Kristen Stoner

Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

An overview of Epigenetics and Chemoprevention

It is now appreciated that both genetic alteration, e.g. mutations, and aberrant epigenetic changes, e.g. DNA methylation, cause cancer. Epigenetic dysregulation is potentially reversible which makes it attractive as targets for cancer prevention. Synthetic drugs targeting enzymes, e.g. DNA methyltransferase and histone deacetylase, that regulate epigenetic patterns are active in clinical settings. In addition, dietary factors have been suggested to have potential to reverse aberrant epigenetic patterns. Uncovering the human epigenome can lead us to better understand the dynamics of DNA methylation in disease progression which can further assist in cancer prevention.

Dietary black raspberries modulate DNA methylation in dextran sodium sulfate (DSS)-induced ulcerative colitis

UNLABELLED Ulcerative colitis (UC) is characterized by chronic inflammation of the colon. During inflammation, NF-κB is increased in colonic epithelial cells and in immune cells, leading to increases in proinflammatory cytokines. These events then increase DNA methyltransferases (DNMTs), which silence a subset of tumor suppressor genes by promoter methylation. Negative regulators of the Wnt pathway are frequently methylated in UC, leading to dysregulation of the pathway and, potentially, to colorectal cancer. We determined if black raspberries (BRBs) influence promoter methylation of suppressors in the Wnt pathway in dextran sodium sulfate (DSS)-induced UC. C57BL/6J mice received 1% DSS and were fed either control or 5% BRB diets. Mice were euthanized on days 7, 14 and 28, and their colons, spleen and bone marrow were collected. Berries reduced ulceration at day 28. This was accompanied by decreased staining of macrophages and neutrophils and decreased NF-κB p65 nuclear localization in the colon at all time points. At day 7, BRBs demethylated the promoter of dkk3, leading to its increased messenger RNA (mRNA) expression in colon, spleen and bone marrow. β-Catenin nuclear localization, c-Myc staining as well as protein expression of DNMT3B, histone deacetylases 1 and 2 (HDAC1 and HDAC2) and methyl-binding domain 2 (MBD2) were all decreased in colon; mRNA expression of these four proteins was decreased in bone marrow cells by BRBs. These results suggest that BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation. SUMMARY Our results suggest that dietary BRBs suppress colonic ulceration by correcting promoter hypermethylation of suppressor genes in the colon, as well as in the spleen and bone marrow that systematically regulate inflammation in DSS-induced UC.

Berry ellagitannins may not be sufficient for prevention of tumors in the rodent esophagus

Biodirected fractionation is used to identify the active inhibitory constituents in berries for esophageal cancer in rats. The present study was undertaken to determine if ellagitannins contribute to the chemopreventive activity of an alcohol/water-insoluble (residue) fraction of berries. Rats consumed diets containing residue fractions of three berry types, that is, black raspberries (BRBs), strawberries (STRWs), and blueberries (BBs), that differ in their content of ellagitannins in the order BRB > STRW > BB. Animals were fed residue diets beginning 2 weeks before treatment with the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA) and throughout the 30-week bioassay. Residue fractions from all three berry types were about equally effective in reducing NMBA tumorigenesis in the rat esophagus irrespective of their ellagitannin content (0.01-0.62 g/kg of diet). These results suggest that the ellagitannins may not be responsible for the chemopreventive effects of the alcohol/water-insoluble fraction of berries.

Abstract 1009: Regression of rectal polyps in familial adenomatous polyposis patients by freeze-dried black raspberries is associated with the demethylation and reactivation of tumor suppressor genes

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

Abstract 1628: Tumor suppressor gene demethylation and reactivation in human colon cancer cells by black raspberry anthocyanins

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. 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 816. doi:10.1158/1538-7445.AM2011-816

Abstract A51: Comparison of different berry types to prevent chemically induced papilloma development in the rat esophagus

