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Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which is responsible for synthesizing a variety of secondary metabolites that participate in development and adaptation. In this study, we isolated a full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene. By comparing the deduced amino acid sequence of Rubus sp. C4H with other sequences reported previously we determined that this sequence was highly conserved among widely divergent plant species. In addition, quantitative real time PCR studies indicated that the C4H gene had a differential expression pattern during fruit development, where gene expression was first detected in green fruit and was then remarkably reduced in yellow fruit, followed by an increase in red and black fruit. To investigate the two peaks in expression observed during fruit development and ripening, we measured the flavonoid content. The content of the major flavanol of Korean black raspberry fruits was determined to be highest at the beginning of fruit development, followed by a gradually decrease according to the developmental stages. In contrast, the content of anthocyanins during the progress of ripening was dramatically increased. Our results suggest that the C4H gene in Korean black raspberry plays a role during color development at the late stages of fruit ripening, whereas the expression of C4H gene during the early stages may be related to the accumulation of flavanols.

Black raspberry juice was fermented to produce gamma-aminobutyric acid (GABA) using lactic acid bacteria (Lactobacillus brevis GABA 100) at different temperatures (25, 30, or 37 degrees C) and pHs (3.5, 4, 4.5, 5, 5.5, or 6) for 15 days. Concentrations of GABA in the juices were determined during fermentation using HPLC. GABA was produced continuously even if the viable bacterial counts markedly decreased. The fermentation at 30 degrees C generally showed higher production of GABA in the juices than those at 25 and 37 degrees C. The GABA in the juices fermented at 30 degrees C reached the maximum levels on the 12th day. The juices fermented at lower pH and lower temperature showed a lower degradation of monomeric anthocyanins. The results suggest that black raspberry juice can be GABA enriched using lactic acid bacteria.

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 17beta-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% (P0.005) and tumor multiplicity by 44% (P0.05). Black raspberry followed closely, with tumor volume diminished by69% (P0.005) 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.

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.

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

The anthocyanin pigment profiles of commercial black currant, blackberry, black raspberry, elderberry, cherry, plum, grape, bilberry, and red cabbage products were characterized by high-performance liquid chromatography (HPLC)/photodiode array detection. Removal of acylated anthocyanins by alkaline hydrolysis and selective removal of anthocyanins on a reversed-phase cartridge with borate buffer were auxiliary techniques that proved helpful in making peak assignments. Both the retention properties on reversed-phase HPLC and the spectral properties by photodiode array detection were used to characterize the anthocyanins. Other properties including tinctoral strength, total anthocyanin concentration, browning, titratable acidity, and Hunter tristimulus values were also determined for these colorants.

In our previous work, a diet containing 5% freeze-dried black raspberries (BRB) was shown to significantly inhibit colorectal cancer in ApcMin mice. Studies in recent years have shown that metabolomics may help to elucidate the anti-cancer effects of dietary factors. Initially, therefore, we evaluated the effects of black raspberries on the metabolomic profiles of C57BL/6J mice, the mouse strain from which ApcMin mice were derived. In this study, we report the effects of the BRB diet on the metabolomic profiles from C57BL/6J mice. After 8 weeks on a 5% BRB or control AIN-76A diet, feces, liver, and colon mucosa tissues from 9 BRB-fed mice and 7 control-diet fed mice were collected and profiled using mass spectrometry. A total of 424 named metabolites were observed in the liver, feces, and colon mucosa, with 115 metabolites whose levels were significantly different between BRB and control-diet mice (P Citation Format: Chad W. Skaer, Pan Pan, Matthew Young, Hsin Tzu Wang, Kiyoko Oshima, Yi-Wen Huang, Gary D. Stoner, John Lechner, Li-Shu Wang. Effects of black raspberry diet on the metabolomic profile of C57BL/6J mouse tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2015-LB-263

BRB powder, extracts and metabolites have been shown to inhibit tumorigenesis in oral and other sites in animal models. Here we examined the effects of an anthocyanin-enriched BRB extract, and protocatechuic acid (PA), a major BRB anthocyanin metabolite, on mutagenesis and DNA adduct formation by DBP-dihydrodiol, and DBP diolepoxide (DBPDE) (the proximate and ultimate carcinogenic metabolites, resp. of DBP) in lacI rat oral fibroblasts. DBP was poorly metabolized to genotoxic metabolites by the oral fibroblasts, but DBP-dihydrodiol was mutagenic and produced DNA adducts. DBP-dihydrodiol (0.5 uM) induced a mutant fraction of 14.1 +/- 3.9 mutants/10E5 pfu in the target cII gene. This was reduced in a concentration-dependent fashion to 9.1 +/- 2.4 by 0.1-1uM PA– a 35% reduction. Mutagenesis by DBP-dihydrodiol was also reduced in a concentration-dependent fashion by BRB extract and at the highest concentration (20ug/ml) mutagenesis was reduced by 68%. The major DNA adduct produced by 0.5 uM DBP-dihydrodiol was anti-DBPDE-dA, as assessed by L.C.-M.S.-M.S. Its levels were reduced from 13.7 +/- 1.2 to 7.8 +/- 1.9 adducts/10E6 dA by 3uM protocatechuic acid, and from 6.8 +/- 0.7 to 3.2 +/- 0.4 by 66 ug/ml BRB extract. The inhibitions were statistically significant. In contrast, no significant effect PA on mutagenesis by 140 nM DBPDE was observed, with mutant fractions similar to that of DBPDE (3.2 +/- 0.45 mutants/10E5 pfu) being obtained over a range of 0.1 - 1 uM PA. Similarly 10-50 ug/ml BRB extract had no effect on DBPDE-induced mutagenesis. Consistent with these results, levels of total DBPDE adducts were unchanged over a range of PA concentrations, with the high dose of 1uM PA yielding, 4.6 +/- 0.7 adducts/10E5 nucleotides and 4.5 +/- 0.9 without PA. Presumably the short halflife of DBPDE precludes detoxification, or scavenging of DBPDE. The effects of PA and BRB extract on oxidative stress in a human oral cancer cell line (SCC 1483, retromolar trigone) were also examined. The redox sensitive fluorescent probe, dihydrodichlorofluorescein diacetate (DHDCF) was added to the cells at 5uM and BRB (0.5-5 ug/ml) or PA (0.02-0.2 uM) was added shortly afterwards. 2 hr later all doses of BRB extract and PA inhibited the oxidation of DHDCF to its fluorescent product, in a dose-dependent manner. At 5ug/ml BRB extract the increase in fluorescence was reduced from 304 +/- 3.3 to 177 +/- 7.1 relative fluorescence units (RFU) and at 0.2 uM PA the increase was reduced to 224 +/- 25 RFU. In summary, BRB extract and PA inhibited mutagenesis and DNA adduct formation by DBP-dihydrodiol, but not DBPDE. BRB extract and PA were also effective inhibitors of oxidative stress. These mechanisms may a play a role in the protective effects of BRB on oral and other cancers in experimental animals. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Seungjin Kim, Ying Zhou, Wieslawa Kosinska, Ana-Vera Golgotiu, Shantu Amin, Gary D. Stoner, Yuan-Wan Sun, Kun-Ming Chen, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by metabolites of the tobacco carcinogen, dibenzo(a,l)pyrene in cultured rat oral fibroblasts; and on oxidative stress in human oral cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2015-915

