N-nitrosomethylbenzylamine Research Articles

Dietary Consumption of Black Raspberries or Their Anthocyanin Constituents Alters Innate Immune Cell Trafficking in Esophageal Cancer.

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.

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

Aberrant methylation of DNA is a common event in the development of cancers, including squamous cell carcinoma (SCC) of the human esophagus. In the present study, we determined: (a) whether aberrant DNA methylation also occurs in the development of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus, a model of human esophageal SCC; and (b) if so, whether dietary black raspberries (BRBs) are capable of preventing this aberrant DNA methylation. A diet containing 5% BRBs inhibited the development of NMBA-induced tumors in the rat esophagus. This inhibition was associated with reduced mRNA levels of the DNA methyltransferases, Dnmt1 and Dnmt3b, in both dysplastic lesions and in papillomas of the esophagus. In addition, promoter methylation of Sfrp4, a WNT pathway antagonist, was significantly reduced by the berry diet, and this was associated with decreased nuclear localization of 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.

Regulation of α-tocopherol on NFκB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine⁃induced human esophageal cell carcinogenesis

To investigate the regulation of α-Tocopherol on NFκB and Nrf2 signaling pathway at early stage of N-nitrosomethylbenzylamine (NMBzA)-induced human esophageal carcinogenesis. Human normal esophageal HET-1A cells were treated with NMBzA at 50 µmol/L, 100 µmol/L for 24 h to intimate the initiation of esophageal carcinogenesis. For intervention groups, HET-1A cells were pre-treated with α-T at 25, 50, 100 µmol/L for 3 h and then co-treated with NMBzA (100 µmol/L) for 24 h. In comparison with HET-1A cells, human esophageal cancer EC109 cells were treated with α-T at corresponding concentrations. Cells treated with 0.1% DMSO were used as negative control. Immunofluorence staining was used for the determination of distribution and activation of NFκB p65 and Nrf2 in the cell. Real time PCR and Western blot were used to determine the expression levels of target genes including cyclinD1, KI67, proliferating cell nuclear antigen (PCNA), cyclo-oxygen-ase 2 (COX2), 5LOX, HO-1, NQO1 and GCLC. Flow cytometry was utilized to analyze the reactive oxygen species contents in the cells. As compared to the control group (1.00 ± 0.08), the expression of CyclinD1 (2.99 ± 0.15), KI67 (2.35 ± 0.38) and PCNA (2.46 ± 0.25) in HET-1A were all markedly increased by NMBzA treatment (F values were 97.23, 65.28, 34.62, P < 0.001). Also, the proportion of cells with nucleus translocation of NFκB p65 (71.0%, 98/138) or Nrf2 (36.3%, 49/135) were significantly increased (χ² values were 194.71, 133.72, P < 0.001), and the expression of COX2 (3.22 ± 0.17), 5LOX (2.87 ± 0.12) as well as HO-1 (1.87 ± 0.22), NQO1 (2.14 ± 0.08), GCLC (2.63 ± 0.41) at protein levels were elevated (F values were 72.35, 43.87, 69.23, 71.34, 85.79, P values were 0.013, 0.015, 0.010, 0.011, 0.002). Under the treatment with 50 µmol/L α-T, comparing with the control group(59.1%,65/110),the nuclear translocation of NFκB p65 (77.7%, 8/104) was clearly inhibited (χ² = 148.1, P < 0.001), and protein expression levels of COX2 (0.74 ± 0.19) and 5LOX (0.42 ± 0.13) were decreased (F values were 56.31, 73.25, P values were 0.003, 0.001). However, no changes on Nrf2 signaling pathway were observed; α-T showed little impact on NFκB or Nrf2 pathway in EC109 cells. At the early stage of NMBz-induced esophageal cancer, α-T could block the initiation of carcinogenesis through suppressing the activation of NFκB signaling pathway. It might be the major mechanism by which α-T is potentially chemopreventive to esophageal cancer. During the progression of esophageal cancer, the cells may acquire the adaptive functions to accommodate oxidative stress via activating Nrf2 pathway.

FBXO4 loss facilitates carcinogen induced papilloma development in mice

Cyclin D1 is frequently overexpressed in esophageal squamous cell carcinoma (ESCC) and is considered a key driver of this disease. Mutations in FBXO4, F-box specificity factor that directs SCF-mediated ubiquitylation of cyclin D1, occur in ESCC with concurrent overexpression of cyclin D1 suggesting a potential tumor suppressor role for FBXO4. To evaluate the contribution of FBXO4-dependent regulation cyclin D1 in esophageal squamous cell homeostasis, we exposed FBXO4 knockout mice to N-nitrosomethylbenzylamine (NMBA), an esophageal carcinogen. Our results revealed that loss of FBXO4 function facilitates NMBA induced papillomas in FBXO4 het (+/−) and null (−/−) mice both by numbers and sizes 11 months after single dose NMBA treatment at 2mg/kg by gavage when compared to that in wt (+/+) mice (P < 0.01). No significant difference was noted between heterozygous or nullizygous mice consistent with previous work. To assess cyclin D1/CDK4 dependence, mice were treated with the CDK4/6 specific inhibitor, PD0332991, for 4 weeks. PD0332991 treatment (150mg/kg daily), reduced tumor size and tumor number. Collectively, our data support a role for FBXO4 as a suppressor of esophageal tumorigenesis.

