Abstract Freeze-dried black raspberry (BRB) powder is considered as a potential cancer chemopreventive agent. In this study, we fed azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated C57BL/6J mice with a diet containing BRB anthocyanins for 12 weeks, and this led to a reduction in colon carcinogenesis. These animals had consistently lower tumor multiplicity compared with AOM/DSS-treated mice not receiving BRB anthocyanins. In AOM/DSS-treated mice, the number of pathogenic bacteria, including Desulfovibrio sp. and Enterococcus spp., was increased significantly, whereas probiotics such as Eubacterium rectale, Faecalibacterium prausnitzii and Lactobacillus were dramatically decreased, but BRB anthocyanins supplement could reverse this imbalance in gut microbiota. BRB anthocyanins also caused the demethylation of the SFRP2 gene promoter, resulting in increased expression of SFRP2, both at the mRNA and protein levels. Furthermore, the expression levels of DNMT31 and DNMT3B, as well as of p-STAT3 were downregulated by BRB anthocyanins in these animals. Taken together, these results suggested that BRB anthocyanins could modulate the composition of gut commensal microbiota, and changes in inflammation and the methylation status of the SFRP2 gene may play a central role in the chemoprevention of CRC. Introduction The human large intestine is an extremely active fermentation site that is also inhabited by different bacterial species, with the highest concentration in the colon (up to 1012 cells per gram of feces) (1). The majority of these bacteria belong to the Firmicutes and Bacteroidetes genera, which participate in many physiological activities and metabolism, including the digestion of food, provision of energy for the colonic epithelial cells and regulation of the immune response (2). Not only does the composition of this bacterial community vary substantially among individuals, but it is also a dynamic community that is susceptible to changes driven by dietary factors and diverse disease conditions. Compositional changes in the gut microbial community can alter the permeability of the intestine, leading to inflammatory bowel disease, and even the progressive development of colorectal cancer (CRC). The mechanism by which intestinal microbiota participates in the formation and development of CRC is still unclear. One generally accepted theory is that intestinal microbiota plays a fundamental role in the development of inflammatory bowel disease, and long-term chronic inflammation can considerably increase the risk of CRC (3). Therefore, improvement in intestinal microbiota is considered a potential therapy for the prevention of inflammatory bowel disease and CRC. DNA methylation at CpG islands is a major epigenetic modification that strongly correlates with carcinogenesis (4). A series of genes have been shown to be hyper- or hypomethylated during tumorigenesis (5). Frequent promoter hypermethylation and gene silencing have previously been observed for the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer (CRC) (6). SFRPs possess a domain similar to the domain in the WNT-receptor frizzled proteins that comprise a group of WNT antagonists, and therefore, it can inhibit the binding of WNT receptor to its ligands and downregulate the Wnt/β-catenin signaling pathway during development (7,8). SFRP2 is a member of the family of SFRP genes. In CRC tissues and certain colon cancer cell lines, hypermethylation of the promoters of SFRP genes can lead to reduced level of SFRPs, like SFRP4 and SFRP5, and the subsequent aberrant WNT signaling (9). Furthermore, DNA methylation can be affected by chronic inflammation. For example, during Helicobacter pylori-induced gastric carcinogenesis, the pro-inflammatory cytokine interleukin-1 beta (IL-1β) enhances DNMT activity via nitric oxide production, which results in CpG methylation-mediated gene silencing (10). The most commonly accepted theory is that an intricate and dynamic relationship might exist among the gut microbiota, immune system and epigenetic modifications (11). Anthocyanins are phytochemicals that are present in abundance in a wide variety of food, including tea, coffee, chocolate, wine, fruit or vegetables with a dark color (2). A diet rich in vegetables and fruits can reduce the risk of various cancers, especially colon cancer. Previous studies have shown that dietary anthocyanins and their metabolites can contribute to the maintenance of gut health by stimulating the growth of beneficial bacteria while inhibiting the growth of pathogenic bacteria (12). Black raspberry (BRB), which belongs to the Rubus occidentalis family of berries, is native to America. The bioactive substances of BRB are divided into phenolic acids, flavonoids, procyanidins, tannic acid, styrene, wooden fat element, terpene and steroid alcohol according to their chemical structures. The most active and abundant active constituents of BRB are ellagic acid and anthocyanins, which can overcome the effects of many chemical carcinogens and efficiently scavenge the free radicals. The freeze-dried BRB powder has been regarded