Research Project: Dietary Strategies for Cancer Prevention

Location: Jean Mayer Human Nutrition Research Center On Aging

2023 Annual Report

Objectives
Vitamins and Carcinogenesis Lab Objective 1: Define the cellular pathways by which obesity, obesigenic diets, and the intake of the 1-carbon nutrients modulate the risk of developing cancers of the colorectum and other common cancers in both animal models and human samples, and exploit these mechanistic insights in order to devise targeted means of mitigating cancer risk. • Sub-objective 1A: Determine whether the pro-inflammatory/pro-carcinogenic NF'B pathway plays the predominant role in mediating the obesity-promoted increased risk of colorectal carcinogenesis. • Sub-objective 1B: Determine whether supplemental levels of dietary vitamin B6 provide additional suppression of obesity-promoted tumorigenesis and colonic inflammation when combined with curcumin + salsalate, beyond that provided by the two latter agents alone. Objective 2: Examine how modifications in the microbiome alter biochemical and molecular processes that lead to colorectal cancer, and explore how intentional manipulations of the microbiome, or its products, can be exploited for cancer prevention. Objective 3: In both genetic and chemically-induced rodent models of colorectal carcinogenesis examine whether select alternative protein sources (e.g. insect-based foodstuffs) suppress pro-carcinogenic pathways and tumorigenesis compared to soy protein and other dietary sources of protein more common in the American diet. Nutrition and Cancer Biology Lab Objective 1: Investigate mechanistically the anti-inflammatory and anti-carcinogenic effect of phytochemical-rich whole food approaches, and purified phytochemicals as well as their derivatives, in preventing inflammation-promoted (e.g., induced by a high-sugar diet, diabetes, and aging) cancer development. Objective 2: Determine the ability of phytochemical-rich whole foods and dietary phytochemicals to prevent cancer development in liver and colon by targeting multiple signaling pathways (e.g. membrane and nuclear receptors) and inter-organ crosstalk (among liver, pancreas, mesenteric adipose tissue, and gut microbiome).

Approach
Vitamins and Carcinogenesis Lab We will identify novel strategies by which colorectal cancer (CRC), and other cancers that commonly afflict elderly Americans, can be prevented. Our aim is to lessen the risk that accompanies cancer-promoting features typifying the U.S. diet, such as its obesigenic character and emphasis on processed animal meat. Using a combination of in vitro experiments and animal models we identify biochemical and molecular pathways that mediate the effects of specific nutrients or dietary patterns on carcinogenesis. We then identify means of modulating those pathways to mitigate cancer risk. We will examine how the inflammatory state created by obesity and high-fat diets activates procancerous pathways in the colon. We are exploring the use of pharmacologic, nutritional, and microbial agents to block those pathways. The third objective is an exploratory aim, designed to generate preliminary data. We will examine whether substituting protein-rich powder derived from roasted crickets attenuates the enhanced risk of CRC that accompanies the habitual consumption of processed meats which are a prominent source of protein in the American diet. This strategy has the added value of promoting food sustainability. Our research will provide novel avenues for reducing the societal burden of common age-related cancers. Nutrition and Cancer Biology Lab We will conduct animal studies to investigate how one dietary phytochemical, xanthophyll beta-cryptoxanthin (BCX), inhibits metabolic syndrome, nonalcoholic fatty liver disease and liver cancer (hepatocellular carcinoma) development in the liver. Of particular interest is understanding how BCX prevents the development of hepatocellular carcinoma in rodents consuming a diet high in refined carbohydrates (HRCD). We will examine the protective effects of intact BCX, independent of its metabolites, regulating key cell signaling pathways in both young and old animals. We will examine multiple organs (liver, pancreas, adipose tissue, and gut) as well as how these organs communicate, while noting gender differences. Specifically, we will use genetically-altered carotenoid cleavage enzyme (beta-carotene 15,15’-oxygenase and beta-carotene 9’,10’-oxygenase) double knockout mice strains to determine whether HRCD-induced liver metabolic syndrome and tumorigenesis can be prevented by intact BCX itself or sweet red pepper extract (SRPE)-rich in BCX. We will treat mice (male and female) with a single injection of a hepatic carcinogen, diethylnitrosamine (DEN), followed by continued exposure to HRCD with or without BCX (or SRPE) intervention. We will examine the effects of dietary BCX intervention against fatty liver, inflammation, fibrosis, and in livers. We will investigate the protective effects of xanthophyll BCX against HRCD-promoted HCC in both young and old mice respectively. We will determine if the BCX protective action process a common mechnism or pathway, such as intestinal permeability/gap junction/adipose/liver axis, salvage pathway of NAD+ biosynthesis enzyme, and circadian transcription factors, and thereby reducing aging/metabolic syndrome-associated liver cancer development.

