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PDF file - 10KB, Supplemental Figure 2. Black raspberry (BRB) powder (A)and BRB suppository (B).

PDF file - 16KB, FAP Supplemental Figure 1, Study flow chart.

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.

PDF file - 24KB, Supplemental Figure 3. APC mutations in 14 FAP patients.

PDF file - 52KB, This table includes the primer sequences used for the qPCR analysis done in this study.

Legend for Supplementary Figure 1.

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Supplementary Figure 1 from Topical Treatment with Black Raspberry Extract Reduces Cutaneous UVB-Induced Carcinogenesis and Inflammation

PDF file - 50KB, Supplemental Table 1. Some chemopreventive agents in black raspberry powder. Supplemental Table 2. Vender information and dilution for primary antibodies. Supplemental Table 3. List of primer sequences for MassARRAY analysis. Supplemental Table 4. Patient characteristics and polyp data from all 14 patients. Supplemental Table 5. Effects of oral BRBs and BRB suppositories on the development of adenomas.

Perspective on This Article from Randomized Phase II Trial of Lyophilized Strawberries in Patients with Dysplastic Precancerous Lesions of the Esophagus

Familial adenomatous polyposis (FAP) is characterized by the early onset of colonic polyposis and a high risk for colorectal cancer. FAP is treated by colectomy followed by lifelong removal of rectal polyps. This study determined whether black raspberries (BRBs) might regress rectal polyps in patients with FAP. Fourteen patients with FAP were treated with BRBs daily for 9 months. Seven patients received BRB powder orally plus two BRB suppositories inserted into the rectum at bedtime. The other 7 received an oral placebo plus the suppositories. Rectal polyp counts and polyp sizes were obtained at time zero and after 9 months of BRB treatment. Polyps and adjacent normal tissue were collected at both time points. The burden (P = 0.036) but not number (P = 0.069) of rectal polyps was significantly decreased. No benefit was noted with the addition of oral BRBs. Three patients were nonresponders. BRBs significantly decreased cellular proliferation, DNA methylation methyl transferase 1 protein expression, and p16 promoter methylation, but not promoter methylation of the Wnt pathway antagonists, SFRP2 and WIF1, in rectal polyps (adenomas) from responders but not from nonresponders. The MBD-seq assay revealed more demethylated transcription start sites (TSS), including those for miRNAs, in BRB-treated adenomas from the responders. In conclusion, BRB suppositories seem sufficient for regressing rectal polyps in patients with FAP. Cancer Prev Res; 7(7); 666–74. ©2014 AACR.

HPLC of 5 ng DBPDE incubated in keratinocyte growth medium overnight at room temperature in the absence (lower trace) and presence of BRB extract (10 ug/ml, middle trace) or (50ug/ml, upper trace).

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response—enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704–12. ©2016 AACR.

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.

Purpose: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions.Experimental Design: This placebo-controlled multicenter study investigated the effects of topical application of bioadhesive gels that contained either 10% w/w freeze-dried black raspberries (BRB) or an identical formulation devoid of BRB placebo to biopsy-confirmed OIN lesions (0.5 g × q.i.d., 12 weeks). Baseline evaluative parameters (size, histologic grade, LOH events) were comparable in the randomly assigned BRB (n = 22) and placebo (n = 18) gel cohorts. Evaluative parameters were: histologic grade, clinical size, and LOH.Results: Topical application of the BRB gel to OIN lesions resulted in statistically significant reductions in lesional sizes, histologic grades, and LOH events. In contrast, placebo gel lesions demonstrated a significant increase in lesional size and no significant effects on histologic grade or LOH events. Collectively, these data strongly support BRB's chemopreventive impact. A cohort of very BRB-responsive patients, as demonstrated by high therapeutic efficacy, was identified. Corresponding protein profiling studies, which demonstrated higher pretreatment levels of BRB metabolic and keratinocyte differentiation enzymes in BRB-responsive lesions, reinforce the importance of local metabolism and differentiation competency.Conclusions: Results from this trial substantiate the LOH reductions identified in the pilot BRB gel study and extend therapeutic effects to significant improvements in histologic grade and lesional size. Clin Cancer Res; 20(7); 1910–24. ©2014 AACR.