with great interest, as a possible cancer chemopreventive agent against cancer of the alimentary canal (13). However, until now, few experimental studies on the effects and mechanisms of action of BRB anthocyanins have been published. In this study, we investigated the effects of BRB anthocyanin-supplemented diet on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC mouse model by examining the compositional changes associated with gut microbiota and the epigenetic status of certain genes involved in the regulation of carcinogenesis in these animals. Materials and methods Cell lines and reagents Human colon cancer cell line HCT116 (CCL-247, American Type Culture Collection, ATCC, Manassad, VA) and LoVo (Cell Bank of Beijing Cancer Institute, Beijing, China) were obtained in 2011 and kept as frozen stocks. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Thermo Fisher Scientific, Logan, UT) and authenticated by their karyotypes and morphologies. The medium was supplemented with 10% fetal bovine serum and antibiotics (10000 U/ml penicillin, 10 μg/ml streptomycin). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Extract containing BRB anthocyanins (purity > 90%) obtained from the ripened fruits of BRB was provided by Jianfeng Nature Products Technology Co., Ltd (Tianjin, China). BRB anthocyanins consist of three major anthocyanins cyanidin-O-glucoside, cyanidin-O-xylosylutinoside and cyanidin-O-rutinoside, and the contents are 2.63, 0.73 and 16.91 mg/g, respectively, and cyanidin-O-sambubioside is the less abundant (14). AOM (A5486) was purchased from Sigma–Aldrich, Saint Louis, MI. DSS (0216011091) was purchased from MP, Santa Ana, CA. Animal care and experimental protocol Five-week-old C57BL/6J mice (18–20 g) were maintained at 12 h/12 h light–dark cycle and given water ad libitum. The animals were housed in cages kept in a room with controlled temperature (21 ± 2.0°C) and humidity (50 ± 5%). All animal experimental protocols were approved by the Ethics Committee of Liaoning University of Traditional Chinese Medicine (Shenyang, China). Establishment of colitis-induced CRC mice model and supplementation of BRB anthocyanins The AOM/DSS model is a well-established chemically induced mice model of colitis-associated cancer. After 1 week of acclimatization, male mice (6 weeks of age) were given a single intraperitoneal injection of AOM (10 mg/kg body weight). At the same time, these animals were administered a diet containing BRB anthocyanins daily. One week after the injection of AOM, the animals were given drinking water containing 2% DSS water for 1 week followed by normal drinking water for 2 weeks, and this step was repeated two more times (15). The mice were divided into four groups: one control group and three AOM/DSS-treated groups. The healthy control group (n = 10) C57/BJ mice were fed with a chow diet over the 12 week period. The three AOM/DSS-treated groups mice (n = 11) were daily fed chow diet containing with or without BRB anthocyanins. The concentrations of BRB anthocyanins in the diet, 3.5 µmol/g (LBA) and 7.0 µmol/g (MBA), corresponded to anthocyanins content in the 5 and 10% freeze-dried BRB powder, respectively, and they were chosen on the basis of our previous study (13). The diets were stored at −20°C before they were used in the experiment. Sample collection At the end of the 12th week, whole blood was collected from the animals and stored at −80°C. All the animals were then sacrificed by CO2 inhalation followed by cervical dislocation. Mouse intestinal epithelial cells were isolated by incubating the dissected, washed intestinal tissues in 15 mM ethylenediaminetetraacetic acid buffer at 37°C for 30 min as described previously (16). The tumors, intestinal tissue, epithelial cells and intestinal content were collected from each of the animals, stored at −80°C immediately and then subjected to a series of analyses. The tumors were examined and accounted under the dissecting microscopy, and the histopathology was identified by the professional pathologist. MTT assay, transwell and colony formation HCT116 and LoVo cells were treated with BRB anthocyanins for 48 h, and cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich) assay as previously described (17), while cell migration and invasiveness were determined by transwell and colony formation assays, respectively. For transwell assay, the cells were seeded on the top chamber of the transwell, while serum-free medium was added to the bottom chamber. After 24 h, the membrane of the transwell insert was fixed with 4% PFA; the cells that had migrated to the bottom side of the membrane were stained with DAPI (Beijing Dingguo Changsheng Biotechnology CO., Ltd, Beijing, China). For each insert, 10 fields were randomly selected, and the cell numbers were quantified using Image-Pro Plus 6.0. To calculate the migration index, the cell number of each treated group was normalized to the cell number of the control group. For colony formation assay, the cells were seeded in a six-well plate at a density of 200 cells/well and grown for 2 weeks. The cell colonies that emerged were washed