Black raspberries demethylate Sfrp4, a WNT pathway antagonist, in rat esophageal squamous cell papilloma.

Aberrant methylation of DNA is a common event in the development of cancers, including squamous cell carcinoma (SCC) of the human esophagus. In the present study, we determined: (a) whether aberrant DNA methylation also occurs in the development of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus, a model of human esophageal SCC; and (b) if so, whether dietary black raspberries (BRBs) are capable of preventing this aberrant DNA methylation. A diet containing 5% BRBs inhibited the development of NMBA-induced tumors in the rat esophagus. This inhibition was associated with reduced mRNA levels of the DNA methyltransferases, Dnmt1 and Dnmt3b, in both dysplastic lesions and in papillomas of the esophagus. In addition, promoter methylation of Sfrp4, a WNT pathway antagonist, was significantly reduced by the berry diet, and this was associated with decreased nuclear localization of ß-CATENIN and reduced expression of c-MYC protein in NMBA-treated esophagi. Decreased promoter methylation of Sfrp4 correlated with decreased expression of Dmnt3b and, ultimately, with increased Sfrp4 mRNA expression. This suggests that epigenetic alterations in NMBA-induced rat esophageal tumorigenesis recapitulate epigenetic events in human esophageal SCC, and that BRBs could be useful in preventing the aberrant DNA methylation involved in the development of human esophageal SCC. © 2015 Wiley Periodicals, Inc.

Rhizoma Paridis Saponins Suppresses Tumor Growth in a Rat Model of N-Nitrosomethylbenzylamine-Induced Esophageal Cancer by Inhibiting Cyclooxygenases-2 Pathway.

Rhizoma Paridis Saponins (RPS), a natural compound purified from Rhizoma Paridis, has been found to inhibit cancer growth in vitro and in animal models of cancer. However, its effects on esophageal cancer remain unexplored. The purpose of this study was to investigate the effects of RPS on tumor growth in a rat model of esophageal cancer and the molecular mechanism underlying the effects. A rat model of esophageal cancer was established by subcutaneous injection of N-nitrosomethylbenzylamine (NMBA, 1 mg/kg) for 10 weeks. RPS (350 mg/kg or 100 mg/kg) was administered by oral gavage once daily for 24 weeks starting at the first NMBA injection. RPS significantly reduced the size and number of tumors in the esophagus of rats exposed to NMBA and inhibited the viability, migration, and invasion of esophageal cancer cells EC9706 and KYSE150 in a dose dependent manner (all P < 0.01). Flow cytometry revealed that RPS induced apoptosis and cell cycle G2/M arrest in the esophageal cancer cells. The expression of cyclooxygenases-2 (COX-2) and Cyclin D1 in rat esophageal tissues and the esophageal cancer cells were also significantly reduced by RPS (all P < 0.01). Consistently, RPS also significantly decreased the release of prostaglandin E2, a downstream molecule of COX-2, in a dose-dependent manner (P < 0.01). Our study suggests that RPS inhibit esophageal cancer development by promoting apoptosis and cell cycle arrest and inhibiting the COX-2 pathway. RPS might be a promising therapeutic agent for esophageal cancer.

Chemoprevention of esophageal cancer with black raspberries, their component anthocyanins, and a major anthocyanin metabolite, protocatechuic acid.

