| Featured Review Cellscience Reviews Vol 1 No.3 ISSN 1742-8130 |
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Introduction
The induction of cancer (carcinogenesis) is a multistage process and its stages have been defined experimentally as initiation, promotion, and progression. Carcinogenesis depends on inherited and acquired susceptibility factors, on exposure to initiation factors, i.e., exogenous and endogenous carcinogens, and on promotion and progression factors. The prevention of cancer, 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). As broadly defined above, cancer prevention applies to the prevention of clinical cancer by the administration of pharmaceuticals (chemoprevention) or dietary constituents.
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. Currently, most people are exposed to different combinations of agents. Because many of those compounds are taken orally, i.e., in diet or as dietary supplements, while some are now present in many cosmetics, the proposed studies are of great practical significance. The objective of this brief review is to pinpoint potential mechanisms of synergistic action of the natural source compounds, known to inhibit one or more stages of skin carcinogenesis, i.e. initiation and promotion/progression. Our hypothesis is that concurrent topical and systemic (i.e. dietary) treatments with selected natural source inhibitors of different stages of skin carcinogenesis result in synergistic effects leading to more efficient prevention of skin cancer. Studies designed to validate the above hypothesis will significantly contribute to better understanding of the role of specific natural source inhibitors and their combinations in prevention of human skin cancer and possibly other cancers.
Mouse Skin Model of Multistage Carcinogenesis
The concept that carcinogenesis is a multistage and multifunctional phenomenon is now widely supported by researchers. With the early experimental design (Berenblum, 1941), which employs a two-step procedure for the treatment of mouse skin, a useful animal model became available to study the multistage 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 (Friedelwald & Rous, 1944; Boutwell, 1964). These sequential events were also found to occur in liver, urinary bladder, breast, cheek pouch, esophagus, colon, stomach, lung, and prostate (Slaga, 1983; Slaga et al., 1995a,b; Walaszek et al., 2004). However, the greatest understanding of the important biologic and cellular events involved in tumor initiation, promotion, and progression, i.e., the last step leading to the conversion of benign tumors into malignant neoplasms, has been provided by studies in the mouse skin carcinogenesis model.
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 (Bishop, 1991; Marshall, 1991). 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 non-initiated cells and mitogenesis of the initiated cells (Slaga, 1984; DiGiovanni, 1992). While promotion occurs over a long period of time, it is reversible in its early stages. 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 (Slaga, 1984; DiGiovanni, 1992).
It is now apparent that the cellular evolution to malignancy involves the sequential alteration of proto-oncogenes (Bishop, 1991) and/or tumor suppressor genes (Marshall, 1991), whose gene products participated in critical pathways for the transduction of signals and/or regulation of gene expression. Skin tumor initiation by chemical carcinogens, such as 7,12-dimethylbenz[a]- anthracene (DMBA), appears to be an irreversible stage that probably involves a somatic mutation, mainly in the Ha-ras oncogene (Slaga et al., 1987). Extensive data has revealed a good correlation between the carcinogenicity of many chemical carcinogens and their mutagenic activity (Slaga et al., 1987). Most tumor-initiating agents either generate or are metabolically converted to electrophilic reactants, which bind covalently to cellular DNA (Slaga et al., 1987; DiGiovanni, 1992). Free radicals and the modified DNA bases by these radicals have also been strongly implicated in carcinogenesis in general (Floyd, 1990; Malins, 1993; Ames et al. 1993). Strong evidence from several laboratories indicates that activation of the Ha-ras gene occurs early in the process of mouse skin carcinogenesis and perhaps is equivalent to the initiation event (Quintanilla et al., 1986; Bizub et al., 1986). Quintanilla et al. (1986) demonstrated the presence of an activated c-Ha-ras gene in mouse skin papillomas and carcinomas induced by DMBA; the activation was associated with a high frequency of A-T transversions at codon 61. Subsequent studies demonstrated that the type of mutation was dependent upon the chemical initiator and independent of the promoter, suggesting a direct effect of the initiator on c-Ha-ras (Brown et al., 1990). Furthermore, infection of mouse skin by a virally activated Ha-ras gene (v-Ha-ras) can serve as the initiating event in two-stage carcinogenesis (Spalding et al.,1993). A large number of carcinogens and tumor initiators are known to be metabolized to electrophilic intermediates, which bind to DNA and cause mutations. There are specific enzymes, which try to detoxify these reactive intermediates directly. Once the genetic material is damaged specific DNA repair enzymes try to reverse the damage (Mitchell et al., 1995).
Selective inflammation and sustained hyperplasia, differentiation alterations, and genetic instability leading to the specific expansion of the initiated cells into papillomas and carcinomas (Slaga et al., 1987; Yuspa & Poirier, 1988, DiGiovanni, 1992) characterize the skin tumor promotion and progression stages. Phorbol esters, especially 12-O-tetradecanoylphorbol-13-acetate (TPA), indole alkaloids, and polyacetate type promoters appear to act through a membrane receptor protein kinase C, whereas benzo[e]pyrene, chrysarobin, and certain peroxides may act through a free radical mechanism (Slaga et al., 1987; DiGiovanni 1992). Histological observations on mice treated topically with tumor promoters have demonstrated that sustained cellular hyperplasia plays a critical role in tumor promotion (Klein-Santo & Slaga, 1981, 1982). Several investigators have concluded that the induction of a sustained hyperplasia correlates well with the skin tumor formation ability of various promoting agents such as phorbol esters, several peroxides, and chrysarobin (Slaga et al., 1976; Naito et al., 1987; Gimenez-Conti et al., 1998). Another important aspect of carcinogenesis, especially during the tumor promotional stage, is that carcinogens and tumor promoters give rise directly to free radicals, or indirectly through the generation of free radicals such as superoxide anion and hydroxyl-radical (Hanausek et al., 2003a).
Radiation alone or in combination with carcinogenic chemicals has been known for a long time to be involved in the etiology of skin cancer. Although both ultraviolet and ionizing radiation have been shown to be either promoting or enhancing agents for chemically promoted tumors, their primary influence appears to be related to their tumor-promoting activities rather than initiation (Haridas et al., 2004). However, recent results indicate (Rebel et al., 2001) that brief exposure of the skin of SKH-1 mice to UV light causes permanent cellular changes that do not result in skin tumors unless the mice are treated with TPA for several weeks. These observations indicate that UV can function as an initiator of tumorigenesis in mouse skin. Exposure of cultured cells or hairless mice to UV causes mutations in a number of genes. The molecular mechanisms of UV-induced skin sunburn lesions and skin tumor initiation are unknown. The most often mutagenized gene in skin cancers is the tumor suppressor gene p53. It is believed that point mutations on p53 are an early event in skin carcinogenesis since one can find them in pretumoral lesions such as keratoacanthomas and actinic keratosis (Rebel et al., 2001). Recently, p53 mutations were shown (Rebel et al., 2001) to be early events in skin carcinogenesis induced by chronic UVB irradiation in SKH-1 mice.
Inhibitors of Skin Tumor Initiation
For a wide variety of chemical carcinogens, it is thought that the carcinogenic process is initiated by covalent interaction of the chemical with critical macromolecules, presumably DNA. Such DNA damage has in general been shown to be mutagenic, and the finding of proto-oncogenes that, when mutated at specific sites become dominantly transforming oncogenes has strengthened the importance of this pathway in carcinogenesis. The realization that the primary covalent interactions between a variety of chemical carcinogens and DNA are mediated by reactive electrophiles (Slaga et al., 1989), either derived directly from carcinogen or produced during cellular metabolism, has served to unify our thinking with regard to the initiation of chemical carcinogenesis. Many classes of chemical carcinogens are currently included among those that work by this general mechanism, including nitrosoamines, nitrogen mustards, mycotoxins, aromatic amines, and polycyclic aromatic hydrocarbons (PAHs). For a number of PAHs, including 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) the ultimate carcinogen is a so-called bay-region dihydrodiol epoxide, produced during cellular metabolism (Kapitulnik et al., 1978; Slaga et al., 1977). Indeed, a general correlation exists between the strength of a series of dihydrodiol epoxides as initiators, and their chemical potential to form the benzylic carbonium ion, which is a reactive, electrophilic intermediate that binds to nucleophiles (Jerina et al., 1976). DMBA and B[a]P covalently modify DNA by reaction of their respective bay region diol epoxides with the exocyclic amino groups of deoxyguanosine and deoxyadenosine. Different factors that affect the production and disposition of the diol epoxides are of interest as possible targets for the inhibition of tumor initiation. The initial activation of PAHs to diol epoxide (Phase I metabolism) is carried out by so called Phase I enzymes. Classical attempts to inhibit carcinogenesis by altering the activities of Phase I enzymes included both inhibition and induction of oxidative enzymes.
A number of potent inhibitors of tumor initiation (Table 1) appear to be effective because they either prevent the formation of the ultimate carcinogen and/or scavenge the reactive ultimate carcinogen. The indoles, aromatic isothiocyanates, coumarines, flavones, di- and triterpenoids, dithiothiones, organosulfides, and D-glucarates have a potent effect on the metabolism of carcinogens (Slaga, 1995; Hursting et al. 1999; Walaszek, 1993). In general, they appear to have a major effect on the detoxification of the carcinogens. Ellagic acid and 2,6-dithiopurine have been shown to be highly potent in scavenging the ultimate (reactive) carcinogenic form of the carcinogen. The majority of these chemicals have properties like phenolic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). They have both antioxidizing activity and influence the metabolism of carcinogens, especially detoxification (Slaga, 1995a,b). Biological systems have developed methods for dealing with electrophilic xenobiotics produced during Phase I metabolisms; these are generally classified as Phase II enzymes. Many antioxidants and anticarcinogenic compounds that block the toxic and neoplastic effects of carcinogen share in common the ability to elevate levels of Phase II detoxification enzymes, i.e., glutathione S-transferases (GSTs), quinone reductase (QR), and UDP-glucuronosylotransferases (UDP-GT) (Wattenberg, 1992; Hanausek et al., 2003b; Walaszek et al., 2004). Substantial evidence has accumulated to suggest that the induction of Phase II enzymes is a causal mechanism for protection since these enzymes divert ultimate carcinogens from reacting with critical cellular macromolecules (Prochaska et al., 1992). Members of the cruciferae and lilliaceae families are rich sources of inducer-activity and can block experimental carcinogenesis. High consumption of these vegetables is associated with the reduction of cancer risk in humans (Block et al., 1992). The elevation of inducers of Phase II enzymes in humans either by dietary or prescriptive interventions is likely to intensify. Anticarcinogenic enzyme inducers have attracted heightened interest as potential human anticarcinogens since they are neither carcinogen- nor organ-specific. Moreover, they are components of human diet (Block et al., 1992). The molecular mechanisms of UV-induced skin tumor initiation and its inhibition have not been studied extensively and remain unknown, however there is some evidence that the inhibition of p53 point mutations may be a critical event in the prevention of initiation of mouse skin carcinogenesis by UV light.
Inhibitors of Skin Tumor Promotion/Progression
In contrast to the initiation stage, the promotion stage is reversible and requires a certain frequency of application to induce benign skin tumors. The progression stage appears to involve the accumulation of additional genetic changes in cells, which form skin papillomas that can be considered as a clone of initiated cells. Skin tumor promoters in general do not bind covalently to DNA and are not mutagenic, but bring about a number of important epigenetic changes (Slaga et al., 1995). In addition to causing inflammation and epidermal hyperplasia, skin tumor promoters produce many other morphologic, biochemical and molecular changes in the skin. Of the observed tumor promoter-related effects on the skin, the induction of epidermal cell proliferation, ornithine decarboxylase and subsequent polyamines, and prostaglandins have the best correlation with promoting activity (DiGiovanni, 1992; Slaga et al., 1995). Overall, the skin tumor promotion is characterized by selective and sustained hyperplasia, leading to the specific expansion of the initiated cells into papillomas. A number of other important epigenetic changes in the skin are induced by tumor promoters, such as membrane and differentiation alterations, an increase in protease activity, and phospholipid synthases (DiGiovanni, 1992; Slaga et al., 1995). In addition, the skin tumor promoters cause a decrease in epidermal superoxide dismutase and catalase activities, as well as decrease in the number of glucocorticoid receptors (Budunova et al., 2003).
UV light and TPA have been shown to damage the immune system (Wang et al., 1992), possibly by increasing the cellular production of active oxygen species that reduce immune function. Antioxidants such as ascorbic acid, alpha-tocopherol, glutathione and avicins can protect the immune system from active oxygen species (Slaga, 1995a,b; Haridas et al., 2004). Topical application of alpha-tocopherol was found to inhibit UV-induced immunosuppression in the skin of mice, and it is possible that other natural source antioxidants may exert a similar effect on UV tumor promotion. For example, the data presented by Wang et al. (1992) indicate that dietary administration of an aqueous green tea extract to SKH-1 mice inhibited UVB-promoted skin tumorigenesis in mice previously initiated with DMBA.
The multistage model of mouse skin tumorigenesis has been extremely useful for studying various factors that inhibit the carcinogenic process (Table 1). Using this model system, one can specifically study the effects of potential modifiers on the initiation and the promotion stages independently. Studies have been performed (DiGiovanni & Fischer, 1995) on many exogenous compounds that have the ability to inhibit the tumor promotion stage of skin carcinogenesis by (a) altering the state of differentiation; (b) inhibiting the promoter-induced cellular proliferation; (c) preventing gene activation by promoters; and (d) scavenging free radicals and reactive oxygen species. Recent studies have also begun to unravel the nature of the tumor progression process in skin carcinogenesis. Several agents have been identified that may inhibit tumor progression in this model system. Free-radical scavenging agents may be worthy of further study as inhibitors of tumor promotion and progression. Although several antioxidants do inhibit skin tumor initiation by procarcinogens, antioxidants are in general much more effective inhibitors of skin tumor promotion. Recently, several new antioxidants such as proanthocyanidins and ursolic acid have been found to inhibit chemical carcinogenesis and skin tumor promotion. 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 also been shown to inhibit chemical carcinogenesis and skin tumor promotion (Slaga, 1995a,b). Their mechanism of action is not definitely known. However, evidence points to several possibilities: (a) they scavenge various radicals generated directly or indirectly by tumor promoters; (b) they increase levels of enzymes that are important in detoxifying cellular radicals; and (c) they have other specific functions. Several antioxidants have been shown to have synergistic activities such as vitamins C and E, vitamin E and selenium, BHA and vitamin E (Slaga, 1995a,b).