with phosphate-buffered saline, fixed with methanol and stained with 0.5% Giemsa. The number of colonies from each well was counted under an inverted microscope. DNA extraction from intestinal contents The DNA of intestine content from three individual mice from each group was extracted by using a Fecal DNA Extraction Kit (Tiangen Biotech Beijing, Co., Ltd, Beijing, China) according to the manufacturer’s instructions and then subjected to 16S rRNA analysis. T-RFLP analysis and assignment of T-RFs detected Analysis of 16S rRNA was performed using the 6-carboxyfluorescein (FAM)-labeled forward primer (5′-FAM-AGAGTTTGATCCTGGCTCAG-3′) and the unlabeled reverse primer 1525r (5′-AAGGAGGTGWTCCARCC-3′). The expected size (1500 bp) and purity of the PCR products were assessed by electrophoresis in 1% agarose gels. The PCR products were then purified using a PCR Purification Kit (Sangon Biotech Co., Ltd, Shanghai, China). The purified PCR products were digested with restriction enzyme HhaI, HaeIII and MspI and then submitted to Sangon Biotech Co., Ltd (Shanghai, China) for capillary sequencing. The major T-RFs were identified by computer simulation, which was performed using an online system at http://mica.ibest.uidaho.edu to analyze the composition of the microbial community (18). RNA extraction and quantitative RT-PCR Total RNA was extracted from the intestinal epithelial cells of each individual mouse (three mice from each group). The quality and quantity of the RNA were determined by UV absorbance measured with a spectrophotometer (Thermo Scientific, NanoDrop-2000c, Gene Co., Ltd, Hong Kong, China). Quantitative real-time PCR analysis was performed using the ABI Prism 7500-HT sequence detection system (96 wells) as described (13). The following primers for mouse cytokine analysis were designed and synthesized as shown in Supplementary Table 1A, available at Carcinogenesis Online. The β-actin gene was used as an internal control. The populations of Eubacterium rectale (19), Faecalibacterium prausnitzii (19), Lactobacillus group (20), Desulfovibrio sp. (21) and Enterococcus spp. (22) in the intestinal content of mice from different treatment groups were also measured by qRT-PCR. The following primers were used as shown in Supplementary Table 1B, available at Carcinogenesis Online, and 16S rRNA was used as an internal control. DNA extraction and methylation-specific PCR The quality and quantity of DNA extracted from the isolated intestinal epithelial cells from three individual mice and from HCT116 and LoVo cells were determined by spectrophotometry. The isolated DNA was subjected to sodium bisulfite modification using a DNA Methylation Kit (ComWin Biotech Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Methylation was then analyzed using methylation-specific PCR. The sample contained 2× GC bufferⅡ, 1.25 mM dNTP mixture, 0.5 mM of each primer and 0.5 U of LA Taq (Takara Bio, Dalian, China), 50 ng bisulphite-treated DNA and water in a final volume of 25 μl. The PCR reaction consisted by a hot started at 95°C for 5 min, followed by 95°C for 45 s, 65°C for 45 s and 72°C for 1 min in the first cycle, with the annealing temperature decreased by 1°C in each succeeding cycle. After 10 cycles, the condition was changed to 95°C for 45 s, 55°C for 45 s, 72°C for 1 min for another 35 cycles followed by a 10 min extension at 72°C. The PCR product was examined by electrophoresis in 2% agarose gel and visualized under UV illumination. The following primers were used as shown in Supplementary Table 2, available at Carcinogenesis Online. Western blotting analyses Protein was extracted from intestinal epithelial cells as previously described (13). The total protein (40 μg) was resolved in 12% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane. The following primary antibodies were used for immunoblotting: anti-p-JNK, anti-p-STAT3, anti-Bcl2, anti-Bax, anti-CDK4, anti-CyclinD1, anti-cMyc，anti-DNMT1, anti-DNMT3A, anti-DNMT3B and anti-β-actin (Bio Basic, Toronto, Canada). After blocking with 5% BSA (Sangon Biotech, Shanghai Co., Ltd) for 2 h at room temperature, the membrane was incubated with the appropriate primary antibody for overnight at 4°C. It was then washed and incubated with the appropriate secondary antibody for 2 h at room temperature, followed by detection using the enhanced chemiluminescence technique (Amersham Life Science). Statistical analyses Data analysis that consisted of one-way analysis of variance (ANOVA) followed by the post hoc test was performed using the SPSS software (SPSS16; Beijing Stats Data Mining, Beijing, China). Results BRB anthocyanins decrease tumor multiplicity in vivo and proliferation, migration and colony formation in vitro The chemoprevention effect of BRB anthocyanins was first confirmed by examining their inhibitory effect on tumorigenesis in mice treated with AOM/DSS. All of the AOM/DSS-treated mice that were fed with the chow diet without BRB anthocyanins developed high intensity tumors (>30 tumors) in the colon and rectum, while only an average 3 or 2.25 tumors per animal appeared in AOM/DSS-treated mice fed with chow