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Systemic delivery of fenretinide in oral cancer chemoprevention trials has been largely unsuccessful due to dose-limiting toxicities and subtherapeutic intraoral drug levels. Local drug delivery, however, provides site-specific therapeutically relevant levels while minimizing systemic exposure. These studies evaluated the pharmacokinetic and growth-modulatory parameters of fenretinide mucoadhesive patch application on rabbit buccal mucosa. Fenretinide and blank-control patches were placed on right/left buccal mucosa, respectively, in eight rabbits (30 min, q.d., 10 days). No clinical or histological deleterious effects occurred. LC-MS/MS analyses of post-treatment samples revealed a delivery gradient with highest fenretinide levels achieved at the patch-mucosal interface (no metabolites), pharmacologically active levels in fenretinide-treated oral mucosa (mean: 5.65 μM; trace amounts of 4-oxo-4-HPR) and undetectable sera levels. Epithelial markers for cell proliferation (Ki-67), terminal differentiation (transglutaminase 1—TGase1) and glucuronidation (UDP-glucuronosyltransferase1A1—UGT1A1) exhibited fenretinide concentration-specific relationships (elevated TGase1 and UGT1A1 levels 5μM) relative to blank-treated epithelium. All fenretinide-treated tissues showed significantly increased intraepithelial apoptosis (TUNEL) positivity, implying activation of intersecting apoptotic and differentiation pathways. Human oral mucosal correlative studies showed substantial interdonor variations in levels of the enzyme (cytochrome P450 3A4—CYP3A4) responsible for conversion of fenretinide to its highly active metabolite, 4-oxo-4-HPR. Complementary in vitro assays in human oral keratinocytes revealed fenretinide and 4-oxo-4-HPR’s preferential suppression of DNA synthesis in dysplastic as opposed to normal oral keratinocytes. Collectively, these data showed that mucoadhesive patch-mediated fenretinide delivery is a viable strategy to reintroduce a compound known to induce keratinocyte differentiation to human oral cancer chemoprevention trials.

Esophageal squamous cell carcinoma (SCC) is responsible for about one-seventh of all cancer-related mortality worldwide. This disease has a multifactorial etiology involving numerous environmental, genetic, and dietary factors. The 5-year survival from esophageal SCC is poor because the disease has usually metastasized at the time of diagnosis. Clinical investigations have shown that primary chemoprevention of this disease is feasible; however, only a few agents have shown efficacy. The Fischer 344 (F-344) rat model of esophageal SCC has been used extensively to investigate the pathophysiology of the disease and to identify chemopreventive agents of potential use in human trials. Multiple compounds that inhibit tumor initiation and/or tumor progression in the rat model have been identified. These include the isothiocyanates which inhibit the metabolic activation of esophageal carcinogens and agents that inhibit the progression of dysplastic lesions to cancer including inhibitors of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and c-Jun (a component of activator protein-1 [AP-1]). The present review deals principally with the use of berry preparations for the prevention of esophageal SCC in rodents, and summarizes recent data from a human clinical trial in China. Our results suggest that the use of berry preparations might be a practical approach to the prevention of esophageal SCC in China and, potentially, other high risk regions for this disease.

Cyclopenta[cd]pyrene (CPP) was examined for its lung tumorigenic activity in strain A/J mice, for the formation and persistence of CPP-induced DNA adducts in lung tissue, and for its induction of mutations in the Ki−ras oncogene from CPP-induced tumors. CPP displayed high tumorigenic activity, inducing 97.7 lung adenomas/mouse at 200 mg/kg. Ki−ras codon 12 mutations in the DNA of induced tumors were: GGT→CGT (50%); GGT→KTT (15%); GGT→TGT (25%); GGT→GAT (10%). All DNA adducts in the lungs of CPP-treated mice were CPP−3,4−oxide derived and most were CPP−3,4−oxide−2′−deoxyguanosine adducts. CPP is highly tumorigenic in the strain A/J mouse lung adenoma model, being 5 times more active than benzo[a]pyrene. The increased activity of CPP may be related to the unique induction of the GGT→CGT, Ki−ras codon 12 mutation.

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.