Diets containing either freeze-dried black raspberries (BRBs) or their polyphenolic anthocyanins (ACs) have been shown to inhibit the development of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer in rats. The present study was conducted to determine whether PCA, a major microbial metabolite of black raspberry (BRB) ACs, also prevents NMBA-induced esophageal cancer in rats. F344 rats were injected with NMBA three times a week for 5 weeks and then fed control or experimental diets containing 6.1% BRBs, an anthocyanin (AC)-enriched fraction derived from BRBs, or protocatechuic acid (PCA). Animals were exsanguinated at weeks 15, 25, and 35 to quantify the development of preneoplastic lesions and tumors in the esophagus, and to relate this to the expression of inflammatory biomarkers. At weeks 15 and 25, all experimental diets were equally effective in reducing NMBA-induced esophageal tumorigenesis, as well as in reducing the expression of pentraxin-3 (PTX3), a cytokine produced by peripheral blood mononuclear cells in response to interleukin (IL)-1ß and TNF-α. All experimental diets were also active at reducing tumorigenesis at week 35; however, the BRB diet was significantly more effective than the AC and PCA diets. Furthermore, all experimental diets inhibited inflammation in the esophagus via reducing biomarker (COX-2, iNOS, p-NF-κB, and sEH) and cytokine (PTX3) expression. Overall, our data suggest that BRBs, their component ACs, and PCA inhibit NMBA-induced esophageal tumorigenesis, at least in part, by their inhibitory effects on genes associated with inflammation.

[Effect of vitamin E and selenium deficiency on esophageal tumorigenesis and its oxidative stress mechanism].

OBJECTIVE: To explore the effect of vitamin E (VE) and selenium (Se) deficiency on esophageal tumorigenesis and its oxidative stress mechanism. METHODS: 110 male F344 rats were randomly divided into 3 groups: VE/Se deficient group (group A), VE/Se normal group (group B) and control group (group C). Animals in group A and group B were subcutaneously injected with N-nitrosomethylbenzylamine (NMBzA) at the dosage of 0.35 mg/kg BW, 3 times per week for 5 weeks. Control group was given the solvent 20% DMSO as negative control. Animals in group A were fed with low-VE/Se diet (46IU/kg VE, 0.05 mg/kg Se), animals in group B and C were fed with normal diet (80IU/kg VE, 0.15 mg/kg Se). Animals were sacrificed to perform macroscopic observation and pathologic inspection at the 25th week of the experiment. Plasma VE level was determined by high-performance liquid chromatography, and Se level was determined by fluorescence method. Cell proliferation and DNA damage in esophagus were detected through immunohistochemistry of BrdU and 8-OH-dG, respectively. Glutathione peroxidase (GPX) and glutathione transferase (GST) activities in plasma, esophagus and liver were analyzed by using test kits. RESULTS: VE/Se levels of group A were significantly lower than those of group B, the later were slightly lower than those of group C. Visible tumor incidence, tumor multiplicity and pathologic lesions in group A were all significantly more than those in group B. Comparing with group B, cell proliferation and 8-OH-dG levels were also significantly increased in group A. GPX and GST activities in plasma, esophagus and liver of group A were significantly higher than those of group B. CONCLUSION: Vitamin E and selenium deficiency significantly promoted the NMBzA-induced esophageal tumorigenesis. Oxidative stress and DNA damage may be one of the critical routes by which NMBzA induces carcinogenesis in rat esophagus.

Time-selective chemoprevention of vitamin E and selenium on esophageal carcinogenesis in rats: the possible role of nuclear factor kappaB signaling pathway.

Previous human intervention trial demonstrated that vitamin E (Ve) and selenium (Se) supplementation decreased esophageal cancer deaths among younger participants, but may have no effect or produce an opposite effect among older ones. In our study, we intended to mimic this human nutritional trial to determine the chemopreventive effects of Ve/Se supplementation at the early or late stage of esophageal carcinogenesis in rats. Esophageal squamous cell carcinoma (ESCC) was induced in Fischer 344 rats with N-nitrosomethylbenzylamine (NMBzA, 0.35 mg/kg BW, s.c., three times per week for 5 weeks). The rats were maintained on a modified AIN-93M diet with low levels of Ve/Se or supplemented with high levels of Ve/Se at different stages. At Week 25, the number and volume of visible tumors, the numbers of dysplasia and ESCC were significantly lower in rats of supplementation during the early stage (Group C) or during the entire experimental period (Group E), but not during the late stage (Group D). Ve/Se supplementation at the early stage also significantly decreased cell proliferation, nuclear factor kappaB (NFκB) activation, protein and mRNA expression of cyclooxygenase 2 and 5-lipoxygenase and biosynthesis of prostaglandin E2 and leukotriene B4 during the carcinogenesis of rat esophagus. Our results demonstrated that the chemopreventive efficacy of Ve/Se supplementation on NMBzA-induced esophageal cancer is time selective and that supplementation during the early stage is clearly effective but probably ineffective during the late stage of carcinogenesis. NFκB signaling pathway activation and aberrant arachidonic acid metabolism might be the underlying mechanism.

