J Cancer Prev 2022; 27(1): 50-57
Published online March 30, 2022
© Korean Society of Cancer Prevention
1Division of Hematology and Oncology, Department of Medicine, Medical College of Wisconsin, 2Division of Biostatistics, Medical College of Wisconsin, 3Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, WI, 4Department of Pathology, Johns Hopkins University, Baltimore, MD, 5Department of Pathology, The Ohio State University, Columbus, OH, 6Department of Hematology & Hematopoietic Cell Transplantation, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA
Correspondence to :
Li-Shu Wang, E-mail: firstname.lastname@example.org, https://orcid.org/0000-0002-6500-6943
Jianhua Yu, E-mail: email@example.com, https://orcid.org/0000-0002-0326-3223
*These authors contributed equally to this work as co-correspondence authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Administration of black raspberries (BRBs) and their anthocyanin metabolites, including protocatechuic acid (PCA), has been demonstrated to exert chemopreventive effects against colorectal cancer through alteration of innate immune cell trafficking, modulation of metabolic and inflammatory pathways, etc. Previous research has shown that the gut microbiome is important in the effectiveness of chemoprevention of colorectal cancer. This study aimed to assess the potency of PCA versus BRB dietary administration for colorectal cancer prevention using an ApcMin/+ mouse model and determine how bacterial profiles change in response to PCA and BRBs. A control AIN-76A diet supplemented with 5% BRBs, 500 ppm PCA, or 1,000 ppm PCA was administered to ApcMin/+ mice. Changes in incidence, polyp number, and polyp size regarding adenomas of the small intestine and colon were assessed after completion of the diet regimen. There were significant decreases in adenoma development by dietary administration of PCA and BRBs in the small intestine and the 5% BRB-supplemented diet in the colon. Pro-inflammatory bacterial profiles were replaced with anti-inflammatory bacteria in all treatments, with the greatest effects in the 5% BRB and 500 ppm PCA-supplemented diets accompanied by decreased COX-2 and prostaglandin E2 levels in colonic mucosa. We further showed that 500 ppm PCA, but not 1,000 ppm PCA, increased IFN-γ and SMAD4 levels in primary cultured human natural killer cells. These results suggest that both BRBs and a lower dose PCA can benefit colorectal cancer patients by inhibiting the growth and proliferation of adenomas and promoting a more favorable gut microbiome condition.
Keywords: Colorectal neoplasms, Genes, APC, Rubus, Gastrointestinal microbiome
Colorectal cancer (CRC), ranked as the third leading cause of new cancer diagnoses and cancer deaths in the United States, has presented recently with increased incidence within the young adult population . Despite improved screening rates, the 5-year relative survival is approximately 65% . As of 2020, it is anticipated that 1.9 million new diagnoses and 935,000 deaths per year worldwide will result from CRC . It is thus critical to pursue improved prophylaxis and treatment of CRC in order to reduce incidence rates and improve patient outcomes.
Current medical recommendations for the prevention of CRC include the consumption of vitamin- and nutrient-rich fruits, whole grains, and vegetables . Our previous research has focused on the utilization of black raspberries (BRBs) in CRC prevention, with demonstrated efficacy in decreasing rectal polyp burden in humans through regulation of metabolic profiles and protective modulation of gene expression [5-7]. The chemopreventive effects of BRBs and underlying mechanisms have been further elucidated in experimentally induced esophageal cancer models. BRBs have been shown to prevent esophageal tumorigenesis in rats by virtue of chemoprotective anthocyanin (AC) components . These BRB-derived increase expression of protective natural killer (NK) cell-associated cytokines, downregulate macrophage-associated cytokines to reduce macrophage accumulation, reduce neutrophil accumulation, and inhibit angiogenesis, overall decreasing the expression of dysregulated inflammatory biomarkers .
One compound of focus that has arisen from these prior studies is protocatechuic acid (PCA), a microbial metabolite of BRB ACs that has been demonstrated to be especially efficacious in reducing neutrophil accumulation in esophageal papilloma tissue, as well as promoting other effects such as the modification of immune cell trafficking and prevention of inflammation. PCA upregulates the expression of the pentraxin 3 (PTX3) promoter, which is silenced via hypermethylation in human esophageal cancer but when active, acts to inhibit angiogenesis and tumorigenesis . Though it is less effective than BRBs in some respects, it has unique advantages as aforementioned in addition to high bioavailability and low cost . However, there is a paucity of studies on the use of PCA in the management of CRC, and this application should be considered as the effects of PCA may not be limited to solely esophageal cancer.
Our more recent studies have focused on the use of multiple intestinal neoplasia (
A BRB-containing diet has been shown to reduce the proliferation of colonic polyps and adenomas within the small intestine and colon of
In addition, the role of gut bacteria is crucial in manifesting the effects of BRBs; while BRBs prevent tumorigenesis, this is no longer evident when antibiotics are simultaneously administered in
All protocols were carried out in accordance with institutional guidelines for animal care dictated by the Medical College of Wisconsin Animal Care and Use Committee (protocol approval number AUA2430). Eight-week-old breeding pairs of
The control diet was an AIN-76A diet from the American Institute of Nutrition (Dyets Inc., Bethlehem, PA, USA). BRB powder was purchased from Berri Products (Corvallis, OR, USA), and stored at 4°C in vacuum-sealed plastic bags at the Medical College of Wisconsin. Water and dietary intake were recorded weekly and there were no differences among groups.
PCA was purchased from Millipore Sigma (St. Louis, MO, USA).