diet containing BRB anthocyanins, either LBA or MBA (Figure 1A). The incidence of tumor was also slightly reduced from 100% (all 11 mice) in the AOM/DSS-treated group not given BRB anthocyanins to about 72.7% (8 of 11) in the AOM/DSS-treated group fed with LBA and 81.8% (9 of 11) in the group fed with MBA group, but the reduction was not significant in both cases (Figure 1B). Figure 1. View largeDownload slide Effect of BRB anthocyanins on tumor multiplicity in vivo and on proliferation, migration and colony formation in vitro. Comparison of the changes in tumor number (A), tumor incidence (B) and cell viability (C) among healthy mice and AOM/DSS-treated mice without and with the supplement of BRB anthocyanins. Attenuation of cell viability, migration rate and colony formation by BRB anthocyanins in LoVo and HCT116 cells (1C-1E). Microscopic examination was at ×20 magnification. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicate significantly different from the solvent control group at the *P < 0.05, **P < 0.01, ***P < 0.001 levels, respectively. Figure 1. View largeDownload slide Effect of BRB anthocyanins on tumor multiplicity in vivo and on proliferation, migration and colony formation in vitro. Comparison of the changes in tumor number (A), tumor incidence (B) and cell viability (C) among healthy mice and AOM/DSS-treated mice without and with the supplement of BRB anthocyanins. Attenuation of cell viability, migration rate and colony formation by BRB anthocyanins in LoVo and HCT116 cells (1C-1E). Microscopic examination was at ×20 magnification. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicate significantly different from the solvent control group at the *P < 0.05, **P < 0.01, ***P < 0.001 levels, respectively. To further confirm the inhibitory effect of BRB anthocyanins on tumorigenesis, HCT116 and LoVo were incubated with BRB anthocyanins, and cell viability was then determined by MTT assay. BRB anthocyanins at a concentration of 25 or 50 µg/ml significantly decreased the viability of both cell lines (Figure 1C), while we also noticed the decrease in viability of the LoVo cell line by the anthocyanins was not dose related. In the case of HCT116 cells, decrease in cell viability was accompanied by 76% reduction in migrating cells and 66% reduction in colony formation after treatment with 25 μg/ml BRB anthocyanins or 92% and 46% reduction, respectively, in migrating cells and colony formation after treatment with 50 μg/ml BRB anthocyanins (Figure 1D). A similar reduction in migrating cells and colony formation was observed for LoVo cells treated with the two different concentrations of BRB anthocyanins (Figure 1E), and the inhibition of colony formation was greater at the lower dose of anthocyanins than at the high dose. The results suggested that treatment of these cancer cells with BRB anthocyanins could inhibit their invasiveness and colony forming ability. T-RFLP analyzes the effects of BRB anthocyanins on gut microbiota composition Changes in the composition of gut microbiota among all the experimental groups were evaluated by T-RFLP. There were no significant differences in community richness, diversity and evenness index gut among the gut microbiota of the different groups of mice, suggesting that BRB anthocyanins might have little effect on the species of intestinal flora (Figure 2A). However, cluster analysis suggested a greater similarity in gut microbiota between AOM/DSS-induced mice given BRB anthocyanins and healthy mice, both of which differed significantly from the AOM/DSS-induced group not given BRB anthocyanins (Figure 2B). Furthermore, the growth of beneficial intestinal microflora including butyrate-producing bacteria and Neisseria was promoted, while the growth of intestinal pathogenic microflora, which included Campylobacter, H. pylori, Bacteroides and Prevotella, was inhibited in AOM/DSS-induced mice given BRB anthocyanins (Figure 2C–D). Figure 2. View large Download slide View large Download slide T-RFLP analysis of the effect of BRB anthocyanins on gut microbiota composition. (A) Changes in flora diversity. (B) Clustering analysis tree. (C and D) Fragment analysis of intestinal flora. ‘*’ indicate significantly different from AOM/DSS-induced mice without administration of BRB anthocyanins at the *P < 0.05, ***P < 0.001 levels, respectively. Figure 2. View large Download slide View large Download slide T-RFLP analysis of the effect of BRB anthocyanins on gut microbiota composition. (A) Changes in flora diversity. (B) Clustering analysis tree. (C and D) Fragment analysis of intestinal flora. ‘*’ indicate significantly different from AOM/DSS-induced mice without administration of BRB anthocyanins at the *P < 0.05, ***P < 0.001 levels, respectively. Changes in differential bacterial strains in stool promoted by BRB anthocyanins Changes in intestinal microflora were further quantitated by qRT-PCR to examine the effects of BRB anthocyanin on the population of individual bacterial species. Compared with the healthy group of mice, the number of intestinal bacteria, including E. rectale, F. prausnitzii and the