A food-based approach that targets interleukin-6, a key regulator of chronic intestinal inflammation and colon carcinogenesis (original) (raw)

Introduction

Alongside the development of technology came a shift in our diet [1]. Westernized populations moved from hunter–gatherer lifestyles with minimal food processing to novel foods such as dairy products, cereals, refined sugars and fatty meats [1], common staples of the current Western diet. Furthermore, foods became easily accessible at low cost and increased convenience. Marked by high fat and high sugar, the Western diet is leading to a greater caloric intake in the society. This high-calorie diet (HCD) coupled with a sedentary lifestyle is linked to multiple diseases, including obesity, type 2 diabetes, cardiovascular disease and certain cancers such as colon cancer [2], [3].

Colon cancer is the second leading cause of cancer-related deaths in the United States. Approximately 5% (1 in 20) of Americans will be diagnosed with colon cancer in their lifetime [4]. Consumption of an HCD has shown to increase the risk for colon cancer in a human model [5], [6], [7]. Recent animal studies also suggest a causal link between HCD and increased colon cancer risk [8], [9], [10], [11], [12], [13]. Eighteen months of consumption of the Western diet induced colonic tumors in normal C57Bl/6 mice [13] in the absence of any carcinogen. Furthermore, a recent study by Erdelyi et al. [14] showed that the high-fat Western diet negatively impacted colonic lipid metabolism, oxidative stress, and immune responses in C57Bl/6 mice. Colonic inflammation plays an important role in elevating the risk for colon cancer with the implication of multiple pathways, including the interleukin-6 (IL-6) signaling pathway [15], [16], [17]. The association of IL-6 signaling with colon cancer was clearly demonstrated in a recent study by Day et al. [10]. These researchers found elevated IL-6 expression in colonic polyps of Apc Min/+ mice fed an HCD compared to those fed a standard diet (SD), suggesting that HCD can drive the increased production of proinflammatory cytokines, such as IL-6, thus elevating the risk for colon cancer.

IL-6 is a proinflammatory cytokine released by myeloid cells in various tissues crucial for immune response, cell survival, apoptosis and proliferation [18], [19], [20], [21]. IL-6 also regulates the proliferation of intestinal epithelial cells [22]. Recent studies propose a link between chronic inflammatory diseases (e.g., irritable bowel syndrome and colon cancer) and IL-6 signaling [16], [18], [20], [23], [24], [25]. A study in C57BL/6 mice found higher IL-6 mRNA and protein expression in the dextran sodium sulfate and azoxymethane (DSS/AOM)-induced colon cancer tumors than surrounding normal colon tissue, suggesting that IL-6 may be responsible for enhanced colon carcinogenesis [26]. IL-6, when bound to its receptor IL-6R, leads to downstream activation of the JAK/STAT3 pathway, inducing expression of genes important in elevation of proliferation and suppression of apoptosis [18]. Recent work by Grivennikov et al. [18] in IL-6 knockout mice reported a decrease in Ki-67-expressing colon crypt cells, an early biomarker for colon cancer. Other studies have added evidence to propose that IL-6 acts in colon cancer by increasing proliferation (reviewed in [27]). Taken together, these studies suggest that IL-6 provides resistance to apoptosis and provides a conducive environment for increased cell proliferation, ultimately leading to enhanced cell growth and survival. Augmented IL-6 and its downstream signaling pathways may provide a proinflammatory milieu favorable for colon cancer development.

Currently, various anti-IL-6 therapeutics are being used or are in clinical trials for multiple diseases and cancers including colon cancer, multiple myeloma, prostate cancer, and Castleman disease [17], [28]. The therapeutics available for colon cancer treatment currently target inhibition of the IL-6/STAT3 signaling pathway with anti-IL-6 receptor antibodies, soluble gp130Fc and small molecule JAK inhibitors [17]. The use of anti-IL-6 receptor antibodies has been shown to suppress the growth of colon tumors and protect against colon carcinogenesis in DSS/AOM-induced colon carcinogenesis in C57BL/6 mice [26]. While these therapeutics provide a potential therapy for cancer patients, therapeutics can be costly and include an array of negative side effects or even lead to drug tolerance [17]. Therefore, there is a growing interest in alternative therapeutics that could alleviate the cost and side effects patients face, reducing the stress of the already existing physical and psychological burden of disease.

Bioactive compounds from plants, including anthocyanins and phenolic acids, are linked to a reduced risk for a variety of cancers, including colon cancer [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]. However, individual phytochemicals have been shown to have proinflammatory and procancerous effects in high doses alone (reviewed in [40]). Plant foods have been illustrated in epidemiological studies and other research to hold a potential for disease prevention as different dietary ingredients can work synergistically to enhance the activity of a single compound, providing a better explanation for the benefits of whole foods observed in epidemiological studies [41], [42], [43].

