Li-Shu Wang

Anthocyanins and their role in cancer prevention

Anthocyanins are the most abundant flavonoid constituents of fruits and vegetables. The conjugated bonds in their structures, which absorb light at about 500 nm, are the basis for the bright red, blue and purple colors of fruits and vegetables, as well as the autumn foliage of deciduous trees. The daily intake of anthocyanins in residents of the United States is estimated to be about 200 mg or about 9-fold higher than that of other dietary flavonoids. In this review, we summarize the latest developments on the anti-carcinogenic activities of anthocyanins and anthocyanin-rich extracts in cell culture models and in animal model tumor systems, and discuss their molecular mechanisms of action. We also suggest reasons for the apparent lack of correlation between the effectiveness of anthocyanins in laboratory model systems and in humans as evidenced by epidemiological studies. Future studies aimed at enhancing the absorption of anthocyanins and/or their metabolites are likely to be necessary for their ultimate use for chemoprevention of human cancer.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Anthocyanins in Black Raspberries Prevent Esophageal Tumors in Rats

Diets containing freeze-dried black raspberries (BRB) suppress the development of N-nitrosomethylbenzylamine (NMBA)–induced tumors in the rat esophagus. Using bioassay-directed fractionation, the anthocyanins in BRB were found to be the most active constituents for down-regulation of carcinogen-induced nuclear factor-κB and activator protein-1 expression in mouse epidermal cells in vitro. The present study was undertaken, therefore, to determine if the anthocyanins contribute to the chemopreventive activity of BRB in vivo. F344 rats consumed diets containing either (a) 5% whole BRB powder, (b) an anthocyanin-rich fraction, (c) an organic solvent-soluble extract (a–c each contained ∼3.8 μmol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 μmol anthocyanins/g diet), (e) a hexane extract, and (f) a sugar fraction (e and f had only trace quantities of anthocyanins), all derived from BRB. Animals were fed diets 2 weeks before treatment with NMBA and throughout the bioassay. Control rats were treated with NMBA only. Animals were killed at week 30, and esophageal tumors were enumerated. The anthocyanin treatments (diet groups a–c) were about equally effective in reducing NMBA tumorigenesis in the esophagus, indicating that the anthocyanins in BRB have chemopreventive potential. The organic-insoluble (residue) fraction (d) was also effective, suggesting that components other than berry anthocyanins may be chemopreventive. The hexane and sugar diets were inactive. Diet groups a, b, and d all inhibited cell proliferation, inflammation, and angiogenesis and induced apoptosis in both preneoplastic and papillomatous esophageal tissues, suggesting similar mechanisms of action by the different berry components.

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.

Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa that can dramatically increase the risk of colon cancers. In the present study, we evaluated the effects of a dietary intervention of freeze-dried black raspberries (BRB), a natural food product with antioxidant and anti-inflammatory bioactivities, on disease severity in an experimental mouse model of UC using 3% dextran sodium sulfate (DSS). C57BL/6J mice were fed either a control diet or a diet containing BRB (5 or 10%) for 7-14 days and then the extent of colonic injury was assessed. Dietary BRB markedly reduced DSS-induced acute injury to the colonic epithelium. This protection included better maintenance of body mass and reductions in colonic shortening and ulceration. BRB treatment, however, did not affect the levels of either plasma nitric oxide or colon malondialdehyde, biomarkers of oxidative stress that are otherwise increased by DSS-induced colonic injury. BRB treatment for up to 7 days suppressed tissue levels of several key pro-inflammatory cytokines, including tumor necrosis factor α and interleukin 1β. Further examination of the inflammatory response by western blot analysis revealed that 7 day BRB treatment reduced the levels of phospho-IκBα within the colonic tissue. Colonic cyclooxygenase 2 levels were also dramatically suppressed by BRB treatment, with a concomitant decrease in the plasma prostaglandin E₂ (276 versus 34 ng/ml). These findings demonstrate a potent anti-inflammatory effect of BRB during DSS-induced colonic injury, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events.

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.

Black raspberry components inhibit proliferation, induce apoptosis, and modulate gene expression in rat esophageal epithelial cells.

We have shown that a diet containing freeze-dried black raspberries (BRB) inhibits the development of chemically induced cancer in the rat esophagus. To provide insights into possible mechanisms by which BRB inhibit esophageal carcinogenesis, we evaluated an ethanol (EtOH) extract of BRB, and two component anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O−rutinoside) in BRB, for their effects on growth, apoptosis, and gene expression in rat esophageal epithelial cell lines. The EtOH extract and both anthocyanins selectively caused significant growth inhibition and induction of apoptosis in a highly tumorigenic cell line (RE-149 DHD) but not in a weakly tumorigenic line (RE-149). The uptake of anthocyanins from the EtOH extract into RE-149 DHD cells far exceeded their uptake into RE-149 cells, which may have accounted for the selective effects of the extract on growth and apoptosis of RE-149 DHD cells. The growth inhibitory and proapoptotic effects were enhanced by the daily addition of the EtOH extract and the anthocyanins to the medium. Interestingly, the EtOH extract did not alter cyclooxygenase-2 (COX-2) and nitric oxide synthase (i-NOS) expression in RE-149 DHD cells, whereas both anthocyanins downregulated the expressions of these genes. This differential effect may have been related to the relative amounts of anthocyanins in the extract vs. when they were added individually to the medium. We conclude that the selective effects of the EtOH extract on growth and apoptosis of highly tumorigenic rat esophageal epithelial cells in vitro may be due to preferential uptake and retention of its component anthocyanins, and this may also be responsible for the greater inhibitory effects of freeze-dried whole berries on tumor cells in vivo.

Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Our recent study identified 2,261 dysregulated genes in the esophagi of rats that received a 1-week exposure to the carcinogen N-nitrosomethylbenzylamine (NMBA). We further reported that 1,323 of these genes were positively modulated to near-normal levels of expression in NMBA-treated animals that consumed dietary phenylethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables. Herein, we report our results with companion animals that were fed a diet containing 5% freeze-dried black raspberries (BRB) instead of PEITC. We found that 462 of the 2,261 NMBA-dysregulated genes in rat esophagus were restored to near-normal levels of expression by BRB. Further, we have identified 53 NMBA-dysregulated genes that are positively modulated by both PEITC and BRB. These 53 common genes include genes involved in phase I and II metabolism, oxidative damage, and oncogenes and tumor suppressor genes that regulate apoptosis, cell cycling, and angiogenesis. Because both PEITC and BRB maintain near-normal levels of expression of these 53 genes, their dysregulation during the early phase of NMBA-induced esophageal cancer may be especially important in the genesis of the disease.

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.

Profiling DNA methylomes from microarray to genome-scale sequencing.

DNA cytosine methylation is a central epigenetic modification which plays critical roles in cellular processes including genome regulation, development and disease. Here, we review current and emerging microarray and next-generation sequencing based technologies that enhance our knowledge of DNA methylation profiling. Each methodology has limitations and their unique applications, and combinations of several modalities may help build the entire methylome. With advances on next-generation sequencing technologies, it is now possible to globally map the DNA cytosine methylation at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes.