Thus, certain strains of lactobacilli might lower the colon cancer risk in humans.     

Inhibition of azoxymethane-induced neoplasia of the large bowel by 3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene (nerolidol)

The inhibitory capacities of four terpenes on azoxymethane (AOM)-induced neoplasia of the large bowel and duodenum was studied in male F344 rats. A complete course of AOM administrations was given and 3 days later the rats were fed a semipurified diet containing 5 mg/g of the test compounds, i.e. 3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene (nerolidol), beta-citronellol, (+/-)-linalool and (1R,2S,5R)-(-)-menthol or a corresponding control diet. The experiment was terminated 22 weeks after the last dose of AOM. Under these conditions, nerolidol showed an inhibitory effect on carcinogenesis of the large bowel. The number of rats bearing large bowel neoplasms (adenomas) was reduced from 82% in the controls to 33% in rats fed nerolidol and the number of tumors/rat from 1.5 in the controls to 0.7 in the nerolidol group. A reduction in adenocarcinomas of the duodenum was found but the data are not statistically significant. The effects of nerolidol are of interest in terms of the identification of a new inhibitor of carcinogenesis of the large bowel. The chemical structure of nerolidol suggests the possibility that the compound might have an impact on protein prenylation or some other aspect of the mevalonate pathway, but this remains to be established.     

Lipoxygenase modulation to reverse carcinogenesis

New studies of the relationship between polyunsaturated fatty acid metabolismand carcinogenesis have led to novel molecular targets for cancer chemoprevention research. These targets include procarcinogenic lipoxygenases (LOXs), including 5-, 8-, and 12-LOX, and anticarcinogenic LOXs, including 15-LOX-1 and possibly 15-LOX-2. Recent studies indicate that 15-LOX-1 is down-regulated in colorectal cancer cells and that the ability of nonsteroidal anti-inflammatory drugs, a class of clinically active cancer chemopreventive agents, to induce apoptosis and growth inhibition in these cells was dependent on the induction of 15-LOX-1 and its metabolic product 13-S-hydroxyoctadecadienoic acid. Consistent with the colorectal studies, 15-LOX very recently has shown anticarcinogenic activity in esophageal and prostatic carcinogenesis. Inhibitors of other LOXs (e.g., 5-LOX) have preclinical anticarcinogenic activity and are being developed for clinical chemoprevention study. These and other LOX data led us to propose that the various LOX pathways exist in a dynamic balance that shifts during carcinogenesis toward 5-, 8-, and 12-LOX (and cyclooxygenase-2) and away from 15-LOX. A novel approach for cancer chemoprevention would involve LOX modulators, i.e., agents that can induce the anticarcinogenic and/or inhibit the procarcinogenic LOXs, thereby shifting the balance of LOX activities from procarcinogenic to anticarcinogenic metabolism of polyunsaturated fatty acids.     

Anticarcinogenic and hepatotoxic interactions between retinyl acetate and butylated hydroxytoluene in rats

The natural retinoid, retinyl acetate (RA), and the phenolic antioxidant, butylated hydroxytoluene (BHT), are both effective inhibitors of mammary carcinogenesis in rats. The present study was designed to determine if an increased inhibition of mammary carcinogenesis is obtained when RA and BHT are administered in combination. At age 50 days (time 0), virgin, female Sprague-Dawley rats received a single intragastric instillation of 16 mg of 7,12-dimethylbenz(a)anthracene dissolved in 1 ml sesame oil. Groups of 30 carcinogen-treated rats received Wayne Lab Chow supplemented with (per kg diet) 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT by the following schedule: -2 to +1 week; +1 week until the end of the experiment; -2 weeks to end; or none. Combined administration of RA plus BHT by the -2 weeks to end schedule was more effective in mammary cancer chemoprevention than was RA alone or BHT alone; the interaction of RA and BHT was additive. Similarly, administration of RA plus BHT by the -2 weeks to end protocol was more active in chemoprevention than was RA plus BHT administered either from weeks -2 to +1 or +1 week to end. Chronic exposure to RA plus BHT induced a high incidence of hepatic fibrosis and bile duct hyperplasia; these changes were not observed in controls and were seen in low incidence in animals exposed to RA only or BHT only. These data indicate that enhanced anticarcinogenic activity can be obtained through the use of "combination chemoprevention" regimens; however, chemopreventive compounds may interact not only to inhibit carcinogenesis but also to induce toxicity.     

Inhibition of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis by concomitant or postcarcinogen antioxidant exposure

When administered prior to or at the time of carcinogen exposure, the phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are effective inhibitors of carcinogenesis in several target organs. However, chronic, postcarcinogen administration of BHT apparently enhances tumorigenesis in certain animal models for liver and lung cancer. The present study was performed to determine the effects of BHA and BHT on mammary carcinogenesis when antioxidant exposure is limited to defined periods encompassing or following carcinogen availability. At 50 days of age (Time O), virgin female Sprague-Dawley rats (25/group) were given a single intragastric dose of 8 mg of 7,12-dimethylbenz(a) athracene . Basal diet (Wayne Lab Meal) was supplemented with 5000 or 2500 mg of BHA or BHT/kg by the following protocol: 2 weeks before until 1 week after carcinogen administration; 1 week after carcinogen administration until the end of the study; or none. The experiment was terminated 210 days after 7,12-dimethylbenz(a)anthracene administration, and all mammary tumors were confirmed histologically. When administered by the 2 weeks before to 1 week after schedule, both BHA and BHT were effective inhibitors of mammary carcinogenesis. However, the compounds also were active in chemoprevention when administered by the 1 week after to end protocol. These data indicate that the anticarcinogenic activity of antioxidants is not limited to influences on carcinogen metabolism, since both BHA and BHT inhibited mammary tumor induction when their administration was begun following clearance of the carcinogen from the mammary gland. The anticarcinogenic activity of postcarcinogen administration of BHA and BHT in the mammary gland is in contrast to the apparent tumor-enhancing activity of BHT in the liver and lung.     

Effects of dairy products on heterocyclic aromatic amine-induced rat colon carcinogenesis

Heterocyclic aromatic amines (HAA) are initiating agents of colon carcinogenesis in animals and are suspected in the aetiology of human colon cancer. In the context of prevention, it seems interesting to test possible protective compounds, such as fermented milk, against HAA food carcinogens. Male F344 rats were used in a model of HAA-induced colon carcinogenesis. The HAA, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (ratio 1:1:1) were administered in food for a 7 week induction period, with a cumulative dose of 250 mg of the HAA, per kg body weight. Four different diets were given to four rat groups: supplemented with 20% water, 30% non-fermented milk, 30% Bifidobacterium animalis DN-173 010 fermented milk and 30% Streptococcus thermophilus DN-001 158 fermented milk. Fecal mutagenicity was quantified during the induction period. At the end of the treatment, DNA lesion levels were determined in the liver and colon using the number of 8-oxo-7,8-dihydro-2'desoxyguanosine (8-oxodGuo) oxidized bases, "3D Test" and comet assay. The metabolic activity of hepatic and colon cytochrome P450 (CYP450) 1A1 and 1A2 was also evaluated. Aberrant colon crypts were scored, 8 weeks after the last HAA treatment. The results showed that dairy products decreased the incidence of aberrant crypts in rats: 66% inhibition with the milk-supplemented diet, 96% inhibition with the B.animalis fermented milk-supplemented diet and 93% inhibition with the S.thermophilus fermented milk-supplemented diet. Intermediate biomarkers showed that there was a decrease in HAA metabolism, fecal mutagenicity and colon DNA lesions. These results demonstrate the early protective effect of milk in the carcinogenesis process. This effect being more pronounced in the case of milk fermented by lactic acid bacteria.     

Anticlastogenic and Anticarcinogenic Potential of Thai Bitter Gourd Fruits

Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possessphase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activatingenzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined theanticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, inmice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesisin rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end ofexperiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. Foranticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% groundfreeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kgonce a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleatedperipheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstratedthat rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as themultiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruitspossesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potentialagainst AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm whengiving during the initiation stage.     

Fatty acid synthase is a potential molecular target for the chemoprevention of breast cancer

The purpose of this investigation was to determine whether fatty acid synthase (FAS) is a potential molecular target for the chemoprevention of breast cancer by evaluating the effect of the FAS inhibitor triclosan on rat mammary carcinogenesis. At 50 days of age, 60 female Sprague-Dawley rats received 50 mg/kg methylnitrosourea (MNU) i.p. to initiate mammary carcinogenesis. One week later, half of the rats were fed triclosan at a level of 1000 p.p.m. in an AIN-93G diet for the remainder of the experiment. The other 30 control rats were fed an AIN-93G diet without triclosan. Twelve weeks after MNU treatment, 70% of control rats had mammary adenocarcinomas compared with only 43.3% of the triclosan group (P < 0.05). The control rats had an average of 2.7 +/- 0.3 tumors/rat compared with 1.8 +/- 0.3 in the triclosan group (P < 0.05). Western analysis showed that the tumors in the control rats expressed high levels of FAS. Immunohistochemistry showed that sections of tumors that stained strongly for FAS also showed strong staining for proliferating cell nuclear antigen. Furthermore, at biologically relevant dose levels, triclosan inhibited the activity of FAS in mammary tumor homogenates. These results indicate that triclosan suppresses rat mammary carcinogenesis by inhibiting FAS and suggest that FAS is a promising molecular target for breast cancer chemoprevention.     

Inhibition of methylnitrosourea-induced large bowel cancer development in rats by sarcophytol A, a product from a marine soft coral Sarcophyton glaucum

An antitumorigenic effect of sarcophytol A (SaA), a simple monohydroxycembratetraene isolated from a marine soft coral Sarcophyton glaucum, was investigated in rat colon carcinogenesis. Three groups (26 rats each) of female CD-Fischer rats given an intrarectal dose of 2 mg of N-methyl-N-nitrosourea 3 times weekly for Wk 1 to 3 were fed standard laboratory chow in the control group or the chow containing 0.01% SaA from Wk 1 or from Wk 4 in experimental groups. The body weight gain and the food intake were not different among all 3 groups, and SaA intake was similar in both experimental groups at a dosage of 6.18 and 6.14 mg/kg of body weight/day at Wk 5 and 3.87 and 3.90 mg/kg of body weight/day at Wk 25. At autopsy at Wk 26, the incidence of large bowel tumors was found to be significantly lower and the mean number of tumors per tumor-bearing rat to be insignificantly smaller in experimental groups than in the control group: 50% and 58% versus 85%, 1.8 and 1.8 versus 2.0. The tumors in both experimental groups were generally smaller. All the tumors except two signet ring cell carcinomas were well-differentiated adenocarcinomas. Induction of ornithine decarboxylase activity, a marker of tumor promotion, in the large bowel mucosa of rats which were fed the SaA chow for 1 wk, then received an intrarectal dose of 12, 6, or 1.2 mumol of deoxycholate, a tumor promoter in large bowel carcinogenesis, and were killed 4 h later was significantly lower than in control rats. Thus, it was concluded that SaA inhibited the development of large bowel cancer, probably through an antipromoting mechanism.     

Distal bowel selectivity in the chemoprevention of experimental colon carcinogenesis by the non-steroidal anti-inflammatory drug nabumetone