The present study compared the ability of seven different berry types [black and red raspberries, strawberries, blueberries, acai, noni, goji (wolfberry)] to inhibit N‐nitrosomethylbenzylamine (NMBA)‐induced tumor development in the F344 rat esophagus. These seven berry types have been shown to differ in their relative content of known chemopreventive agents such as anthocyanins, ellagitannins and carotenoids. Each berry type was picked when ripe, freeze‐dried under anoxic conditions to prevent decomposition of their chemical components, and ground into a powder. Beginning at 4 weeks of age, 120 F344 rats were injected with NMBA (0.3 mg/kg, 3x/wk for 5 weeks). Control rats (15) were injected with DMSO/water (80:20), the solvent for NMBA. Beginning one week after treatment with carcinogen, individual berry types were administered at 5% of the diet to groups of 15 NMBA‐treated rats until the end of the bioassay (35 weeks). At necropsy, the number of esophageal papillomas was enumerated in all groups of rats. No papillomas were seen in control rats treated with DMSO/water. All berry types caused a significant reduction in the number of NMBA‐induced papillomas when compared to rats treated with NMBA only. There was no significant difference in the relative ability of the different berry types to reduce tumor incidence, multiplicity or size. Histopathological studies showed that each berry type was about equally effective in preventing the conversion of preneoplastic lesions (dysplasias) to papillomas. These results suggest that the chemopreventive potential of berries for the rat esophagus is not restricted to black raspberries and strawberries as shown previously in our laboratory. They also suggest that berries that vary markedly in their content of anthocyanins, ellagitannins and carotenoids all exhibit chemopreventive potential in the rat esophagus. Supported by NCI grant CA103180 and USDA grant 38903‐03560. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A51.

Abstract A50: Black raspberry treatment in FAP patients shows re‐expression of methylation‐silenced genes

In a clinical trial, we found that freeze‐dried black raspberry (BRB) powder reduced the number of rectal polyps in patients with familial adenomatous polyposis (FAP). The present study was undertaken to determine if the berries influenced the growth and apoptosis of cells in rectal polyps. In addition, because the p16 tumor suppressor gene is inactivated by methylation in greater than 60 percent of colon tumors, we examined the levels of both p16 and of DNA methyltransferase 1 (DNMT1) expression in normal and polyp specimens. DNMT1 has been shown to be overly expressed in cultured human colon cancer cell lines and this correlated with silencing of the p16 gene. Polyps and normal tissues were obtained from FAP patients before and after 36 weeks of berry treatment. Immunohistochemical staining for Ki‐67 and c‐MYC showed that cell proliferation was significantly reduced by berry treatment in both normal rectum and in rectal polyps. TUNEL staining for apoptosis was observed to increase significantly in both normal rectum and in rectal polyps. There was a significant decrease in DNMT1 expression and an increase in p16 expression in berry‐treated normal and polyp specimens. These results suggest that nine months of black raspberry treatment resulted in reduced cell proliferation, increased apoptosis and p16 gene expression and decreased DNMT1 expression in both normal and polyp specimens taken from FAP patients. The p16/DNMT1 data suggest that black raspberries may act as a demethylating agent and should be further evaluated for their effects on the methylation status of other candidate tumor suppressor genes in colonic polyps and tumors. This research was supported by NCI grant CA103180 and USDA grant 38903‐03560. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A50.

Abstract A49: Evidence for gene demethylation in human colon cancer by black raspberries

Black raspberries (BRBs) exhibit cancer preventative effects in the human colon, in part, by reducing the growth rate and increasing the apoptosis of human colon tumor cells. Recently, we have initiated investigations to determine whether BRBs also cause re‐expression of genes in human colon cells that are silenced by aberrant hypermethylation. Cultured human colon cancer cell lines HCT116, Caco2 and SW480, were treated with an anthocyanin‐rich (AC) fraction of BRBs every day for five days. AC treatment caused a decreased activity of DNA methyltransferases in a substrate‐antibody assay (p These in vitro observations were confirmed by the examination of normal and tumorous tissue samples taken from 20 colon cancer patients who received daily dietary intervention of freeze‐dried BRB powder (60g/day) in drinking water for an average of 3 weeks. DNA from the tissue samples was analyzed by MassARRAY which revealed an overall decrease in p16 methylation in both normal and tumor tissues (p Immunohistochemical analysis of p16 and DNMT1 in the same tissue samples showed that BRB treatment increased p16 expression and decreased DNMT expression (p Citation Information: Cancer Prev Res 2010;3(1 Suppl):A49.