Research Project: Dietary Strategies for Cancer Prevention

Location: Jean Mayer Human Nutrition Research Center On Aging

2021 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 LAB The investigators previously demonstrated, in an animal model of obesity-promoted colon cancer, that the combination of supplemental curcumin and vitamin B6 suppresses tumorigenesis by 60-80%, and is far superior to either agent alone. Over the past year, the investigators have continued to elaborate the biochemical and molecular basis of the synergy through which curcumin and B6 interact to produce this synergy. These observations are immediately relevant to Objective #1. The investigators previously found that gut bacterium P. distasonis (Pd) suppressed the formation of chemical and obesity-induced colon tumors in mice, reduced inflammatory cytokines in mice and cell culture and increased gut barrier integrity in vitro and in vivo. Several studies following up on these findings, or related to the role of gut bacteria in colorectal cancer (CRC) and aging, are underway. Specifically: -Rodent studies are underway to determine if Pd can suppress age-elevated colonic inflammation and strengthen intestinal barrier integrity. -Methods have been established to deplete the gut microbiota mouse and then replenish with material from another donor animal. -Aging is a risk factor for CRC. Animal studies are underway to understand how age-altered gut microbial communities can promote CRC. -In Drosophila it is established that increased gut permeability is associated with shorter lifespan. Because Pd reduced gut permeability we sought to test if Pf could do the same in flies and also affect lifespan. In collaboration with a Biologist at Tufts University, we fed Pf to Drosophila and found that it increased lifespan and slowed age related loss of physical vigor. -Cell and molecular studies are ongoing to identify anti-inflammatory proteins from Pd. An extramural grant was secured to expand the scope of mechanistic questions that could be explored. These studies are immediately relevant to Objective #2. The investigators continued to work with a rodent diet company to design diets that could be used to determine whether a diet whose protein is sourced from cricket powder is less tumorigenic than one whose protein is sourced from processed pork, soy flour or milk solids. Diets were formulated and the nutrient compositions were analyzed to ensure that diet components that were potentially confounding effect modifiers were normalized. Further refining of these diets is underway to complete normalization of any confounding factors. An extramural grant was secured to expand the scope of the study to encompass additional mechanistic questions. This work is immediately relevant to Objective #3. NUTRITION AND CANCER BIOLOGY LAB In support of Objective 1 and Objective 2, we have conducted three animal studies: In the first study, we investigated whether phytochemical xanthophyll beta-cryptoxanthin (BCX) prevent nonalcoholic fatty liver disease (NAFLD) development dependent on carotenoid cleavage enzyme, beta-carotene 9’,10’-oxygenase (BCO2). Male BCO2-/- knock out (KO) mice at 1 month and 5 months of age were fed with or without BCX concentrate (10 mg/kg diet, equivalent to the daily human consumption of 3-4 ounces of sweet red peppers) for 3 months. Results showed that BCO2-/- KO mice developed NAFLD at 8 months old of age, but not at 4 months old of age. Moreover, BCX feeding significantly reduced the severity of NAFLD of 8 months old BCO2 KO mice, which was associated with increased master metabolic regulators (SIRT1 activity/PGC1a/PPARa expression), inhibition of fatty acid synthesis genes (FAS and SCD1) and up-regulation of fatty acid oxidation gene (MCAD) in the liver of BCO2 KO mice. There were no differences in the hepatic levels of vitamin A (retinol and retinyl palmitate) among all groups. The present study supports that the aging process is related to lipid metabolism alterations in the liver. This study also suggests that BCX at physiologic dosage can protect against NAFLD development and activate SIRT1 pathways in the absence of carotenoid cleavage enzyme BCO2. In the second study, we investigated whether dietary phytochemical lycopene can inhibit cigarette smoke (CS)-induced NAFLD development using ferret model to mimick cigarette smokers and second-hand smokers. Ferrets were divided into 4 groups: control, CS exposed, CS plus low-dose lycopene [2.2 mg/kg BW/day, which is equivalent to 30 mg/day lycopene intake in humans (one cup of canned tomato paste provides 75 mg of lycopene)], and CS plus high-dose lycopene (6.6 mg/kg BW/day) groups, for 26 weeks. Results showed that CS-exposed ferrets developed nonalcoholic steatohepatitis,together with key genes involved in hepatic fibrogenesis as compared to the control group. Furthermore, lycopene feeding at low and high lycopene doses significantly reversed these detrimental changes by CS to the levels of the control group. Interestingly, a major lycopene cleavage enzyme, beta-carotene 9’,10’-oxygenase (BCO2) was down-regulated by CS exposure, but this decrease was prevented by lycopene feeding. CS exposure also downregulated liver expression of antioxidant enzymes and upregulated oxidative stress marker, which were all prevented by lycopene. The present study suggest that CS can promote development of nonalcoholic steatohepatitis and liver fibrosis in ferrets, which is associated with downregulation of BCO2 and impairment of antioxidant system in liver; and dietary lycopene may inhibit CS-promoted nonalcoholic steatohepatitis by preventing suppression of BCO2 and decline in antioxidant network. In the third study, we used Sirt1y/y homozygous male mice carrying a point mutation (H355Y) that ablates the deacetylase activity, along with their wild type littermates (Sirt1+/+), and followed them for 6, 10 and 18 months of age. Results showed that Sirt1y/y homozygous mice developed severe pulmonary emphysema at the ages of 6, 10 and 18 months, with the respective incidences of 33%, 100% and 100%, while the Sirt1+/+ wild-type mice only developed emphysema (13% incidence) at 18 months of age. The present study indicated that the lack of SIRT1 enzymatic activity plays a major role in the susceptibility of organs to aging and pulmonary emphysema development by inducing cellular senescence and disrupting circadian clock genes. The development of emphysema in Sirt1y/y mice was accompanied with higher protein levels of matrix metalloproteinase (MMP)-2, MMP9, and tissue inhibitor of metalloproteinase-1, and ratio of cleaved/total anti-poly (ADP-ribose) polymerase. The ablation of SIRT1 activity significantly up-regulated mRNA expression of hypoxia-inducible factor-1a and retinoic acid receptor-beta, while p21 protein and phosphorylated AMPK increased and phosphorylated ribosomal S6 decreased, suggesting the association of ablation of SIRT1 activity with cellular quiescence and senescence. Additionally, the lack of SIRT1 activity down-regulated the mRNA expression of circadian clock genes (BMAL1, NPAS2, CRY1, CRY2) in the lungs of Sirt1y/y mice, as compared with that of Sirt1+/+ mice.