This study was undertaken to evaluate the carcinogenic potential of 5-methylchrysene (5-MeC) in strain A/J mouse lung and to correlate the 5-MeC-DNA adduct profile in lung tissue with the mutation spectrum in the K-ras gene of lung tumors. Strain A/J mice received a single i.p. injection of 5-MeC at doses of 10, 50, 100 and 200 mg/kg and after 24, 48 and 72 h their lungs were collected for DNA adduct analysis. Eight months later, lungs from the remaining mice were harvested and the lung tumors counted and collected for subsequent mutational analysis of the K-ras gene. 5-MeC was found to be a potent lung carcinogen in strain A/J mice, inducing more than 100 tumors/mouse at a concentration of 200 mg/kg. Six 5-MeC-DNA adducts were observed; one adduct comigrated with the standard N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I [1R,2S,3S-trihydroxy-4R-(N2-deoxy-guanosyl-3'-phosphate)- 1,2,3,4-tetrahydro-5-methyl-chrysene], derived from the bay-region diol-epoxide of 5-MeC. DNAs isolated from 5-MeC-induced lung tumors were evaluated for activating mutations in the K-ras gene by polymerase chain reaction-single strand conformation polymorphism and direct DNA sequencing analysis. Mutations were detected in 44 of 49 (90%) 5-MeC-induced tumors and the mutations were GGT-->TGT (50%), GGT-->GTT (23%) and GGT-->CGT (27%) in codon 12 of the gene. These results suggest that the N2-deoxyguanosine adduct of 5-MeC-diol-epoxide I may be one of the promutagenic adducts of 5-MeC in strain A/J mouse lung.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous cyclopenta-fused polycyclic aromatic hydrocarbon. CPP is highly genotoxic in bacterial and mammalian systems inducing gene mutations, sister chromatid exchanges and morphological transformation. CPP is a mouse skin carcinogen, a mouse skin tumor initiator and induces pulmonary tumors in newborn mice. We have examined the tumorigenic activity of CPP in strain A/J mice, have determined the formation and persistence of CPP-induced DNA adducts in lung tissue, and analyzed the mutational spectrum in the Ki-ras oncogene from CPP-induced tumors. CPP dissolved in tricaprylin was administered by i.p. injection to male A/J mice (20 mice/dose) at 0, 10, 50, 100 and 200 mg/kg. Animals were killed 8 months later and the lungs removed, fixed, and surface adenomas enumerated. CPP proved to be highly tumorigenic in A/J mice in terms of inducing lung adenomas. The observed tumor multiplicities (lung adenomas/mouse) were: 97.7 +/- 28.7 at 200 mg/kg, 32.8 +/- 15.4 at 100 mg/kg, 4.63 +/- 2.11 at 50 mg/kg and 0.58 +/- 0.82 at 10 mg/kg. Tricaprylin-treated controls produced 0.60 +/- 0.58 lung adenomas/mouse. Groups of mice treated under the same dosing conditions as those in the tumor studies were killed 1, 3, 7, 14 and 21 days after treatment. The lungs were removed, and the DNA was subjected to DNA adduct analysis by the 32P-postlabeling method. Total CPP-DNA adducts in mouse lung peaked at day 3 with 5870 amol CPP adducts/micrograms DNA after a single dose of 200 mg/kg. DNA adduct levels decreased to 1800 amol CPP adducts/micrograms DNA at day 21. Qualitative DNA adduct analysis revealed four major adducts and one minor adduct. Co-chromatography of the lung DNA from CPP-treated mice with calf thymus DNA treated with CPP-3,4-oxide indicated that all DNA adducts were oxide derived and comparison with CPP-3,4-oxide-treated polydeoxyguanylic acid suggests that almost all of these adducts are CPP-3,4-oxide-2'-deoxyguanosine adducts. Ki-ras codon 12 mutation analysis of the DNA from tumors taken from the 100 and 200 mg/kg CPP dose groups demonstrated the following patterns: GGT-->CGT (50%); GGT-->GTT (15%); GGT-->TGT (25%); GGT-->GAT (10%). We conclude that CPP is highly tumorigenic in the A/J mouse lung adenoma model, being five times more active than benzo[a]pyrene. This is unlike the result of CPP as a mouse skin tumorigen or tumor initiator in which CPP is considerably less potent than benzo[a]pyrene.(ABSTRACT TRUNCATED AT 400 WORDS)

Objective/Hypothesis: Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis. Study Design: Translational research study. Methods: Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry, and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens taken from cancer patients and normal control subjects were analyzed for the presence of pepsin by Western blot. Results: Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time- and dose-dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens. Conclusions: These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx. Laryngoscope, 2012

Dysplasia is a histologic precursor of esophageal squamous cell carcinoma (SCC). We previously showed that dietary freeze-dried, or lyophilized, strawberry powder inhibits N-nitrosomethylbenzylamine-induced SCC in the rat esophagus. On the basis of this observation, we conducted a randomized (noncomparative) phase II trial in China to investigate the effects of two doses of freeze-dried strawberries in patients with esophageal dysplastic lesions in a high-risk area for esophageal cancer. We randomly assigned 75 patients identified by endoscopy to have dysplastic esophageal premalignant lesions to receive freeze-dried strawberry powder at either 30 g/d (37 patients) or 60 g/d (38 patients) for six months; the powder was mixed with water and drunk. After six months, we assessed the changes in histologic grade of these lesions (primary endpoint) in a blinded fashion. The dose of 30 g/d, did not significantly affect histology or any other measured parameter. The dose of 60 g/d, however, reduced the histologic grade of dysplastic premalignant lesions in 29 (80.6%) of the 36 patients at this dose who were evaluated for histology (P < 0.0001). The strawberry powder was well tolerated, with no toxic effects or serious adverse events. Strawberries (60 g/d) also reduced protein expression levels of inducible nitric oxide synthase (iNOS) by 79.5% (P < 0.001), cyclooxygenase-2 (COX-2) by 62.9% (P < 0.001), phospho-nuclear factor kappa B (NFκB)-p65 (pNFκB-p65) by 62.6% (P < 0.001), and phospho-S6 (pS6) by 73.2% (P < 0.001). Freeze-dried strawberries (60 g/d) also significantly inhibited the Ki-67 labeling index by 37.9% (P = 0.023). Our present results indicate the potential of freeze-dried strawberry powder for preventing human esophageal cancer, supporting further clinical testing of this natural agent in this setting. Cancer Prev Res; 5(1); 41–50. ©2011 AACR.

Anthocyanins are members of a class of flavonoid compounds commonly known as plant polyphenols. They are responsible for the blue, purple, red and intermediate colors of many flowers, leaves, vegetables and fruits, especially berries. The daily intake of anthocyanins in the United States diet is estimated to be about 200 mg or about 9-fold higher than that of other dietary flavonoids. Anthocyanins have been evaluated for chemopreventive potential, both in cell cultures and in animal model tumor systems. Limited information on their chemopreventive effects is also available from epidemiological studies. This chapter summarizes the chemistry, synthesis and bioavailability of anthocyanins and the latest developments regarding their anti-carcinogenic effects in cell cultures and in animal model systems. The chemopreventive activity of protocatechuic acid (PCA), one of the most abundant metabolites of anthocyanins, is also discussed due to its prominent role in the overall inhibitory effects of anthocyanins. We suggest that PCA be further evaluated for chemoprevention in animal model systems and in humans in view of its relatively low toxicity and commercial availability.

Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried components of black raspberries (BRB) affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRB's chemopreventive effects. Consequently, functional assays, immunoblotting, and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were conducted. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles showed the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated, whereas immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. β-Glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC/MS-MS analyses showed retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, postrinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data show that comparable to the small intestine, the requisite hydrolytic, phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, interpatient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness. Cancer Prev Res; 4(8); 1209–21. ©2011 AACR.

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.

Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to manage preinvasive and invasive lesions. Food-based cancer prevention entities, such as black raspberries and their derivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cell growth and tumor formation. Here, we extend the role of black raspberry-mediated chemoprevention to that of cervical carcinogenesis.Three human cervical cancer cell lines, HeLa (HPV16-/HPV18+, adenocarcinoma), SiHa (HPV16+/HPV18-, squamous cell carcinoma) and C-33A (HPV16-/HPV18-, squamous cell carcinoma), were treated with a lyophilized black raspberry ethanol extract (RO-ET) at 25, 50, 100 or 200μg/ml for 1, 3 and 5days, respectively. Cell proliferation was measured by WST1 (tetrazolium salt cleavage) assays. Flow cytometry (propidium iodide and Annexin V staining) and fluorescence microscopy analysis were used to measure apoptotic cell changes.We found that non-toxic levels of RO-ET significantly inhibited the growth of human cervical cancer cells, in a dose-dependent and time-dependent manner to a maximum of 54%, 52% and 67%, respectively (p0.05). Furthermore, cell growth inhibition was persistent following short-term withdrawal of RO-ET from the culture medium. Flow cytometry and fluorescence microscopy demonstrated RO-ET-induced apoptosis in all cell lines.Black raspberries and their bioactive components represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies.

Berries and cancer prevention , Berries and cancer prevention , کتابخانه دیجیتال جندی شاپور اهواز

The purpose of this investigation was to establish a dose response for the effects of dietary phenethyl isothiocyanate (PEITC) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis and DNA methylation. Groups of 13-27 rats were randomly assigned to AIN-76A diets containing 0, 0.325, 0.75, 1.5 or 3.0 mumol PEITC/g. Two weeks later, rats were administered NMBA subcutaneously at a dose of 0.5 mg/kg once a week for 15 weeks. Animals were maintained on control or experimental diets for an additional 8 weeks and were terminated at week 25 of the experiment. No significant effects on weight gain or food intake were noted for any of the experimental diets when compared with control values. Animals receiving only NMBA developed 9.3 +/- 0.9 tumors/rat, with an incidence of 100%. Dietary PEITC at concentrations of 0.75, 1.5 and 3.0 mumol/g inhibited NMBA-induced esophageal tumor multiplicity by 39%, 90% and 100%, respectively. Esophageal tumor incidence in these groups was reduced by 0%, 40% and 100%, respectively. The 0.325 mumol/g PEITC diet did not significantly affect NMBA-induced esophageal tumorigenesis. These results indicate that the minimum inhibitory dietary concentration of PEITC is between 0.325 and 0.75 mumol/g. Groups of 20 rats were assigned to diets containing 0-3.0 mumol PEITC/g for two weeks as described above, and then sacrificed 24 hours after administration of [3H-methyl]NMBA. The esophageal DNA was isolated, purified, hydrolyzed, and analyzed by HPLC. PEITC inhibited DNA methylation in a dose-dependent manner, as was found in the tumor bioassay. The inhibition of tumor incidence was highly correlated with the percentage inhibition of either 7-methylguanine or O6-methylguanine. These latter results suggest that the inhibitory activity of PEITC in this model is manifested, at least in part, during the functional equivalent of tumor initiation.

The effects of phenethyl isothiocyanate (PEITC) on benzo[a]pyrene (B[a]P)-induced DNA adducts and pulmonary adenomas in A/J mice were investigated. Groups of 24 male and 24 female A/J mice were administered PEITC by gavage at doses of 0.075, 0.25, 0.50, and 0.75 mmol/kg (12, 41, 82, 122 mg/kg) for 6 consecutive days. A single dose of 0.40 mmol/kg (100 mg/kg) B[a]P was given by i.p. injection after the fourth dose of PEITC. A positive control received only a single i.p. injection of B[a]P, 100 mg/kg body weight. The vehicle control group was administered corn oil by gavage for 6 consecutive days and a single i.p. injection of tricaprylin following 4 doses of corn oil. In addition, 2 groups of 24 male and 24 female mice each were administered PEITC at dose levels of 12 and 122 mg/kg body weight to evaluate the effects of this compound alone. Body weight loss occurred in both males and females in the 0.25, 0.50, and 0.75 mmol/kg PEITC groups relative to B[a]P controls and to untreated controls during the first week of the study. Tumor incidence and multiplicity in the PEITC-treated groups, evaluated 7 months after B[a]P administration, were not significantly different when compared with the B[a]P group. The results of a subsequent DNA adduct bioassay, using similar dose levels of PEITC and B[a]P, correlated with the results of the tumorigenesis study, indicating that pretreatment with PEITC did not inhibit the formation of B[a]P-DNA adducts in the lungs of A/J mice.

