Epidemiologic Reviews Copyright © 2001 by the Johns Hopkins University Bloomberg School of Public Health All rights reserved
Vol. 23, No. 1 Printed in U.S.A.
Role of Phytochemicals in Prevention and Treatment of Prostate Cancer
Stephen Barnes
INTRODUCTION
WHY ARE PHYTOCHEMICALS IN PLANTS?
Prostate cancer risk is much lower in the peoples of Southeast Asia compared with Americans (1). In Japanese men, this difference rapidly disappears on immigration to the United States (2). It begs answers to the question of what is the role of diet in prostate cancer risk. While some dietary components, such as fat, have been implicated in the "causation" of prostate cancer, there has been an increasing interest in dietary factors that "prevent" prostate cancer; "phytochemicals", plant-derived non-nutritive compounds, are one type of these dietary factors. "Bioflavonoids" are a wellknown subclass of a phytochemical group known as polyphenols, and have a wide range of properties. Those with estrogen-like activities ("phytoestrogens") have received a great deal of attention in recent years, and this review is focused on them. The reader is referred to an excellent review of the literature by Griffiths et al. (3) for the period up to 1998 concerning the role of phytoestrogens in prevention of prostate cancer, as well as a broader review by Kurzer and Xu (4) of the effects of dietary phytoestrogens. Historically, when humans selected a plant as being suitable for eating they did so based on the nutritious value it provided, its edibility, and its lack of toxicity. Depending on the plant, it was often necessary to cook it first to break down its macromolecules to alter its texture, or to heat it to inactivate toxins or enzymes that may have led to undesirable taste phenomena. Accordingly, fire and then cuisine have been an important part of the emergence of the human race. Phytochemicals for humans are, therefore, "Trojan horses", and have not been ingested intentionally. However, even two and half centuries ago it was realized by Captain James Cook that the health of the men on his ship Endeavour depended on taking fresh fruit and vegetables with them as they explored the South Pacific. He had discovered the value of a well-known phytochemical, vitamin C. In the first half of the 20th century, other familiar phytochemicals (vitamins A through K) were isolated and their identities determined.
Besides the more familiar vitamins, plants contain quite large amounts of phytochemicals (0.1 to 5 percent wet weight) that are important to the plant. For instance, the isoflavones present in soy are the molecular signals secreted by the roots of the soybean that attract Rhizobium sp. bacteria to form nodules in the roots of the plant and allow nitrogen fixation to occur (5). The soybean, therefore, has its own supply of fixed nitrogen that it also provides to the surrounding soil. Isoflavones are part of the broader class of bioflavonoids; the latter are found in most plants, whereas the isoflavones are largely restricted to tropical legumes. Of the commonly eaten plants, the soybean is the richest source of isoflavones (6). However, it should be noted that the Native Americans who populate the eastern United States have for many centuries consumed the tubers of the American groundnut {Apios americana) that have high levels of a glucosylglucoside of genistein (7, 8). Other related polyphenols found in significant quantities in plant foods include the lignans. As for the isoflavones, they are phenolic compounds, but with fourcarbon instead of two-carbon bridges between their two phenolic groups. They are the building blocks of lignan biosynthesis in plants. Flaxseed is a particularly rich source of the lignan monomers (9). PHYTOCHEMICALS AND PROSTATE CANCER RISK
The puzzle modern-day scientists are trying to unravel is what is the role of phytochemicals in the prevention of chronic diseases such as prostate cancer. The interest in the phytoestrogens (plant estrogens) goes back over 30 years. Initially, research was directed at the estrogenic effects of this class of compounds, particularly in the breeding of farm animals (10). For some toxicologists there has been an implicit assumption that the estrogenic effects due to phytoestrogens observed in sheep (11) and cheetahs (12) would apply to humans. However, the extensive consumption of soy phytoestrogens (isoflavones) by Southeast Asians in their diet, and by commercially bred rodents, are without a conspicuous effect on fertility, suggesting that such an assumption is overstated. Indeed, it could be equally argued that the consumption of soy phytoestrogens is preventing chronic disease (5). Epidemiologic studies have hinted at the association between soy phytoestrogens and reduction in prostate cancer risk, although there have been inconsistent results between studies. In Japanese men living in Hawaii, con-
Received for publication September 1, 2000, and accepted for publication March 21, 2001. Abbreviations: DMAB, 3,2'-dimethyl-4-aminobiphenyl. From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, AL. Reprint requests to Dr. Stephen Barnes, Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1670 University Boulevard, VH G009, Birmingham, AL 35294-0019 (e-mail:
[email protected]).