Abstract 1247: Assessment of the roles of prostaglandin E2 receptors in a rodent model of chemically induced esophageal carcinogenesis

Abstract Esophageal carcinogenesis is a multistage process characterized by morphological changes from normal esophagus to basal cell hyperplasia, dysplasia, carcinoma in situ and squamous cell carcinoma (SCC). Our laboratory has used a rodent model of N-nitrosomethylbenzylamine (NMBA)-induced esophageal SCC to investigate molecular carcinogenesis and identify putative chemopreventive agents. In our previous studies, we found that the expression of cyclooxygenase-2 and the production of prostaglandin E2 (PGE2) in rat esophageal epithelium were elevated in papillomatous lesions relative to precancerous and normal tissues. PGE2 exerts its cellular effect by binding to its cognate receptors. There are four subtypes of PGE2 receptors, EP1, EP2, EP3 and EP4, which are involved in carcinogenesis depending on the cell or tissue types. The overall objective of the current study was to assess the roles of PGE2 receptors in NMBA-induced rat esophageal tumorigenesis. F344 rats were injected subcutaneously with NMBA (0.30 mg/kg b.w.) three times per week for five weeks. One week after NMBA treatment, animals were randomized into five experimental groups and fed 300 ppm celcecoxib plus 50 ppm S,S'-1,4-phenylenebis(1,2-ethanediyl)bisisothiourea (PBIT) starting from week 6, 9, 12 , or 15, respectively, or 5% freeze-dried black raspberries (BRB) starting from week 6. Esophagi were collected at 29 weeks. Results of real-time PCR, western blot analysis and immunohistochemistry demonstrated a correlation between the upregulation of EP1, EP2, EP3 and EP4 and neoplastic progression in the rat esophagus. The expression of the PGE2 receptors in preneoplastic tissues and papillomas was significantly elevated when compared to normal tissues. Moreover, we observed that celecoxib + PBIT and 5% BRB significantly reduced EP2 expression. Our data suggest, therefore, that the production of PGE2 receptors by the esophageal epithelium is associated with the development of NMBA-induced esophageal tumorigenesis in rats, and EP2 antagonist may have chemopreventive potential in esophageal cancer. Research efforts are needed to elucidate the function of PGE2 receptors and related oncogenic pathways in esophageal SCC. (Supported by NIH/NCI R01 CA131073-01A1). Citation Format: Ni Shi, Tong Chen. Assessment of the roles of prostaglandin E2 receptors in a rodent model of chemically induced esophageal carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1247. doi:10.1158/1538-7445.AM2014-1247

Abstract 1253: Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells

Abstract The long-term goal of our research is to investigate the molecular carcinogenesis of esophageal squamous cell carcinoma and to develop strategies for esophageal cancer prevention. Previous studies in our laboratory demonstrated an association between activation of PI3K/AKT/mTOR pathways and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. The objective of current study is to evaluate the effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, and MK2206, an AKT inhibitor, alone or in combination in esophageal cancer cell lines, KYSE-70, -150, -270 and -410. The cancer cell lines were treated with different doses of NVP-BEZ235 (1, 10, 50, 100, 1000 nMol/L) and MK2206 (0.5, 1, 3, 10, 20 uMol/L), or NVP-BEZ235 100 nMol/L plus MK2206 3 uMol/L. Western blot was performed to assess the activation of PI3K/AKT/mTOR pathways. Cell proliferation, apoptosis and cell cycle were analyzed by WST1 and flow cytometry. We found that the combination of NVP-BEZ235 and MK2206 significantly reduced the phosphorylation of p-AKTT308, p-AKTS473, p-mTORS2448, p-S6S235/236 and p-4EBP1T37/46, when compared to NVP-BEZ235 (1000 nMol/L) and MK2206 (20 uMol/L) alone (p&amp;lt;0.05). The cell proliferation rate was reduced by NVP-BEZ235 1000 nMol/L, MK2206 20 uMol/L, or NVP-BEZ235 plus MK2206 to 20% (p&amp;lt;0.01), 24% (p&amp;lt;0.01) and 17% (p&amp;lt;0.01), respectively. The NVP-BEZ235 1000 nMol/L and MK2206 10 uMol/L induced apoptosis in esophageal cancer cells to 30% and 20% (p&amp;lt;0.01), respectively. NVP-BEZ235 plus MK2206 induced apoptosis in esophageal cancer cells to 45% (p&amp;lt;0.01). The combination treatment increased the proportion of cells at G0/G1 phase compared to cells treated with NVP-BEZ235 or MK2206 alone. Our results indicate that co-targeting the PI3K/mTOR and AKT signaling pathways produces synergistic activity against esophageal carcinoma cell growth. This in-vitro study provides evidence to test the efficacy of NVP-BEZ235 in combination with MK2206 in inhibition of esophageal tumor development in vivo. (Supported by NIH/NCI R01 CA131073-01A1). Citation Format: Hao Yu, Ni Shi, Zui Pan, Tong Chen. Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2014-1253