Ellagic acid (EA) is a naturally occurring plant phenol that was recently demonstrated to protect cultured rat embryos from the embryotoxic effects of N-methyl-N-nitrosourea (MNU). The teratogenic mechanism of action of MNU, as well as other methylating agents, is debated: both cell death and mutation have been proposed. In some model systems, EA has been reported to selectively decrease the mutagenic DNA adduct O6-methylguanine (O6MG) when compared to the cytotoxic DNA adduct N7-methylguanine (N7MG). The present study was initiated to determine 1) the distribution of 14C-EA and 3H-MNU in the rat whole embryo culture model system and 2) the effects of EA on MNU-induced DNA adduct formation in this model system. 14C-EA (50 microM for 2 hr, known embryoprotective concentration; no MNU added) was used to demonstrate access of EA to the embryo within the 2 hr exposure period. The majority of EA (99.5%) remained in the media while tissue concentrations of 57.0 and 47.9 pmol/mg were attained in the yolk sacs and embryos, respectively. Similarly, MNU (75 microM for 1 hr, known effective concentration; no EA added) was distributed between the media, yolk sacs, and embryos at 99.7%, 73.7 and 112.9 pmol/mg, respectively. When non-radiolabeled EA (50 microM for 2 hr) was used to protect embryos prior to exposure to 3H-MNU (75 microM for 1 hr), the distribution of MNU in the model system was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three non-tumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, with RF = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%-90% more mAb-5G-reactive antigen than the non-tumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.

Strain A/J mice received intraperitoneal injections of benz[j]aceanthrylene (B[j]A) or benzo[a]pyrene (B[a]P). At 24, 48, and 72 h, lung tissues were removed for analysis of B[a]P- or B[j]A-derived DNA adduct formation during the first 3 d of exposure. One group of mice exposed to these hydrocarbons was kept for 8 mo to determine lung tumor multiplicity, the occurrence of mutations in codons 12 and 61 of the Ki-ras gene in the tumors that arose, the relationship between Ki-ras oncogene mutations in tumors, and the presence and quantity of genomic DNA adducts. The major DNA adduct in the lungs of mice exposed to B[a]P was N2-(10 beta-[+B, 7 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl)-deoxyguanosine (BPDE-I-dGuo) arising from bay-region diolepoxide activation of B[a]P and was consistent with the occurrence of tumors with mutations GGT-->TGT (56%), GGT-->GTT (25%), and GGT-->GAT (19%) in codon 12, all involving mutations of a guanine. B[j]A, a demethylated analogue of 3-methylcholanthrene (3-MCA) with an unsaturated cyclopenta ring, produced 16-to 60-fold more tumors at equivalent doses than did B[a]P; the mutations in tumors were GGT-->TGT (4%), GGT-->GTT (30%), and GGT-->CGT (65%). Analysis of adduction patterns in DNA suggested that B[j]A was activated to form DNA-binding derivatives in A/J mouse lungs primarily at the cyclopenta ring even though B[j]A contains a bay region. As reported in the published literature, the mutation spectrum induced by 3-MCA in Ki-ras codon 12 of mouse cells is similar to that of B[a]P but not to that of its close relative B[j]A. In contrast to B[j]A, 3-MCA is activated mostly via a bay-region diol-epoxide since its cyclopenta ring is saturated and not easily epoxidates. Therefore, we propose that the GGT-->CGT mutations produced by B[j]A in Ki-ras codon 12 were mostly the result of cyclopenta-ring-derived adducts.

Purpose: This study evaluated the effects of black raspberries (BRBs) on biomarkers of tumor development in the human colon and rectum including methylation of relevant tumor suppressor genes, cell proliferation, apoptosis, angiogenesis, and expression of Wnt pathway genes. Experimental Design: Biopsies of adjacent normal tissues and colorectal adenocarcinomas were taken from 20 patients before and after oral consumption of BRB powder (60 g/d) for 1–9 weeks. Methylation status of promoter regions of five tumor suppressor genes was quantified. Protein expression of DNA methyltransferase 1 (DNMT1) and genes associated with cell proliferation, apoptosis, angiogenesis, and Wnt signaling were measured. Results: The methylation of three Wnt inhibitors, SFRP2, SFRP5, and WIF1, upstream genes in Wnt pathway, and PAX6a, a developmental regulator, was modulated in a protective direction by BRBs in normal tissues and in colorectal tumors only in patients who received BRB treatment for an average of 4 weeks, but not in all 20 patients with 1–9 weeks of BRB treatment. This was associated with decreased expression of DNMT1. BRBs modulated expression of genes associated with Wnt pathway, proliferation, apoptosis, and angiogenesis in a protective direction. Conclusions: These data provide evidence of the ability of BRBs to demethylate tumor suppressor genes and to modulate other biomarkers of tumor development in the human colon and rectum. While demethylation of genes did not occur in colorectal tissues from all treated patients, the positive results with the secondary endpoints suggest that additional studies of BRBs for the prevention of colorectal cancer in humans now appear warranted. Clin Cancer Res; 17(3); 598–610. ©2010 AACR.

Colorectal cancer (CRC) is the leading digestive tract cancer in the Western world, particularly in the United States where it accounts for approximately 10% of all cancer deaths. The multistage development of CRC is associated with chromosomal instability, DNA-repair defects, aberrant DNA methylation, and mutational events in oncogenes and tumor suppressor genes. Preclinical studies using cultured human colon tumor cells and animal models of colorectal cancer indicate that black raspberries (BRBs) and their constituent anthocyanins and ellagitannins elicit chemopreventive effects against CRC. A 7-day Phase I trial indicated that BRBs are well tolerated by humans and their component anthocyanins and ellagic acid are absorbed into blood. The results of a Phase Ib trial in which BRBs were administered daily to colorectal cancer patients from the time of diagnosis of the disease until surgical removal of the tumor (average = 3 weeks) are presented in this chapter. BRB treatment resulted in reduced proliferation and increased apoptosis of CRC cells. In addition, tumor angiogenesis was inhibited by berries. BRB treatment reduced s-catenin expression and enhanced E-cadherin expression in CRC indicating a protective effect on the Wnt signaling pathway. DNA methylation assays showed that BRBs are capable of demethylating tumor suppressor genes associated with the Wnt pathway, in part, through inhibiting the expression of DNMT1, a DNA methyltransferase that is commonly overexpressed in CRC. These data suggest that BRBs should be further examined for chemopreventive effects against CRC.