Colonic mucosa was used to measure COX-2 mRNA expression using primers purchased from Integrated DNA Technologies (IDT), Inc. (Coralville, IA, USA); prostaglandin E2 (PGE2) levels were measured using an ELISA kit (MyBioSource, San Diego, CA, USA).
Human NK cells were isolated from fresh peripheral blood from healthy subjects and PBMC from healthy subjects using the NK Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s protocol but with minor modifications, as described previously . Freshly isolated NK cells were cultured in RPMI1640+10% FBS+1% P/S (RPMI, Roswell Park Memorial Institute; FBS, Fetal bovine serum; P/S, Penicillin-Streptomycin) and immediately treated with PCA at 500 ppm or 1,000 ppm or dimethylsulfoxide (DMSO) (Millipore Sigma) for 16 hours. The cells were collected for real-time PCR using the primers purchased from IDT, Inc.
GraphPad Prism was used to analyze tumor data and inflammatory markers (un-paired, two-tailed
A diet containing AIN-76A (control), 5% BRBs, 500 ppm PCA, or 1,000 ppm PCA was administered to
Twenty six percent of AIN-76A diet-treated
As previously noted, gut bacteria are necessary for the effects of BRBs and their metabolites to manifest . Samples were taken from
Mice given the AIN-76A control diet demonstrated a prevalence of
In order to better define the anti-inflammatory effects of BRBs and PCA, further analysis was conducted on specific inflammatory and immune-mediating markers. The enzyme COX-2 responds to inflammatory stimuli by promoting the synthesis of pro-inflammatory prostaglandins including PGE2 . Both COX-2 and PGE2 are related to colorectal tumorigenesis . The consumption of BRBs and 500 ppm PCA significantly (
Primary cultured human natural killer cells were treated with 500 ppm and 1,000 ppm PCA. Only 500 ppm PCA, but not 1,000 ppm PCA, significantly increased both IFN-γ (
In our previous studies, it was determined that BRBs and PCA exert protective effects against both esophageal and colorectal cancer by modulating innate inflammatory and metabolic pathways. Using
According to a pharmacokinetic study in humans, about 70% of ingested cyanidin-glucosides are converted to PCA in the human gut . Therefore, for comparative purposes, we fed mice an amount of PCA (500 ppm in the diet) equivalent to about 70% of the anthocyanin content in a 5% BRB diet. According to our results, PCA was effective at this dose, suggesting that it could be responsible for part of the chemopreventive activity of whole BRBs. PCA supplementations at both 500 and 1,000 ppm had similar effects, albeit in the small intestine only. PCA did not have significant activity in the colon. This may be reflective of determinations made in previous research where PCA had lesser anti-inflammatory effects than whole BRBs . This discrepancy may also be due to the route of administration. However, it is important to note that while it was hypothesized that direct administration of PCA, as opposed to metabolism of whole BRBs into PCA, would improve PCA bioavailability, whole BRBs still had an overall greater breadth of effects. Thus, another option for future consideration may be examining adenoma response to different percentage supplementations of whole BRBs.
This study also aimed to examine the changes in gut microbiota that result from BRB and PCA administration, based on the knowledge that gut bacteria play a vital role in the effectiveness of BRBs . The inflammatory qualities of bacteria in the colon and intestine are critical to consider in the context of their role in CRC development as chronic inflammation is a significant risk factor for tumorigenesis; it can mediate DNA damage via the release of reactive oxygen species and allow invasion of malignant bacteria through the weakened gut barrier . It is therefore pertinent to examine changes in the gut microbiome that might reduce the capacity for inflammation and thus tumorigenic damage.
Our results demonstrated that
Although both 5% and 10% BRBs suppressed rat esophageal tumor development, the cancer-inhibiting effects of 5% BRBs were better than those exerted by 10% BRBs, suggesting that more is not always better . Likewise, the current study suggests that 1,000 ppm PCA is not more effective than 500 ppm PCA. One thousand ppm PCA did not offer a significant advantage over the other experimental diets in prevention of adenoma development, so further investigation may be needed in order to determine the optimal dosage of PCA for maximal benefit.
BRBs suppressed adenoma development partly via alteration of gut microbiome profiles, particularly by replacing pro-inflammatory bacteria with anti-inflammatory bacteria. We further demonstrated that whole BRBs and 500 ppm PCA decreased inflammatory markers such as COX-2 and PGE2. In addition, 500 ppm PCA, but not 1,000 ppm PCA, enhanced IFN-γ and SMAD4 expression in primary cultured natural killer cells; both IFN-γ and SMAD4 are involved in crucial anti-inflammatory signaling in suppressing colorectal cancer development [18,33,34]. Our results suggest that an enhanced anti-inflammatory gut microbiome by virtue of whole BRBs and 500 ppm PCA could result in an anti-inflammatory tumor microenvironment and thereby suppress tumor development.
Overall, this study confirms the effectiveness of 5% BRB and 500 ppm PCA dietary supplementation in reducing the extent of small intestinal and colonic adenomas in a murine CRC model, and suggests that these regimens have the potential to modify the gut bacterial profile in beneficial ways that enhance the proportion of anti-inflammatory species over pro-inflammatory ones. The results of this study suggest BRBs and PCA as alternative chemopreventive treatments for CRC and possibly other malignancies, and support previous literature regarding the effectiveness of BRBs.
This work was supported by NIH grants CA148818 and USDA/NIFA 2020-67017-30843 (to L.-S. Wang), and CA185301, AI129582 and NS106170 (to J. Yu).
No potential conflicts of interest were disclosed.
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