Progress Report
Vitamins and Carcinogenesis. A. Vitamins and Carcinogenesis: Over the past year, we completed an experiment that demonstrated--within the confines of a laboratory rodent model—that the inflammatory cytokine TNF-alpha is a key mediator by which obesity promotes pro-cancerous pathways in the colon and the risk of colorectal cancer. These observations are immediately relevant to Objective #1. B. Vitamins and Carcinogenesis: In a collaboration with the Tufts University Center for Cellular Agriculture we examined the nutritional aspects of laboratory-grown meat from insect muscle cells. These observations are immediately relevant to Objective #3 since diets based on these cultured meats will eventually be examined for cancer-mitigating properties. C. Vitamins and Carcinogenesis: In collaboration with Texas A&M Agrilife program, we are sharing a new post-doctoral trainee who is pursuing studies designed to examine the biochemical, molecular, metabolomic and microbiomic effects on the colon produced by a cricket powder-based diet. These observations are immediately relevant to Objective #3. D. Vitamins and Carcinogenesis: The investigator conducted a pilot experiment utilizing highly novel diets to test the hypothesis as to whether a cricket powder-based diet conveys cancer preventive properties in a mouse model of obesity-promoted colon carcinogenesis compared to diets whose protein is derived from more conventional sources of protein. Much was learned about limitations of the design, enabling the investigator to correct these limitations and to set up a repeat experiment of improved design. These observations are immediately relevant to Objective #3. Cancer and Biology. We carried out the following studies: Study 1) To determine if the ablation of sirtuin 1 (SIRT1) activity promotes nonalcoholic fatty liver disease (NAFLD) and liver cancer (hepatocellular carcinoma, HCC) development in catalytically inactive (sirt1y/y) mice relative to wild type (WT) mice at 6, 10 and 18 months; Study 2) To determine if mice with systemic ablation of SIRT1 activity display distinctive phenotypes in response to a carcinogen (diethylnitrosamine (DEN)-induced HCC development compared to WT mice at 18 months; and Study 3) To determine whether isolated ß-cryptoxanthin (BCX) has protective effects dependent or independent of BCO1/BCO2 and their metabolites in BCO1/BCO2-/- double knockout (DKO) mice. Cancer and Biology. A. In Study 1, we found that sirt1y/y mice developed very mild NAFLD as compared to WT littermates at 6, 10 months and 18 months. There were no significant differences between hepatic lipid biomarkers and several genes involved in lipid metabolism as well as inflammatory biomarkers in livers. However, all sirt1y/y mice developed pulmonary emphysema at the ages of 10 and 18 months, with the respective incidences of 100%, while the WT mice did not developed emphysema until 18 months of age (with 13% incidence only). The development of emphysema in sirt1y/y mice was associated with cellular senescence as evidenced by increased protein levels of p21, ratio of phosphorylate- and total-AMPK, and MMP-2, MMP9, and tissue inhibitor of metalloproteinase-1, and decreased the ratio of phosphorylate- and total ribosomal S6. The ablation of SIRT1 activity significantly up-regulated mRNA expressions of RARß2, VEGFa, HIF1a, and oxidative stress responsive genes (SOD1, CAT, GPX1, GPX2) in the lungs of sirt1y/y mice. However, there were no inflammatory cell infiltrations and cytokine responses (IL-6 and TNFa) in the lungs of Sirt1y/y mice, as compared with that of WT mice. These data indicated that lack of SIRT1 activity plays a vital role in the susceptibility of lungs to age-associated pulmonary emphysema, which is associated with increased cellular senescence independent of inflammatory response. Cancer and Biology. B. In Study 2, male and female sirt1y/y mice and respective WT mice were injected intraperitoneally with a liver-specific carcinogen, diethylnitrosamine (DEN), and fed a high sugar diet at 18 months. Results showed that in female and male mice, Sirt1y/y mice developed more severity of NAFLD compared with WT mice. However, there were no significant differences in liver tumor (HCC) incidence, HCC numbers, HCC volume between Sirt1y/y and its corresponding WT mice. Sirt1y/y mice exhibited significant increased protein levels of acetylated-p53 and its downstream target, p27 in the livers compared with WT mice. Moreover, Sirt1y/y mice showed significant decreased protein levels of cyclin D1 and anti-apoptotic marker Bcl-2. We also examined sirt1y/y mice and WT mice injected with DEN and fed a high fat diet (HFD) for 8-10 months. Although there was no difference on HCC multiplicity and HCC volume between WT and sirt1y/y mice, sirt1y/y mice showed substantial increase in hepatic actylated-FoxO1, and both mRNA expression and protein levels of p21, indicating a strong association between decrease of SIRT1 deacetylase activity and hepatic cellular senescence. Both hepatic inflammatory foci and mRNA expression of inflammatory markers (IL-6 and IL-1beta significantly decreased in sirt1y/y mice. These data suggest that lack of SIRT1 may promote steatosis by inducing hepatic cellular senescence, rather than by inducing inflammation. Cancer and Biology. C. In Study 3, male and female BCO1-/-/BCO2-/- double knockout mice (DKO) and wild type (WT) littermates were supplemented with BCX, an oxygenated provitamin A carotenoid and then exposed to cigarette smoke (CS). We observed that CS-exposure significantly induced macrophage and neutrophil infiltration in the lung tissues of mice, regardless of genotypes, compared to the non-exposed littermates. BCX treatment significantly inhibited CS-induced-inflammatory cell infiltration, hyperplasia in the bronchial epithelium, and enlarged alveolated airspaces in both WT and DKO mice, regardless of sex. The protective effects of BCX were associated with the lower expression of IL-6 and TNF-a, and matrix metalloproteinases-2 and -9. BCX treatment led to a significant increase of hepatic BCX levels in DKO mice, but not in WT mice with significant increase of hepatic vitamin A concentration. No apo-10’-carotenoids were detected in any of the groups. We conducted an in vitro experiment using human bronchial epithelial cell line BEAS-2B cells, which were pre-treated with BCX and subsequently, stimulated with lipopolysaccharide (LPS). We found that BCX, at comparable doses of 3-OH-ß-apo-10’-carotenal (a cleavage metabolite of BCX), inhibited LPS-induced elevation of IL-6 and TNF-a mRNA levels in a dose-dependent manner. The effect of BCX was associated with reduced AKT phosphorylation and stabilized IBa protein in BEAS-2B cells. We did not detect any cleavage metabolites of BCX in this culture system. These data indicate that BCX can serve as an effective protective agent against inflammation, independent of its cleavage metabolites including vitamin A.