Recent studies demonstrate that plants are rich in compounds such as avicins, i.e., triterpenoid saponins that inhibit oxidative stress and induce programmed cell death (apoptosis) of premalignant and malignant cells (Hanausek et al., 2001, Haridas et al., 2004). These studies indicate that avicins could develop as important chemopreventive agents in many conditions where chronic inflammation and oxidative and nitrosative stress may lead to tumorigenicity.
Natural Source Inhibitors and Their Combinations
Scores of epidemiologic studies have noted a lower risk of cancer among persons whose diet include a relatively large amount of vegetables, fruits and other plant products (Block et al., 1992). 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 non-toxic or markedly less toxic then synthetic chemopreventive agents.
The studies on the mechanism(s) of anti-tumor initiating and anti-tumor promoting properties of a variety of naturally occurring inhibitors and other beneficial phytochemicals suggest that they are very important for the prevention of skin cancer as well as for the prevention of other epithelial cancers in humans (Table 2). The mouse skin cancer model relates very well to other models where squamous cell carcinomas are induced. It is however important to choose for such studies compounds that act through one or more different mechanisms.
Thus, the well established mouse skin model may be used to measure the inhibitors' ability to modify the carcinogen activation, enhance Phase II enzymes detoxification, modify antioxidant enzymes, prevent oxidative damage to DNA bases and mutations, decrease inflammation and hyperplasia and modulate the immune response, induced by (a) the short-term DMBA treatment (high dose); (b) short-term UV-irradiation; (c) the DMBA initiation (low dose) followed by the TPA promotion; and (d) the UV irradiation followed by the TPA promotion. One may first test individual compounds at various topical or dietary doses, and then combinations of the most promising inhibitors for potential synergistic effects.
Conclusions
We conclude that in the end the results of the combination studies should support the theory underlying such studies that the concurrent topical and dietary treatments with carefully selected natural source inhibitors or their combinations yield impressive synergistic effects. The natural source inhibitors should be selected on the basis of their overall efficacy and lack of toxicity. Specifically, these agents should be chosen based on their overall efficacy in inhibiting both the initiation and promotion stages. Alternatively, one compound may be selected because it is a very good inhibitor of the initiation stage and this compound will be tested with another compound that very efficiently inhibits the promotional stage of carcinogenesis. Obviously, some of the compounds will prove to have both anti-initiating and anti-promotional activity, but they will most likely inhibit the initiation and promotion stages to a different extent. Specifically, such compounds may inhibit one stage to a greater degree than the other. In the DMBA/TPA model, the chemopreventive agents that affect the Phase I and Phase II metabolism will probably be more important. On the other hand, in the UV/TPA model, natural source antioxidants will most likely prove to be the most effective chemopreventive agents. It is probable that two good inhibitors with different mechanisms of action will be more effective in inhibiting the development of skin tumors compared to one inhibitor alone. In the future, combinations employing three or more agents and the mechanisms of their synergistic actions may be investigated if it appears that this would, in fact, be a highly effective strategy. The carefully designed human studies should then follow.
The overall conclusion of this brief review is that there is now growing evidence from both animal and human studies indicating that while individual cancer preventive agents are effective, combinations of agents, especially natural source compounds are much more potent in preventing cancer. The constituents of diets as well as diets as a whole have a part to play in all the stages of carcinogenesis. Dietary microconstituents such as vitamins and minerals as well as bioactive compounds mostly found in plant foods are of particular importance. Vitamins include carotenoids, vitamin C and E, and folate. Minerals include selenium, calcium, iodine, and iron. Cereals (grains), vegetables, fruits, pulses (legumes) and other plant foods contain many microconstituents, other than vitamins and minerals, that are known to be biologically active. These bioactive compounds include D-glucarate, allium compounds, dithiolthiones, isothiocyanates, terpenoids, isoflavones, protease inhibitors, phytic acid, polyphenols, glucosinolates and indoles, flavonoids, plant sterols, saponins and coumarins. It is likely that further research will produce evidence indicating that diets rich in various bioactive compounds as well as nutritional supplements containing combinations of the most active natural source compounds protect against a number of cancers.
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