2008 Annual Report 1a.Objectives (from AD-416) LAB:Cancer Biology To establish the dose response effects of lycopene in plasma and lung tissue with and without smoke exposure. To establish the potential for lycopene inhibition of smoke-induced lung lesions and mechanism(s) of action. To characterize both the expression and activity of carotene 9', 10'-monooxygenase in the metabolism of lycopene with and without smoke exposure, and the biological effect of the metabolite(s) of lycopene. 1b.Approach (from AD-416) LAB:Nutrition and Cancer Biology We will investigate the chemopreventive efficacy of lycopene against lung and prostate carcinogenesis in a ferret model (which is highly analogous to humans) under a variety of different circumstances (low and high doses of lycopene, with and without exposure to tobacco smoke, lung vs. prostate). We then would like to extend our work to an in vitro cell culture model, which allows in-depth mechanistic studies and examines whether the induction of IGFBP-3 with lycopene or its metabolites affect kinase activity in the IGF-I/Ras/PI3K/Akt or MAP Kinase pathways. Lastly, we will examine lycopene metabolism in different tissues (lung, liver, prostate, colon and stomach) and in the lycopene treated lung cells (normal cells vs. transformed cells) by examining carotene 9',10'-monooxygnase expression/function and its regulation by varying treatments. 3.Progress Report Investigated the chemopreventive efficacy of lycopene, a major carotenoid from tomato and tomato products, against cancer risk in animal models and cell culture models. Since this research is aimed at defining the relationship between lycopene, genetic signaling pathway and environmental factors (e.g. tobacco smoking, alcohol and chemical carcinogen exposures) and the risk of cancer, the progress of this research is highly aligned with components 6 and 7 of the National ARS National Program #107 Action Plan that are to examine the ‘Relationship between diet, genetics and lifestyle and the risk for chronic disease’ as well as ‘Health promoting properties of plant and animal foods'. During fiscal year 2008, we have investigated the metabolism and molecular biological functions of lycopene and its metabolite, particularly in terms of potential interaction with exogenous factors (e.g. tobacco smoking, alcohol and chemical carcinogen exposures):. 1)We conducted both animal and cell culture studies to determine whether apo-10’-lycopenoids have biological activities against lung carcinogenesis. We report that apo-10’-lycopenoic acid inhibited the growth of NHBE (normal human bronchial epithelial) cells, BEAS-2B immortalized normal bronchial epithelial cells, and A549 non-small cell lung cancer cells, as well as the carcinogen (NNK) induced lung cancer in an animal model;. 2)We examined the effects of apo-10’-lycopenoids on the expression of Nrf2-regulated phase II detoxifying/antioxidant enzymes, intracellular levels of glutathione and H2O2-induced oxidative damage in an immortalized human bronchial epithelial cell line, BEAS-2B. We report that apo-10’-lycopenoic acid, in a time- and dose-dependent manner, results in the nuclear accumulation of transcription factor Nrf2 protein in BEAS-2B cells, which is associated with the induction of phase II detoxifying/ antioxidant enzymes;. 3)We investigated potential interactions between alcohol ingestion and lycopene supplementation and their effect on hepatic lycopene concentration, cytochrome P4502E1 (CYP2E1) induction, and inflammation. We observed that lycopene supplementation at the higher dose significantly induced hepatic CYP2E1 protein, TNFa mRNA, and the incidence of inflammatory foci in the alcohol-fed rats, but not in the control rats;. 4)We investigated potential interactions between carcinogen (NNK)/smoking exposure and lycopene supplementation in the ferret model. We observed that the NNK treatment alone induced lung cancer; however, lycopene supplementation had no significant effect on lung tumor incidence and also was associated with higher mortality compared to the other groups; and. 5)We have explored the active form of vitamin D with or without in the presence of active form of vitamin A in a carcinogen (NNK)-initiated lung cancer model. We demonstrate that adding 9-cis retinoic acid to 1a,25-dihydroxyvitamin D3 treatment can eliminate symptoms of hypercalcemic toxicity and body weight loss, while maintaining tumor preventive effects. 4.Accomplishments 1. Anti-carcinogenic and antioxidant functions of lycopene are mediated by lycopene metabolite, apo-10’-lycopenoids. Lycopene is a major carotenoid mostly obtained by consuming tomato and tomato products. Previously we demonstrated that lycopene can be converted into its oxidative metabolites, called apo-lycopenoids, in biological tissues. However, the potential chemopreventive effect of apo-lycopenoids against lung cancer development is unknown. We demonstrated that apo-10’-lycopenoic acid inhibited the growth of lung cancer cells and significantly reduced lung tumor multiplicity in A/J mice in a dose-dependent manner without any apparently adverse effect. In addition, we demonstrated that the anti-carcinogenic and antioxidant functions of lycopene may be mediated by apo-10’-lycopenoids via inducing certain detoxifying/antioxidant enzymes. Our findings suggest the apo-10’-lycopenoic acid is a potential chemopreventive agent against lung tumorigenesis. This research is highly aligned with components 6 and 7 of the National ARS Program #107 Action Plan, that are to examine the ‘Relationship between diet, genetics and lifestyle and the risk for chronic disease’ as well as ‘Health promoting properties of plant and animal foods’. 