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

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

Abstract 3686: Microarray analysis of dynamic regulation of genes in early and late stages of chemically induced esophageal carcinogenesis in rats.

Abstract Esophageal carcinogenesis is a multistage process characterized by morphological changes from normal esophagus to basal cell hyperplasia, dysplasia, carcinoma in situ and squamous cell carcinoma (SCC). Our laboratory has used the model of N-nitrosomethylbenzylamine (NMBA)-induced esophageal SCC in rats to investigate carcinogenesis and identify putative chemopreventive agents for this disease. The current study is part of large scale investigations on carcinogenesis of esophageal SCC. F344 rats were treated with NMBA (0.30 mg/kg b.w.) or a solution of 20% dimethyl sulfoxide (DMSO) in water (the solvent for NMBA) three times per week for five weeks. The esophageal tissues were collected at 6 and 29 weeks. The RNA pools from 3 individual RNA samples each were prepared for microarray analysis using Affymetrix Rat Genome 230 2.0 Array. Triplicate microarrays were completed for total 27 rats/9 microarrays. A P-value cutoff of 0.001 with a minimum 2.0-fold change was used to define the differently expressed genes. Ingenuity Pathway Analysis (IPA) software was used to identify cancer-related pathways and cellular functions. At 6 weeks (tumor incidence = 20%), 173 genes were modulated by NMBA when compared to control animals. Among which, 20 genes were associated with cancer, cellular development, cellular assembly and organization network; 34 genes were associated with inflammatory disease. The top five cellular functions altered by NMBA were cellular development, carbohydrate metabolism, cell death and survival, cellular growth and proliferation, and cellular function and maintenance. At 29 weeks (tumor incidence = 100%), we identified 1628 genes which were significantly up- or down-regulated by NMBA treatment when compared to normal animals and among which, 466 genes were involved in tumor development. In addition, we identified four cancer-related pathways (nuclear factor erythroid 2-related factor 2 mediated oxidative stress response, protein kinase A signaling, mitochondrial dysfunction, and AMP-activated kinase signaling) and five cellular functions (cellular development, cell morphology, cell death and survival, carbohydrate metabolism and molecular transport). Among genes altered during NMBA-induced esophageal carcinogenesis, 16 genes differently expressed only at 6 weeks including Gbp2, Psmb, Psmb9, Lgals3bp, Id1, Igfbp3, Agpat4, rCG_35099, Tes, RGD1563091, Cyp26b1, Itga1, Scarb1, LOC682861, Serpinb2 and Slfn3. The fold changes of Plcd4, Hbb, Bmper, Hba-a2, Defb4, LOC100134871 and LOC364653 increased from 6 weeks to 29 weeks. In conclusion, we identified differently expressed genes in early and late stages of NMBA-induced esophageal SCC by microarray analysis. Further investigations on biomarkers and cellular/molecular events during multistage of carcinogenesis in NMBA-rat preclinical model of esophageal SCC are underway. Citation Format: Ni Shi, Ronald G. Nines, Lianbo Yu, Michael J. Nicholl, Tong Chen. Microarray analysis of dynamic regulation of genes in early and late stages of chemically induced esophageal carcinogenesis in rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2013-3686

The enhancing effect of ethanol on the mutagenic activation of N-nitrosomethylbenzylamine by cytochrome P450 2A in the rat oesophagus

Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B(1) or other N-nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Abstract A49: Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins

Abstract Background: We have shown that black raspberries (BRB) as well as their component anthocyanins (AC) inhibit N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. Like many polyphenolic compounds, the bioavailability of BRB anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to produce protocatechuic acid (PCA). PCA has been shown to be bioavailable and also to be chemopreventive. Furthermore, we have shown that BRB and their anthocyanins decrease methylation and increase tumor suppressor gene (TSG) expression; e.g., SFRP and WIF1, Wnt pathway antagonists, as well as p16 and PAX6a, in human colon tumors. The current study was designed to determine if PCA is effective in preventing NMBA-induced rat esophageal tumorigenesis by reducing DNA methylation and increasing expression of TSG(s). Materials and Methods: Male Fischer 344 rats aged 3-5 weeks were randomized into five groups and injected s.c. with either the vehicle (20% DMSO/water) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. During this time all rats were fed control diet (AIN-76A). At the beginning of week 6, all rats were started on their study diets (Group 1: vehicle + AIN-76A, Group 2: NMBA + AIN-76A, Group 3: NMBA + 3.8 umoles AC/g of AIN-76A, Group 4: NMBA+ 6% BRB in AIN-76A, Group 5: NMBA+ 500 ppm PCA in AIN-76A). The concentration of 3.8 umole AC/g of diet for Group 3 was chosen to compare with past studies, 6% BRB were used to match the anthocyanin content of Group 3, and 500 ppm PCA is the estimated amount of anthocyanins converted to PCA from 3.8 umole AC/g diet in the gut. A total of 9 animals per group were harvested at weeks 15 and 25, and 30 rats per group will be harvested at week 35. Esophagi were harvested and esophageal tumors counted and sized. After counting the tumors, one half of the esophagus was used for histological analysis and the other half for DNA/RNA extraction to investigate relevant TSG expression and methylation. Results: The study is still on going. H&amp;E slide scoring revealed significantly reduced hyperplasia in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group at week 15. At week 25, the test diets had no effect on dysplasia frequency, however, all test diets reduced tumor incidence. NMBA treatment led to an increase in dimethyltransferase 3b (DNMT3b) mRNA expression at week 15 and week 25, and 500 ppm PCA decreased DNMT3b expression at both time points. NMBA treatment caused increased methylation and down-regulated mRNA expression of SFRP4 when compared to the vehicle control group at week 25 but not at week 15. None of the test diets influenced DNA methylation or mRNA expression of SFRP4 in NMBA-treated rats at either week 15 or 25. No differences in methylation status of PTX3 (Pentraxin 3), a cytokine released by phagocytic and dendritic cells and shown to be methylated in human esophageal cancer, was observed across all groups at either time point. Conclusion: These results suggest for the first time that PCA may be protective against NMBA-induced esophageal tumorigenesis in rats. PCA significantly reduced tumor incidence, similar to that of BRB and their anthocyanins, by preventing the conversion of dysplastic lesions into papillomas. Preliminary evidence suggests that PCA influences the expression of a key methyltransferase (DNMT3b) in NMBA-treated rat esophagus. Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. Chemoprevention of esophageal cancer using PCA, a metabolite of black raspberry-derived anthocyanins. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A49.

Abstract B26: The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats

Abstract Background: Black raspberries (BRB) and their component anthocyanins (AC) inhibit esophageal cancer in rats induced by N-nitrosomethylbenzylamine (NMBA). Previous studies indicate that the bioavailability of the anthocyanins is low. The majority of BRB anthocyanins are metabolized by gut bacteria to protocatechuic acid (PCA). PCA has greater bioavailability than the AC and is chemopreventive in several organ sites. The current study was undertaken to determine if PCA is also effective against NMBA-induced esophageal cancer and if it influences inflammatory cytokine expression. Cytokines were measured using a simultaneous quantification bead assay. The pro-inflammatory cytokines measured were IL-1 β, IL-5, IL-7, erythropoietin (EPO), TNF- α, &amp; interferon-gamma (IFN- γ), and the anti-inflammatory cytokines were IL-10 and T cell-derived IL-2 and the chemokines CCL5 &amp; CCL20. Methods and Materials: Male Fischer 344 rats aged 3-5 weeks were randomized into groups by body weight &amp; age. Rats were given s.c. injections of either the vehicle (20% DMSO/H2O) or NMBA (0.35 mg/kg b.w.) three times a week for five weeks. All rats were fed AIN-76A control diet during injections. Starting at week 6, groups of rats were fed experimental diets. These include Group 1 (DMSO + AIN-76A diet), Group 2 (NMBA + AIN-76A), Group 3 (NMBA + 3.8 umoles AC/ g AIN-76A), Group 4 (NMBA + 6% BRB in AIN-76A), &amp; Group 5 (NMBA + 500 ppm PCA in AIN-76A). To match previous studies, the Group 3 diet contained 3.8 umoles of AC/g diet. A 6% BRB diet for Group 4 matched the anthocyanin content of Group 3, &amp; the Group 5 diet contained 500 ppm PCA to match the estimated amount of Group 3 AC converted to PCA in the gut. 9 animals from each group were sacrificed at weeks 15 &amp; 25, and 30 will be sacrificed at week 35. The esophagus was taken for tumor counts &amp; used for H&amp;E histological scoring. Rat blood was collected in heparin coated tubes, centrifuged, &amp; stored at −80°C. Cytokines and chemokines were quantified using a bead array. Results: The study is ongoing. At week 15 H&amp;E slide scoring revealed hyperplasia was significantly reduced in all test diet groups (BRB, AC, PCA) when compared to the NMBA control group. No tumors were present at week 15 in any of the NMBA-treated rats. At week 25 there were significant decreases in tumor count, hyperplasia, &amp; dysplasia in Groups 3-5 when compared to Group 2. Preliminary results showed that pro-inflammatory IL-1 β expression was significantly higher in Group 2 when compared to Groups 1, 4, &amp; 5 at week 25. Anti-inflammatory IL-10 expression was decreased in all NMBA treated groups at weeks 15 &amp; 25. At week 15, T-cell derived IL-2 expression was significantly lower in Groups 1, 4, &amp;5 when compared to Group 2, whereas at week 25, IL-2 expression was significantly lower only in Groups 1 &amp; 5 when compared to Group 2. Conclusions: PCA is chemopreventive against NMBA-induced esophageal tumorigenesis in rats. Similar to BRB and their AC, PCA significantly reduced tumor incidence and the conversion of dysplastic lesions to papillomas. Cytokine quantification assays suggest that PCA, like BRB and AC, significantly influence the expression levels of certain proinflammatory and anti-inflammatory cytokines in a protective manner. Further observations will be presented at the meeting. Citation Format: Dan Peiffer, Jibran H. Siddiqui, Chieh-Ti Kuo, Yi-Wen Huang, Noah P. Zimmerman, Li-Shu Wang, Gary D. Stoner, Steven G. Carmella, Ben Ransom, Stephen S. Hecht. The anti-inflammatory effects of black raspberries and the anthocyanin metabolite protocatechuic acid in N-nitrosomethylbenzylamine-induced esophageal cancer in rats. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B26.

