Harini S. Aiyer

Influence of Berry Polyphenols on Receptor Signaling and Cell-Death Pathways: Implications for Breast Cancer Prevention

Breast cancer is the most commonly diagnosed cancer among women worldwide. Many women have become more aware of the benefits of increasing fruit consumption, as part of a healthy lifestyle, for the prevention of cancer. The mechanisms by which fruits, including berries, prevent breast cancer can be partially explained by exploring their interactions with pathways known to influence cell proliferation and evasion of cell-death. Two receptor pathways, estrogen receptor (ER) and tyrosine kinase receptors, especially the epidermal growth factor receptor (EGFR) family, are drivers of cell proliferation and play a significant role in the development of both primary and recurrent breast cancer. There is strong evidence to show that several phytochemicals present in berries such as cyanidin, delphinidin, quercetin, kaempferol, ellagic acid, resveratrol, and pterostilbene interact with and alter the effects of these pathways. Furthermore, they also induce cell death (apoptosis and autophagy) via their influence on kinase signaling. This review summarizes in vitro data regarding the interaction of berry polyphenols with the specific receptors and the mechanisms by which they induce cell death. This paper also presents in vivo data of primary breast cancer prevention by individual compounds and whole berries. Finally, a possible role for berries and berry compounds in the prevention of breast cancer and a perspective on the areas that require further research are presented.

Dietary berries and ellagic acid diminish estrogen-mediated mammary tumorigenesis in ACI rats.

Estrogen acts as a complete mammary carcinogen in ACI rats. Prevention studies in this model allowed us to identify agents that are effective against estrogen-induced mammary carcinogenesis. In this study, we investigated efficacy of dietary berries and ellagic acid to reduce estrogen-mediated mammary tumorigenesis. Female ACI rats (8–9 wk) were fed either AIN-93M diet (n = 25) or diet supplemented with either powdered blueberry (n = 19) and black raspberry (n = 19) at 2.5% wt/wt each or ellagic acid (n = 22) at 400 ppm. Animals received implants of 17β-estradiol 2 wk later, were palpated periodically for mammary tumors, and were euthanized after 24 wk. No differences were found in tumor incidence at 24 wk; however, tumor volume and multiplicity were reduced significantly after intervention. Compared with the control group (average tumor volume = 685 ± 240 mm3 and tumor multiplicity = 8.0 ± 1.3), ellagic acid reduced the tumor volume by 75% (P < 0.005) and tumor multiplicity by 44% (P < 0.05). Black raspberry followed closely, with tumor volume diminished by > 69% (P < 0.005) and tumor multiplicity by 37% (P = 0.07). Blueberry showed a reduction (40%) only in tumor volume. This is the first report showing the significant efficacy of both ellagic acid and berries in the prevention of solely estrogen-induced mammary tumors.

Berries and Ellagic Acid Prevent Estrogen-Induced Mammary Tumorigenesis by Modulating Enzymes of Estrogen Metabolism

To determine whether dietary berries and ellagic acid prevent 17β-estradiol (E2)–induced mammary tumors by altering estrogen metabolism, we randomized August-Copenhagen Irish rats (n = 6 per group) into five groups: sham implant + control diet, E2 implant + control diet (E2-CD), E2 + 2.5% black raspberry (E2-BRB), E2 + 2.5% blueberry (E2-BB), and E2 + 400 ppm ellagic acid (E2-EA). Animals were euthanized at early (6 wk), intermediate (18 wk), and late (24 wk) phases of E2 carcinogenesis, and the mammary tissue was analyzed for gene expression changes using quantitative real-time PCR. At 6 weeks, E2 treatment caused a 48-fold increase in cytochrome P450 1A1 (CYP1A1; P < 0.0001), which was attenuated by both BRB and BB diets to 12- and 21-fold, respectively (P < 0.001). E2 did not alter CYP1B1 levels, but both berry and EA diets significantly suppressed it by 11- and 3.5-fold, respectively, from baseline (P < 0.05). There was a 5-fold increase in 17β-hydroxysteroid dehydrogenase 7 (17βHSD7), and this was moderately abrogated to ∼2-fold by all supplementation (P < 0.05). At 18 weeks, CYP1A1 was elevated by 15-fold in E2-CD and only E2-BB reduced this increase to 7-fold (P < 0.05). Catechol-O-methyltransferase expression was elevated 2-fold by E2 treatment (P < 0.05), and all supplementation reversed this. At 24 weeks, CYP1A1 expression was less pronounced but still high (8-fold) in E2-treated rats. This increase was reduced to 3.2- and 4.6-fold by E2-BRB and E2-EA, respectively (P < 0.05), but not by E2-BB. Supplementation did not alter the effect of E2 on steroid receptors. The diets also significantly suppressed mammary tumor incidence (10-30%), volume (41-67%), and multiplicity (38-51%; P < 0.05). Berries may prevent mammary tumors by suppressing the levels of E2-metabolizing enzymes during the early phase of E2 carcinogenesis. Cancer Prev Res; 3(6); 727–37. ©2010 AACR.

