Translating molecular targets for chemoprevention into interventions for at-risk populations
Authors: Thomas Kensler, J. Groopman, B. Roebuck, T. Sutter, P. Egner, P. Dolan, M. Yamamoto, G. Bailey, J. Fahey, L. Jacobson, S. Gange, A. Munoz, G-s. Qian, Y. Zhu, B. Zhang, J-g. Chen, Paul Talalay
Journal: Cancer Research (2007)

LE16-01   Unlike many human cancers, the etiology of hepatocellular carcinoma (HCC) is well understood. Infection with hepatitis viruses coupled with dietary exposure to the fungal toxin, aflatoxin, results in multiplicative increases in risk, and accounts for much of the disease. While primary prevention entailing vaccination against hepatitis viruses and avoidance of aflatoxin exposure is appealing, these strategies will require considerable time and resources to be successful. In the developing world, where the burden of HCC is highest, immediate, practical, and economical approaches are mandatory. Thus, targeted chemoprevention may be most appropriate for the current generation of individuals at risk (1).  Chemoprevention involves the use of natural or synthetic agents to block, retard, or even reverse the carcinogenic process. Two approaches have been evaluated for reducing the body burden of aflatoxins _ use of interceptor molecules and inducers of carcinogen detoxication pathways. Chlorophylls and their water soluble salts (chlorophyllins) are constituents of the human diet and have been found to be effective anti-carcinogens in several animal models. Chlorophyllin is a mixture of sodium-copper salts of chlorophyll that is marketed as an over-the-counter drug for controlling odor and an accelerant in wound healing, and is extensively used as a food additive for coloration. Chlorophyllin can act as an ‘interceptor molecule’ through the formation of tight molecular complexes with carcinogens such as aflatoxin, thereby diminishing bioavailability by impeding their absorption. In a clinical trial performed in Qidong, China, chlorophyllin consumption at each meal led to an overall 55% reduction in median urinary levels of excreted aflatoxin-DNA adducts compared to placebo (2). However, supplementation of diets with foods rich in chlorophylls might represent a more practical means of administration.  >Activities of enzymes that are involved in the metabolic detoxication of aflatoxin are influenced by nutritional status, age, hormones and exposure to drugs or other xenobiotics. Oltipraz, a drug originally developed for the chemotherapy of schistosomiasis,  is an effective inducer of enzymes that detoxify carcinogens ( e.g.,  glutathione  S -transferases and UDP-glucuronosyltransferases) and is a potent anticarcinogen in animals (3). A common  cis -acting sequence, the Antioxidant Response Element (ARE), is found in the promoter regions of these protective genes. Several transcription factors are known to bind to this motif, such as members of the basic leucine zipper NF-E2 family. One such factor, Nrf2, appears to be a critical molecular target for the regulation of inducible and/or basal expression of genes by the ARE (4). An actin-binding protein, Keap1, sequesters Nrf2 in the cytoplasm by binding to its amino-terminal regulatory domain. Keap1 is a sulfhydryl-rich protein, and several cysteine residues mediate the Keap1-inducer interaction. Treatment with oltipraz or other inducers such as sulforaphane alters the interaction between Keap1 and Nrf2, allowing Nrf2 to translocate to the nucleus. In the nucleus, Nrf2 forms heterodimers with small Maf-family proteins to activate gene expression. Highlighting the importance of this signaling pathway,  Nrf2 -deficient mice are considerably more sensitive to carcinogenesis than wild-type mice, perhaps reflecting a lower constitutive expression of carcinogen detoxication enzymes. Moreover, the cancer chemopreventive efficacies of oltipraz and sulforaphane are completely lost in the knockout mice (5). Genomic, proteomic and biochemical analysis indicate that Nrf2 regulates the expression of carcinogen detoxication and antioxidative genes, as well as those affecting glutathione homeostasis, NADPH generation, solute transporters, and proteasome function. Monitoring for induction of Nrf2-regulated genes led to the original identification of oltipraz as a potential chemopreventive agent (3) and, more recently, the recognition that some triterpenoids are exceptionally potent inhibitors of aflatoxin carcinogenesis  in vivo  (6). As proof of principle for the merit of targeting Nrf2, administration of oltipraz in a placebo-controlled, randomized, double-blind clinical trial also conducted in Qidong resulted in a 2.6-fold increase in the excretion of aflatoxin-mercapturic acid, a detoxication product of the reactive, DNA-damaging intermediate of aflatoxin  (7). However, pharmaceutical-based interventions for populations at highest risk for HCC, such as those in Southeast Asia, China and sub-Saharan Africa, might not be a very practical approach to chemoprevention. Drugs like oltipraz are typically expensive and therefore beyond the reach of those who would benefit the most.  >Natural products are not  de facto  safer than synthetic agents, although patterns of long-term ingestion of certain food types provide guides for identifying promising compounds or foods themselves. A decrease in risk of HCC is associated with increased consumption of yellow-green vegetables, which contain a range of biologically active phytochemicals. Particularly encouraging are the findings that edible plants belonging to the family Cruciferae and genus  Brassica  ( e.g. , broccoli, cauliflower, Brussels’ sprouts) contain substantial quantities of isothiocyanates  - mostly in the form of their glucosinolate precursors. The major isothiocyanate in 3-day old broccoli sprouts, sulforaphane, is an exceedingly potent inducer of protective enzymes and inhibitor of carcinogenesis in rats. Studies of such plant materials in high-risk cohorts for HCC are in progress. Initial studies indicate inverse associations between urinary levels of sulforaphane metabolites and aflatoxin-DNA adducts (8).  >Although cancer prevention, in its ideal form, entails permanent reduction or elimination of tumor development, short of successful universal vaccination and eradication of carcinogenic exposures, such a goal is not realistic. Chemoprevention provides opportunities to create molecular detours, but not necessarily roadblocks, to impede the carcinogenic process. Numerous animal studies have demonstrated that chemopreventive interventions act to not only reduce cancer incidence, but to significantly extend tumor latency as well. These latter effects alone presage strong public health benefit.  >1. Kensler, T.W., et al. (2003) Translational strategies for cancer prevention in liver.  Nature Rev. Cancer  3: 321-329.  >2. Egner, P., et al.  (2001)  Chlorophyllin intervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer.  Proc. Natl. Acad. Sci USA  98: 14601-14606.  >3. Kensler, T.W., et al. (1999) Perspective: development of cancer chemopreventive agents - oltipraz as a paradigm.  Chem. Res. Toxicol . 12:113-126.  >4. Kensler, T.W., et al. (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway.  Ann. Rev. Pharmacol. Toxicol . 47: 89-116.  >5. Yates, M.S.,et al. (2006) Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole.  Cancer Res.  66: 2488-2494.  >6. Wang, J-S., et al.  (1999) Protective alterations in phase 1 and 2 metabolism of aflatoxin B 1  by oltipraz in residents of Qidong, People9s Republic of China.  J.  Natl. Cancer Inst.  91:347-354.  >7. Ramos-Gomez, et al. (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.  Proc. Natl. Acad. Sci. (USA)  98: 3410-3415.  >8. Kensler, T.W., et al.. (2005) Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthene tetraols in a randomized clinical trial in He Zuo Township, Qidong, PRC.  Cancer Epidemiol. Biomarkers Prev . 14:2605-2613.