We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to carcinogenesis induced by certain carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value < 0.01 and percent methylation difference > 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the environmental carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955

Black raspberry (BRB) seeds are a major waste product after fruit processing. The seeds are abundant in ellagitannins (ET), a class of hydrolysable tannins, which are hydrolyzed to ellagic acid (EA) and further metabolized to urolithin A (UA) and urolithin B (UB), known to be bioavailable in the colon and the prostate. In this study, the anti-cancer activities of these compounds were evaluated on HT-29 colon cancer cells. ET, EA, UA and UB inhibited the proliferation of the cancer cells. EA caused a slight, but significant cell cycle arrest at the G1 phase, and urolithins caused cell cycle arrest at the G2/M phase and upregulated p21 expression. Apoptotic cells were detected by Annexin V-FITC/PI assay when treated with the compounds. Disruption in mitochondrial membrane potential and activation of caspases 8 and 9 suggest that both extrinsic and intrinsic apoptotic pathways may be involved. Activation of caspase 3 and cleavage of PARP further confirmed the induction of the apoptosis. ET, EA, UA and UB showed anti-cancer activity by arresting the cell cycle and inducing apoptosis on HT-29 human colon cancer cells. This study suggests that the BRB seeds could be a potential source of anti-cancer ET.

Berry fruits are widely consumed in our diet and have attracted much attention due to their potential human health benefits. Berries contain a diverse range of phytochemicals with biological properties such as antioxidant, anticancer, anti-neurodegerative, and anti-inflammatory activities. In the current study, extracts of six popularly consumed berries--blackberry, black raspberry, blueberry, cranberry, red raspberry and strawberry--were evaluated for their phenolic constituents using high performance liquid chromatography with ultraviolet (HPLC-UV) and electrospray ionization mass spectrometry (LC-ESI-MS) detection. The major classes of berry phenolics were anthocyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthocyanidins, and phenolic acids. The berry extracts were evaluated for their ability to inhibit the growth of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT116), and prostate (LNCaP) tumor cell lines at concentrations ranging from 25 to 200 micro g/mL. With increasing concentration of berry extract, increasing inhibition of cell proliferation in all of the cell lines were observed, with different degrees of potency between cell lines. The berry extracts were also evaluated for their ability to stimulate apoptosis of the COX-2 expressing colon cancer cell line, HT-29. Black raspberry and strawberry extracts showed the most significant pro-apoptotic effects against this cell line. The data provided by the current study and from other laboratories warrants further investigation into the chemopreventive and chemotherapeutic effects of berries using in vivo models.

Despite focused efforts to improve therapy, 5-yr survival rates for persons with advanced-stage oral squamous cell carcinoma (SCC) remain discouragingly low. Clearly, early detection combined with strategies for local intervention, such as chemoprevention prior to SCC development, could dramatically improve clinical outcomes. Previously conducted oral cavity human chemoprevention trials, however, have provided mixed results. Although some therapies showed efficacy, they were often accompanied by either significant toxicities or circulating antiadenoviral antibodies. It is clearly apparent that identification of nontoxic, effective treatments is essential to prevent malignant transformation of oral epithelial dysplasias. This study employed cell lines isolated from human oral SCC tumors to investigate the effects of a freeze-dried black raspberry ethanol extract (RO-ET) on cellular growth characteristics often associated with a transformed phenotype such as sustained proliferation, induction of angiogenesis, and production of high levels of reactive species. Our results demonstrate that RO-ET suppresses cell proliferation without perturbing viability, inhibits translation of the complete angiogenic cytokine vascular endothelial growth factor, suppresses nitric oxide synthase activity, and induces both apoptosis and terminal differentiation. These data imply that RO-ET is a promising candidate for use as a chemopreventive agent in persons with oral epithelial dysplasia.

Seed flours from black raspberry, red raspberry, blueberry, cranberry, pinot noir grape, and chardonnay grape were examined for their total fat content, fatty acid composition, total phenolic content (TPC), total anthocyanin content (TAC), radical scavenging capacities against the peroxyl (ORAC) and stable DPPH radicals, chelating capacity against Fe(2+), and antiproliferative activities using the HT-29 colon cancer cell line. Significant levels of fat were detected in the fruit seed flours and their fatty acid profiles may differ from those of the respective seed oils. Cranberry seed flour had the highest level of alpha-linolenic acid (30.9 g/100 g fat) and the lowest ratio of n-6/n-3 fatty acids (1.2/1). The ORAC value of the chardonnay seed flour was 1076.4 Trolox equivalents mumol/g flour, and its TPC was 186.3 mg gallic acid equivalents/g flour. These values were 3-12 times higher than the other tested fruit seed flours. Furthermore, the ORAC value was significantly correlated to the TPC under the experimental conditions (P0.05). These fruit seed flours also differed in their TAC values and Fe(2+)-chelating capacities. In addition, black raspberry, cranberry, and chardonnay grape seed flour extracts were evaluated for their antiproliferative effects using HT-29 colon cancer cells. All three tested seed flour extracts significant inhibited HT-29 cell proliferation. The data from this study suggest the potential of developing the value-added use of these fruit seed flours as dietary sources of natural antioxidants and antiproliferative agents for optimal human health.

Anthocyanins are the most abundant phenolic compounds, widely distributed in fruits and vegetables, and exhibit potent antioxidant capacity. Humans ingest a significant amount of anthocyanins in the daily diet. The objective of the current study was to examine human absorption and metabolism of black raspberry anthocyanins when administered at high doses (2.69 +/- 0.085 g/day). Ten healthy men consumed 45 g of freeze-dried black raspberries daily for 1 week. Urine samples were collected over a 12 h period in 4 h intervals at day 1 and day 7. Urinary anthocyanins were analyzed by high-performance liquid chromatography coupled to a photodiode array detector and a tandem mass spectrometer using precursor ion and product ion analyses. Anthocyanins were excreted in intact forms and metabolized into methylated derivatives in human urine. The urinary excretion of anthocyanins reached a maximum concentration (1091.8 +/- 1081.3 pmol/L, n = 10) during the 4-8 h period after black raspberry ingestion. As compared to the anthocyanin distribution in black raspberries, urinary cyanidin 3-xylosylrutinoside was detected at a higher concentration than that of cyanidin-3-rutinoside.