Effect on pro-inflammatory and antioxidant genes and bioavailable distribution of whole turmeric vs curcumin: Similar root but different effects.

Curcuma longa is a perennial member of the Zingiberaceae family, and cultivated mainly in India, and Southeast Asia. The hypothesis for this study is that turmeric will have distinctive effects from curcumin due to the presence of other bioactive compounds. Thirty Eight-week old Sprague-Dawley rats were separated into three oral feeding groups. Group 1, standard rat chow, Control diet - AIN 93M, group 2 - Curcumin - 700ppm or 0.7g/kg diet, and group 3 - Turmeric - 14,000ppm or 14g/kg diet for a total of 3weeks. One group of rats were feed all three diets only and another group underwent esophagoduodenal anastomosis to evaluate the effects of bioavailability. Curcumin diet did not increase the transcription of mRNA of TNF-alpha, IL-6, iNOS, and COX-2. The average fold change in the mRNAs level was not significant. Whereas turmeric diet increases the levels of IL-6 (1.9-fold, p=0.05), iNOS (4.39-fold, p=0.02), IL-8 (3.11-fold, p=0.04), and COX-2 (2.02-fold, p=0.05), suggesting that turmeric either was more bioavailable or had more affect on pro-inflammatory genes compare to curcumin diet. We have demonstrated the molecular effects of curcumin and turmeric in the role as an anti-inflammatory therapy. However, significant bioavailable differences do occur and must be considered in further chemopreventative investigative trials the setting of reflux esophagitis, Barretts esophagus, and other upper gastrointestinal cancers.

Prevention of Oxidative DNA Damage by Bioactive Berry Components

The hormone 17ß-estradiol (E2) causes oxidative DNA damage via redox cycling of its metabolites such as 4-hydroxy estradiol (4E2). In this study, ACI rats (8 wk old) were fed either AIN-93M diet or diets supplemented with 0.5% each of mixed berries (strawberry, blueberry, blackberry, and red and black raspberry), blueberry alone (BB; 2.5%), or ellagic acid (EA; 400 ppm) from 2 wk prior to and up to 12 wk of E2 treatment. The liver DNA was analyzed for the presence of 8-oxo-7,8-dihydroguanine (8-oxodG) and other polar adducts by 32P-postlabeling. Compared to sham treatment, E2 significantly increased the levels of both 8-oxodG and P-1 subgroup (259% and 214%, respectively; P< 0.05). EA diet significantly reduced E2-induced levels of 8-oxodG, P-1, P-2, and PL-1 by 79, 63, 44, and 67%, respectively (P< 0.001). BB diet also significantly reduced the levels of P-1, P-2, and PL-1 subgroups by 77, 43, and 68%, respectively (P< 0.001). Mixed berries were, however, ineffective. In addition, aqueous extracts of berries (2%) and EA (100 μM) were tested for their efficacy in diminishing oxidative DNA adducts induced by redox cycling of 4E2 catalyzed by copper chloride in vitro. EA was the most efficacious (90%), followed by extracts of red raspberry (70%), blueberry, and strawberry (50% each; P< 0.001).

