Claire Seguin

Modulation of Genetic and Epigenetic Biomarkers of Colorectal Cancer in Humans by Black Raspberries: A Phase I Pilot Study

Purpose: This study evaluated the effects of black raspberries (BRBs) on biomarkers of tumor development in the human colon and rectum including methylation of relevant tumor suppressor genes, cell proliferation, apoptosis, angiogenesis, and expression of Wnt pathway genes. Experimental Design: Biopsies of adjacent normal tissues and colorectal adenocarcinomas were taken from 20 patients before and after oral consumption of BRB powder (60 g/d) for 1–9 weeks. Methylation status of promoter regions of five tumor suppressor genes was quantified. Protein expression of DNA methyltransferase 1 (DNMT1) and genes associated with cell proliferation, apoptosis, angiogenesis, and Wnt signaling were measured. Results: The methylation of three Wnt inhibitors, SFRP2, SFRP5, and WIF1, upstream genes in Wnt pathway, and PAX6a, a developmental regulator, was modulated in a protective direction by BRBs in normal tissues and in colorectal tumors only in patients who received BRB treatment for an average of 4 weeks, but not in all 20 patients with 1–9 weeks of BRB treatment. This was associated with decreased expression of DNMT1. BRBs modulated expression of genes associated with Wnt pathway, proliferation, apoptosis, and angiogenesis in a protective direction. Conclusions: These data provide evidence of the ability of BRBs to demethylate tumor suppressor genes and to modulate other biomarkers of tumor development in the human colon and rectum. While demethylation of genes did not occur in colorectal tissues from all treated patients, the positive results with the secondary endpoints suggest that additional studies of BRBs for the prevention of colorectal cancer in humans now appear warranted. Clin Cancer Res; 17(3); 598–610. ©2010 AACR.

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.

A Phase Ib Study of the Effects of Black Raspberries on Rectal Polyps in Patients with Familial Adenomatous Polyposis

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

Mechanistic basis for the chemopreventive effects of black raspberries at a late stage of rat esophageal carcinogenesis

The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA‐ and NMBA + BRB‐treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA‐treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17 846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17 846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell–cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis.

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.

Chemopreventive Effects of Berries and Berry Components in the Rodent Esophagus

Esophageal squamous cell carcinoma (ESCC) is the 7th leading cause of cancer death worldwide. Its etiology and late detection rate make it an important target for intensive studies in chemoprevention and treatment. The Fischer-344 rat model of esophageal squamous cell carcinoma has been used extensively to evaluate the ability of black raspberries (BRBs) and other berry types to inhibit esophageal tumorigenesis. In this model, tumors are induced by the nitrosamine carcinogen, N-nitrosomethylbenzylamine (NMBA). Dietary BRBs inhibit both the initiation and promotion/progression stages of esophageal tumorigenesis in the rat. They inhibit initiation events by influencing the metabolic activation and detoxification of NMBA, and promotion/progression events by reducing cell proliferation, inflammation, and angiogenesis and by stimulating apoptosis and differentiation. Genes associated with these cellular functions are positively modulated by BRBs. Biofractionation studies indicate that amongst the most active chemopreventive agents in BRBs are the anthocyanins. An anthocyanin-enriched fraction of BRBs was nearly as active as whole BRBs themselves in preventing esophageal tumorigenesis. However, the ellagitannins and other constituents in the fiber fraction of BRBs are also active and studies are underway to further identify these constituents. In a recent study, 7 different berry types, including the “exotic” berries (acai, noni and wolfberry [goji]), were compared for their ability to inhibit NMBA-tumorigenesis in the rat esophagus. All seven types were active, irrespective of their content of anthocyanins and ellagitannins. It appears that each berry type has unique constituents with cancer preventive potential.

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 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.