PDF file - 59KB, Function of proteins evaluated by immunoblotting.

Purpose: The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene–associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties.Experimental Design: Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes.Results: Confirming earlier phase I data, none of the 27 participants developed FBR gel–associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene–associated loci.Conclusions: These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.

PDF file - 63KB, Long term follow up of clinical trial patients.

Supplementary Table S1 from Effects of a Topically Applied Bioadhesive Berry Gel on Loss of Heterozygosity Indices in Premalignant Oral Lesions

PDF file - 1911KB, Immunoblot analyses of proteins associated with either keratinocyte differentiation (TGase 1, Involucrin, Loricrin, Cytokeratin 10/13) or local enteric recycling (UDP-Glc DH, UGT1A) in biopsy tissues of clinical trial participants. As not all OIN lesions had sufficient size to obtain tissue for Western analyses, only samples from the larger OIN lesions are represented. We have previously confirmed that these enzymes of interest reside almost exclusively in the epithelia. Pan cytokeratin was therefore used as the protein loading control.

Supplementary Table 4 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

Supplementary Table 2 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

There is little information on early molecular events in the development of N-nitrosomethylbenzylamine (NMBA)–induced rat esophageal tumorigenesis and of the effects of chemopreventive agents on these events. In this study, we identified genes in rat esophagus that were differentially expressed in response to short-term NMBA treatment and modulated by cotreatment with phenylethyl isothiocyanate (PEITC). Rats were fed AIN-76A diet or AIN-76A diet containing PEITC for 3 weeks. During the 3rd week of dietary treatment, they were administered three s.c. doses of NMBA (0.5 mg/kg body weight). Rats were sacrificed 24 h after the last treatment; esophagi were excised and processed for histologic grading, microarray and real-time PCR analysis. Histopathologic analysis showed that treatment of rats with PEITC had a protective effect on NMBA-induced preneoplastic lesions in the rat esophagus. We identified 2,261 genes that were differentially expressed in the NMBA-treated versus control esophagi and 1,936 genes in the PEITC + NMBA versus NMBA-treated esophagi. The intersection of these two sets resulted in the identification of 1,323 genes in NMBA-treated esophagus, the vast majority of which were modulated by PEITC to near-normal levels of expression. Measured changes in the expression levels of eight selected genes were validated using real-time PCR. Results from 12 microarrays indicated that PEITC treatment had a genome-wide modulating effect on NMBA-induced gene expression. Samples obtained from animals treated with PEITC alone or cotreated with PEITC + NMBA were more similar to controls than to samples treated with NMBA alone. [Cancer Res 2007;67(13):1–9]

Supplementary Table 1 from Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Supplementary Table 5 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

Supplementary Table 1 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

Supplementary Table 6 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

Supplementary Table 7 from Effects of Phenylethyl Isothiocyanate on Early Molecular Events in N-Nitrosomethylbenzylamine–Induced Cytotoxicity in Rat Esophagus

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]

More than 50% of the world’s population is infected with Helicobacter pylori. H. pylori is the major causative agent of gastric ulcers and gastric cancer. H. pylori eradication using antibiotics either alone or together with a proton pump inhibitor is the primary strategy to decrease the incidence of gastric cancer. Although eradication therapy is effective, there are significant adverse effects and more importantly, resistance to antibiotics occurs, which represents a major therapeutic challenge. Multiple natural products have been shown to suppress H. pylori both in vitro and in animal model systems. However, only a handful of natural products have been evaluated in human clinical trials. The focus of this review is to summarize the results of published human clinical trials to assess the ability of natural products to reduce or eliminate H. pylori infections. Current evidence suggests that these products appear to have great potential to be developed as pharmaceutical candidates for eradication of H. pylori, hopefully both antibiotic-sensitive and antibiotic-resistant strains. Frequent consumption of locally produced foodstuff for controlling H. pylori infection in different countries around the world may well be a feasible long-term solution to fight against this worldwide prevalent pathogen.