2. Interaction between chronic alcohol ingestion and lycopene supplementation. Recent in vitro evidence suggests that the antioxidant lycopene can prevent alcohol induced oxidative stress and inflammation. However, in vivo knowledge of possible interactions between escalating doses of lycopene and chronic alcohol ingestion are lacking. We investigated potential interactions between alcohol ingestion and lycopene supplementation and their effect on hepatic lycopene concentration and inflammation. We observed that both plasma and hepatic lycopene concentrations were greater in alcohol-fed rats vs. control rats supplemented with identical doses of lycopene. Notably, lycopene supplementation at the higher dose significantly induced hepatic inflammation in the alcohol-fed rats, but not in the control rats. These data indicate an interaction between chronic alcohol ingestion and lycopene supplementation and suggest a need for caution among individuals consuming high amounts of both alcohol and lycopene. This research is aligned with Components 6 and 7 of the National ARS Program #107 Action Plan, that are to examine the ‘Relationship between diet, genetics and lifestyle and the risk for chronic disease’ as well as ‘Health promoting properties of plant and animal foods’. 3. Evidence of active form of vitamin A as antagonist of vitamin D toxicity. There is currently scientific debate about whether vitamin A synergizes or antagonizes the effects of vitamin D against lung cancer development. In order to determine this, we have explored the active form of vitamin D (1a, 25-dihydroxyvitamin D3) with or without in the presence of active form of vitamin A (9-cis retinoic acid) in a carcinogen-initiated lung cancer model. We demonstrate that adding 9-cis retinoic acid to 1a, 25-dihydroxyvitamin D3 treatment can eliminate symptoms of hypercalcemic toxicity (elevated serum calcium levels) and body weight loss, while maintaining tumor preventive effects. Our results are important to cancer prevention using vitamin A and/or vitamin D and will stimulate more research into the mechanism(s) behind this antagonistic interaction. The progress of this research is highly aligned with Components 6 and 7 of the National ARS Program #107 Action Plan, that are to examine the ‘Relationship between diet, genetics and lifestyle and the risk for chronic disease’ as well as ‘Health promoting properties of plant and animal foods’. 4. Interaction of carcinogen/smoke exposure and lycopene supplementation. In this study, ferrets were divided into six groups and were supplemented with placebo or two doses of lycopene with or without carcinogene (NNK). The NNK treatment alone induced lung cancer; however, lycopene supplementation had no significant effect on lung tumor incidence. The NNK treatment was associated with higher mortality and higher incidence of hepatic lesions, but both were reduced with lycopene supplementation. Ferrets were then divided into four groups - control, NNK and smoke alone or NNK + smoke supplemented with two doses of lycopene. Lycopene supplementation in smoke exposed ferrets was associated with higher mortality compared to the other groups. There were no significant effects on lung tumor incidence or lung inflammation. We conclude that the development of lung tumors in ferret lungs by injecting with NNK alone provides an excellent model for studying lung carcinogenesis; however, the effect of lycopene against lung lesions is inconclusive. The progress of this research is highly aligned with Components 6 and 7 of the National ARS Program #107 Action Plan, that are to examine the ‘Relationship between diet, genetics and lifestyle and the risk for chronic disease’ as well as ‘Health promoting properties of plant and animal foods’. 6.Technology Transfer Review Publications Lian, F., Smith, D., Ernst, H., Russell, R., Wang, X. 2007. Apo-10'-lycopenoic acid suppresses lung cancer cell growth by activating retinoic acid receptor Beta in vitro, and inhibits lung tumorigenesis in vivo in the A/J mouse model. Carcinogenesis. 28:1567-1574. Kim, Y., Lian, F., Yeum, K., Chongviriyaphan, N., Choi, S., Russell, R., Wang, X. 2007. The Effects of Combined Antioxidant Supplementation on Antioxidant Capacity, DNA Single-Strand Breaks and Regulation of Insulin Growth Factor-1/IGF-Binding Protein 3 in the Ferret Model of Lung Cancer. International Journal of Cancer. 120(9):1847-1854. Veeramachaneni, S., Ausman, L.M., Choi, S., Russell, R., Wang, X. 2008. High Dose Lycopene Supplementation Increases Hepatic CYP2E1 Protein and Inflammation in Alcohol-Fed Rats. Journal of Nutrition. 138(7):1329-1335. Mernitz, H., Wang, X. 2007. Beta-Carotene. In: Golson, J.G., Colditz, G.A. Encyclopedia of Cancer and Society. Sage Publications. p. 94-99. Lian, F., Wang, X. 2008. Apo-10'-lycopenoic acid, an enzymatic metabolite of lycopene, induces Nrf2-mediated expression of phase II detoxifying/antioxidant enzymes in human bronchial epithelial cells. International Journal of Cancer. 123:1262-1268. Lee, C., Wang, X., Chien, K., Ge, Z., Rickman, B.H., Rogers, A.B., Varro, A., Whary, M.T., Wang, T.C., Fox, J.G. 2008. Vitamin C supplementation does not protect L-gulono-gamma-lactone oxidase-deficient mice from Helicobacter pylori-induced gastritis and gastric premalignancy. International Journal of Cancer. 22(5):1068-1076. Wang, X., Mernitz, H. 2007. The Bioconversion of Carotenoids into Vitamin A: Implications for Cancer Prevention. In: Columbus, F., Loessing, I. T. editors. Vitamin A: New Research. Hauppauge, NY: Nova Science Publishers, Inc. p. 1-19.