There have been very limited reports on the effects of commercial fruit juices on human CYP3A activity. Therefore, the inhibitory effects of readily available commercial fruit juices on midazolam 1′-hydroxylase activity, a marker of CYP3A, were evaluated in pooled human liver microsomes. The fruit juices investigated were black raspberry, black mulberry, plum, and wild grape. White grapefruit, pomegranate, and orange juice were used as positive and negative controls. The black mulberry juice showed the most potent inhibition of CYP3A except for grapefruit juice. The inhibition depended on the amount of a fruit juice added to the incubation mixture. The inhibitory potential of human CYP3A was in the order: grapefruit > black mulberry > wild grape > pomegranate > black raspberry. The IC50 values of all fruit juices tested were reduced after preincubation with microsomes in the presence of the NADPH-generating system, suggesting that a mechanism-based inhibitory component was present in these fruit juices, as in the case of grapefruit. The results suggest that, like grapefruit juice, commercial fruit juices also have the potential to inhibit CYP3A-catalzyed midazolam 1′-hydroxylation. Therefore, in vivo studies investigating the interactions between fruit juices such as black mulberry and wild grape and CYP3A substrates are necessary to determine whether inhibition of CYP3A activity by fruit juices is clinically relevant.

Si is one of the most widely distributed elements in the Earth’s crust, and in turn soil is the most enriched with silica layer of the Earth’s crust – 40 to 70% of SiO2 contain in the clay soils and 90–98% in the sandy soils. Mainly, Si is present as quartz, alkali and aluminum silicates. They usually form the soil skeleton and are chemically or biochemically inert (Perelman, 1986; Reims, 1990; Sokolova, 1985). In the classification of element mobility, Si is defined both as an inert and as a mobile element (Perelman et al., 1989). Mobile Si substances represent monosilicic acid, polysilicic acid, organosilicon compounds and complex compounds with organic and inorganic substances (Matichenkov et al., 2001). Beginning in 1848, numerous laboratory, greenhouse and field experiments have shown benefits of silicon fertilization for rice, corn, wheat, barley, sugar cane and other crops and benefits for maintaining a sustainable agriculture. Silicon fertilization has a double effect on the soil-plant system. Firstly, improved plant Si nutrition reinforces plant protective properties against diseases, insect attack and unfavorable climatic conditions. Secondly, the soil treatment with Si biogeochemically active substances optimizes soil fertility through improved water, physical, and chemical soil properties and maintaining nutrients in a plant-available form. The role of Si in the nutrition of plant species used in horticulture has not been well investigated in comparison to agricultural crops like rice or sugarcane. Some authors have demonstrated the relevant uptake of this element for some plant species used in horticulture. Si supplements have been used for production of greenhouse crops in Europe. Now some growers and researchers consider Si as a ‘quasi-essential’ element for plant growth and development.

Targeted therapies, such as agents that inhibit angiogenesis, offer hope as complementary agents in cancer therapy. Angiogenesis-inhibiting agents have the potential for inhibiting tumor growth and limiting the dissemination of metastasis, thus keeping cancers in a static growth state for prolonged periods. Black raspberry (Rubus occidentalis) extract was discovered to be antiangiogenic (0.1% w/v) in a novel human tissue-based in vitro fibrin clot angiogenesis assay. Assay-guided fractionation of a crude black raspberry extract resulted in a highly potent antiangiogenic fraction that accounted for only 1% of the fresh weight of whole black raspberries. At 0.075% (w/v), the active fraction completely inhibited angiogenic initiation and angiogenic vessel growth. Further subfractionation of this active fraction revealed the coexistence of multiple antiangiogenic compounds, one of which has been identified as gallic acid. However, the individual subfractions did not outperform the active whole fraction. These findings suggest that an active black raspberry fraction may be a promising complementary cancer therapy. It is natural and potent enough for manageable dosing regimens. These extracts contain multiple active ingredients that may be additive or synergistic in their antiangiogenic effects. These observations warrant further investigations in animals and human trials.

Black raspberries are a rich natural source of chemopreventive phytochemicals. Recent studies have shown that freeze-dried black raspberries inhibit the development of oral, esophageal, and colon cancer in rodents, and extracts of black raspberries inhibit benzo(a)pyrene-induced cell transformation of hamster embryo fibroblasts. However, the molecular mechanisms and the active components responsible for black raspberry chemoprevention are unclear. In this study, we found that 2 major chemopreventive components of black raspberries, ferulic acid and beta-sitosterol, and a fraction eluted with ethanol (RO-ET) during silica column chromatography of the organic extract of freeze-dried black raspberries inhibit the growth of premalignant and malignant but not normal human oral epithelial cell lines. Another fraction eluted with CH2Cl2/ethanol (DM:ET) and ellagic acid inhibited the growth of normal as well as premalignant and malignant human oral cell lines. We investigated the molecular mechanisms by which ferulic acid and beta-sitosterol and the RO-ET fraction selectively inhibited the growth of premalignant and malignant oral cells using flow cytometry and Western blotting of cell cycle regulatory proteins. There was no discernable change in the cell cycle distribution following treatment of cells with the RO-ET fraction. Premalignant and malignant cells redistributed to the G2/M phase of the cell cycle following incubation with ferulic acid. beta-sitosterol treated premalignant and malignant cells accumulated in the G0/G1 and G2/M phases, respectively. The RO-ET fraction reduced the levels of cyclin A and cell division cycle gene 2 (cdc2) in premalignant cells and cyclin B1, cyclin D1, and cdc2 in the malignant cell lines. This fraction also elevated the levels of p21waf1/cip1 in the malignant cell line. Ferulic acid treatment led to increased levels of cyclin B1 and cdc2 in both cell lines, and p21waf1/cip1 was induced in the malignant cell line. beta-sitosterol reduced the levels of cyclin B1 and cdc2 while increasing p21waf1/cip1 in both the premalignant and malignant cell lines. These results show for the first time that the growth inhibitory effects of black raspberries on premalignant and malignant human oral cells may reside in specific components that target aberrant signaling pathways regulating cell cycle progression.

Fragaria (strawberry) and Rubus species (blackberry, wild blackberry, red raspberry and black raspberry) were thought to be infected with distinct isolates of Tobacco streak virus (TSV). Employing serology and nucleic acid hybridization it has been shown that these isolates form a cluster distinct from other strains of TSV. In this study we have cloned and sequenced the complete RNA 3 of an isolate of TSV from strawberry (Fragaria) as well as the coat protein (CP) gene of 14 additional isolates of TSV originating from Fragaria and Rubus species. Our data suggest that the isolates of TSV that infect Fragaria and Rubus belong to a distinct virus for which we propose the name Strawberry necrotic shock virus (SNSV). The RNA 3 of SNSV contains 2248 nucleotides, 43 more than the type isolate of TSV from white clover (TSV-WC), with a CP gene that is 669 nucleotides long, in contrast to the 714–7 nucleotides of the TSV CP sequences found in the database. The movement protein gene of SNSV is 897 nucleotides in length, 27 more than that of the TSV-WC isolate of TSV. The CP genes of the 15 Fragaria and Rubus isolates that we studied form two distinct phylogenetic clusters that share about 95% amino acid sequence identity, while they only share 60–65% amino acid sequence identity with TSV-WC.

