(Chest. 2004;125:128S-133S.)
© 2004
American College of Chest Physicians
Mechanisms of Chemoprevention*
Zbigniew Walaszek, PhD;
Margaret Hanausek, PhD and
Thomas J. Slaga, PhD
* From the AMC Cancer Research Center, Denver, CO.
Correspondence to: Thomas J. Slaga, PhD, AMC Cancer Research Center, 1600 Pierce St, Denver, CO 80214; e-mail: slagat{at}amc.org
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Introduction
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The induction of cancer (carcinogenesis) depends on inherited and acquired susceptibility factors, on exposure to initiation factors (exogenous and endogenous carcinogens), and on promotion and progression factors. Chemoprevention, namely inhibition or reversal of carcinogenesis, may be conducted at variety of time points in this process to reduce occurrence of in situ or invasive cancers (primary intervention at earlier stages in the process) or cancer morbidity and/or mortality (secondary intervention at later stages in the process). As broadly defined above, chemoprevention applies to the prevention of clinical cancer by the administration of pharmaceuticals or dietary constituents. The efficacy of such prevention interventions is evaluated in clinical trials. Phase I trials determine the dose-related safety of drugs and frequently include pharmacokinetic studies. Phase II and III trials are used to test drug activity. Agents used in successful phase II clinical trials have evidence of chemopreventive efficacy and the high likelihood of the agent preventing cancer at the target site. They must have a high margin of safety and a logical presumed mechanism of chemopreventive activity. The availability of effective chemoprevention agents is only one component of a full chemoprevention program. Another important component is the availability of a marker or markers, which can help evaluate the effects of chemopreventive agents early during the prevention trials and evaluate individuals as to the magnitude of general or site-specific risk to cancer. Many of the molecular markers, such as activating mutations in oncogenes and inactivating mutations in tumor suppressor genes, can often be detected in earlier stages of cancer development. The development of new cancer preventive agents and the evaluation of efficacy of novel biological or molecular markers as intermediate end points in prevention trials are important avenues in cancer research.
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Multistage Carcinogenesis
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The concept that carcinogenesis is a multistep and multifunctional phenomenon is now widely supported by researchers. With the early experimental design,1 which employs a two-step procedure for the treatment of mouse skin, a useful animal model became available to study the multistep nature of carcinogenesis. In fact, some early investigators began to analyze the process, and they defined the concepts of tumor initiation and promotion as well as co-carcinogenesis in operational terms.2 For much of the time during which initiation-promotion studies in mouse skin were being pursued by a number of researchers,3 little experimental evidence existed, other than hormone-dependent tumors, that similar processes occurred at other organ sites. Later, however, these sequential events were also found to occur in liver, urinary bladder, breast, cheek pouch, esophagus, colon, stomach, lung, and prostate.45 The generality of the sequential nature of biological events in carcinogenesis is especially true in those tumors induced by exogenous chemical agents. In addition to extensive studies in the mouse skin model, several approaches to the sequential evolution of liver tumors by multistep protocols have been studied.67 The greatest understanding of the important biological and cellular events involved in tumor initiation, promotion, and progression has been provided by studies in the skin and liver models.5 Understanding the mechanism(s) by which an agent induces, promotes, or enhances cancer is important in overall risk assessment. Although one can question the relevance to humans of tumor promotion in experimental animals, it is important to emphasize that tumor promoters, in general, induce the cell proliferation, which is critically involved in selectively expanding initiated cells into tumors.
Malignant neoplasia is the result of multiple genetic defects successively accumulating over a period of time.8 By providing confirmation of molecular defects, human cancer genetics strongly, though indirectly, supports the concept of multistage carcinogenesis. However, only animal models can provide direct information on the underlying mechanisms and enable a final proof of the multistage concept. The results from the skin model, in particular, appear to be of relevance for a more in-depth understanding of the human epithelial cancers including colorectal cancer.8 Thus, the induction of a neoplasm is a multistage process that occurs over a long period of time. These stages have been defined experimentally as initiation, promotion, and progression.