Tolfenamic acid suppresses cytochrome P450 2E1 expression in mouse liver

Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg(-1), 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 μM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.

Inhibitory effects of lupeal acetate of Cortex periplocae on N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis

Lupeal acetate of Cortex periplocae (CPLA), a triterpene compound extracted from a traditional Chinese herb, has been identified as an inhibitor of cancer cell growth. The objective of the present study was to evaluate the potential mechanisms through which CPLA inhibits N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. We treated F344 rats subcutaneously with the esophageal carcinogen NMBA (0.5 mg/kg body weight) and intramuscularly with CPLA (20 mg/kg), 3 times a week for 5 weeks. Rats were then sacrificed at weeks 9, 15 or 25, esophageal tissues were collected and tumor data were recorded. To investigate the mechanisms by which CPLA modulates tumorigenesis in esophagus, we evaluated the protein expression of glycogen synthase kinase-3β (GSK-3β) and β-catenin and the gene expression of c-myc. CPLA significantly (P<0.05) reduced the incidence of esophageal tumors observed at 25 weeks from 93.3% in NMBA-treated controls to 33.3% in the NMBA- and CPLA-treated rats. CPLA reduced β-catenin and c-myc expression, but increased GSK-3β expression, in preneoplastic lesions of the esophagus. These results suggest a novel tumor-suppressive role of CPLA through the activation of GSK-3β expression and the inhibition of β-catenin and c-myc expression. Therefore, CPLA is a potential therapeutic candidate for esophageal squamous cell carcinoma.

Abstract 1623: Epigenetic regulation of tumor suppressor genes in esophageal cancer by berries