Accomplishments
1. Certain gut bacteria reduces the risk of colon cancer. While colon cancer continues to be the third most common cause of cancer death in U.S. adults, cost-effective and non-invasive means of prevention are still lacking. ARS-funded researchers in Boston, Massachusetts, built on studies of the gut bacterium, P. distasonis, which they had previously shown to suppress colon cancer formation in mice. They found the anti-cancer effect is related to the suppression of inflammation and strengthening gut barrier integrity. Further, the investigators also found that feeding this bacterium to fruit flies can also increase gut barrier integrity, increased median lifespan and retard age-related loss of physical vigor.

2. Pigment abundant in tomato reduces cigarette smoking-related fatty liver and inflammation. Cigarette smoke is an independent risk factor in development of nonalcoholic fatty liver, inflammation, and fibrosis. ARS-funded researchers in Boston, Massachusetts, using a ferret model to simulate cigarette smokers and people exposed to second-hand smoke, provided strong experimental evidence that feeding lycopene, a red pigment abundant in tomato and tomato products was effective in reducing the effect of cigarette smoke-induced liver inflammation. The protective effects of lycopene were associated with increased antioxidant enzymes activity and carotenoid cleavage enzyme in the liver. This study indicates that cigarette smokers and people exposed to second-hand smoke may benefit from the protective effects of increased consumption of foods rich in lycopene.

Review Publications
Mason, J.B. 2020. Nutritional principles and assessment of the gastroenterology patient. In: Feldman, M., Lawrence, Friedman, S., Brandt, L.J. Sleisenger and Fordtran's Gastrointestinal and Liver Disease. 11th edition. Philadelphia, PA: Elsevier. Volume 1. p.52-73.
Mustra Rakic, J., Liu, C., Veeramachaneni, S., Wu, D., Paul, L., Ausman, L., Wang, X. 2021. Dietary lycopene attenuates cigarette smoke-promoted nonalcoholic steatohepatitis by preventing suppression of antioxidant enzymes in ferrets. Journal of Nutritional Biochemistry. 91(2021):108596. https://doi.org/10.1016/j.jnutbio.2021.108596.
Zhu, R., Chen, B., Bai, Y., Miao, T., Rui, L., Zhang, H., Xia, B., Li, Y., Gao, S., Wang, X., Zhang, D. 2020. Lycopene in protection against obesity and diabetes: A mechanistic review. Pharmacological Research. 159:104966. https://doi.org/10.1016/j.phrs.2020.104966.