Over the past 10 years, several commercial producers of black raspberry in Ohio have experienced a decline in plant vigor and survival in their plantings. In 2004, a survey of several black and red raspberry plantings in Ohio was conducted to determine if viruses were present. Of the 115 samples taken in the survey, Raspberry bushy dwarf (RBDV), Tomato ringspot (ToRSV), and Tobacco ringspot virus (TRSV) were detected in 21, 5, and 2, respectively. This is the first report of RBDV in Ohio.Accepted for publication 27 April 2005. Published 10 May 2005.

BRB powder and extracts have been shown to inhibit tumorigenesis in oral and other alimentary-tract sites in animal models; and have promising effects on markers of carcinogenesis in several human trials. The purpose of this study was to compare the inhibitory effects of BRB and two related compounds (protocatechuic acid and kaempferol) on mutagenesis and DNA adduct formation induced by the tobacco carcinogen dibenzo[a,l]pyrene (DBP) and its metabolites, DBP-diol and DBP-diol epoxide (DBPDE), in a rat oral epithelial cell line. Mutagenesis and DNA adduct formation induced by DBP, DBP-diol and DBPDE were monitored in rat oral epithelial and fibroblast cells, containing a lacI mutagenesis reporter gene; and the order of potency in both adduct formation and mutagenesis was DBPDE>>DBP-diol>>DBP in both cell types. In the fibroblasts for mutagenesis, the potencies (in units of mutants/10E5 pfu /uM) were: DBPDE, 16,320; DBP-diol, 25; DBP1.7, (above background); background was 2.9., Similar results were obtained for epithelial cells. Analysis of DNA adducts levels is still in progress and results thus far (in units of cis + trans isomers of anti-DBPDE-dA/10E6 A, are: DBPDE, 2096, DBP-diol ≈1.8, DBP < 1.0. The above results are consistent with mutagenesis being dependent on DNA adducts and indicate this cell culture model reflects initiation of carcinogenesis. The background level of mutagenesis was greater in the epithelial cells, so the fibroblasts were then used for subsequent mutagenesis experiments. As initiation by DBP and DBP-diol requires biotransformation to an ultimate mutagen/carcinogen (in this case DBPDE) we initially assayed inhibition of DBP-diol induced mutagenesis, so that effects of BRB on processes involved in activation, as well as effects on steps after formation of DBPDE would be detected. The parent compound, DBP, was not employed because it was very weakly mutagenic, and studies with DBPDE are still in progress. Treatment of cells with one µM DBP-diol led to an increase in mutant fraction from 2.9 +/- 0.8 to 25 +/- 2.3 mutants/10E5 pfu. When pretreated with BRB and its related compounds 4 hours before the addition of DBP-diol, significant inhibition of mutagenesis was observed. With 3 uM protocatechuic acid mutagenesis was reduced to 15.6 +/- 3.7 and with 3uM kaempferol to 13.3 +/- 4.1 mutants/10E5 pfu. Pretreatment of the cells with 25 ug/ml of an anthocyanin-enriched BRB extract (a concentration previously used in colon cancer cells), reduced mutagenesis to 9.8 +/- 0.35 mutants/10E5 pfu. These results suggest that BRB and specific related components will be protective against carcinogenesis induced by certain tobacco carcinogens. Supported by NIH grant #CA173465. Citation Format: Joseph B. Guttenplan, Wieslawa Kosinska, Tianzhen Han, Kun-Ming Chen, Shangmin Zhang, Krishnegowda Gowdahalli, Amin Shantu, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by the tobacco carcinogen, dibenzo(a,l)pyrene and its metabolites in cultured rat oral fibroblasts. [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 252. doi:10.1158/1538-7445.AM2014-252

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. However, DMBA is not present in the environment and the hamster cheek pouch model may not be applicable to humans. We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, in mouse oral cavity. Recently, we demonstrated that administration of BRB at 5% in the diet can significantly reduce DNA adducts resulted from the administration of DB[a,l]P. It has also been shown that anthocyanins are the most abundant compounds in BRBs that can account for much of their antioxidant activity. In the current study, under identical conditions we compare the inhibitory effects of an anthocyanin enriched fraction extracted from black raspberry (BRBE), protocatechuic acid (PA, a major metabolite of anthocyanins) or ferulic acid (FA, a major phenolic acid in raspberries shown to reduce oral carcinogenesis) with BRB on DNA adducts induced by DB[a,l]P in mouse oral cavity. Levels of DB[a,l]PDE-DNA adducts were quantified by a LC-MS/MS method. We demonstrated that the administration of BRBE (1.6 %), protocatechuic acid (0.2%) or ferulic acid (0.05%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) significantly resulted in 29.6%, 23.0% or 25.3% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively, compared to a 19.2% reduction of BRB. Our results clearly showed that the anthocyanin components of BRB and its metabolite, PA, are responsible for the inhibitory effects of BRB on the DB[a,l]P- induced DNA adducts formation; in addition, BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Supported by NIH grant #CA173465. Citation Format: Kun-Ming Chen, Shangmin Zhang, Yuan-Wan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [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 230. doi:10.1158/1538-7445.AM2014-230

Bioactive phyotochemicals from natural products, such as black raspberries have direct anti-cancer properties on malignant cells in culture and in xenograft models. In addition, black raspberry components inhibit cancer progression in more complex rodent carcinogenesis models. Although mechanistic targets for black raspberry phytochemicals in cancer cells are beginning to emerge, the potential role in modulating host immune processes impacting cancer have not been systematically examined. We hypothesized that black raspberries contain compounds capable of eliciting potent immunomodulatory properties that impact cellular mediators relevant to chronic inflammation and tumor progression. In the present report, we studied both an ethanol extract from black raspberries (BRB-E) containing a diverse mixture of phytochemicals and two abundant phytochemical metabolites of black raspberries produced upon ingestion (Cyanidin-3-Rutinoside, C3R; Quercitin-3-rutinoside, Q3R). BRB-E inhibited proliferation of CD3/CD28 activated human CD4+ and CD8+ T lymphocytes. BRB-E also limited the viability of CD3+ T cells cultured in IL-2, and were potent inhibitors of IL-2-induced STAT5 phosphorylation. Conversely, BRB-E significantly enhanced the monokine-induced by gamma (MIG)-mediated chemotaxis of CD8+ T lymphocytes in transwell assays. This enhanced chemotaxis was associated with upregulated expression of CXCR3 on T lymphocytes. BRB-E also inhibited the in vitro expansion of myeloid-derived suppressor cells (MDSC). Consistent with the reduction in MDSC, pre-treatment of immune cells with BRB E attenuated IL-6-mediated signal transducer and activator of transcription-3 (STAT3) phosphorylation. Finally, the C3R and Q3R metabolites were also found to be immunomodulatory, as C3R inhibited proliferation of CD3/CD28 activated T lymphocytes and STAT5 phosphorylation, while Q3R inhibited MDSC expansion and STAT3 signaling but not STAT5. Together these data indicate that black raspberries or their physiologically-relevant metabolites contain phytochemicals that affect immune processes relevant to carcinogenesis. Furthermore, specific components and their metabolites may be a source of lead compounds for drug development that exhibit targeted immunological outcomes or inhibition of specific STAT-regulated signaling pathways. Citation Format: Thomas A. Mace, Samantha King, Zeenath Ameen, Omar Elnaggar, Gregory Young, Kenneth Riedl, Steven Schwartz, Steven Clinton, Thomas Knobloch, Christopher Weghorst, Gregory B. Lesinski. Bioactive compounds or metabolites from black raspberries modulate T lymphocyte function, myeloid cell differentiation and Jak/STATsignaling. [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 2585. doi:10.1158/1538-7445.AM2014-2585

The Korean black raspberry (Rubus coreanus Miquel, KB) on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq (TM) 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3'H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2) gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.