Relevance of drug metabolizing enzyme activity modulation by tea polyphenols in the inhibition of esophageal tumorigenesis.

Tea is a popular, socially accepted, drink that is enjoyed by millions of people. A growing body of evidence suggests that moderate consumption of tea may protect against several forms of cancer. It is also known that bioactivation of precarcinogens and detoxification of ultimate carcinogens is carried out mainly by drug metabolizing enzymes such as cytochrome P450 (CYP). The present study investigates the effect of tea consumption on modulating CYP and phase II conjugating enzymes, and their association in the chemopreventive effect against esophageal tumorigenesis using both in vitro and in vivo techniques. Female Wistar rats were given aqueous solutions (2% w/v) of six different teas, standard green tea extract (GTE) (0.5% w/v), and dandelion tea (2% w/v) as the sole source of fluid for two weeks prior to and during the entire period of tumour induction (12 weeks). Animals were gavaged with 0.5 mg/kg N-nitrosomethylbenzylamine (NMBA) twice weekly for 12 weeks for esophageal tumor induction and the activities of different CYP isoforms and phase II enzymes were determined in the liver microsomes or cytosols. GTE, green tea and Dandelion tea caused decrease in tumour multiplication, tumour size and tumour volume; however, none of these tea preparations altered tumour incidence. No appreciable changes in drug metabolizing enzyme activity were observed in the treatment groups. Thus, the modulations in the activities of CYP 1A1/ 1A2 and CYP2E enzymes, by pre-treatment with green and dandelion teas, observed in our earlier experiments, seem to be compensated by the tumor inducing agent, NMBA. The balance between phase I carcinogen-activating enzymes and phase II detoxifying enzymes could be important in determining the risk of developing chemically-induced cancer and the present study in conjunction with the previous observations suggest a possible role of drug metabolizing enzymes in the anticancer effect of tea.

Modulating effects of rooibos and honeybush herbal teas on the development of esophageal papillomas in rats.

Widespread consumption of herbal teas has stimulated interest in their role as cancer preventive agents. The present investigation monitored the modulation of methylbenzylnitrosamine (MBN)-induced esophageal squamous cell carcinogenesis by rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal and Camellia sinensis teas in male F344 rats. The tumor multiplicity was significantly (P < 0.05) inhibited by unfermented honeybush (45.5%), green (50%), and black (36%) teas, while the other teas exhibited weaker effects (<30% inhibition). The mean total papilloma size was reduced by unfermented rooibos (87%), unfermented honeybush (94%), and fermented honeybush (74%) due to the absence of large papillomas (>10 mm(3)). Reduction of the mean total papilloma number correlated with the total polyphenol (TPP) (r = 0.79; P < 0.02) and flavanol/proanthocyanidin (FLAVA) (r = 0.89; P < 0.008) intake (mg/100 g body weight) of the teas and the FLAVA (r = 0.89; P < 0.04) and flavonol/flavones/xanthones (r = 0.99; P < 0.002) intake when considering only the herbal teas. A daily TPP intake threshold of 7 mg/100 g body weight existed below where no inhibition of papilloma development was observed. Fermentation of herbal teas reduced the inhibitory effects on papilloma development associated with a reduction in the polyphenolic constituents. The inhibitory effect of herbal teas on papilloma development is associated with different flavonoid subgroups and/or combination thereof.

Mechanistic basis for the chemopreventive effects of black raspberries at a late stage of rat esophageal carcinogenesis.

The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA- and NMBA + BRB-treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA-treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17 846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17 846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell-cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis.