Lactobacillus group in the AOM/DSS-treated group, were dramatically reduced, but this trend was reversed when these mice were also given BRB anthiocyanins (MBA group), resulting in significant increases in the population of these bacteria (Figure 3). The number of Desulfovibrio sp. and Enterococcus spp. were dramatically increased in the AOM/DSS-treated group compared with the healthy group of mice, and in the AOM/DSS-treated group given MBA, the number of Desulfovibrio sp. and Enterococcus spp. was significantly decreased. Figure 3. View largeDownload slide Changes in differential bacterial strains in the intestine contain caused by BRB anthocyanins. Quantitation of stool bacteria in healthy control, AOM/DSS-treated mice without and with administration of BRB anthocyanins. 16S rRNA was used as a loading control. Values shown are the means ± SEMs of three determinations. ‘*’ and ‘**’ indicate significantly different from the healthy control group at the P < 0.05 and P < 0.01 levels, respectively. Figure 3. View largeDownload slide Changes in differential bacterial strains in the intestine contain caused by BRB anthocyanins. Quantitation of stool bacteria in healthy control, AOM/DSS-treated mice without and with administration of BRB anthocyanins. 16S rRNA was used as a loading control. Values shown are the means ± SEMs of three determinations. ‘*’ and ‘**’ indicate significantly different from the healthy control group at the P < 0.05 and P < 0.01 levels, respectively. BRB anthocyanins ameliorate AOM/DSS-induced intestinal inflammation To further investigate the effect of BRB anthocyanin supplement on chronic inflammation-initiated intestinal tumorigenesis induced by AOM/DSS in mice, the levels of cytokines, IL-1β, IL-6, IL-10, COX2 and TNF-α were measured. Compared with AOM/DSS-treated group, the levels of inflammatory cytokines like IL-1β, IL-6, COX2 and TNF-α in mice given the higher dose of BRB anthocyanins (MBA group) were significantly decreased (P < 0.05). Decreased expression levels of IL-1β, IL-6, COX2 and TNF-α, but not the anti-inflammatory cytokine IL-10, were also observed in AOM/SDD-treated mice given a lower dose of BRB anthocyanins (LBA group) (Figure 4). The data suggested that BRB anthocyanins supplement significantly decreased the elevation of intestinal inflammatory cytokines in mice induced by AOM/DSS. Figure 4. View largeDownload slide Changes in mRNA levels of various cytokines in the intestinal epithelial cells induced by BRB anthocyanins. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates significantly different from the healthy control group, and ‘#’ indicate significantly different from with AOM/DSS-induced mice without administration of BRB anthocyanins at the * or #P < 0.05, ##P < 0.01 levels, respectively. Figure 4. View largeDownload slide Changes in mRNA levels of various cytokines in the intestinal epithelial cells induced by BRB anthocyanins. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates significantly different from the healthy control group, and ‘#’ indicate significantly different from with AOM/DSS-induced mice without administration of BRB anthocyanins at the * or #P < 0.05, ##P < 0.01 levels, respectively. BRB anthocyanins downregulate the expression of DNA methyltransferase and demethylate the hypomethylated promoters of SFRP2 gene Much evidence has revealed a close relationship between gut microbiota and DNA methylation, both in the laboratory and clinical findings. Changes in the epigenetic status associated with intestinal microbiota disorder as revealed by changes in DNA methylation during the process of CRC formation induced by AOM/DSS were also investigated in this study. DNA methylation in mammalian cells is regulated by a family of highly related DNA methytransferase enzymes (DNMT1, DNMT3A and DNMT3B). The expression of DNA methyltransferases (DNMTs) in the intestinal epithelial cells was analyzed using western blot. The levels of the DNMT1 were decreased in mice that were fed with LBA or MBA, with MBA having the most significant effect on the expression of DNMT1. Ten-eleven translocation (TET) proteins are Fe(II)- and 2-oxoglutarate (2OG)-dependent dioxygenases that mediate active DNA demethylation. To further investigate whether the demethylation process is passive or active, the influence of BRB anthocyanins on TET was determined. No changes in mRNA levels of TET1, TET2 and TET3 were observed in the intestinal epithelial cells of mice given BRB anthocyanins (Figure 5B), suggesting that demethylation regulated by BRB anthocyanins was a passive process. Figure 5. View largeDownload slide Analysis of the changes in DNMTs, TET and the methylation of SFRP2 and WIF1 promoters by BRB anthocyanins. DNMTs and TET regulated by BRB anthocyanins (A–B). Methylation status of SFRP2 and WIF1 promoters in human CRC cells (C–E) and mouse intestinal epithelial cell from healthy mice and AOM/DSS-treated mice with and without administration of BRB anthocyanins (E). SFRP2 protein expression level was upregulated by BRB anthocyanins in vivo (F). M represents the methylated area of promoter; U represents the unmethylated area of the promoter. A representative blot is shown. The plots show the relative intensity of each band in grey scale. Data are shown as the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates a significant difference from the healthy control group, whereas ‘#’ indicates a significant difference between AOM/DSS-treated group given BRB anthocyanins and AOM/DSS-treated group without BRB anthocyanins given at the * or #P < 0.05 levels, respectively. Figure 5. View largeDownload slide Analysis of the changes in DNMTs, TET and the methylation of SFRP2 and WIF1 promoters by BRB anthocyanins. DNMTs and TET regulated by BRB anthocyanins (A–B). Methylation status of SFRP2 and WIF1 promoters in human CRC cells (C–E) and mouse intestinal epithelial cell from healthy mice and AOM/DSS-treated mice with and without administration of BRB anthocyanins (E). SFRP2 protein expression level was upregulated by BRB anthocyanins in vivo (F). M represents the methylated area of promoter; U represents the unmethylated area of the promoter. A representative blot is shown. The plots show the relative intensity of each band in grey scale. Data are shown as the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates a significant difference from the healthy control group, whereas ‘#’ indicates a significant difference between AOM/DSS-treated group given BRB anthocyanins and AOM/DSS-treated group without BRB anthocyanins given at the * or #P < 0.05 levels, respectively. Previous studies have reported that aberrant activation of the Wnt signaling pathway can occur as a result of its upstream inhibitors being hypermethylated (23,24). Methylation status of the promoters of SFRP2 and WIF1 (Wnt inhibitory factor 1), both of which are antagonists of Wnt signaling pathway, was quantified in HCT116 and LoVo cells in vitro and in vivo using methylation-specific PCR (Figure 5C–E). HCT116 and LoVo cells were treated with BRB anthocyanins at a concentration of 25 or 50 µg/ml. The positive control used is 5-Aza (5-aza-2′-deoxycytidine), a DNA methylation transferase inhibitor. The methylation level of the SFRP2 promoter was decreased in both cell lines (Figure 5C–D). However, change in the methylation of the WIF1 promoter in HCT116 and LoVo cells by BRB anthocyanins was not obvious under the same condition. The impact of BRB anthocyanins on SFRP2 methylation was also evaluated by examining the extent of SFRP2 methylation in intestinal epithelial cells isolated from mice subjected to the different treatments. The CpG regions of SFRP2 in intestinal epithelial cells of AOM/DSS-treated mice were hypermethylated, but this methylation was reduced when the mice were given BRB anthiocyanin-supplemented diets (Figure 5E), consistent with the results obtained with HCT116 cells. Furthermore, BRB anthiocyanin-supplemented diets also upregulated the protein expression of SFRP2 (Figure 5F). The data suggested that BRB anthocyanins could lead to the demethylation of the hypomethylated promoter of the SFRP2 gene, but not of the WIF1 gene, both in vivo and in vitro. BRB anthocyanins downregulate the expression of tumorigenesis-associated genes AOM/DSS-induced CRC is a process characterized by multiple stages, which involve the interaction of multiple genes. To obtain further insight into the mechanism by which BRB anthocyanins could protect the mice against the carcinogenic effect of AOM/DSS, the protein levels of various genes associated with the tumorigenesis were investigated. BRB anthocyanin significantly suppressed AOM/DSS-induced decreased level of p-STAT3 (signal transducer and activator of transcription-3, STAT3) in the intestinal epithelial cells. Furthermore, the genes involved in the β-catenin signaling pathway, including p-JNK, Bcl2, CDK4, CyclinD1 and c-Myc, which promote tumor proliferation and inhibit apoptosis, were significantly downregulated by BRB anthocyanins. The expression of Bax, the apoptotic inducing factor, was upregulated by BRB anthocyanins. Discussion Accumulated evidence indicates that the complex community of gut microbiota that inhabits the gastrointestinal tract is strongly associated with the development of CRC (25). The beneficial roles of probiotics in lowering the gastrointestinal inflammation and preventing CRC have been frequently demonstrated, but their immunomodulatory effects and mechanisms in the suppression of tumor growth still remain unclear. The current study was designed to investigate the role of gut microbiota in the chemopreventive effect of BRB anthocyanins against CRC. Our results clearly demonstrated that supplementing the diet of mice with BRB anthocyanins could significantly modulate the disruption of gut microbiota, which in turn induced the alteration in tumor epigenetic, genetic or exacerbated inflammation in AOM/DSS-treated mice. By regulating the composition of gut microbiota via increasing the amount of probiotics and decreasing the amount of pathogenic bacteria, BRB anthocyanins exerted their chemopreventive effects against tumor formation in the AOM/DSS-induced CRC mouse model. The imbalance of gut microbiota manifested as