Initiation involves mutation of cellular DNA resulting in the activation of oncogenes and the inactivation of tumor suppressor genes. Initiation is thought to be irreversible and consist of a single gene mutation that is caused in most cases by environmental genotoxic agents such as chemicals, radiation, and viruses. Oncogenes can also be activated by chromosomal translocations and gene amplifications. Studies in the human colon indicate that the carcinogenic process involves multiple genetic alterations in a staged fashion.9
Promotion follows initiation and involves the process of gene activation, such that the latent phenotype of the initiated cell becomes expressed through cellular selection and clonal expansion. This can occur through a variety of mechanisms, including toxicity, terminal differentiation or mitoinhibition of the noninitiated cells, and mitogenesis of the initiated cells.34 While promotion occurs over a long period of time, it is reversible in its early stages. Proof that promotion is reversible in humans is supported by the observation that the rate of lung cancer induction in individuals who quit smoking approaches that of nonsmokers.10 The breadth of the available data, as well as the multistage nature of tumor promotion, suggests that this process, which is now thought to occur in most tissues in which cancer can be induced or in which it occurs spontaneously, may involve the interaction of a number of endogenous factors as well as environmental factors such as chemicals, radiation, viruses, bacteria, and diet and nutrition, thus unifying all current areas of cancer research.45 In human cancer, smoking; environmental factors such as asbestos, hydrocarbons, radiation, and hormones; alcoholic beverages; and diet and nutrition are now thought to have more of a promotional influence in the multistage carcinogenesis process.11
The last step leading to cancer is called progression. Progression involves genetic damage that results in the conversion of benign tumors into malignant neoplasms capable of invading adjacent tissues and metastasizing to distant sites. The additional genetic alterations thought to be required for neoplastic progression often occur faster than would be expected from the statistics of accidental genotoxic insults due to so-called genetic instability. The concept of genetic instability implies that while environmental genotoxic agents generally cause cancer initiation, the additional mutations required for neoplastic progression may be attributed to endogenous reactions and factors such as detoxification and removal of damaged cells by programmed cell death. Genetic instability may happen due to the errors in DNA replication, spontaneous hydrolytic alterations of DNA such as depurination and deamination in combination with an impaired ability of premalignant cells to repair DNA damage or due to oxidative DNA damage.12 Modified DNA bases, especially 8-hydroxy-2'-deoxyguanosine, produced by oxygen-free radicals have been implicated in the genesis of cancer.13 The importance of free radicals in radiation carcinogenesis and oxygen-free radicals and electrophiles in chemical carcinogenesis is also well recognized.
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Inhibition of Different Stages of Carcinogenesis
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There has been significant progress in the understanding of the multistage nature of carcinogenesis, and the mechanisms of cancer prevention.14 It is now apparent that the cellular evolution to malignancy involves the sequential alteration of proto-oncogenes15 and/or tumor suppressor genes,16 whose gene products participated in critical pathways for the transduction of signals and/or regulation of gene expression. One of the goals of current studies in cancer prevention is to determine the mechanisms of synergistic action of the natural source compounds and/or synthetic chemopreventive agents, known to inhibit one or more stages of carcinogenesis, ie, initiation, promotion, and progression. The basic theory underlying these studies is that concurrent treatment with various natural source or synthetic inhibitors of different stages of carcinogenesis results in synergistic effects leading to more efficient prevention of cancer.
The mechanisms that focus on initiation events (Table 1
) include the following: inhibition or alteration of phase I enzymes responsible for the formation of reactive carcinogenic metabolites, ultimately leading to their reduced formation; inhibition or induction of oxidative enzymes pathways that produce products of lower carcinogenic potential; induction of detoxification enzymes (phase II enzymes) and pathways (nonoxidative) for both proximate and ultimate carcinogen; scavenging of reactive, carcinogenic intermediates through direct chemical interaction; inhibition or enhancement of DNA repair mechanisms; and inhibition of cell proliferation and DNA synthesis. A number of potent inhibitors of tumor initiation appear to be effective because they either prevent the formation of the ultimate carcinogen and/or scavenge the ultimate carcinogen. Some phenolic antioxidants have been studied extensively, primarily because of their use as food preservatives. In addition to the alterations in oxidative