Chemopreventive effects of early-stage and late-stage supplementation of vitamin E and selenium on esophageal carcinogenesis in rats maintained on a low vitamin E/selenium diet.

Low vitamin E and selenium (Ve/Se) nutritional status is known to be associated with increased risk of esophageal squamous cell carcinoma (ESCC). A previous human intervention trial demonstrated that Ve/Se supplementation decreased the occurrence of esophageal cancer death among younger participants but not among older ones. In this study, we intended to mimic this human nutritional status to determine the chemopreventive effects of Ve/Se supplementation at the early or late stage of esophageal carcinogenesis in rats maintained on a low Ve/Se diet. ESCC was induced in F344 rats with N-nitrosomethylbenzylamine (NMBzA) (0.35 mg/kg body wt, subcutaneously, three times per week for 5 weeks). The rats were maintained on a modified AIN-93M diet with low levels of Ve/Se or supplementation to the normal level by using the AIN-93M diet. At Week 25, the numbers of visible tumors and ESCC were significantly lower in rats on AIN-93M diet during the entire experimental period (Group D) or during the early stage (Group B) but not during the late stage (Group C). Ve/Se supplementation (switching from the low Ve/Se diet to the AIN-93M diet) also decreased cell proliferation, angiogenesis, 8-hydroxy-2'-deoxyguanosine, biosynthesis of prostaglandin E2 and leukotriene B4, expression of cyclooxygenase 2 and 5-lipoxygenase in the esophagus. Our results demonstrated that Ve/Se supplementation inhibited NMBzA-induced esophageal carcinogenesis in rats on low Ve/Se diet, and supplementation during the early stage is more effective than during the late stage of carcinogenesis.

Effect of zinc supplementation on N-nitrosomethylbenzylamine-induced forestomach tumor development and progression in tumor suppressor-deficient mouse strains.

Zinc deficiency is associated with high incidences of esophageal and other cancers in humans and leads to a highly proliferative hyperplastic condition in the upper gastrointestinal tract in laboratory rodents. Zn replenishment reduces the incidence of lingual, esophageal and forestomach tumors in Zn-deficient rats and mice. While previous animal studies focused on Zn deficiency, we have investigated the effect of Zn supplementation on carcinogenesis in Zn-sufficient mice of wild-type and tumor suppressor-deficient mouse strains. All mice received N-nitrosomethylbenzylamine and half the mice of each strain then received Zn supplementation. At killing, mice without Zn supplementation had developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice developed an average of 7.0 versus 5.0 tumors for Zn supplemented (P < 0.05); Zn-supplemented Fhit-/- mice averaged 5.7 versus 8.0 for control mice (P < 0.01); Zn-supplemented Fhit-/-Nit1-/- mice averaged 5.4 versus 9.2 for control mice (P < 0.01) and Zn-supplemented Fhit-/-Rassf1a-/- (the murine gene) mice averaged 5.9 versus 9.1 for control mice (P < 0.01). Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit-/-Nit1-/-). Histological analysis of forestomach tissues also showed significant decreases in severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse strain. Thus, Zn supplementation significantly reduced tumor burdens in mice with multiple tumor suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final carcinogen dose, the reduction in tumor burden was the same as observed when supplementation began immediately after carcinogen dosing, suggesting that Zn supplementation may affect tumor progression rather than tumor initiation.

Drinking water with red beetroot food color antagonizes esophageal carcinogenesis in N-nitrosomethylbenzylamine-treated rats.

This study was undertaken to determine if the oral consumption of red beetroot food color would result in an inhibition of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. Rats were treated with NMBA and given either regular water ad libitum or water containing 78 microg/mL commercial red beetroot dye, E162. The number of NMBA-induced esophageal papillomas was reduced by 45% (P < .001) in animals that received the food color compared to controls. The treatment also resulted in reduced rates of cell proliferation in both precancerous esophageal lesions and in papillomas of NMBA-treated rats, as measured by immunohistochemical staining of Ki-67 in esophageal tissue specimens. The effects of beetroot food color on angiogenesis (microvessel density by CD34 immunostaining), inflammation (by CD45 immunostaining), and apoptosis (by terminal deoxynucleotidyl transferase dUTP nick end-labeling staining) in esophageal tissue specimens were also determined. Compared to rats treated with NMBA only, the levels of angiogenesis and inflammation in the beetroot color-consuming animals were reduced, and the apoptotic rate was increased. Thus, the mechanism(s) of chemoprevention by the active constituents of red beetroot color include reducing cell proliferation, angiogenesis, and inflammation and stimulating apoptosis. Importantly, consumption of the dye in the drinking water for a period of 35 weeks did not appear to induce any overt toxicity. Based on the fact that red beetroot color contains betanins, which have strong antioxidant activity, it is postulated that these effects are mediated through inhibition of oxygen radical-induced signal transduction. However, the sum of constituents of E162 has not been determined, and other components with other mechanisms may also be involved in antagonizing cancer development.

Multiple berry types prevent N-nitrosomethylbenzylamine-induced esophageal cancer in rats.

PURPOSE: The present study compared the ability of different berry types to prevent chemically-induced tumorigenesis in the rat esophagus. We also determined if berries influence the levels of inflammatory cytokines in the serum of carcinogen-treated rats. METHODS: Rats were treated with the carcinogen N-nitrosomethylbenzylamine (NMBA) for 5 weeks, then placed on diets containing 5% of either black or red raspberries, strawberries, blueberries, noni, açaí or wolfberry until the end of the study. The effects of the berries on tumor incidence, multiplicity and size were determined, as well as their effects on the levels of selected inflammatory cytokines in serum. RESULTS: All berry types were about equally effective in inhibiting NMBA-induced tumorigenesis in the rat esophagus. They also reduced the levels of the serum cytokines, interleukin 5 (IL-5) and GRO/KC, the rat homologue for human interleukin-8 (IL-8), and this was associated with increased serum antioxidant capacity. CONCLUSIONS: Seven berry types were about equally capable of inhibiting tumor progression in the rat esophagus in spite of known differences in levels of anthocyanins and ellagitannins. Serum levels of IL-5 and GRO/KC (IL-8) may be predictive of the inhibitory effect of chemopreventive agents on rat esophageal carcinogenesis.

[Natural compounds in chemoprevention of esophageal squamous cell tumors--experimental studies]. / Srodki pochodzenia naturalnego w chemoprewencji raka plaskonablonkowego przelyku--badania doswiadczalne.

Chemoprevention refers to prevention of tumor formation due to administration of nontoxic synthetic or natural compounds, which can block or weaken the influence of carcinogens on target cells or even reverse previously formed lesions. In esophageal squamous cell carcinoma--which belongs to the group of the most aggressive tumors of digestive system with poor prognosis--an effective prevention would be strongly expected. Chemopreventive properties of many complex diets and pure natural compounds were evaluated until now. Most studies were performed on rats exposed to chemical carcinogens, usually N-nitroso-N-methylbenzylamine (NMBA). The best effects were achieved after administration of diallyl sulfide and phenethyl isothiocyanate. Lyophilized black raspberries, blackberries and strawberries, as well as products obtained from leaves and buds of tea plant, elagic acid and resveratrol were also very effective. Mechanisms of chemopreventive action were associated with, i.e., activity of enzymes involved in carcinogen metabolic activation (mostly cytochrome P450 isoenzymes), inhibition of DNA adducts formation and modulation of gene expression involved in regulation of proliferation, apoptosis and angiogenesis. However, some agents e.g., perillyl alcohol, could enhance development of proliferative lesions in esophageal epithelium.

Protection against esophageal cancer in rodents with lyophilized berries: potential mechanisms.