Abstract Squamous cell carcinoma (SCC) is one of two major types of esophageal cancer. Major etiological factors for this disease include the use of tobacco and alcohol, and the consumption of salt-cured and moldy foods that are frequently contaminated with N-nitrosamine carcinogens. Using a rat model of esophageal SCC, we have demonstrated the ability of freeze-dried black raspberries (BRBs) to inhibit tumors induced with the nitrosamine carcinogen, N-nitrosomethylbenzylamine (NMBA). BRBs inhibit the progression of NMBA-induced dysplastic lesions to papillomas and carcinomas by reducing cell proliferation, inflammation and angiogenesis and promoting apoptosis and cell differentiation. However, there is no information regarding the potential effects of BRBs on epigenetic events in NMBA-induced rat esophageal tumorigenesis. F344 male rats were given s.c. injections of NMBA, at 0.3 mg/kg, 3 times/wk for 5 wks, and their esophagi harvested at 9, 15, 17 and 35 wks. Histopathological studies revealed the presence of small dysplastic esophageal lesions at 17 wks but not at 15 wks. Immunohistochemical analysis of Ki-67 stained esophageal epithelial cells indicated that cell proliferation at 15 wks was significantly higher than at 9 wks. Genome-wide microarray analysis indicated that more genes were downregulated than upregulated by NMBA (n=153 vs. 69) in papillomas at 35 wks when compared to whole esophagus at 9 wks. Some of these genes such as Sfrp4, a soluble frizzled-related protein, are associated with Wnt signaling and cell proliferation. To investigate whether BRBs elicit effects on DNA methylation of putative tumor suppressor genes, genome-wide methylation analysis was applied using normal esophageal tissues and papillomas collected at the end of the bioassay (35 wks). BRBs significantly demethylated the promoter CpG islands of 81 genes, some of which are involved in the regulation of important cellular functions such as proliferation and Wnt signaling. Included among these is Sfrp4; NMBA treatment induced its promoter methylation in both preneoplastic lesions (a mixture of normal, hyperplastic and dysplastic epithelium) and in papillomas and reduced its mRNA expression. In contrast, BRBs decreased promoter methylation and increased mRNA expression of Sfrp4 in preneoplastic lesions and in papillomas, suggesting that BRBs might reactivate tumor suppressor genes silenced by methylation. Dnmt3b mRNA expression was induced in both preneoplastic lesions and in papillomas of NMBA-treated rat esophagus suggesting that Dnmt3b could be a major contributor to promoter hypermethylation in NMBA-treated tissues. In conclusion, DNA methylation of tumor suppressor genes appears to contribute to the development of NMBA-induced rat esophageal tumorigenesis and another mechanism for the protective effects of BRBs may be to reduce DNA methylation. This research was supported, in part, by NCI grant R01 CA103180 to GDS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1623. doi:1538-7445.AM2012-1623

Abstract 1624: Black raspberries suppress phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in carcinogen-induced esophageal cancer in rats

Abstract Esophageal squamous cell carcinoma (SCC) is one of the most common malignant neoplasms worldwide. The overall 5-year survival rate of esophageal SCC in the United States is only 13%, which is close to the observed rates in high-risk countries including China and other global regions. To decrease the incidence of esophageal cancer, cancer chemoprevention through dietary and/or chemical intervention would be a logical and practical approach. Our laboratory and others have used the N-nitrosomethylbenzylamine (NMBA)-induced rat preclinical model of esophageal cancer to investigate the mechanisms of esophageal carcinogenesis and to evaluate the efficacy of potential chemopreventive agents. Studies found that dietary freeze-dried black raspberries (BRBs) significantly inhibited NMBA-induced tumor development in rat esophagus, at least in part by inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)), c-Jun, vascular endothelial growth factor (VEGF), and activation of mitogen-activated protein kinase (MAPK) and nuclear factorβB (NFκB) pathways. The phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays important roles in certain aspects of carcinogenesis. The principal objective of this study is to assess the roles of this signaling cascade in preclinical esophageal cancer model and examine the mechanistic actions of BRBs against this disease. In the present study, F344 rats were treated with NMBA (0.30 mg/kg b.w.) three times per week for 5 weeks. After 72 hours, animals were fed AIN-76A diet or AIN-76A diet containing 5% BRBs. At week 29, rats were sacrificed and esophageal tumors were counted. Western blot assays were conducted to detect the expression levels of some proteins involved in PI3K/AKT/mTOR pathway. Our data show that in esophageal preneoplastic lesions, BRBs significantly decreased expression levels of p-AKTSer478, p-PI3K-p83, p-S6 and p-mTORSer2448 from 3.2-fold, 5.9-fold, 5.4-fold and 4.4-fold in rats fed control diet to 0.9-fold (P &amp;lt; 0.005), 1.1-fold (P &amp;lt; 0.001), 1.2-fold (P &amp;lt; 0.001) and 1.3-fold (P &amp;lt; 0.001), in rats fed 5% BRBs, respectively. In papillomatous lesions, the expression levels of the above proteins were also reduced by BRBs from 5.3-fold to 1.5-fold (p-AKTSer478; P &amp;lt; 0.001), 6.7-fold to 1.7-fold (p-PI3K-p83, P &amp;lt; 0.001), 8.4-fold to 1.5-fold (p-S6, P &amp;lt; 0.001) and 7.9-fold to 1.7-fold (p-mTORSer2448, P &amp;lt; 0.001). Our results indicated that the PI3K/AKT/mTOR signaling pathway was activated in NMBA-induced tumor development in rats and the activation was significantly suppressed by BRBs. Further investigation of this signaling cascade in esophageal carcinogenesis is underway in our laboratory. (Supported by NCI RO1 CA131073-01A1). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1624. doi:1538-7445.AM2012-1624

Dietary zinc deficiency fuels esophageal cancer development by inducing a distinct inflammatory signature