an overall reduction in microbial diversity, reduced abundance of the phylum Firmicutes, including F. prausznitzii as well as concurrent increases in Bacteroidetes, will lead to the simulation of chronic intestinal inflammation and increase the susceptibility to CRC (26). In this study, we found 100% tumor incidence was observed in AOM/DSS-treated mice, and BRB could significantly reduce the tumor size (Figure 1A), consistent with the findings previously reported for APC1638 and MUC2 mice (13), while reduction in tumor incidence, an important index of prevention, caused by BRB anthocyanins was not as significant as in the previous study. The duration in which the animals were given BRB anthocyanins, as well as the differences in genetic background, diet composition and the physical environment in which the animals were kept could have contributed to the different results obtained in the current study compared with the previous study. What was impressing in the current finding was the significant increase in the number of pathogenic bacteria (including Haemophilus, Escherichia coli and Salmonella enterica) in addition to the 100 % tumor incidence detected in the 12th week of the experimental period in AOM/DSS-induced animal models. Eubacteria exhibited a significant correlation with CRC. Eubacteria are bacteria with increased ability to harvest energy from the diet administered to the host (27), and their reduction in number has been found in the intestinal flora of CRC patient (28). On the contrary, Proteobacteria, which is usually abundant in CRC patients, are generally regarded as gut commensals with potential pathogenic features (29). At the phylum level, Firmicutes were the most dominant bacteria in the intestinal content of AOM/DSS-treated mice not given BRB anthocyanins, with the pathogenic subgroups, Haemophilus, E. coli and S. enterica, being more abundant. Significant reduction in the number of these bacteria occurred in the intestinal content of AOM/DSS-treated mice given BRB anthocyanins when compared with AOM/DSS-treated mice without BRB anthocyanins given (Figure 3). Butyrate, a short chain fatty acid generated in the colon by bacterial fermentation of unabsorbed carbohydrate, provides energy for colonic epithelial cells, promotes epithelial cell differentiation, ameliorates inflammation and hastens the repair of colon tissue (30). E. rectale and F. prausnitzii are two major butyrate-producing bacteria in the intestine (31). Lactobacillus can ameliorate colonic carcinogenesis, inhibit preneoplastic lesions and reduce tumor load and size (32). The present results suggested the beneficial modulation effects of dietary BRB anthocyanin supplement could indeed be the enhancement of the growth of E. rectale, F. prausnitzii and Lactobacillus and inhibition of the growth of Desulfovibrio sp. and Enterococcus spp. Furthermore, the releases of inflammatory cytokines, including IL-1β, IL-6, COX2 and TNF-α, were increased significantly in the AOM/DSS-treated mice. BRB anthocyanins could decrease the level of four aforementioned inflammatory cytokines. The most significant ones were TNF-α and IL-6, consistent with our previous finding in Muc2−/− mice (13). During inflammation, NF-κB level is increased in the colon epithelial cells and immune cells, leading to increases in pro-inflammatory cytokines (33). These events would then increase the activity of DNMTs, which can silence a subset of tumor suppressor genes via methylation at the promoters. DNMT1 is essential for the maintenance DNMTs in mammalian cells and is responsible for accurately replicating the genomic DNA methylation patterns during the S phase of the cell cycle (34). In contrast, de novo methylation of DNA is mediated by DNMT3A and DNMT3B, which have both maintenance and de novo DNA methylation activities (35). All three DNMTs are overly expressed in tumors (36). The effects of BRB anthocyanins on the methylation of DNA in human colon cancer cells, Ulcerative colitis (UC)-associated mice models and human CRC patients after they had consumed BRBs have been studied by Wang et al. earlier (37–40), who demonstrated that BRB anthocyanins not only can inhibit the expression of DNMT3B and DNMT1 in human CRC cell, the specimens from UC-associated mice models, also the biopsies collected from human CRC patients after they had consumed BRBs for an average of 4 weeks had lower levels of DNMT1 protein. Meanwhile, their data also demonstrated that BRB anthocyanins could colocalize with DNMT3B and DNMT1 in HCT116 cells. Our data provided additional evidence for the inhibitory effect of BRB anthocyanins on DNMT1 in this AOM/DSS-induced UC-associated CRC mouse model. DNMT3B also exhibited a decreased pattern; however, this was not significant. It is known that all three TET genes are mutated and show reduced expression at the mRNA level, and the corresponding proteins have impaired activity in a wide range of different cancer types, including CRC (41). Our data suggested that BRB anthocyanins regulated the methylation of SFRP2 in mouse CRC cells via a passive mode, as virtually no changes in the mRNA