For several years, our laboratory has been evaluating the ability of lyophilized (freeze-dried) black raspberries (Rubus occidentalis, BRBs), blackberries (R. fructicosus, BBs), and strawberries (Fragaria ananasia, STRWs) to inhibit carcinogen-induced cancer in the rodent esophagus. To assure "standardized" berry preparations for study, each berry type is of the same cultivar, picked at about the same degree of ripeness, washed and frozen within 2-4 h of the time of picking, and freeze-dried under conditions that preserve the components in the berries. Some of the known chemopreventive agents in berries include vitamins A, C, and E and folic acid; calcium and selenium; beta-carotene, alpha-carotene, and lutein; polyphenols such as ellagic acid, ferulic acid, p-coumaric acid, quercetin, and several anthocyanins; and phytosterols such as beta-sitosterol, stigmasterol, and kaempferol. In initial bioassays, freeze-dried STRW, BRB, and BB powders were mixed into AIN-76A synthetic diet at concentrations of 5% and 10% and fed to Fischer 344 rats before, during, and after treatment with the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA). At 25 wk of the bioassay, all three berry types were found to inhibit the number of esophageal tumors (papillomas) in NMBA-treated animals by 24-56% relative to NMBA controls. This inhibition correlated with reductions in the formation of the NMBA-induced O6-methylguanine adduct in esophageal DNA, suggesting that the berries influenced the metabolism of NMBA leading to reduced DNA damage. Studies are ongoing to determine the mechanisms by which berries influence NMBA metabolism and DNA adduct formation. BRBs and STRWs were also tested in a postinitiation scheme and were found to inhibit NMBA-induced esophageal tumorigenesis by 31-64% when administered in the diet following treatment of the animals with NMBA. Berries, therefore, inhibit tumor promotion and progression events as well as tumor initiation. In vivo mechanistic studies with BRBs indicate that they reduce the growth rate of premalignant esophageal cells, in part, through down-regulation of cyclooxygenase-2 leading to reduced prostaglandin production and of inducible nitric oxide synthase leading to reduced nitrate/nitrite levels in the esophagus. Based upon the preclinical data on rodents, we have initiated prevention trials in humans to determine if berries might exhibit chemopreventive effects in the esophagus.

Black raspberries inhibit N-nitrosomethylbenzylamine (NMBA)-induced angiogenesis in rat esophagus parallel to the suppression of COX-2 and iNOS.

Angiogenesis, the formation of new blood vessels, is critical to tumor growth and metastasis. Vascular endothelial growth factor (VEGF), an important angiogenic activator, is essential for angiogenesis. Our laboratory has used a rodent model of human esophageal squamous cell carcinoma (ESCC) to identify putative chemopreventive agents for this disease and determine their mechanisms of action. We reported that dietary black raspberry powder (BRB) inhibits N-nitrosomethylbenzylamine (NMBA)-induced tumor development in the rat esophagus by inhibiting the formation of DNA adducts and reducing the proliferation rate of preneoplastic cells. On a molecular level, BRB downregulates the expression of c-Jun, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). In this study we analyzed the effect of BRB on angiogenesis. VEGF expression was determined by real-time RT-PCR and immunohistochemical analysis of microvessel density (MVD). BRB significantly suppressed VEGF-C expression from a 2.38 (+/- 0.34)-fold increase in animals treated with NMBA alone to a 1.08 (+/- 0.22)-fold increase in animals treated with NMBA plus BRB (P < 0.005). The MVD of esophagus was decreased from 53.7 +/- 5.6 vessels/cm in animals treated with NMBA alone to 22.6 +/- 2.6 vessels/cm in animals treated with NMBA plus BRB (P < 0.0001). Our data also suggest that downregulation of VEGF is correlated with suppression of COX-2 (r2 = 0.86, P < 0.001) and iNOS (r2 = 0.81, P < 0.005). As high vascularity is a risk factor for metastasis and tumor recurrence, BRB may have cancer therapeutic effects in human esophageal cancer.

Chemoprotection against N-nitrosomethylbenzylamine-induced mutation in the rat esophagus.