Use of non-steroidal anti-inflammatory drugs (NSAIDs) for chemoprevention of colon cancer has been hindered by their potential gastro-intestinal toxicity. Nabumetone, which is approximately 10 to 36 times safer than conventional NSAIDs, was evaluated in 2 models of experimental colon carcinogenesis. In azoxymethane (AOM)-treated Fisher 344 rats, nabumetone caused dose-dependent inhibition of aberrant crypt foci (ACF), with 750 and 1,500 ppm resulting in 15% and 37% reductions, respectively (p < 0.05). Moreover, complex ACF were reduced by 48% in the latter group. MIN mice studies confirmed the chemopreventive efficacy of nabumetone, with 900 ppm suppressing approximately half of the intestinal tumors. Interestingly, inhibition of intermediate biomarkers in both models was markedly greater in the distal than the proximal bowel. To mechanistically evaluate this regional selectivity, we assessed cyclo-oxygenase-2 (COX-2) expression in the uninvolved mucosa and demonstrated a 3- to 4-fold excess in the distal relative to the proximal bowel in both MIN mice and AOM-treated rats. We then investigated another putative NSAID target, peroxisome proliferator-activated receptor-delta (PPAR-delta) and demonstrated up-regulation during AOM-induced colonic tumorigenesis. Furthermore, in pre-neoplastic mucosa, there was a 3-fold excess of PPAR-delta in the distal colon. We demonstrate that nabumetone is an effective protective agent in both experimental models of colon carcinogenesis. The striking distal predilection of nabumetone may be, at least partially, explained by distal bowel over-expression of COX-2 and PPAR-delta.     

The effect of interferon on carcinogenesis by N-ethyl-N'-nitro-N-nitrosoguanidine in the duodenum of mice

The inhibitory effect of murine interferon alpha/beta (Mu-IFN) on the induction of adenocarcinoma of the duodenum in C57BL/6 mice given N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) was examined. ENNG was given continuously in drinking water for 4 weeks and Mu-IFN was given intraperitoneally for 12 weeks. Then, the duodenal tumors of mice were examined stereomicroscopically and histologically. The level of IFN activity and natural killer (NK) activity were evaluated after an intraperitoneal single injection of Mu-IFN, and the level of NK activity was evaluated 2, 5 and 8 weeks after giving ENNG and Mu-IFN. In the mice given Mu-IFN, the incidence of duodenal tumors was significantly decreased (P less than 0.01), compared with that in mice given ENNG alone. Further, anti-asialo GM1 was given intraperitoneally every 5 days for 8 weeks to depress NK function and the incidence and size of duodenal tumors were examined. The results indicated that NK cells also have an important effect on the process of carcinogenesis. These data suggest that chemoprevention with IFN may be feasible.     

The Effect of Interferon on Carcinogenesis by N-Ethyl-N'-nitro-N-nitrosoguanidine in the Duodenum of Mice

The inhibitory effect of murine interferon α/β (Mu-IFN) on the induction of adenocarcinoma of the duodenum in C57BL/6 mice given N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) was examined. ENNG was given continuously in drinking water for 4 weeks and Mu-IFN was given intraperitoneally for 12 weeks. Then, the duodenal tumors of mice were examined stereomicroscopically and histologically. The level of IFN activity and natural killer (NK) activity were evaluated after an intraperitoneal single injection of Mu-IFN, and the level of NK activity was evaluated 2, 5 and 8 weeks after giving ENNG and Mu-IFN. In the mice given Mu-IFN, the incidence of duodenal tumors was significantly decreased ( P < 0.01), compared with that in mice given ENNG alone. Further, anti-asialo GM1 was given intraperitoneally every 5 days for 8 weeks to depress NK function and the incidence and size of duodenal tumors were examined. The results indicated that NK cells also have an important effect on the process of carcinogenesis. These data suggest that chemoprevention with IFN may be feasible.     

Inhibition by long-term fermented miso of induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine in CD (SD) rats

The present study was designed to investigate the effects of fermented miso in the diet on the induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in male CD (SD) rats. A total of 120 animals, 6 weeks of age, were divided into 6 groups and given MNNG (100 ppm) in the drinking water for 16 weeks. Starting 1 week before the carcinogen treatment the rats were fed a normal control MF solid diet, or the same diet containing 10% long-term fermented, medium- or short-term fermented miso, or 1% NaCl until the end of the MNNG exposure period. They were then maintained on the MF control diet and normal tap water until the autopsy time point at 52 weeks. The long-term fermented miso significantly reduced the size of the gastric tumors as compared with the other groups. The present results thus indicate that dietary supplementation with long-term fermented miso could act as a chemopreventive agent for gastric carcinogenesis.     

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.     

Effects of multiple putative anticarcinogens on the carcinogenicity of trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-oxadiazole

In an attempt to dissociate the chemotherapeutic from the carcinogenic properties of the antischistosomal and antitrypanosomal nitrovinylfuran trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]1,2,4-oxadiazole [(SQ18506) CAS: 28754-68-9; (E)-5-amino-3-(2-(5-nitro-2-furyl)vinyl)-1,2,4-oxadiazole], potential inhibitors of carcinogenesis were administered to female outbred CD-1 mice before and during exposure to SQ18506. The compounds tested were ascorbic acid, etretinate, butylated hydroxyanisole (BHA), cysteamine hydrochloride, cysteine hydrochloride, dimercaprol, disulfiram, 1,4-dithiothreitol, reduced glutathione, and spermidine phosphate. The primary types of tumors observed were squamous cell carcinomas of the stomach and thymic and nonthymic lymphomas. BHA reduced the incidence of malignant tumors to control levels, whereas cysteine hydrochloride, spermidine phosphate, and disulfiram reduced the incidence of chemically induced tumors by 42, 34, and 32%, respectively. Although cysteamine hydrochloride and disulfiram had no or only a modest effect on the overall incidence of tumors, the data suggested possible tissue-specific anticarcinogenic properties for these agents. Of the 8 antioxidants tested, only 1 had marked anticarcinogenic properties against SQ18506. These data indicate that antioxidant properties alone cannot account for the anticarcinogenic activity of the compounds tested. Coadministration of the anticarcinogen BHA with SQ18506 also blocked the chemotherapeutic effects of this agent on female CD-1 mice infected with Schistosoma mansoni.     

Chemopreventive Potential of Neem Flowers on Carcinogen-induced Rat Mammary and Liver Carcinogenesis

We have previously reported that dietary neem flowers (Azadirachta indica A. Juss var. siamensis Valeton) caused ‍a marked increase in glutathione S-transferase (GST) activity in the liver, while resulting in a significant reduction ‍in the activities of some hepatic P450-dependent monooxygenases. These results strongly indicate that neem flowers ‍may have chemopreventive potential. In the present study, we examined the inhibitory effects of neem flowers on ‍9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague Dawley rats ‍and on aflatoxin B ‍1 ‍(AFB1)-induced hepatocarcinogenesis in male Wistar rats. Young animals were fed with AIN-76 ‍purified diets containing either 10-12.5% ground freeze-dried neem flowers for 1 week prior to, during, and for 1 ‍week after the administration of each carcinogen. Interestingly, it was found that neem flowers resulted in a marked ‍reduction of the incidence of mammary gland (about 35.2%) and liver tumors (61.7% and 80.1% for benign and ‍malignant tumors, respectively). Furthermore, the multiplicity of tumors per rats was also lower in the neem flower ‍groups, i.e. those for mammary gland tumors and benign and malignant liver tumors were reduced to 44.0%, ‍87.9% and 88.9%, respectively. These results clearly demonstrated that neem flowers contain some chemopreventive ‍agents capable of inhibiting AFB1 and DMBA induced liver and mammary gland carcinogenesis in rats.     

Decrease in size of azoxymethane induced colon carcinoma in F344 rats by 180-day fermented miso

The present study was designed to investigate the effects of fermented miso (fermented soybean paste) on the induction of colon tumors by azoxymethane (AOM) in male F344 rats. A total of 91 rats, 6 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. The animals were placed on diets one week before the first AOM dose: commercial normal control MF diet or a diet containing 10% 2-year, 180-day fermented, or 3-4-day fermented miso. There were no differences in body and organ weights, and no aberrant crypt foci (ACF) among carcinogen-treated groups at week 25. The rates of tumor incidence were 45%, 85%, 75% and 60% with the 2-year, 180-day, and 3-4-day fermented miso and MF, respectively, and those for colon tumors were 34%, 55%, 60% and 55%, respectively. The size of well-differentiated adenocarcinomas and total (well differentiated and signet ring cell) adenocarcinomas in the 180-day fermented miso group was significantly smaller than that in the 2-year fermented miso and MF+AOM groups. Nuclear staining of beta-catenin in colon tumors was increased for the 3-4-day fermented miso compared to the 180-day fermented miso. Cdx2 staining tendency was decreased in colon tumors and adenocarcinomas compared to normal mucosa and ACF, which stained in 100% of cases. In addition, the PCNA index was significantly reduced in the 180-day group compared with those groups receiving the 3-4-day fermented miso and MF diet. The germinal region was also decreased. The present results indicate that dietary supplementation with 180-day fermented dietary miso could act as a chemopreventive agent for colon carcinogenesis.     