Color-fleshed potatoes contain a variety of secondary metabolites, including polyphenols. Specifically, purple-fleshed potatoes are rich in phenolic acids and anthocyanins. Our previous studies linked the anti-colon-cancer properties of color-fleshed potatoes to their bioactive compounds [44], [45], [46]. Consumption of color-fleshed potatoes has been on the rise in the past 10 years, likely due to their putative health benefits. While studies on individual compounds and their anticancer effects have been performed, the benefits of plant foods such as anthocyanin-containing purple-fleshed potatoes, rich in anthocyanins, against colonic inflammation/cancer in humans or in a human-relevant model are lacking.

Developing an appropriate model for in vivo studies is crucial to best understand and extrapolate the data to human models. While mice are popular models for studying a variety of diseases, the anatomical and physiological differences between rodents and humans are significant. In addition, wide differences in dietary patterns exist between the two groups. The human is an omnivore, whereas rats and mice were originally granivores. Other models exist, including cats/dogs, but these models have differing diet and meal patterns than humans. Primates are rarely used in food intake studies due to their expense and scarcity [47]. In contrast, the pig is an excellent model to study the nutrition and food intake in humans. This study used a pig model because it is experimentally tractable and it is a clinically relevant model of the human gastrointestinal tract [47], [48], [49].

Using a human-relevant porcine model, we investigated the effect of HCD on colonic inflammation. We screened a panel of inflammatory biomarkers involved in colonic inflammation/cancer and identified the IL-6 signaling as the prominently altered pathway using quantitative polymerase chain reaction (qPCR) and proteomics analysis. Proteins in the IL-6 signaling pathway as well as IL-6 significantly correlated with Ki-67 proliferative index and zone, early biomarkers of colon cancer. Thus, our data revealed the role of IL-6 and its signaling pathway in enhancing colonic proliferation in colon cancer development in a human-relevant model. We further evaluated if dietary intervention of purple-fleshed potatoes could alleviate HCD-induced colonic inflammation. We witnessed suppression in IL-6 expression with the supplementation of only 10% w/w purple-fleshed potatoes, even after baking, in HCD-consuming pigs. Our study suggests staple crops that contain anthocyanins should be further as potential dietary interventions in the prevention and treatment of gastrointestinal inflammation/cancers.

Section snippets

Diet-induced inflammation

Male pigs (6 weeks old) were obtained from Smithfield Premium Genetics (Rose Hill, NC, USA) and housed individually in indoor pens at the North Carolina State University Swine Educational Unit (Clayton, NC, USA). The animals were allocated into different treatment groups by body weight so that mean initial body weight was similar among the treatment groups (_N_=8 animals/treatment).

Experimental diets

Animals were provided with one of the following diets: a SD (~5% fat), an HCD (17% added dry fat and ~3%–5%

Pathway screening

We used a pig model to test HCD-induced inflammatory pathways, as it closely resembles human gastrointestinal anatomy and physiology. To understand alterations in inflammatory pathways caused by HCD, we screened 10 different pathways including 23 genes (Table 1) known for their involvement in inflammation. These pathways have been previously linked to inflammatory disorders and colon cancer risk.

Previous studies have suggested that elevated IL-6 expression plays a critical role in multiple

Discussion

Although evidence links HCD to colonic inflammation and colon cancer, there have been no studies on this aspect in a human-relevant porcine model. Previous research focused on HCD-induced inflammation in mouse models. Further, studies that evaluated dietary interventions of bioactive compound (anthocyanin) containing foods often evaluate extracts rather than whole foods. Therefore, the purpose of this study was to use a human-relevant porcine model to investigate the anti-inflammatory role of

Author contributions

S.R. conducted the pig study under the supervision of J.V., L.R. and S.W.K. A.S. performed the qPCR (and analyses) and statistical analysis, and correlations. E.E. performed the Ki-67 proliferative index and zone experiments used for correlations. S.R. and A.S. performed the extraction of the samples for proteomics. S.R. worked with J.V. and V.B. for proteomic analysis data collection and analysis. A.S. wrote the manuscript with help from S.R. and J.V. F.S and Q.L conducted the statistical

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgments

J.V. is the PI of USDA-NIFA NRI Integrated Grant 2009-55200-05197 that supported this work. We are grateful to Johnston Farms in Edison, CA, USA, who provided the potatoes for the pig study, and World Wide Foods, Burley, ID, USA; Van Drunen Farms, Momence, IL, USA; and Mr. Andrew Kester who helped with processing the potatoes for the pig study. We thank members of S.W.K laboratory at North Carolina State University for their assistance with pig sample collection. We also appreciate the help for