Helicobacter pylori infection is commonly treated with a combination of antibiotics and proton pump inhibitors. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed the antibacterial effects of black and red raspberries and blackberries on H. pylori. Freeze-dried powders and organic extracts from black and red raspberries and blackberries were prepared, and high-performance liquid chromatography was used to measure the concentrations of anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, a high-throughput metabolic growth assay based on the Biolog system was developed and validated with the antibiotic metronidazole. Biocompatibility was analyzed using human gastric organoids. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. Antimicrobial activity was higher for extracts than powders and appeared to be independent of the anthocyanin concentration. Importantly, human gastric epithelial cell viability was not negatively impacted by black raspberry extract applied at the concentration required for complete bacterial growth inhibition. Our data suggest that black and red raspberry and blackberry extracts may have potential applications in the treatment and prevention of H. pylori infection but differ widely in their MICs. Moreover, we demonstrate that the Biolog metabolic assay is suitable for high-throughput antimicrobial susceptibility screening of H. pylori.

Helicobacter pylori is an important bacterial pathogen that causes chronic infection of the human stomach, leading to gastritis, peptic ulcer disease and gastric cancer. Treatment with appropriate antibiotics can eliminate H. pylori infection and reduce the risk for severe disease outcomes. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed organic extracts and powders from black and red raspberries and blackberries and determined their antibacterial effects on multiple H. pylori strains. We used high-performance liquid chromatography to measure berry anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, we developed a high-throughput metabolic growth assay based on the OmniLog™ system. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. We next used human gastric epithelial organoids to evaluate biocompatibility of the berry preparations and showed that black raspberry extract, which had the strongest antimicrobial activity, was non-toxic at the concentration required for complete bacterial growth inhibition. To determine whether dietary black raspberry application could eliminate H. pylori infection in vivo, mice were infected with H. pylori and then were placed on a diet containing 10% black raspberry powder. However, this treatment did not significantly impact bacterial infection rates or gastric pathology. In summary, our data indicate that black and red raspberry and blackberry products have potential applications in the treatment and prevention of H. pylori infection, because of their antibacterial effects and good biocompatibility. However, delivery and formulation of berry compounds needs to be optimized to achieve significant antibacterial effects in vivo.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of two subtypes of esophageal cancer, with high incidence and mortality rates in developing countries. OBJECTIVE: The current study investigated the potential chemoprotective effects of strawberries and aspirin against the development of rat esophageal papillomas, the precursors to ESCC. METHODS: Using a prevention model, we administered study diets to rats before, during, and after N-nitrosomethylbenzylamine (NMBA) treatment. The effects of the four diets were evaluated: the control diet, 5% strawberry powder in the control diet, 0.01% aspirin in the drinking water, and the combination of strawberries and aspirin. At week 25, we euthanized all the rats and collected their esophagi to quantify tumor incidence, multiplicity, and burden, as well as for molecular analysis. RESULTS: Both strawberries and aspirin significantly decreased esophageal tumor multiplicity, with the combination causing the most robust suppression. Aspirin alone and the combination decreased the total tumor burden in the esophagus. None of the diets had a significant effect on tumor incidence or the expression of COX-1 and COX-2. Strawberries and aspirin, alone and in combination, significantly suppressed squamous epithelial cell proliferation (PCNA). CONCLUSIONS: Strawberries, aspirin, and their combination exhibit chemoprotective effects against NMBA-induced esophageal tumors in rats.