Dietary freeze-dried black raspberry's effect on cellular antioxidant status during reflux-induced esophagitis in rats

INTRODUCTION Esophageal cancer consists of two distinct types, esophageal adenocarcinoma (EAC) and squamous cell carcinoma, both of which differ significantly in their etiology. Freeze-dried black raspberry (BRB) has been consistent in its ability to modulate the biomarkers and reduce the incidence of carcinogen-induced squamous cell carcinoma in rats. In our previous studies in the esophagoduodenal anastomosis (EDA) model, we have shown that the early modulation of manganese superoxide dismutase (MnSOD) significantly correlates with the development of reflux-induced EAC in rats. In this study we looked at the short-term effects of a BRB-supplemented diet on the modulation of antioxidant enzymes in reflux-induced esophagitis. METHODS Male SD rats (8 wk old; n = 3-5) were randomized into three groups--sham-operated, fed control AIN-93M diet (SH-CD), EDA operated and fed either control diet (EDA-CD) or 2.5% (w/w) BRB diet (EDA-BRB). The effect of both reflux and dietary supplementation was analyzed 2 and 4 wk after EDA surgery. RESULTS Animals in the EDA groups had significantly lower weight gain and diet intake compared to SH-CD (P < 0.05). The sham-operated animals received an average esophagitis score of 0.1 ± 0.1; this increased significantly in EDA-CD animals to 1.8 ± 0.14 (P < 0.001 versus SH-CD) and in EDA-BRB group to 1.7 ± 0.06 (P < 0.001 versus SH-CD), with BE changes also present. However, dietary supplementation of BRB did not alter or ameliorate the grade of esophagitis or the induction of BE. BRB diet caused a 43% increase in MnSOD levels compared to EDA-CD (0.73 ± 0.16; P = 0.09); however, this effect was not statistically significant and at 4 wk, EDA-CD (0.58 ± 0.12) showed an increase in MnSOD expression compared to SH-CD (0.34 ± 0.01). CONCLUSIONS In conclusion, our data suggest that dietary BRB does not increase the levels of cellular antioxidant enzymes or reduce the levels of lipid peroxidation compared to a control diet, in a short-term study of gastroesophageal reflux induction in the EDA animal model. However, it remains to be tested whether this is indicative of its ineffectiveness to inhibit reflux-induced EAC incidence over the long term.

The Resistance of Esophageal Adenocarcinoma to Bile Salt Insult is Associated with Manganese Superoxide Dismutase Expression

BACKGROUND Bile acids are implicated as etiologic agents in esophageal cancer. We sought to analyze the impact of bile acid exposure on esophageal epithelial cells, Barretts metaplastic cells (BE), esophageal adenocarcinoma cells (EAC), and esophageal squamous carcinoma cell (ESC). We sought to determine if cellular resistance is related to manganese superoxide dismutase expression. METHODS Cells were exposed to sodium choleate (CA), sodium deoxycholate (DCA), sodium glycocholate (GCA), sodium taurocholate (TCA), or a 1:1 mixture (MIX) of reagents at concentrations in the range 0.2-0.8 mM. Cell viability was evaluated by MTT assay. Manganese superoxide dismutase (MnSOD) expression was analyzed by Western blot. Statistical analysis was performed using SPSS ver. 17.0, SPSS Inc., Chicago, IL. RESULTS Bile salt exposure inhibited cell viability in esophageal squamous cells in time- and growth-dependent manner. There was a 50% decrease in cell viability from 4 to 24 h. BE, EAC, and ESC cell lines were more resistant to bile insult. In untreated cell lines, MnSOD expression was significantly decreased in EAC and ESC cell lines compared with esophageal squamous epithelial cells and BE cells (P=0.002). Exposure of ESC cells to bile salt increased MnSOD expression. CONCLUSION The confirmation of the role of reactive oxygen species (ROS) and bile acids in esophageal carcinogenesis has interesting implications for chemoprevention in patients with reflux esophagitis and Barretts esophagus. Further studies are necessary to assess the preventative role of antioxidant supplementation.