Accomplishments

Review Publications
Chiaverelli, R.A., Hu, K., Liu, C., Lim, J., Daniels, M.S., Xia, H., Mein, J., Lintig, J.V., Wang, X. 2023. Beta-Cryptoxanthin attenuates cigarette smoke-induced lung lesions in the absence of carotenoid cleavage enzymes in mice. Molecules. https://doi.org/10.3390/molecules28031383.
Neeson, C., Wang, X. 2023. Implication of SIRT1 on development of nonalcoholic fatty liver disease and impact of carotenoid intervention. JSM Gastroenterology and Hepatology. 10(1):1114.
Guo, C., Kim, S.J., Frederick, A., Li, J., Jin, Y., Zeng, H., Mason, J.B., Liu, Z. 2019. Genetic ablation of tumor necrosis factor-alpha attenuates colonic Wnt-signaling associated with obesity. Journal of Nutritional Biochemistry. https://doi.org/10.1016/j.jnutbio.2019.108302.
Fedosov, S.N., Nexo, E., Heegaard, C.W., Goldin, J., Mason, J.B. 2023. Protein binding assays for an accurate differentiation of vitamin B12 from its inactive analogue. A study on edible cricket powder. Food Chemistry. https://doi.org/10.1016/j.fochx.2023.100824.