levels of TET1, TET2 and TET3 were observed in the intestinal epithelial cells of AOM/DSS-treated mice receiving BRB anthocyanins (Figure 5B). Negative regulators of the Wnt pathway are frequently methylated in UC, leading to dysregulation of the pathway and potentially to CRC (23). SFRPs have been postulated to serve as tumor suppressors, due to their putative WNT-inhibitory activity. The expression of SFRPs has been shown to be affected by epigenetic silencing (24,42). BRBs anthocyanin-mediated demethylation of the SFRP2 gene led to its increased expression level in HCT116 and LoVo human CRC cells and mouse intestinal epithelial cells (Figure 5F), which is consistent with previous findings in human colon cancer specimens reported by Wang et al. (39). At the same time, this also led to decreased levels of downstream factors such as β-catenin, CDK4, CyclinD1, c-Myc. These results suggested that BRB anthocyanins may suppress colonic ulceration by regulating the hypermethylation of the promoters of the genes involved in the suppression of inflammation in the colon. Increased multiplication of CRC cells triggered by the activity of STAT3 via IL-6 or a constitutively active STAT3 mutant has been reported (43). In CRC patients, the levels of STAT3 and p-STAT3 are significantly raised compared with healthy individuals, and this may be accompanied by an increased level of Bcl-xl, which can also promote tumor proliferation (44). STAT3 can actively contribute to malignancy, and downregulating its expression could be considered as a potential therapeutic approach to combating CRC. Suppression of IL-6/STAT3 trans-signaling in mice can effectively inhibit the growth of colon cancer (29). The significant decrease in p-STAT3, p-JNK1 and Bcl2 expression and increase in Bax expression observed in mice receiving BRB anthocyanins in the form of diet supplement (Figure 6) indicated that inhibition of the STAT3 signaling might indeed constitute part of the effect of BRB anthocyanins on the modulation of gut microbiota, regulation of inflammation and the eventual prevention of CRC formation and development. This finding was consistent with the previous report regarding the promotion of STAT3 signaling pathways by gut microbiota being a factor that could accelerate tumor growth in CRC mice (45). Figure 6. View largeDownload slide Effect of BRB anthocyanins on various genes associated with tumorigenesis of CRC in intestinal epithelial cells. A representative blot is shown. The plots show the relative intensity of each band in grey scale. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates a significant difference between the AOM/DSS-treated group and healthy control group, whereas ‘#’ indicates a significant difference between AOM/DSS-treated group given BRB anthocyanins and AOM/DSS-treated group without BRB anthocyanins given at the *P < 0.05, ** or ##P < 0.01 levels, respectively. Figure 6. View largeDownload slide Effect of BRB anthocyanins on various genes associated with tumorigenesis of CRC in intestinal epithelial cells. A representative blot is shown. The plots show the relative intensity of each band in grey scale. Data are the means ± SEMs of triplicate experiments performed independently. ‘*’ indicates a significant difference between the AOM/DSS-treated group and healthy control group, whereas ‘#’ indicates a significant difference between AOM/DSS-treated group given BRB anthocyanins and AOM/DSS-treated group without BRB anthocyanins given at the *P < 0.05, ** or ##P < 0.01 levels, respectively. In conclusion, during the process of AOM/DSS-induced UC-associated CRC, disruption of gut microbiota in the intestinal tract might be an early event, which could trigger the onset of inflammation and epigenetic alteration in intestinal epithelial cells, eventually leading to the tumor formation and development. BRB anthocyanins might act as efficient prebiotics by sustaining the growth of protective bacteria but not of the pathogenic bacteria, as well as by modulating the composition and commensal of gut microbiota. The chemoprevention effect of BRB anthocyanins could therefore center on the modulation of inflammation and aberrant epigenetic status of SFRP2 induced by an imbalance of intestinal flora homeostasis. Supplementary material Supplementary Table 1 and 2 can be found at Carcinogenesis online. Funding This work was mainly supported by the grants from National Natural Science Foundation of China (81272333 to Bi, X), Program of Liaoning Excellent Talents in University (LETU#LR2014001 to Bi, X) and in part from Innovation Team Project (No: LT2015011) from the Education Department of Liaoning Province. Conflict of Interest Statement: None declared. Abbreviations AOM azoxymethane BRB black raspberry CRC colorectal cancer DNMTs DNA methyltransferases DSS dextran sodium sulfate IL-1β interleukin-1 beta SFRPs secreted frizzled-related proteins TET Ten-eleven translocation UC Ulcerative colitis. 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Carcinogenesis – Oxford University Press
Published: Mar 1, 2018
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