Diet Composition Affects Surgery-Associated Weight Loss in Rats with a Compromised Alimentary Tract

BACKGROUND Esophageal adenocarcinoma (EAC) is the fastest growing cancer in terms of incidence and has a high mortality rate. The animal model to study EAC uses esophagoduodenal anastomosis (EDA) to induce mixed-reflux (bile/acid) causing esophagitis, Barretts esophagus, and EAC sequence within 6 mo. However, the lack of fully functional stomach in these rats leads to the development of malnutrition. METHODS We have assessed the ability of a chemically pure, purified ingredient diet (AIN-93M) to reduce surgery-associated malnutrition in rats that have undergone the EDA-surgery. Animals were either sham- (SH) or EDA-operated and fed either a grain-based rodent diet (RD) (SH-RD, n=3; EDA-RD, n=10) or a purified diet (PD) (SH-PD, n=4; EDA-PD, n=11). The animals were weighed periodically for assessment of weight gain and euthanized at the end of 24 wk to measure esophageal tumor incidence. RESULTS Animals that underwent sham surgery continued to gain weight throughout the study period and no tumors were detected. The EDA-operated animals had significantly lower weight gain compared with sham animals. There was no significant difference in weight gain among EDA animals fed two different types of diets until 9 wk after the surgery. After 9 wk, EDA-RD continued to lose weight significantly, whereas the weight loss leveled in EDA-PD (P<0.001). At termination, neither tissue histopathology nor tumor incidence was significantly different between the groups. CONCLUSION These results show that compared with a natural ingredient diet, a purified ingredient diet can reduce surgery-associated weight loss in rats with a compromised alimentary tract. This reduction in malnutrition has the potential to reduce the confounding effects of weight loss on future animal studies reported.

Interaction of dietary polyphenols with molecular signaling pathways of antiestrogen resistance: possible role in breast cancer recurrence

Abstract Breast cancer is the most common cancer diagnosed in women and its global incidence is rising rapidly. Adjuvant hormonal therapy, with antiestrogens (AE) such as tamoxifen and fulvestrant, is highly effective in the treatment of estrogen receptor-positive (ER+) breast cancers and is largely responsible for the increase in survival rates seen in the past four decades. However, nearly 50% of women with ER+ cancer display de novo or acquired resistance to AE therapies. Potential molecular mechanisms driving the resistance phenotype are beginning to be elucidated, allowing further development of more effective therapeutic and preventive strategies to reduce the overall mortality due to breast cancer. Over 70% of breast cancer survivors surveyed report increasing their comsumption of fruits, vegetables, and natural product supplements upon diagnosis. These are rich sources of dietary polyphenols (PPs) that can interact with cell-signaling pathways involved in the development of AE resistance. However, research on mechanisms by which these agents may affect AE resistance and whether PP intake can significantly change breast cancer recurrence is limited. We summarize the available data on the effects of PPs on breast cancer recurrence and the interactions of these compounds with some of the signaling pathways hypothesized to drive cell death and survival involved in the development of AE resistance in breast cancer.

Molecular changes in the esophageal epithelium after a subchronic exposure to cigarette smoke in the presence of bile-acid reflux.

Background: Gastroesophageal reflux of bile acids plays an important role in the development of Barrett’s esophagus (BE)–associated esophageal adenocarcinoma (EAC). Cigarette smoke has been demonstrated to exacerbate the effects of reflux and thus the initial stages of EAC carcinogenesis. To date, no in vivo studies have been conducted to look at the concomitant effects of cigarette smoke and bile acids on EAC incidence. Methods: In this pilot study, rats that underwent esophagoduodenal anastomosis (EDA) surgery to induce reflux were exposed to whole-body cigarette smoke 3 weeks after surgery. Smoke exposure (135 mg/m3/day) was done for 4 h/day for 5 consecutive days and animals were euthanized after a 48-h recovery period. Results: Exposure to EDA-smoke accelerated the development of BE when compared to EDA-air. The presence of reflux caused a significant 3.5-fold increase in nuclear factor-κB-inducing kinase (NIK) staining (1.47 ± 0.6; p = 0.01). Animals with both reflux and smoking had the highest (10-fold; 4 ± 0.9) induction of cyclooxygenase-2 (COX-2) expression (p < 0.05). Similarly, there was a 10-fold increase in 4-aminobiphenyl (4-ABP) protein adducts identified in all smoke-exposed animals (p < 0.01). Conclusion: Cigarette smoke aggravates reflux-induced BE and potentially accelerates the progression of BE to EAC through the loss of manganese superoxide dismutase (MnSOD), and overexpression of NF-κB- and COX-2-mediated factors.