Esophageal squamous cell carcinoma (ESCC) is the 7th leading cause of cancer death worldwide. Its etiology and late detection rate make it an important target for intensive studies in chemoprevention and treatment. The Fischer-344 rat model of esophageal squamous cell carcinoma has been used extensively to evaluate the ability of black raspberries (BRBs) and other berry types to inhibit esophageal tumorigenesis. In this model, tumors are induced by the nitrosamine carcinogen, N-nitrosomethylbenzylamine (NMBA). Dietary BRBs inhibit both the initiation and promotion/progression stages of esophageal tumorigenesis in the rat. They inhibit initiation events by influencing the metabolic activation and detoxification of NMBA, and promotion/progression events by reducing cell proliferation, inflammation, and angiogenesis and by stimulating apoptosis and differentiation. Genes associated with these cellular functions are positively modulated by BRBs. Biofractionation studies indicate that amongst the most active chemopreventive agents in BRBs are the anthocyanins. An anthocyanin-enriched fraction of BRBs was nearly as active as whole BRBs themselves in preventing esophageal tumorigenesis. However, the ellagitannins and other constituents in the fiber fraction of BRBs are also active and studies are underway to further identify these constituents. In a recent study, 7 different berry types, including the “exotic” berries (acai, noni and wolfberry [goji]), were compared for their ability to inhibit NMBA-tumorigenesis in the rat esophagus. All seven types were active, irrespective of their content of anthocyanins and ellagitannins. It appears that each berry type has unique constituents with cancer preventive potential.

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.

1. Berries have been part of the human diet for many centuries. They are a rich source of known chemopreventive agents including provitamin A carotenoids, C, E, and folate, calcium and selenium, simple and complex phenols, and phytosterols.

A monoclonal antibody, designated MAb-5A IIgG1), was generated against a tumorigenic rat esophageal epithelial cell line, B-2T. MAb-5A reacted with a series of non-tumorigenic and tumorigenic epithelial cell lines derived from F-344 rat esophagi or tracheas. However, the highest level of antigen expression was detected on tumorigenic rat epithelial cell lines. A trace amount of antigen was detected in primary cultures of normal rat epithelial cells derived from either esophagi or tracheas. MAb-5A did not react with rat fibroblasts. MAb-5A reacted with B-2T derived tumor tissues propagated in vivo but showed only slight reactivity with normal rat esophageal epithelial tissues. Cell surface radioiodination, extraction and immunoprecipitation experiments were conducted to characterize the antigen molecule. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed two protein bands with mobilities corresponding to approximately 140 and 120 kDa, respectively. Under reducing conditions the 140 kDa band shifted to approximately 120 kDa whereas the 120 kDa band shifted upward to approximately 130 kDa. The non-reduced/reduced mobilities of these bands suggest that they may be members of the integrin family of matrix receptors. A polyclonal antibody to the beta 1 subunit immunoprecipitated two similar bands detected by MAb-5A, further suggesting that the antigen complex may be related to members of the integrin superfamily.

Ellagic acid is a naturally occurring plant phenol that has demonstrated anticarcinogenic and antimutagenic activity in several test systems. Given the common proposed etiopathogenic processes of mutagenesis, carcinogenesis, and teratogenesis induced by genotoxic chemicals, the present study was initiated to determine whether ellagic acid would protect rat embryos in culture from the teratogenic effects of N-methyl-N-nitrosourea (MNU). Ellagic acid alone (as used in these experiments; 50 microM in DMSO) was not embryotoxic. Ellagic acid (50 microM) significantly (P less than 0.01) prevented MNU (75 microM)-induced effects including mortality (absence of heart beat), abnormal formation of the cephalic neural tube derivatives, and delayed differentiation as assessed by a morphological scoring system. These embryoprotective effects were dose responsive. Sequential treatment of embryos with ellagic acid followed by MNU in fresh media also was embryoprotective with no diminution of effect. The site at which ellagic acid interrupts the critical teratogenic events induced by MNU is apparently within the embryo and/or placenta. This model of chemical embryoprotection may be useful in determining the role of cell death and/or mutation in the teratogenic mechanism of action of methylating agents.

On April 20, 2009, we lost a true gentleman and cancer researcher with the passing of Dr. Bandaru S. Reddy. Bandaru was a dear friend and distinguished colleague of many in the scientific community. To those fortunate enough to be included among Bandaru's “second family” of postdocs and visiting

Cyanidin 3-glucoside (C3GLU), cyanidin 3-rutinoside (C3RUT), cyanidin 3-sambubioside (C3SAM) and cyanidin 3-(2G-xylosyl) rutinoside (C3XRUT) are the four constituent black raspberry anthocyanins that contribute significantly to the chemopreventive effects of freeze dried black raspberries (FBR). A highly sensitive and specific LC-MS/MS assay was developed and validated to simultaneously quantify these four FBR anthocyanins in human saliva, plasma and oral tissue homogenates. In saliva, the lower limit of quantification (LLOQ) for these anthocyanins was 1.0 ng/mL. The within-run and between-run coefficients of variations (CVs) at the quality control concentrations (1.0, 5.0, 50 and 500 ng/mL) were all

Light in the UVB spectrum (280-320 nm) induces a number of changes in the epidermis and dermis of mice and humans, resulting in a robust inflammatory response. A standardized black raspberry extract (BRE) has been effective in reducing signaling pathways commonly initiated by inflammatory stimuli. In this study, we determined whether this extract could reduce cutaneous UVB-induced inflammation and carcinogenesis. In our carcinogenesis model, female SKH-1 hairless mice were exposed to one minimal erythemal dose of UVB thrice weekly on nonconsecutive days for 25 weeks. Immediately after each exposure, the mice were treated topically with either BRE dissolved in vehicle or with vehicle only. Beginning on week 19, mice treated with BRE had a significant reduction in tumor number and in average tumor size. This reduction correlated with a significant reduction in tumor-infiltrating CD3+foxp3+ regulatory T-cells. In the acute model, mice were exposed to a single minimal erythemal dose of UVB and treated topically with BRE or with vehicle. At 48 hours post-UVB exposure, topical BRE treatment significantly reduced edema, p53 protein levels, oxidative DNA damage, and neutrophil activation. The ability of topical BRE to reduce acute UVB-induced inflammation and to decrease tumor development in a long-term model provides compelling evidence to explore the clinical efficacy of BRE in the prevention of human skin cancers.

Diets containing freeze-dried black raspberries (BRB) suppress the development of N-nitrosomethylbenzylamine (NMBA)–induced tumors in the rat esophagus. Using bioassay-directed fractionation, the anthocyanins in BRB were found to be the most active constituents for down-regulation of carcinogen-induced nuclear factor-κB and activator protein-1 expression in mouse epidermal cells in vitro. The present study was undertaken, therefore, to determine if the anthocyanins contribute to the chemopreventive activity of BRB in vivo. F344 rats consumed diets containing either (a) 5% whole BRB powder, (b) an anthocyanin-rich fraction, (c) an organic solvent-soluble extract (a–c each contained ∼3.8 μmol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 μmol anthocyanins/g diet), (e) a hexane extract, and (f) a sugar fraction (e and f had only trace quantities of anthocyanins), all derived from BRB. Animals were fed diets 2 weeks before treatment with NMBA and throughout the bioassay. Control rats were treated with NMBA only. Animals were killed at week 30, and esophageal tumors were enumerated. The anthocyanin treatments (diet groups a–c) were about equally effective in reducing NMBA tumorigenesis in the esophagus, indicating that the anthocyanins in BRB have chemopreventive potential. The organic-insoluble (residue) fraction (d) was also effective, suggesting that components other than berry anthocyanins may be chemopreventive. The hexane and sugar diets were inactive. Diet groups a, b, and d all inhibited cell proliferation, inflammation, and angiogenesis and induced apoptosis in both preneoplastic and papillomatous esophageal tissues, suggesting similar mechanisms of action by the different berry components.

Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells.

This study was undertaken to measure the liberation in vitro of ellagic acid [2], a naturally occurring inhibitor of carcinogenesis, from precursor ellagitannins under conditions found in the gut tract. Enzymes, namely beta-glucosidase, esterases, and alpha-amylase, were incubated with raspberry extract. In addition, raspberry extract and casuarictin [1] were treated at different pH's and with the contents of small intestine and cecum from rats fed AIN-76A diet. The esterase activity of the enzyme samples was measured spectrophotometrically using p-nitrophenol acetate as the substrate, and the amount of ellagic acid [2] released from all samples was analyzed by hplc. The hydrolysis of the ellagitannins was not catalyzed by any of the purified enzymes tested, and components of the raspberry extract were found to inhibit the purified esterases noncompetitively. Casuarictin [1] was hydrolyzed to yield high quantities of ellagic acid [2] when placed in buffer at pH 7 and 8, or when incubated with cecal contents for two hours. The release of ellagic acid [2] from the raspberry extract was optimal at pH 8, and maximal release in cecal contents occurred with 1 h. Small intestinal contents had no significant effect on ellagic acid liberation from either casuarictin [1] or raspberry extract.

[14CO]Ellagic acid with a chemical purity of 98.9% and radiochemical purity of 99.9% was synthesized with an overall yield of 16% (both chemically and radiochemically). Reaction of 14CO2 with lithiated 3,4,5-trimethoxybenzene and demethylation of the resulting 3,4,5-trimethoxybenzoic acid was followed by esterification and coupling of methyl gallate into ellagic acid. Two efficient coupling methods were employed: direct aeration and aeration of methyl gallate in the presence of the phenolic oxidase, tyrosinase. The latter method produced the highest yield and purity. This preparation produced [14CO]ellagic acid with a specific activity of 20 mCi/mmol. The yields of labeled 3,4,5-trimethoxybenzoic acid and ellagic acid based on Ba14CO3 were 65% and 16%, respectively.

A gas chromatography, negative ion chemical ionization mass spectrometry (GC-NICI-MS) based assay for tobacco-specific nitrosamine adducts of DNA is described. The assay is based on the observation that acid hydrolysis of DNA from animals treated with tobacco-specific nitrosamines releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). HPB and the internal standard [4,4-D2]HPB are derivatized with pentafluorobenzoyl chloride and the resulting HPB-pentafluorobenzoate is purified by high-performance liquid chromatography prior to GC-NICI-MS analysis. DNA from human peripheral lung and tracheobronchial tissue, collected at autopsy, was analyzed for acid-released HPB. The mean HPB level (fmol/mg of DNA) for peripheral lung DNA was 11 +/- 16 (SD, n = 9) for smokers and 0.9 +/- 2.3 (n = 8) for nonsmokers. Mean adduct levels in tracheobronchus were 16 +/- 18 (n = 4) for smokers and 0.9 +/- 1.7 (n = 4) for nonsmokers. These are the first measurements of tobacco-specific nitrosamine-DNA adducts in humans. Further studies comparing the levels of DNA and globin adducts will provide a better understanding of the metabolic activation of tobacco-specific nitrosamines in humans and may provide a more accurate indication of an individual's risk of developing tobacco-related cancer.