We previously showed that metabolic activation of the environmental and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) to its active fjord region diol epoxide (DB[a,l]PDE) is required to induce DNA damage, mutagenesis, and squamous cell carcinoma (SCC) in the mouse oral cavity. In contrast to procarcinogens, which were employed previously to induce SCC, DB[a,l]PDE does not require metabolic activation to exert its biological effects, and thus, this study was initiated to examine, for the first time, whether black raspberry powder (BRB) inhibits postmetabolic processes, such as DNA damage, mutagenesis, and tumorigenesis. Prior to long-term chemoprevention studies, we initially examined the effect of BRB (5% added to AIN-93M diet) on DNA damage in B6C3F1 mice using LC/MS-MS and on mutagenesis in the lacI gene in the mouse oral cavity. We showed that BRB inhibited DB[a,l]PDE-induced DNA damage (P < 0.05) and mutagenesis (P = 0.053) in the oral cavity. Tumor incidence in the oral cavity (oral mucosa and tongue) of mice fed diet containing 5% BRB was significantly (P < 0.05) reduced from 93% to 66%. Specifically, the incidence of benign tumor was significantly (P < 0.001) reduced from 90% to 31% (62% to 28% in the oral cavity and 28% to 2% in the tongue), a nonsignificant reduction of malignant tumors from 52% to 45%. Our preclinical findings demonstrate for the first time that the chemopreventive efficacy of BRB can be extended to direct-acting carcinogens that do not require phase I enzymes and is not just limited to procarcinogens. Cancer Prev Res; 11(3); 157–64. ©2017 AACR.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Worldwide, cancers of the oral cavity and pharynx comprise the sixth most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol, and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful, and survival rates have not improved despite advances in therapeutic approaches. Therefore, new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal, several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12-dimethylbenz[a]anthracene in hamster cheek pouch and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N'-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that (1) utilize environmental carcinogens, (2) reflect tumor heterogeneity, and (3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective, we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance the repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

Carcinogenesis is the process whereby a normal cell is transformed into a neoplastic cell. This action involves several steps starting with initiation and followed by promotion and progression. Driving these stages are oxidative stress and inflammation, which in turn encompasses a myriad of aberrant gene expressions, both within the transforming cell population and the cells within the surrounding lesion. Chemoprevention of cancer with bioreactive foods or their extracted/purified components occurs via normalizing these inappropriate gene activities. Various foods/agents have been shown to affect different gene expressions. In this review we discuss whereby the chemoprevention activities of the red beetroot itself may disrupt carcinogenesis and the activities of the water soluble betalains extracted from the plant.

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response—enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704–12. ©2016 AACR.

// Laura A. Kresty 1 , John J. Fromkes 2 , Wendy L. Frankel 3 , Cynthia D. Hammond 2 , Navindra P. Seeram 4 , Maureen Baird 3 , Gary D. Stoner 1 1 Division of Hematology & Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA 2 Department of Internal Medicine, The Ohio State University, Columbus, Ohio, USA 3 Department of Pathology, The Ohio State University, Columbus, Ohio, USA 4 Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island, USA Correspondence to: Laura A. Kresty, email: Lkresty@mcw.edu Keywords: Barrett’s esophagus, black raspberry, esophageal adenocarcinoma, chemoprevention, oxidative stress Received: February 18, 2016 Accepted: May 23, 2016 Published: July 7, 2016 ABSTRACT Black raspberries inhibit a broad range of cancers in preclinical models which has led to clinical evaluations targeting premalignant lesions of the colon, oral cavity and esophagus. A phase I pilot study was conducted in twenty Barrett’s esophagus (BE) patients to investigate the effect of lyophilized black raspberries (LBR) on urinary metabolites and markers of lipid peroxidation, DNA damage and tissue markers of cellular proliferation, detoxification, and inflammation. Surveys, biopsies, blood and urine samples were collected before and after 6 months of LBR treatment (32 or 45 g). LBR significantly reduced urinary excretion of 8-epi-prostaglandin F2α, a marker of lipid peroxidation linked to oxidative stress and free radical damage. Urinary levels of the ellagitannin metabolites, urolithin A-glucuronide, urolithin A-sulfate and dimethylellagic acid glucuronide were significantly increased following 12 and 26 weeks of LBR consumption and may prove useful as indicators of compliance in future clinical studies. Immunohistochemical staining of BE biopsies following LBR treatment showed significant increases in mean GST-pi levels, with 55.6% of subjects responding favorably. In summary, LBR significantly decreased urinary lipid peroxidation levels and significantly increased GST-pi, a marker of detoxification, in BE epithelium. Still, LBR may need to be formulated differently, administered at higher concentrations or multiple times a day to increase efficacy.