Most leafhopper vectors transmit viruses in a circulative manner and many of these viruses are propagative in their vector. For some of the viruses (Wound tumor and Potato yellow dwarf viruses, for example) there is proof of transovarial passage (Sinha and Shelley, 1965; Black, 1953). Viruses of this group are characterized by having a defined latent period (LP) during which the leafhopper cannot transmit virus after an acquisition feed (AF). AF with these viruses is of at least 30 minutes. The LP may be relatively short (hours) or long (at least a week). Most of the leafhopper species which transmit the circulative/propagative viruses remain viruliferous for most of their lives after a single AF on a virus source (Harris, 1979). With a few exceptions (e.g., Sugar beet curly top and Potato yellow dwarf viruses) none of these viruses is mechanically transmitted. Viruses such as Rice tungro and Maize chlorotic dwarf viruses are transmitted by their leafhopper vectors in a semipersistent manner (Ling, 1966; Nault et al. 1973) without a detectable LP or evidence for their circulation within the insect bodies; they enter the hemocoele and are transmitted via the salivary glands. The vectors of these two viruses become non-viruliferous within a very short time. Inoculation access (IA) of more than 30 minutes was needed for a reasonable rate of transmission by the vector.

Isoenzyme staining was used to characterize 55 of 78 raspberry cultivars (Rubus idaeus L., R. × neglectus Peck, and R. occidentalis L.). Six enzymes were needed to achieve this characterization: isocitrate dehydrogenase, malate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, shikimic acid dehydrogenase, arid triose phosphate isomerase. The 23 cultivars that were not uniquely characterized were grouped into eight groups of two and two groups of three and four. Two of these groups comprised black raspberry cultivars, all of which were similar isozymically. Isoenzymes could not distinguish between the cultivar Willamette and a spine-free mutant of the cultivar. Analysis of cultivars obtained from several sources revealed that raspberry cultivar mislabeling exists but is not very prevalent. Regular isoenzyme analysis of raspberry cultivars held by germplasm repositories, certified and other propagators, and breeders is both feasible and advisable for early detection of cultivar mislabeling.

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. DMBA, however, is not present in the environment and the hamster cheek pouch model may not be applicable to humans. Recently, we developed a relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. In the current study, we evaluated the effects of BRB and related components including kaempferol and resveratrol on DNA adducts induced by DB[a,l]P in mouse oral cavity as an initial step to explore the potential preventive activity of BRB and its related components on DB[a,l]P-induced oral cancer. The DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We demonstrated that the administration of BRB (5%), kaempferol (0.025%) or resveratrol (0.002%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) resulted in 27%, 36% or 65% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively. In conclusion, our results support that dietary consumption of BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Citation Format: Kun-Ming Chen, Shang-Min Zhang, Cesar Aliaga, Yuan-Wan Sun, Arthur Berg, Arun Sharma, Shantu Amin, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [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 3708. doi:10.1158/1538-7445.AM2013-3708

Black raspberries are being developed to chemoprevent GI tract cancers in humans and they have been shown to prevent breast cancer development in a rat model. Therefore, it is important to determine whether they can also prevent other major human cancers such as prostate cancer. We induced prostate cancer in Wistar HsdCpb:WU rats by treatment with Flutamide (20 mg/kg BW by gavage daily for 20 days), a single SC injection with testosterone propionate (100 mg/kg) on day 21, and single IP injection of N-methyl-N-nitrosourea (MNU; 30 mg/kg) 52-56 hrs later. Two weeks later, rats received two SC Silastic tubing implants (ID 0.078 in; OD 0.125 in.) containing crystalline testosterone tightly packed over 3 cm length. One week later, rats were started on AIN-93M diet (control) or isoenergetic AIN-93M diets containing 5% or 10% lyophilized black raspberry powder (exchanged for starch). The powder was from a single batch of freshly harvested black raspberries (BerriProducts, Corvallis, OR), stored at -20oC until mixed into the diet, and provided fresh to the rats thrice weekly. The treatment did not affect body weight gain or survival (mean 49-52 wks). The study was terminated 58 weeks after MNU and at necropsy prostate complexes and all tumor-like lesions were collected for histology. Prostate cancer incidence was determined by enumerating grossly observed tumors and histopathologic evaluation using step sections. Overall incidence of large prostate tumors, histologically confirmed as adenocarcinomas, was reduced from 16/31 rats in the control group (52%) to 14/30 (46%) and 8/30 (27%) rats in the low and high dose black raspberry groups, resp., which trend was significant (p = 0.049; X2 test). The difference in incidence between control and high dose groups was borderline significant (p = 0.067, 2-sided; p = 0.41, 1-sided Fisher exact test). In subgroups of 4-5 rats, the prostate was collected after 3 weeks of feeding the diets and the concentrations of the black raspberry anthocyanin metabolite protocatechuic acid (PCA) were measured in the lateral prostate using LC-MS/MS. Mean (± SD) prostatic concentrations of PCA (ng/g wet tissue) were 0.5 ± 0.0 (detection limit), 3.64 ± 1.35 (p Citation Format: Maarten C. Bosland, Michael J. Schlicht, Jillian Eskra, Zhongfa Liu. Inhibitory effects of black raspberry consumption on prostate 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 LB-185. doi:10.1158/1538-7445.AM2013-LB-185

In the 1930s and 1940s, Lloyd George became one of the most widely planted cultivars of red raspberry (Rubus idaeus) in Britain. It was grown for its flavor and quality and, in Scotland, for its resistance to soil-borne viruses. However, by the late 1950s, it had virtually disappeared from commercial production. Its rapid demise was attributed to the widespread incidence of “bushy dwarf” disease (Cadman and Harris, 1951), also known as “symptomless decline” (Cadman, 1952), which seriously decreased plant size, vigor, and fruit yield. Cadman (1961) detected in diseased plants a sap-transmissible virus that, because of its consistent association with the disease, he named raspberry bushy dwarf virus (RBDV). However, Barnett and Murant (1970) found that, on its own in young Lloyd George raspberry plants, RBDV did not induce obvious disease. Jones (1979) confirmed this by detailed observations of the growth, over 2 years, of RBDV-infected Lloyd George raspberry plants grown in pots in a gauze house. Nevertheless, he showed in the same experiment that, in mixed infection with black raspberry necrosis virus (BRNV), an aphid-borne virus, RBDV intensified the degeneration in vigor [characterized by stunting and proliferation (bushiness) of canes] caused by infection with BRNV alone. RBDV therefore does play a causal part in the total bushy dwarf syndrome.