Involvement of the 5-lipoxygenase/leukotriene A4 hydrolase pathway in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch, and inhibition of carcinogenesis by its inhibitors

Previous studies have shown that aberrant arachidonic acid (AA) metabolism, especially cyclooxygenase-2 (Cox-2) and 5-lipoxygenase (5-Lox) pathways, are activated during oral carcinogenesis, and can be targeted for cancer prevention. This study was designed to investigate the importance of 5-Lox/leukotriene A4 hydrolase (LTA4H) pathway of AA metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch carcinogenesis. In a short-term study, topical application of DMBA for 3 weeks induced severe inflammation and aberrant AA metabolism. Subsequent topical treatment with zileuton, celecoxib, or their combination for 1 week significantly suppressed aberrant AA metabolism and cell proliferation in the oral epithelium. Interestingly, zileuton was effective in inhibiting biosynthesis of multiple AA metabolites, including leukotriene B4 (LTB4), 5-, 12-, 15-hydroxyeicosatetraenoic acid and prostaglandin E2 (PGE2), while celecoxib only suppressed PGE2 biosynthesis significantly at a high dose. In a long-term carcinogenesis study topical application of LTB4 or PGE2 enhanced oral carcinogenesis by increasing the incidence and volume of visible tumors, and the incidence of squamous cell carcinoma (SCC). To further examine the role of LTB4 in oral carcinogenesis, two LTA4H inhibitors, bestatin and SA6541, were evaluated in a long-term chemoprevention experiment. Both agents significantly inhibited SCC, and such an inhibition correlated with reduced levels of LTB4 in hamster cheek pouch. In summary, our studies have demonstrated that 5-Lox/LTA4H pathway is one of the major AA-metabolizing pathways involved in DMBA-induced oral carcinogenesis in hamsters, and may be targeted for chemoprevention.     

Tamoxifen resistance and Her2/neu expression in an aged, irradiated rat breast carcinoma model

Clear links have been established between occupational or therapeutic radiation exposure and breast cancer. Tamoxifen chemoprevention following radiation exposure may be able to reduce the risk of developing breast cancer later in life. In order to model carcinogenesis in this setting, an in vivo model of tamoxifen chemoprevention and tamoxifen failure in a radiation-induced rat mammary carcinoma model was characterized. Two hundred and twenty-seven 60-day-old female rats received whole body or sham exposure to ionizing radiation. Thirty days later long-term, continuous, tamoxifen chemoprevention was initiated in half the population and all animals were monitored over three and a half years for the development of mammary tumors. Mammary tumors were surgically removed and carcinomas were histologically identified and characterized. Results showed that tamoxifen chemoprevention decreased the incidence and prolonged the latency of radiation-induced mammary carcinomas. However, many individuals receiving tamoxifen chemoprevention developed their first carcinoma very late in life. These carcinomas shared morphological features distinct from the majority of carcinomas that developed in the absence of tamoxifen chemoprevention. Analyses of cell lines established from these carcinomas and immunohistochemistry of tumor sections revealed that the highest levels of Her2/neu expression were associated with in vivo tamoxifen exposure. Treatment of rat mammary carcinoma cells with an anti-rat Her2/neu monoclonal antibody (MAb 7.16.4) inhibited cell growth and this effect was more pronounced in the presence of tamoxifen. These studies suggest that carcinoma growth driven by the Her2/neu pathway may be associated with tamoxifen chemoprevention failure in the rat mammary carcinoma model. Additionally, strategies combining targeted Her2/neu antibodies, vaccines or drugs with estrogen pathway modification may be more effective in reducing breast cancer chemoprevention failures.