This study was a 14-day, outpatient, open-label randomized crossover trial of lyophilized black raspberries (BRBs) in older overweight or obese males to determine whether BRB consumption affects postprandial inflammation associated with consumption of a high-fat high-calorie (HFHC) meal. Ten study participants consumed 45 g/d of lyophilized BRBs for 4 days, followed by a HFHC breakfast plus BRBs on day 6 or consumed the HFHC breakfast on day 6 without previous consumption of BRBs and then crossed over to the other treatment after a 2-day washout period. Blood samples were obtained before and 1, 2, 4, 8, and 12 hours after consumption of the HFHC breakfast. The primary study outcomes were changes in area under the concentration-time curve (AUC) for interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α). The secondary outcomes were safety and tolerability of lyophilized BRB powder. The chronology and values of measured serum concentrations for IL-6, TNF-α, and CRP were consistent with those described previously by other investigators. The AUC of serum IL-6 was lowered significantly (P = 0.03, n = 10) with BRB consumption (34.3 ± 7.6 pg·mL⁻¹·h⁻¹ compared with 42.4 ± 17.9 pg·mL⁻¹·h⁻¹ for consumption of the HFHC meal alone). However, no significant differences (change in AUC) were calculated for serum CRP and TNF-α. The findings of this pilot study suggest that consumption of lyophilized BRBs may attenuate postprandial inflammation in overweight or obese males consuming a HFHC meal. Further investigation of BRBs is warranted to better elucidate their inflammomodulatory potential.

Freeze-dried black raspberries (BRB), their component anthocyanins (AC), and a metabolite of BRB ACs, protocatechuic acid (PCA), inhibit the development of esophageal cancer in rats induced by the carcinogen, N-nitrosomethylbenzylamine (NMBA). All three components reduce inflammation in the esophagus and in plasma. The present study determined the relation of changes in inflammatory markers to infiltration of innate immune cells into NMBA-treated esophagus. Rats were injected with NMBA (0.35 mg/kg) for 5 weeks while on control diet. Following NMBA treatment, rats were fed diets containing 6.1% BRB powder, an AC-rich fraction of BRBs (3.8 μmol/g), or 500 ppm PCA. At weeks 15, 25, and 35, inflammatory biomarker expression in the plasma and esophagus was quantified, and infiltration of immune cells in the esophagus was examined. At all three time points, BRB, AC, and PCA similarly affected cytokine production in the esophagus and plasma of NMBA-treated rats, relative to the NMBA-only control. These included decreased expression of the proinflammatory cytokine IL1β and increased expression of the anti-inflammatory cytokine IL10. Moreover, all three diets also increased the expression of IL12, a cytokine that activates both cytolytic natural killer and CD8+ T cells. In addition, the three diets also decreased infiltration of both macrophages and neutrophils into the esophagus. Overall, our results suggest that another mechanism by which BRBs, ACs, and PCA inhibit NMBA-induced esophageal tumorigenesis is by altering cytokine expression and innate immune cell trafficking into tumor tissues. Cancer Immunol Res; 4(1); 72–82. ©2015 AACR.

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 β-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.