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&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.

Random amplification of polymorphic DNA (RAPD) and NMR techniques were used to differentiate and identify species of black raspberry (BR) of different geographic origins. BR leaf samples from five geographic origins were identified as Rubus japonicus and R. coreanus by RAPD. 1H-NMR analysis was also performed for BR fruit extracts from different geographic origins. Major compounds assigned in 1H-NMR spectra of BR fruits were amino acids, organic acids, sugars, phenolic acids, and purine derivatives. In addition, relative levels of total phenolic compounds, flavanols, flavonoids, and anthocyanins in the BR fruits were further analyzed. Hierarchical cluster analyses (HCA) based on the genetic (RAPD of leaf samples) and metabolic (1HNMR, total phenolic compounds, flavanols, flavonoids and anthocyanins of fruit samples) datasets were independently performed. The HCA dendrogram pattern derived from RAPD genetic fingerprinting of BR leaf samples matched that from the 1H-NMR data of BR fruit samples. This research demonstrates that metabolic profiling of BR fruit based on 1H-NMR is a promising method for differentiating BR fruits of various species and geographic origins.

We previously reported in a Phase I clinical trial that black raspberry (BRB) powder is well tolerated by humans when administered in a slurry of water at 45g/day for 7 days. We then undertook a study to determine if BRB might regress rectal polyps in familial adenomatous polyposis (FAP) patients. Fourteen FAP patients who had undergone a colectomy were treated with BRB daily for a period of nine months. Seven patients received BRB powder (20g/3x/day) orally in water plus two suppositories (each composed of 700 mg BRB) that patients inserted into the rectum one hour before bedtime. The other seven patients were randomized to receive an oral placebo plus the two rectal suppositories. Rectal polyp counts were taken at time zero and after nine months of BRB treatment. The number of rectal polyps was reduced by a median of 43% at nine months overall including a median reduction of 59% in patients treated by both routes and 36% in patients treated with the suppositories only. Polyps and aberrant crypt foci, both were histopathologically identified as tubular adenomas, and adjacent normal tissues were collected both before and after berry treatment. Promoter methylation of CDKN2A, also known as p16, in collected specimens was measured using MassARRAY. Global methylation LINE-1 was determined by Pyrosequencing. MBDCap-seq genome-wide methylation analysis was used to discover other genes demethylated by berries. Further, the specimens were evaluated for p16, Ki-67, TUNEL, DNMT1 and DNMT3B expression by semi-quantitative immunohistochemistry. Our results showed that treatment with BRB powder, both orally and in the form of a BRB suppository, significantly reduced cell proliferation in the tubular adenomas. The suppository alone was sufficient to decrease promoter methylation of p16 leading to an increase in p16 protein expression in adjacent normal rectum and in tubular adenomas. This was associated with decreased protein expression of DNMT1 and DNMT3B. Based on MBDCap-seq data, BRBs significantly demethylated promoter CpGs of 44 genes in tubular adenomas. These genes are involved in the regulation of important cellular functions; e.g., cell proliferation, Wnt signaling and Notch pathway, etc. Lastly, berry treatment did not induce changes in global methylation LINE-1. In conclusion, our data provide evidence that one of the mechanisms for BRB-induced rectal polyp regression in FAP patients is through the demethylation and reactivation of tumor suppressor genes.(Supported by NCI grants CA148818 and CA103180, and USDA grant 38903-03560). 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 1009. doi:1538-7445.AM2012-1009

Dark-colored berries are gaining increasing attention lately for their chemopreventive and chemotherapeutic activities. Jamun (Syzygium cumini L.), the Indian blackberry is perhaps the only berry that has the diversity of anthocyanins of blueberry and abundance of ellagic acid present in black raspberry. We previously reported chemoprotective activities in jamun pulp extract and powder in vitro and short-term in vivo studies (Aqil et al. AACR 2010, Vol. 51, Abst. # 5688). Here we report anti-cancer potential of jamun pulp powder against estrogen mediated breast cancer and the role of miRNAs and other targets in the disease inhibition. Groups of female ACI rats were given AIN-93M diet or diet supplemented with jamun pulp powder (5%, w/w). Two weeks later, animals from each group were treated with subcutaneous silastic implants of 17β-estradiol (1.2 cm; 9 mg). Additional groups of animals received control or experimental diet in the absence of estrogen. Diet and water were provided ad libitum. Twelve weeks later animals were palpated weekly for the mammary tumors, and all groups were euthanized when tumors incidence reached over 90% in control group. Compared to control, jamun-supplemented diet delayed the first tumor appearance by 21 days, and significantly reduced the tumor incidence (65% vs 96%), tumor burden (313±95 vs 661±123 mm3) and tumor multiplicity (1.8±0.3 vs 4.2±0.4 tumors/rat). The experimental diet also significantly reduced the estrogen-associated growth of pituitary prolactinomas, circulating prolactin and estradiol levels and the mammary cell proliferation (PCNA) markers. Mechanistically, the jamun diet significantly offset estrogen-associated increases in mammary cell proliferation, ER-α and cyclin D1 as determined by western blot analysis. We have recently identified ‘miRNA signature’ associated with estrogen treatment (Munagala et al. AACR 2011, Vol. 52, Abst. # 155). All miRNAs that were either significantly overexpressed (miR-182 and miR-375) or underexpressed (miR-127 and miR-206) as a result of estrogen treatment were significantly protected by intervention with the jamun diet. Targets for these miRNAs remain to be identified. Together, our data show that jamun significantly offset estrogen-mediated alterations in i) mammary cell proliferation; ii) ER-α; iii) cyclin D1, and iv) candidate miRNAs, and that the modulation of these biomarkers correlated with the reduction in mammary carcinogenicity. (Supported by CA-125152, CA-118114 and Agnes Brown Duggan Endowment). 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 5447. doi:1538-7445.AM2012-5447

Ascertaining the potentials of diet-derived phenolics in eradicating cancer stem cells (CSCs), that proliferate and self-renew extensively leading to the initiation and progression of tumor, may escort significant clinical implications. Recently, we have shown that black raspberry extract (RSE) potentiates pancreatic cancer (PC) cell killing by targeting transcriptional machinery. Herein, we investigated ex vivo the effect of RSE on the regulation of EMT, pluripotency maintaining and other stem cell related molecules in PC-CSCs. CD133+CD44+CD24+ESA+ PC-CSCs isolated from human PC xenografts developed in athymic NCr-nu/nu nude mice with human MiaPaCa2, BXPC3, Panc-1 or Panc-3.27 cells were maintained in vitro. PC-CSCs exposed to RSE (2.0μg) for 3h were assessed for transcriptional regulation of 93 stem cell related molecules using QPCR profiling. Expression and cellular localization of epithelial-to-mesenchymal transition (EMT) markers and pluripotent maintaining factors including ABCG2, E-Cadherin, N-Cadherin, MYB, MYC, Nanog and SOX2 in MiaPaCa2, BXPC3 xenografts were assessed using immunohistochemistry (IHC). The results revealed that RSE significantly inhibited 83, 50 and 54 stem cell related molecules in PC-CSCs isolated from xenografts developed using MiaPaCa2, Panc-1 and Panc 3.27 respectively. Of these, 27 genes were commonly suppressed by RSE in all three PC-CSCs population. Further, IHC staining showed complete repression of EMT and pluripotent markers in both MiaPaCa2 and BXPC3 xenografts. Together, these data imply that RSE may exert selective inhibition of EMT and pancreatic CSCs’ self renewal capacity and, may thus serve as a potential “deliverable” to negate PC initiation and progression. 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 1615. doi:1538-7445.AM2012-1615

Our laboratory has shown that the anthocyanins (ACs) in black raspberries (BRBs) are responsible for much of their chemopreventive effects in the rat esophagus. The present study was undertaken to determine the effects of BRB ACs on human colon cancer cells in vitro. Three days of AC treatment at 5 and 25 μg/ml medium significantly decreased cell proliferation and increased apoptosis in HCT116, Caco2, and SW480 human colon cancer cell lines. The ACs also reduced the activities and protein expression levels of DNA methyltransferases 1 and 3 (DNMT1, DNMT3) in all three cell lines. Promoter methylation of p16 (CDKN2A) and the Wnt pathway inhibitors, Sfrp2, Sfrp5 and Wif1, was decreased by AC treatment resulting in increased mRNA expression levels of all three Wnt inhibitors. ACs did not alter global methylation LINE-1. DNMT1 and DNMT3B knockout (KO) HCT116 cells were generated using siRNA to determine the role of these methyltransferases in AC-induced demethylation. In DNMT1 KO cells, the promoter methylation of Sfrp5 was reduced when compared with wild type HCT116 cells. AC treatment further reduced promoter methylation of Sfrp5 in DNMT1 KO cells suggesting that the ACs targeted protein(s) other than DNMT1 to regulate methylation. Promoter methylation of Sfrp5 was decreased to almost zero by treatment of DNMT3B KO cells with ACs. Interestingly, promoter methylation of p16 was reduced in both DNMT1 and DNMT3B KO cells, and AC treatment further reduced p16 methylation in both lines. In conclusion, these results suggest that ACs demethylated and reactivated tumor suppressor genes through regulation of DNMT1 and DNMT3B. This research was supported by NCI grant CA148818. 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 1628. doi:1538-7445.AM2012-1628

A number of otolaryngologic inflammatory and neoplastic diseases are associated with gastric reflux extending proximal to the esophagus. Extraesophageal reflux, also known as laryngopharyngeal reflux (LPR), increases the risk of developing laryngeal cancer by approximately two-fold and analyses of neoplastic development in patients with prior gastrectomy suggest that nonacid components of refluxate promote laryngeal carcinogenesis as well. Clinical and basic science research implicates gastric pepsin as a key deleterious agent of LPR. Our previous research demonstrates that pepsin at pH7 induces hyperproliferation and inflammatory and cell cycle regulatory gene expression changes in airway epithelial cells in vitro. Acid suppression therapy through proton-pump inhibitors (PPI's), the most commonly prescribed therapeutic for reflux, has demonstrated minimal efficacy in resolution of LPR-attributed disease. Phytochemicals derived from algae, pine, and most recently Noni fruit, however, have been shown to not only attenuate reflux-attributed lesions and inflammation in rat models of disease but also directly inhibit peptic activity. This study examines the potential of plant polyphenols curcumin, ecabet sodium (ES) and an anthocyanin-enriched fraction of freeze-dried black raspberry (BRB) to inhibit peptic activity and pepsin-mediated cell damage and proliferative changes in cultured human laryngeal and pharyngeal cells. Cultured human laryngeal epithelial cells from punch biopsy, pharyngeal carcinoma (FaDu) and gastric adenocarcinoma (AGS) cells were pre-treated with/without curcumin (10uM), ES (125ug/ml) and BRB (100ug/ml) for 30 minutes, rinsed and subsequently treated with/without nonacid pepsin (0.1mg/ml, pH7.0) for 1 hour. Morphological changes were examined by transmission electron microscopy and proliferative changes by cell count assay and realtime PCR array (curcumin only). Direct inhibition of peptic activity was determined by in vitro kinetic assay. Curcumin, ES and BRB significantly inhibited peptic activity (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B168.

Berries, particularly blueberry (BB), black raspberry (BRB), are gaining increased attention for their chemopreventive and chemotherapeutic activities and have been tested in limited clinical studies with mixed results. Previous studies with strawberry and BRB failed to elicit protection against NNK/PAH-induced lung carcinogenicity in the A/J mice (the Stoner studies). However, a low dose of a mixture of different berries (BB, BRB, blackberry, and strawberry) elicited modest inhibition of cigarette smoke-mediated lung carcinogenicity in our study. In this study we investigated chemopreventive and chemotherapeutic activities of BB and BRB using two different animal models: To determine the DNA adduct-inhibiting potential female A/J mice were provided AIN-93M diet or diets supplemented with lyophilized powder of BB or BRB (5% each, w/w) and then challenged with continuous low doses of dibenzo-[a,l]pyrene (DBP) by subcutaneous polymeric implants (two, 20 mm long, 1.4 mm dia; DBP load 5%). Three weeks later animals were euthanized and the target tissue (lung) DNA was analyzed by 32P-postlabeling assay. The DNA adduct burden (as adducts/109 nucleotides) in animals receiving the BB (67 ± 16), BRB (68 ± 9) diets was found to be unaffected compared with control diet (54 ± 17), indicating that the berry bioactives were ineffective against the DNA adduct-forming ability of DBP under the conditions used. To determine the chemotherapeutic potential, female nude mice received AIN-93M (control) diet or diet supplemented with a mixture of BB and BRB (2:1) and 4 days later inoculated with human lung cancer A549 cells (3×106 cells). Four weeks into the ongoing study, the berry diet has shown significant (nearly 60%) inhibition of the lung tumor xenograft (186 ± 117 versus 79 ± 47). The anti-tumor activity elicited by the berry diet in xenograft model is consistent with the anti-tumor activity of anthocyanidins reported by us in cell culture and nude mice (Kausar et al. AACR 51: 1884, 2010). In conclusion, our data suggest that the anti-lung cancer potential of the berries may be associated with mechanisms other than inhibition of carcinogen-induced DNA adducts. (Supported from CA-118114, KLCRP and Agnes Brown Duggan Endowment). 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 4597. doi:10.1158/1538-7445.AM2011-4597

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 < 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 < 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 < 0.005), 1.3-fold (P < 0.005), 1.3-fold (P < 0.005), 1.4-fold (P < 0.005) and 1.4-fold (P < 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 < 0.005), 1.5-fold (P < 0.005), 1.6-fold (P < 0.005), 1.5-fold (P < 0.005) and 1.6-fold (P < 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