Chronic inflammation is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC). The causes of inflammation in ESCC, however, are undefined. Dietary zinc (Zn)-deficiency (ZD) increases the risk of ESCC. We have previously shown that short-term ZD (6 weeks) in rats induces overexpression of the proinflammatory mediators S100a8 and S100a9 in the esophageal mucosa with accompanying esophageal epithelial hyperplasia. Here we report that prolonged ZD (21 weeks) in rats amplified this inflammation that when combined with non-carcinogenic low doses of the environmental carcinogen, N-nitrosomethylbenzylamine (NMBA) elicited a 66.7% (16/24) incidence of ESCC. With Zn-sufficiency, NMBA produced no cancers (0/21) (P<0.001). At tumor endpoint, the neoplastic ZD esophagus, as compared with Zn-sufficient esophagus, had an inflammatory gene signature with upregulation of numerous cancer-related inflammation genes (CXC and CC chemokines, chemokine receptors, cytokines and Cox-2) in addition to S100a8 and S100a9. This signature was already activated in the earlier dysplastic stage. Additionally, time-course bioinformatics analysis of expression profiles at tumor endpoint and before NMBA exposure revealed that this sustained inflammation was due to ZD rather than carcinogen exposure. Importantly, Zn replenishment reversed this inflammatory signature at both the dysplastic and neoplastic stages of ESCC development, and prevented cancer formation. Thus, the molecular definition of ZD-induced inflammation as a critical factor in ESCC development has important clinical implications with regard to development and prevention of this deadly disease.

Relevance of Drug Metabolizing Enzyme Activity Modulation by Tea Polyphenols in the Inhibition of Esophageal Tumorigenesis

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

Abstract 819: Black raspberries inhibit N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis through down-regulation of MAPK and NFκB pathways

Abstract Esophageal cancer is the 6th most common cancer worldwide. Our laboratory has used a rodent preclinical model of esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In our previous studies, we found that freeze-dried black raspberry (BRB) significantly suppressed N-nitrosomethylbenzylamine (NMBA)-induced tumor development in rat esophagus, at least in part by inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), c-Jun and vascular endothelial growth factor (VEGF). The mitogen-activated protein kinase (MAPK) and nuclear factor κB (NFκB) pathways play important roles in certain aspects of carcinogenesis. However, their roles and effects of BRB on these two pathways in the NMBA model of rat esophageal tumorigenesis have not been investigated yet. In the present study, F344 rats were treated with NMBA (0.30 mg/kg b.w.) three times per week for 5 weeks. After 72 hours, animals were fed AIN-76A diet or AIN-76A diet containing 5% BRB. At week 35, rats were sacrificed and esophageal tumors were counted. The incidence of esophageal tumors was decreased from 100% in NMBA-treated rats to 81.48% (P &amp;lt; 0.05) in rats treated with NMBA+5% BRB. The tumor multiplicity was reduced in rats fed 5% BRB to an average of 1.44 ± 0.25 (P &amp;lt; 0.001) tumors per rat compared with 4.73 ± 2.70 in rats fed the control diet. In preneoplastic lesions, BRB decreased protein expression of phospho-NFκB-p65, phospho-IkBα, phospho-MAPK-p38, phospho-MAPK-p44/42 and phosphor-SAPK/JNK from 4.2-fold, 3.9-fold, 3.1-fold, 4.7-fold and 5.5-fold in rats fed the control diet to 1.2-fold (P &amp;lt; 0.005), 1.3-fold (P &amp;lt; 0.005), 1.3-fold (P &amp;lt; 0.005), 1.4-fold (P &amp;lt; 0.005) and 1.4-fold (P &amp;lt; 0.005), respectively, in rats fed 5% BRB. In papillomatous lesions, BRB reduced protein expression of phospho-NFκB-p65, phospho-IkBα, phospho-MAPK-p38, phospho-MAPK-p44/42 and phosphor-SAPK/JNK from 5.9-fold, 4.7-fold, 5.7-fold, 5.9-fold and 6.1-fold in rats fed the control diet to 1.3-fold (P &amp;lt; 0.005), 1.5-fold (P &amp;lt; 0.005), 1.6-fold (P &amp;lt; 0.005), 1.5-fold (P &amp;lt; 0.005) and 1.6-fold (P &amp;lt; 0.005), respectively, in rats fed 5% BRB. Our results indicated that both MAPK and NFκB pathways were activated in NMBA-induced tumor development in rats and these activations were significantly inhibited by BRB. Collectively, our data suggest that BRB may offer a relatively nontoxic alternative to the prevention of esophageal cancer in humans. (Supported by NCI RO1 CA131073-01A1). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 819. doi:10.1158/1538-7445.AM2011-819