Prevention of esophageal cancer may be possible through dietary modification or supplementation. In this study we have investigated the mutation preventive properties of ellagic acid, green tea, and diallyl sulfide (DAS) against the mutagenicity of the nitrosamine N-nitrosomethylbenzylamine (NMBA) in the esophagus of the rat. In addition, the effect of the consumption of ethanol on the mutagenicity of NMBA was examined. NMBA is specific in inducing tumors in the rat esophagus and has been used in many studies investigating the mechanism and the prevention of this cancer. We found that the type of mutations induced by two 2-mg/kg subcutaneous injections of NMBA in the lacI gene of "Big Blue" rats is consistent with that found previously for nitrosamines in other systems and consists of G:C-->A:T transitions. We report that the addition of ellagic acid to the feed, replacing drinking water with green tea, and gavage with DAS significantly reduced the mutagenicity of NMBA. In contrast, the addition of 5% ethanol to the drinking water increased the mutagenicity of NMBA. This is consistent with findings that these compounds modulate NMBA-induced carcinogenesis in the rat.

Preventive effect of fermented brown rice and rice bran on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats.

Modifying effect of fermented brown rice by Aspergillus Oryzae (FBRA) during the initiation or post-initiation phase of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis was investigated in rats. Five-week-old male F344 rats were divided into 7 groups, and groups 1-5 were given subcutaneous injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks starting at 7 weeks of age. Groups 2 and 4 were fed the diet containing 5 and 10% FBRA during the initiation phase, respectively, whereas groups 3 and 5 were fed these diets during the post-initiation phase. Group 6 was given the diet containing 10% FBRA throughout the experiment, and group 7 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 89% and 1.63+/-1.01/rat, respectively. Those of groups 3 (65%, 1.04+/-1.04) and 5 (58%, 0.77+/-0.86) were significantly less than those of group 1. Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (dysplasia) of group 5 were less than those of group 1. Post-initiation feeding of 10% FBRA significantly decreased BrdU incorporation in the non-lesional esophageal tissues when compared to that of the control. In addition, the analysis of expression levels of phase I enzymes of livers at the termination of experiment showed no clear differences among the groups. These observations indicate for the first time that FBRA inhibits NMBA-induced esophageal tumor development in rats possibly through inhibition of cell proliferation in the post-initiation phase, and suggest that FBRA is a promising dietary agent for prevention of human esophageal cancer.

Rapid in vivo assay for topical oral cancer chemopreventive agents.

The cancer chemopreventive effect of topically applied phenethyl isothiocyanate (PEIT) was examined in a hamster buccal pouch model, in which squamous cell carcinomas (SCC) are induced at high frequency, by topical application of N-methyl-N-benzylnitrosamine (MBN). The buccal pouches of eleven hamsters were pretreated thrice-weekly for two weeks with corn oil (CO) containing 50 mM PEIT, followed by 22 weeks of twice-weekly application of CO containing MBN and PEIT, both at 50 mM. Under similar conditions, twelve hamsters were pretreated with CO for 2 weeks, followed by MBN in CO. Tumor analysis was performed 19 days after the last application of PEIT and MBN. The incidence of tumors (approximately 90% SCC) in the unprotected and protected groups was 100% and 73%, respectively. Although total tumor incidence was marginally decreased, PEIT inhibited significantly the tumor frequency and tumor burden by 79% and 74%, respectively. In both groups, 43% of the carcinomas exhibited p53 immunohistochemical activity. A short-term experiment was performed to determine whether PEIT inhibits MBN-induced cellular foci expressing gamma-glutamyltranspeptidase histochemical activity (gamma-GT foci). Buccal pouches of four protected hamsters received 50 mM PEIT pretreatment on days 1 and 4, followed on days 6 and 9 by application of CO containing MBN and PEIT, both at 50 mM. Four unprotected hamsters were similarly pretreated with CO, followed by MBN in CO. gamma-GT foci were enumerated in buccal pouch epithelial whole mounts prepared from pairs of protected and unprotected hamsters on days 10 through 13. Whereas the number of gamma-GT foci in unprotected hamsters ranged from 98 to 356 per 10 cm2, the protected hamsters exhibited no more than one focus per 10 cm2. This model may be useful for rapid identification of chemopreventive agents, and combinations of agents, which inhibit initiation and promotion stages of oral carcinogenesis.