Chemoprevention by polyphenolics in fruits, particularly in berries and pomegranate, is gaining increased attention. To determine bioavailability of ellagic acid/ ellagitannins, first, we fed ACI rats with dietary black raspberry powder (5% w/w; 75 ppm ellagic acid in diet). Analysis of plasma samples by solvent extractions and HPLC showed that the levels of ellagic acid in plasma had plateaued at 3 weeks: 399 ± 85, 249 ± 98 and 344 ± 23 ng/ml at 3, 12 and 28 weeks, respectively. In the second study, we fed ACI rats with ellagic acid at 400 ppm in diet. In order to increase bioavailability, we also delivered ellagic acid systemically by subcutaneous silastic implants. The circulating levels of ellagic acid were found to plateaue at 8 weeks in the experimental diet group: 1,167 ± 171, 1,458 ± 302 and 1,272 ± 192 ng/ml at 8, 16 and 28 weeks, respectively. The ellagic acid levels, however, continued to increase when delivered by silastic implants: 53 ± 15, 130 ± 30 and 185 ± 72 ng/ml at 8, 16 and 28 weeks, respectively. Notably, it required nearly 130-fold higher dose via diet (4 mg/day) compared with implant route (0.03 mg/day) to achieve nearly 7-fold higher plasma ellagic acid levels. The oral bioavailability of ellagic acid was estimated at only 0.2%, whereas over 5% was bioavailable when administered by sustained systemic delivery. To determine if oral bioavailability of ellagic acid can be increased by administering it in the form of ellagitannins, tissues were taken from another study where female S/D rats were fed diet supplemented with equimolar doses of punicalagins (1,500 ppm) or ellagic acid (430 ppm) and treated with benzo[a]pyrene. Groups of animals were also administered punicalagins and ellagic acid via subcutaneous polycaprolactone implants (20% and 10% load, respectively). Punicalagins were undetectable by either route of administration, but its break down product ellagic acid was detected in the plasma. Dietary administration of punicalagins (17 mg/day) resulted in plasma ellagic acid levels of 3.84 ± 1.37 ng/ml corresponding to only slight (150-fold higher plasma ellagic acid levels (589 ± 78 ng/ml), resulting in much higher bioavailability (0.85%). Ellagic acid administration via the diet (5 mg/day) and polycaprolactone implants (about 0.16 mg/day) showed 317 ± 39 and 673 ± 101 ng/ml plasma ellagic acid, respectively. In conclusion, i) when administered via the diet either as pure compound or as a component of black raspberry, there was no significant difference in the bioavailability of ellagic acid; however sustained, systemic delivery enhanced the bioavailability of ellagic acid by 25-fold; and ii) oral bioavailability of ellagic acid when administered as free ellagic acid was much higher than when administered as ellagitannins. (USPHS CA-18114 (RCG), CA-143676 (MVV) and the Duggan Endowment (RCG)). 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 4603. doi:10.1158/1538-7445.AM2011-4603

This study investigated the relationship between the anthocyanin concentration in a series of organic fruit species and the photoelectrochemical performance of the TiO2 dye-sensitized solar cells (DSSCs) sensitized by these dyes. It was hypothesized that organic dyes with a greater anthocyanin concentration have higher fill factors (FFs) and maximum output power (PMAX), which would lead to higher energy conversion efficiency. Anthocyanin dye solution was extracted from eight test fruits using water as the extracting solvent. Using these test organic dyes, multiple DSSCs were assembled such that light entered through the TiO2 side of the cell. The full current-voltage (I-V) covariations were measured at various incremental resistance values. Defining PMAX as the dependent variable, a series of linear, semi-logarithmic, quadratic, and finally power law regression models were used to investigate the relationship between anthocyanin concentration and photoelectrochemical performance. Regression analysis demonstrated that the power law model (R2 = 0.86) was the best fit and experimentally sound model to predict the relationship between PMAX and anthocyanin concentration. Based on this model, anthocyanin content and PMAX relation approaches zero for zero concentration and follows a sublinear increase for higher concentration. Dyes extracted from blueberry and black raspberry with the highest anthocyanin content generated higher PMAX with better FF and conversion efficiency. Estimating these linear and power law models is the first step in finding organic anthocyanin sources in nature that have the highest dye content to efficiently generate energy.

Introduction Cervical cancer is the 3rd most common cancer globally. The long latency from dysplasia to invasive cancer makes it amenable to prevention strategies. Phytochemicals, including those in lyophilized black raspberries (LBRs), are of great interest due to their growth inhibitory and anti-tumorigenic effects in preclinical models. Previously we showed that LBR extracts inhibited the proliferation of human cervical cancer cells in vitro in a dose- and time-dependent manner. Angiogenesis is an important part of carcinogenesis. We hypothesized that LBRs modulate the expression of an angiogenesis associated signaling cascade. Methods Human cervical cancer cells (SiHa) were treated with vehicle control (.02% DMSO) or LBR extract (200 µg/mL) for 1 or 3 days (d). Cells were harvested at 1d or 3d and RNA was extracted. Gene expression profiling was performed using a TaqMan Gene Signature Angiogenesis Array, which simultaneously screens 94 genes involved in angiogenesis. Genes demonstrating ≥2-fold change in expression following geNorm transformation were used for Ingenuity Pathway Analysis (IPA) network modeling. Biomarkers modulated in a manner favorable for cancer prevention were validated by real time RT-PCR. Results Short-term LBR exposure of SiHa cells in vitro resulted in inhibition of cell proliferation, and reduced expression of IL8 and MMP2 genes detected using the Angiogenesis Array. IPA network modeling predicted that MMP2 and NFkB would be part of the IL8 signaling cascade and this is supported in the literature. Validation with real time RT-PCR showed a 24-fold (1d) and 28-fold (3d) reduction in IL8 expression following LBR exposure. However, MMP2 and NFkB did not demonstrate a significant differential expression at either time point. Conclusion Short-term exposure of cervical cancer cells to LBR results in a striking reduction of IL8 gene expression. Although MMP2 and NFkB are associated with known IL8 signaling mechanisms, they are not modulated by LBR extract in this in vitro model. Therefore, the growth inhibitory activity and transcriptional repression of IL8 following LBR extract exposure proceed via an MMP2-NFkB-independent cascade. Other signaling mechanisms within the IL8 signaling cascade (HIF, AP1, STAT3) may be involved and will be further explored. Since IL8 plays an important role in tumor growth, metastasis, and angiogenesis, it supports the potential for phytonutrient-based chemoprevention of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2904.

We previously demonstrated protection with berries in diet, against estrogen-induced ACI rat mammary tumorigenesis, by inhibition of proliferation as early as 3 weeks of estrogen administration. In this study, blueberry (BB) and black raspberry (BRB) were administered in diet at 5% 2 weeks prior to implantation with 1.2 cm silastic implants containing 9 mg 17β-estradiol. Rats were killed at 3 weeks, 3 months and ∼7 months, when the tumor incidence in control group reached 80%. Analysis revealed significant reduction (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1887.