Abstract 811: Co-inhibition of cyclooxygenase-2 and inducible nitric oxide synthase prevents N-nitrosomethylbenzylamine-induced esophageal squamous cell carcinoma in rats

Abstract Hyperactivation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) has been observed separately in esophageal squamous cell carcinoma (ESCC) in rats that is induced by N-nitrosomethylbenzylamine (NMBA), a powerful carcinogen found in some human diets. In approach to discover new prevention strategies for ESCC, celecoxib and S,S′-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), selective inhibitors respectively to COX-2 and iNOS, were evaluated in the NMBA-rat preclinical esophageal cancer model. F344 rats were treated with NMBA (0.30mg/kg b.w.) three times per week for five weeks. Seventy-two hours after the last NMBA treatment, rats were fed with control diet with or without different doses of celecoxib and PBIT. At week 35, rats were sacrificed and esophageal tumors were counted. Celecoxib (1000 ppm) or PBIT (100 ppm) alone, and a combination treatment with celecoxib (500 ppm) and PBIT (50 ppm), significantly decreased the NMBA-induced tumor multiplicity (from 4.73 ± 0.45 to 2.00 ± 0.25, 1.89 ± 0.23, and 1.61 ± 0.27, respectively), which were accompanied by the inhibition of transcription, translation and kinase activity of both COX-2 and iNOS in all these treatments. By using this COX-2/iNOS co-inhibition strategy, celecoxib/PBIT combination inhibited cancerous esophagus cell proliferation by immunohistochemistry staining of PCNA, and decreased c-Myc expression in NMBA-induced papillomas by Western blot analysis. In addition, the histopathological examination of rat esophagus showed that celecoxib/PBIT combination significantly prevented NMBA-induced tumor transformation (from 8.373% to 1.765%). In conclusion, our results indicated that co-inhibition of COX-2 and iNOS by Celecoxib/PBIT combination prevented NMBA-induced ESCC in rats, which represents a new means of esophageal cancer prevention. (Supported by NIH/NCI RO1 CA131073-01A1). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2011-811

Abstract LB-465: Investigation on chemopreventive effect of lyophilized strawberries in human subjects with precancerous lesions in esophagus: a phase 1b study in China

Abstract Esophageal squamous cell carcinoma (SCC) is the third most common gastrointestinal malignancy and the sixth most frequent cause of cancer death in the world. Nitrosamine carcinogens in tobacco smoke, in the diet, and those produced in the acidic conditions of the stomach, appear to be important causative agents of esophageal SCC. Among these is N-Nitrosomethylbenzylamine (NMBA), present in the diet in China, and likely the most potent nitrosamine carcinogen for the rat esophagus. Our laboratory has used the NMBA-induced esophageal cancer model in rats to identify putative chemopreventive agents, including natural food products, to prevent this disease. Dietary freeze-dried strawberries were found to significantly inhibit tumor development in the rat esophagus by inhibiting the metabolism of NMBA to DNA damaging species and by reducing the growth rate of premalignant cells. Based on these observations, we conducted a Phase I trial in China to investigate the effects of freeze-dried strawberries in a cohort of adult individuals with esophageal dysplastic lesions in a high-risk area for esophageal cancer. In this study, 60 grams of freeze-dried strawberries were given to participants who have been identified by endoscopy to have dysplastic lesions, every day for six months. Biopsy specimens were obtained by endoscopy before and after strawberry treatment. Overall, 36 patients (94.7%) have successfully completed the study, including proper completion of the pre- and post-esophageal endoscopy and biopsy, and the dietary strawberry diary. Twenty patients are females and fifteen patients are males. The average of age is 54.6 ± 1.28. Among 36 patients, 31 patients were diagnosed with mild dysplasia (86.11%) and 5 patients were diagnosed with moderate dysplasia (13.89%). After 6-month strawberry treatment, in patients with mild dysplasia, there was no change in 4 patients, decrease in 26 patients (P &amp;lt; 0.0001) and an increase in 1 patient in histological grade. In patients with moderate dysplasia, there was no change in 2 patients and a decrease in 3 patients in histological grade. Strawberries reduced protein expression levels of inducible nitric oxide synthase (iNOS; 77.3% reduction, P = 0.03), cyclooxygenase-2 (COX-2; 47.3% reduction, P = 0.11), phospho-NFκB-p65 (63.1% reduction, P = 0.23) and phospho-S6 (87.9% reduction, P = 0.02). In addition, we evaluated the effects of strawberries on cell proliferation rate by Ki-67 staining. Strawberries significantly inhibited Ki-67 labeling index by 37.9% (P = 0.023). Our results suggest that strawberries may be a useful alternative to chemopreventive drugs for the prevention of human esophageal cancer. (Supported by California Strawberry Commissions) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-465. doi:10.1158/1538-7445.AM2011-LB-465