Chemoprevention with theaflavins of rat esophageal intraepithelial neoplasia quantitatively monitored by image tile analysis.

The objective of the study was to compare three methods of monitoring the inhibition by dietary theaflavins of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal intraepithelial neoplasia: the mean tile grade, measured by computer-assisted quantitative image tile analysis; tumor multiplicity; and mean tumor size. A "tile" is defined as a small portion of a microscopic image at x 40, 87 x 292 microm in size. The computer divided the image of esophageal intraepithelial neoplasia into a grid of contiguous tiles and measured four tissue features within each tile based on cytonuclear and tissue architectural changes used by pathologists to diagnose intraepithelial neoplasia. The tile grade is defined as the weighted sum of the four feature measurements within a tile, the weights being determined by Fisher linear discriminant analysis. The mean tile grade of 300 tiles is used to grade rat esophageal intraepithelial neoplasia. NMBA was given s.c., 0.5 mg/kg, three times a week for 5 weeks. Theaflavins were given in the drinking water at 360 ppm (low dose) and 1200 ppm (high dose) throughout the experiment. In a given set of four groups of rats, one group received theaflavins alone, one NMBA alone, one NMBA plus low-dose theaflavins, and one NMBA plus high-dose theaflavins. One set of four groups, four rats/group, was sacrificed at the 15th week and another at the 20th week after starting NMBA; a final set with 15 rats/group was sacrificed at 25 weeks. At the 15th and 20th weeks, the mean tumor grade was the only variable that responded significantly (P < 0.01) to the low dose of dietary theaflavins. In fact, tumor multiplicity and mean tumor size sometimes showed enhancement at these doses. At the 25th week, when there were 15 instead of 4 rats/group, the mean tile grade, tumor multiplicity, and mean tumor size were all significantly (P < 0.01) decreased by both low and high doses of theaflavins. The mean tile grade is a more sensitive and reproducible variable than tumor multiplicity and mean tumor size in detecting the chemopreventive effects of theaflavins on intraepithelial neoplasia in the rat esophagus. This suggests that the mean tile grade may be a useful intermediate end point for use in human chemoprevention trials.

Chemopreventive effect of curcumin on N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats.

Modifying effects of curcumin (derived from the rhizome of Curcuma longa L.) during the initiation or post-initiation phase of N-nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis were investigated in male F344 rats. Five-week-old rats were divided into 5 groups, and groups 1, 2 and 3 were given intraperitoneal injections of NMBA (0.5 mg / kg body weight / injection 15 times) for 5 weeks from 7 weeks old to induce esophageal neoplasms. Groups 2 and 3 were fed the diet containing 500 ppm curcumin during the initiation and post-initiation phases, respectively. Group 4 was given the diet containing curcumin throughout the experiment, and group 5 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 66.7% and 0.83 +/- 0.70, respectively. Those of groups 2 and 3 were significantly less than those of group 1 (39.3%, 0.46 +/- 0.64, P < 0.05; 33.3%, 0.36 +/- 0.56, P < 0.05, respectively). Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (moderate or severe epithelial dysplasia) of group 2 (57.1%, 0.61 +/- 0.57; 40%, 0.29 +/- 0.46) or 3 (56.7%, 0.67 +/- 0.66; 23.3%, 0.23 +/- 0.43) were less than those of group 1 (100%, 1.67 +/- 0.70; 70.8%, 0.92 +/- 0.72) (P < 0.05). In this experiment, feeding of curcumin significantly decreased the expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling index) in the non-lesional esophageal epithelium (P < 0.01). These findings indicate that curcumin inhibits NMBA-induced esophageal carcinogenesis when given during the post initiation as well as initiation phase. This inhibition may be related to suppression of the increased cell proliferation induced by NMBA in the esophageal epithelium.