metabolism, the phenolic antioxidants have also been shown to increase the detoxification pathways for many chemical carcinogens.11 The indoles, aromatic isothiocyanates, coumarines, flavones, di- and triterpenoids, dithiothiones, organosulfides, chlorophyllin, and D-glucarates have a potent effect on the metabolism of carcinogens.111718 In general, they appear to have a major effect on the detoxification of carcinogens.19 Ellagic acid, 2,6-dithiopurine, and chlorophyllin have been also shown to be highly potent in scavenging the ultimate (reactive) carcinogenic form of carcinogens. Disulfiram, vitamin C, and vitamin E appear to inhibit chemical carcinogenesis in a similar manner to the phenolic antioxidants by their effect on the metabolism of the carcinogen, their antioxidizing activity, and preventing the formation of carcinogens.171819 The mechanism by which selenium inhibits chemically induced tumors may be related to its effect on glutathione peroxidase since it is a cofactor for this enzyme.11
The process of tumor promotion/progression involves a combination of several mechanisms. Among antitumor-promoting mechanisms (Table 1), the ones that are most promising include the following: inhibition of inflammation; inhibition of cell proliferation and hyperplasia; modulation of cell differentiation and apoptosis; scavenging of reactive oxygen species and preventing depletion of antioxidant defense systems; and enhancement of tumor promoters detoxification pathways. The natural and synthetic cancer preventive agents that exert their effects against tumor promotion usually inhibit one, more, or even all events involved in the tumor promotion process.81418 It is unclear, however, whether inhibition of one particular event involved in promotion by chemopreventive agents is sufficient and/or necessary to exert their maximum to complete antitumor promoting effects. Selenium was found to be effective inhibitor of skin tumor promotion by croton oil. Selenium is a necessary cofactor for the enzyme glutathione peroxidase that detoxifies hydrogen peroxide and hydroperoxides within the cell. The possibility exists that Se-dependent glutathione peroxidase lowers the level of potentially dangerous and damaging peroxide radicals generated from various co-carcinogenic and promoting chemicals. 
-Tocopherol and L-ascorbic acid are also known to significantly reduce tumor formation induced by 7,12-dimethylbenz[a]anthracene and croton oil. -Tocopherol also reduced the number of fibrosarcomas induced by 3-methylcholanthrene and mammary gland adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene.11 Since the inhibitory effect of L-ascorbic acid was observed in some cases long after the carcinogen was administered, this observation indicates it was active during promotional stage or possibly during progression.18
In recent years, several novel antioxidants such as proanthocyanidins, ursolic acid, and chlorophyllin also have been found to inhibit chemical carcinogenesis and mouse skin tumor promotion.1118 Caventol was found to inhibit skin tumor promotion through inhibition of tumor necrosis factor- release and protein isoprenylation. Several polyphenolic antioxidants in green tea have been shown to inhibit chemical carcinogenesis and mouse skin tumor promotion.1118 A number of other antioxidants have also been found to inhibit tumor promotion and/or progression in mouse skin. Although their mechanisms of action are not definitely known, evidence points to several possibilities: they scavenge various radicals generated directly or indirectly by tumor promoters, they increase levels of enzymes that are important in detoxifying cellular radicals, or they have other specific functions.18 Several antioxidants have been shown to have synergistic activities such as vitamins C and E, and vitamin E, selenium, and -lipoic acid.11
Compounds such as the very effective anti-inflammatory steroids inhibit early in the pathway and consequently inhibit the formation of all the important end products of arachidonic acid metabolism.20 The most consistent inhibitors of tumor promotion/progression are nonsteroidal anti-inflammatory drugs that inhibit both the cyclo-oxygenase and lipoxygenase pathways. Recent findings, reviewed by Marks and Furstenberger,8 strongly indicate that both cyclo-oxygenase and lipoxygenase may represent novel targets of cancer chemoprevention.
Glucocorticoids affect cellular functions via the glucocorticoid receptor, a well-known transcription factor. Recent results21 provide in vivo evidence that the glucocorticoid receptor plays a tumor suppressor role in skin carcinogenesis induced by oncogenic ras, possibly through transrepression of nuclear factor-
B–dependent genes and alteration of proliferation/apoptosis/differentiation balance in transformed keratinocytes.
Studies demonstrate that plants are rich in compounds, such as avicins, ie, triterpenoid saponins that inhibit oxidative stress and induce programmed cell death of premalignant and malignant cells.22 These studies indicate that avicins could develop as important chemopreventive agents in many conditions in which chronic inflammation and oxidative and nitrosative stress may lead to tumorigenicity.22 Table 2
summarizes a current rational approach to cancer prevention.