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

The detection of activated protooncogenes in mouse lung tumors has led to a major advance in our understanding of carcinogenesis of the lung at the molecular level. A high frequency of activated K-ras protooncogenes has been detected in tetranitromethane (TNM)- and 1,3-butadiene-induced lung tumors in B6C3F1 mice. In the past several years, we have pursued protooncogene activation in spontaneous and chemically induced tumors of strain A mice. The strain A mouse has a high incidence of spontaneous lung tumors and is susceptible to tumor induction by chemical carcinogens. We have detected and characterized the activated protooncogenes in the DNA of both spontaneously occurring and chemically induced lung tumors of strain A mice. Activated K-ras genes were detected using the NIH/3T3 transfection assay, and the activating mutations were identified by utilizing the polymerase chain reaction (PCR) and direct sequence analysis. A strong selectivity of mutations in the K-ras genes were observed in chemically induced lung tumors, as compared to spontaneous tumors, indicating that the carcinogens directly induced point mutations in the K-ras protooncogene. These findings suggest that the strain A mouse lung tumor model appears to be a very sensitive system to identify the mechanism by which chemical carcinogens activate the K-ras gene in lung tissue in vivo.

The progression of pulmonary neoplasia was examined in strain A/J male mice treated with a single dose of vinyl carbamate (60 mg/kg, i.p.) 6 weeks after birth. Interim sacrifices were performed at 7, 8, 10, 12, or 14 months. Proliferative lesions of the lung were divided into four categories: hyperplasias, adenomas, carcinomas arising within adenomas, and carcinomas. Grossly visible surface tumor counts, histologic diagnoses, and morphometric measurements of histologic lesions were used to evaluate progression. Vinyl carbamate-treated mice showed increased mean surface tumor counts at all time points. Diagnostic evaluation suggested that as a function of time, the relative frequency of hyperplasias decreased and the relative frequency of adenomas increased. The relative frequency of adenomas subsequently decreased, whereas the relative frequency of carcinomas increased. At all time points, carcinomas arising within adenomas were present. As time progressed, the number of carcinomas arising within adenomas decreased, whereas the number of "pure" carcinomas increased. Morphometric analysis of lesions indicated hyperplasias to be small, that adenomas were larger than hyperplasias, and carcinomas were larger than adenomas and hyperplasias, suggesting that few adenomas or carcinomas arise de novo. Collectively, these data suggest that the majority of pulmonary tumors in A/J mice treated with vinyl carbamate arise as hyperplasias, progress to adenomas, and ultimately result in carcinomas.

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

The effect of phenethyl isothiocyanate (PEITC) on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured rat oral tissue was investigated. Two protocols were used. In one, oral tissue from untreated rats was cultured in the presence of 10 or 50 microM PEITC and either NNN or NNK. The levels of NNN and NNK metabolites released into the culture media were determined by HPLC analysis. The presence of 10 microM PEITC inhibited the formation of all NNN metabolites from 45 to 70% when the concentration of NNN was 1 microM or 10 microM. When the concentration of PEITC was 50 microM the extent of inhibition was from 70 to 90%. alpha-Hydroxylation of NNK was inhibited 70 to 90% and N-oxidation of NNK was inhibited 80 to 90% by 10 microM PEITC. Carbonyl reduction of NNK to NNAL was unaffected by 10 microM PEITC and only slightly inhibited by 50 microM PEITC. In the second protocol, rats were fed NIH-07 diet containing 3 mumol PEITC/g for 1-14 days. The metabolism of NNN by cultured oral tissue from these rats was decreased from 40 to 90% relative to that by tissue from control rats. NNK metabolism was inhibited 40 to 60%. The extent of inhibition was the same when rats were fed PEITC containing diet for 1 or 14 days. NNN and NNK are the only tobacco constituents which induce oral cavity cancer in an animal model. The results of this study suggest the possibility that PEITC may be useful as a chemopreventive agent for oral cavity cancer.

A diet containing 5% freeze-dried black raspberries (BRB) markedly inhibits esophageal cancer in rats treated with the carcinogen, N-Nitrosomethylbenzylamine (NMBA). We previously identified esophageal genes that become dysregulated after short-term treatment of rats with NMBA and determined which genes are maintained at near-normal levels of expression if the animals were fed 5% BRB prior to and during NMBA treatment. In this study, we report the effects of the BRB diet on gene expression in esophagi from untreated (control) animals. After 3 wk on a 5% BRB diet, control esophagi were excised, stripped of the submucosal and muscularis layers, and processed for histology and microarray profiling. RNA microarrays revealed that the BRB altered the expression levels of 36 genes; 24 were upregulated, and 12 were downregulated. Among the upregulated genes are genes associated with cellular matrix, signaling cascades, transcription regulation, apoptosis, metabolism, and intriguingly, contraction. Most of the downregulated transcripts are involved in cell regulation, signal transduction, and metabolism. Histopathological analyses revealed that the BRB have little or no effect on esophageal morphology. In conclusion, histological and molecular studies indicate that a 5% BRB diet produces only modest effects on the esophagus, the target tissue for NMBA carcinogenesis in the rat.

Our recent study identified 2,261 dysregulated genes in the esophagi of rats that received a 1-week exposure to the carcinogen N-nitrosomethylbenzylamine (NMBA). We further reported that 1,323 of these genes were positively modulated to near-normal levels of expression in NMBA-treated animals that consumed dietary phenylethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables. Herein, we report our results with companion animals that were fed a diet containing 5% freeze-dried black raspberries (BRB) instead of PEITC. We found that 462 of the 2,261 NMBA-dysregulated genes in rat esophagus were restored to near-normal levels of expression by BRB. Further, we have identified 53 NMBA-dysregulated genes that are positively modulated by both PEITC and BRB. These 53 common genes include genes involved in phase I and II metabolism, oxidative damage, and oncogenes and tumor suppressor genes that regulate apoptosis, cell cycling, and angiogenesis. Because both PEITC and BRB maintain near-normal levels of expression of these 53 genes, their dysregulation during the early phase of NMBA-induced esophageal cancer may be especially important in the genesis of the disease. [Cancer Res 2008;68(15):6460–7]

Anthocyanins are the most abundant flavonoid constituents of fruits and vegetables. The conjugated bonds in their structures, which absorb light at about 500 nm, are the basis for the bright red, blue and purple colors of fruits and vegetables, as well as the autumn foliage of deciduous trees. The daily intake of anthocyanins in residents of the United States is estimated to be about 200 mg or about 9-fold higher than that of other dietary flavonoids. In this review, we summarize the latest developments on the anti-carcinogenic activities of anthocyanins and anthocyanin-rich extracts in cell culture models and in animal model tumor systems, and discuss their molecular mechanisms of action. We also suggest reasons for the apparent lack of correlation between the effectiveness of anthocyanins in laboratory model systems and in humans as evidenced by epidemiological studies. Future studies aimed at enhancing the absorption of anthocyanins and/or their metabolites are likely to be necessary for their ultimate use for chemoprevention of human cancer.

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation. [Cancer Res 2008;68(12):4945–57]

The rat zona glomerulosa has a renin-angiotensin system that appears to function as an autocrine or paracrine system in the regulation of aldosterone production. To further investigate dynamic changes of production of renin and aldosterone in vitro we developed a primary monolayer culture of rat adrenal glomerulosa cells in serum-free medium. Collagenase-dispersed glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 48 hours; the medium was then replaced with serum-free PFMR-4 medium. The cell viability and the aldosterone secretion were stable over the additional 48 hours in the serum-free control medium. After incubation for 24 hours in the serum-free medium, the cells were exposed to high K+ or adrenocorticotropic hormone (ACTH) for another 24 hours. ACTH stimulated aldosterone secretion, and this increased secretion was associated with an increase in renin activity (cell active renin, from 15.56 +/- 0.71 to 45.75 +/- 5.69; cell inactive renin, from 0.67 +/- 0.54 to 8.75 +/- 3.40; medium inactive renin, from 5.58 +/- 1.16 to 106.20 +/- 14.01 pg angiotensin I (Ang I)/micrograms protein/3 hr). Aldosterone was also stimulated by high K+. This increase was also associated with an increase in active renin in the cells (from 15.08 +/- 1.80 to 23.26 +/- 2.15 pg Ang I/micrograms protein/3 hr) and an increase in inactive renin in the medium (from 10.87 +/- 1.62 to 21.37 +/- 3.20 pg Ang I/micrograms protein/3 hr). Addition of the angiotensin converting enzyme inhibitor lisinopril attenuated both ACTH- and high K(+)-stimulated aldosterone secretion significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of acrylonitrile to induce cytotoxicity, sister-chromatid exchanges and DNA single-strand breaks was studied in cultured human bronchial epithelial cells. The toxic effect as determined by cloning efficiency was observed at a dose of 600 micrograms/ml but not at doses of both 150 and 300 micrograms/ml. The frequency of sister-chromatid exchange in untreated cells was 3.7 +/- 1.3 per cell. In contrast, cells treated with acrylonitrile at 150 and 300 micrograms/ml exhibited 6.6 +/- 1.3 and 10.7 +/- 1.7 sister-chromatid exchanges per metaphase, respectively. DNA single-strand breaks were induced by acrylonitrile at dose levels of 200 and 500 micrograms/ml. The genotoxic effects on human bronchial epithelial cells that were directly exposed to acrylonitrile are of interest in relation to evidence for the higher lung cancer incidence of acrylonitrile workers in epidemiological studies.

Naturally-occurring aromatic isothiocyanates, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), were tested for their post-treatment effects on lung tumorigenicity by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A J mice. Mice at 7 weeks of age were administered a single i.p. dose of NNK (10 μmol/mouse). One week after NNK dosing, mice were placed on AIN-76A diet containing 1 or 3 μmol/g diet of BITC or PEITC. The control group was maintained on AIN-76A diet after NNK administration. Mice were killed 16 week after NNK treatment and lung adenomas were counted. The results showed mice fed control diet developed 7.8 tumors/mouse. Mice fed PEITC at concentrations of 1 or 3 μmol/g diet had 8.2 or 6.1 tumors/mouse, respectively. Feeding BITC at 1 μmol/g diet resulted in a tumor yield of 8.0 tumors/mouse, whereac BITC diet at 3 μmol/g diet gave 5.2 tumors/mouse, a small but significant inhibition. However, in the high BITC dose group, a loss in weight gain due to reduced food intake was noted. The results of this study showed that post-treatment of aromatic isothiocyanates had little, if any, effect on NNK lung tumorigenicity in A J mice. This is in contrast to our previous findings in which pretreatment with PEITC greatly inhibited lung tumor induction by NNK in A J mice and suggests that tumor inhibition by PEITC is due to inhibition of NNK metabolic activation.