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Chemoprevention of Lung Cancer
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The -Tocopherol, ß-Carotene Cancer Prevention Study enrolled > 29,000 male smokers in Finland, using a two-by-two factorial design with the above agents individually, in combination, and with placebo.23 Individuals randomized to receive beta-carotene had a statistically significant 18% increased risk for acquiring lung cancer. Following the announcement on these results, an interim analysis was performed on the ß-Carotene and Retinol Efficacy Trial, which was terminated early when the analysis showed a 28% increase in lung cancer incidence in the beta-carotene study arm.24 A study by Clark et al25 showed a striking reduction in lung cancer, as well as other epithelial cancers, in individuals with prior skin cancers treated with selenomethionine. A study by Keith et al26 demonstrated that manipulation of pulmonary prostacyclin synthase expression prevents murine lung cancer. In addition, both steroidal and nonsteroidal anti-inflammatory agents are very effective inhibitors of lung cancer development induced by several different lung carcinogens. The steroidal anti-inflammatory agent dexamethasone is also very effective against cigarette smoke-induced lung cancer. (See page 128S in this supplement.)
There is now extensive evidence2728 from short-term and long-term models for the possible control of different stages of carcinogenic process by the ß-glucuronidase inhibitor D-glucaro-1,4-lactone, and specifically by its precursors such as D-glucaric acid salts (D-glucarates). D-Glucaric acid has been found in some vegetables and fruits.29 Thus, the consumption of fruits and vegetables naturally rich in D-glucaric acid, or self-medication with D-glucaric acid derivatives such as calcium D-glucarate or potassium hydrogen D-glucarate, offers a promising chemopreventive approach. A phase I clinical trial30 was undertaken to begin to explore the potential role of D-glucaric acid in the prevention of lung cancer in tobacco smokers. Current smokers and nonsmokers, both men and women, were assigned to escalating doses of calcium D-glucarate administered over a 6-week period. Blood levels of D-glucaric acid and ß-glucuronidase were determined at baseline and every 2 weeks. In addition, lymphocytes and sputum specimens were collected for K-ras oncogene determination. A consistent suppression of ß-glucuronidase levels was achieved by increasing doses of calcium D-glucarate that, in turn, correlated well with increasing D-glucaric acid blood levels. DNA was isolated from the lymphocyte fraction of the blood and sputum obtained from male and female smokers and nonsmokers (baseline). Mutated K-ras, an oncogene linked to lung cancer, was found in the DNA isolated from baseline blood lymphocytes and from sputum of some male smokers. No K-ras mutations were found in nonsmokers. The baseline D-glucaric acid level in the blood of smokers with mutated K-ras was significantly lower (ie, by 
34%, p < 0.05) than in other smokers or nonsmokers. No unusual toxicity was encountered in this phase I study, and calcium D-glucarate was well tolerated, even at the highest dose levels.31 Thus, calcium D-glucarate supplementation has potential for reducing the risk of lung cancer development in tobacco smokers.
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Conclusions
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Scores of epidemiologic studies have noted a lower risk of cancer among persons whose diet includes a relatively large amount of vegetables, fruits, and other plant products. A popular explanation, both within the scientific community and among members of the public, is that different vitamins and other micronutrients in vegetables, fruits, and other natural plant products prevent carcinogenesis by interfering with detrimental actions of mutagens, carcinogens, and tumor promoters. These natural inhibitors of carcinogenesis are apparently nontoxic or markedly less toxic than many drugs. While it is generally accepted that a diet of large amounts of vegetables, fruits, and other plant products lowers cancer incidence, there is still a need to identify the most effective constituents of the diet as well as to elucidate their mechanisms of action. In conclusion, many natural and synthetic cancer preventive agents are effective inhibitors of tumor initiation, promotion, and progression. In a number of cases, the mechanism(s) of action are related to their abilities to prevent critical carcinogen metabolism, and to increase detoxification pathways for carcinogens and free radicals as well as to their antioxidizing activity. Table 3
summarizes critical mechanisms involved in effective cancer prevention. Usually, cancer preventive agents inhibit the initiation and promotion/progression stages to a different degree. Therefore, a combination(s) of various natural and synthetic cancer preventive agents, with different mechanisms of action, will most likely prove to be more effective in inhibiting the development of cancer compared to one agent alone. Table 4
summarizes important chemopreventive agents that are effective against the induction of cancer in both animal and human studies. These compounds should be very effective in combination against lung cancer as well as other cancers.
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Introduction
Multistage Carcinogenesis