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Phytoestrogens and Prostate Cancer
sumption of tofu led to a threefold reduction in prostate cancer risk (13). However, dietary associations are easily confounded by other components of diet. In this same study, miso, a fermented form of soy that also contains isoflavones, was associated with a small increase of prostate cancer risk. On the other hand, a cross-national study of prostate cancer mortality found that there was a significant inverse association between soy consumption and the risk of prostate cancer (14). In an epidemiologic study to investigate dietary soy/phytoestrogen and prostate cancer risk in Australia, a significant inverse relation was observed between coumestrol (p = 0.03), another phytoestrogen, with weaker inverse associations with daidzein (p = 0.07) and genistein (p = 0.25) (15). In an Adventist population in the United States, soy milk (more than one glass per day) was associated with a 70 percent reduction in risk of prostate cancer (while impressive, not statistically significant); however, when analyzed for trend on the basis of how much soy milk was consumed, statistical significance was achieved (16). In contrast, a study of soy intake in 47 prefectures, conducted in Japan from 1980 to 1985, found a positive significant association between soy intake and prostate cancer. However, when adjustments were made for mean age, proportion of smokers, and alcohol intake, this positive association was reduced (17). To sort out the role of phytoestrogens on prostate cancer risk, it is necessary to demonstrate that their addition to the diet of men or suitable animals leads to reduction in prostate cancer risk or progression. However, it is quite difficult to assess this in men because of the very slow development of prostate cancer. Direct evaluation of phytoestrogens will, therefore, take large and expensive clinical trials, albeit very important since the changing age distribution of most populations (and, hence, susceptibility) is leading to increasing cases of prostate cancer. However, it is possible that identification and validation of surrogate endpoint biomarkers for prostate cancer will facilitate future studies. EFFECTS OF PHYTOCHEMICALS IN CELL CULTURE
Preclinical data connecting phytochemicals and reduction of prostate cancer risk are relatively sparse. Using cell culture approaches with established human prostate cancer cell lines, it was shown that the soy isoflavone genistein inhibits both serum and growth factor-stimulated proliferation of these cells (18). Although this result has been confirmed by others, the concentrations needed to achieve this inhibition (>50 |iM) are two to three orders of magnitude greater than blood concentrations observed in Asian men eating soy as a significant part of their diet (19). On the other hand, concentrations of genistein in the low |j.M range inhibit DNA synthesis in thin sections of prostate tissue in histoculture (20). These results suggest that if isoflavones have an effect in vivo, then they do so via secondary mechanisms. These could include regulation of growth factor secretion by neighboring cells in the prostate or by growth factor receptors in the tumor cell itself (21), or subtleties in the metabolism of genistein that are not modeled in cell culture. A possible example of the latter is the recent discovery in the rat Epidemiol Rev Vol. 23, No. 1, 2001
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that the principal metabolite of genistein that accumulates in the prostate is 2-(4-hydroxyphenyl)-propionic acid (22). This compound is not formed in cell culture experiments and, therefore, its effects on cell growth must be investigated directly. EFFECTS OF PHYTOCHEMICALS IN ANIMAL MODELS OF PROSTATE CANCER
In the past 2 years, there have been several studies using animal models of prostate cancer to explore the chemopreventive effects of soy, extracts of soy, and purified isoflavones. In each case, a protective effect of isoflavones has been observed. One study examined the role of soy and soy extracts on the growth of a transplantable prostate tumor in rats (23). In a similar animal model, dietary genistein was also shown to inhibit prostate tumor growth (24). In the other studies, the effects of soy and soy extracts on human prostate cancer cell xenografts was investigated in nude mice (25, 26). Soy protein had a mild effect in these studies (20 percent inhibition of growth) that was enhanced up to twofold by co-administration of a soy phytochemical extract (25, 26). One cannot dismiss the possibility that soy protein may also contribute to the anti-cancer effect. The anticancer effect is not confined to the isoflavone-containing foods. Rye enriched in lignans had a larger inhibitory effect than soy protein using a human LNCaP xenograft model of prostate cancer in nude mice (27). These results suggest that soy and lignan phytochemicals have the potential for slowing the growth of prostate cancer cells, perhaps including the role of apoptosis (27). The latter data are consistent with an anecdotal report of the effect of phytoestrogens on apoptosis in the prostate (28). A newly diagnosed prostate cancer patient was treated with isoflavone preparation from red clover (four doses of 40 mg each of isoflavones per day for 1 week). Histology of the prostate at the time of surgical resection revealed evidence of substantial apoptosis in the region of the tumor. Two other models of prostate cancer have been used to look at whether phytoestrogens can prevent the appearance of prostate tumors. Lobund-Wistar rats are spontaneously susceptible to the risk of prostate cancer—in these animals prostate cancer can be more rapidly induced by administration of Af-methylnitrosourea and testosterone propionate. Soy fractions added to the diet of these animals have a small protective effect on prostate cancer risk (29). The carcinogen 3,2'-dimethyl-4-aminobiphenyl (DMAB) can also be used to induce prostate cancer when it is injected into the prostate of male rats. Animals consuming genistein in their diet had a small but significant reduction (29 percent) in ventral prostate carcinoma number (30). In a second study with DMAB using the P-glucosides daidzin and genistin (0.1 percent in the diet), both isoflavones significantly reduced ventral prostate carcinoma number by 80 percent (31). However, they had no effect when invasive tumor growth was stimulated with testosterone propionate. In a recent development, the chemopreventive effect of genistein has been examined using the transgenic adenocarcinoma of mouse prostate (TRAMP) model of prostate cancer.
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Genistein both reduced the number of prostate tumors and the size of the tumors (32). MECHANISMS OF ACTION OF PHYTOCHEMICALS
Phytoestrogens may prevent prostate cancer because of a dose-dependent reduction in serum 17(i-estradiol and testosterone levels, as recently observed in Japanese men (33). The presence of estrogen receptor P in the prostate (34), which binds the isoflavone genistein with an affinity similar to that of 17(3-estradiol (35), suggests that certain phytoestrogens may mediate their anti-cancer effect through this target. However, other biomarkers and cellular mechanisms for their action have been suggested; these include inhibition of serum prostate-specific antigen (36) and induction of p21 by a p53-independent pathway (37), inhibition of epidermal growth factor autophosphorylation, and inhibition of NF-K B activation (38). Whether all these mechanisms operate in vivo remains to be determined. In a recent study, a soy protein beverage was shown to have no effect in elderly men with elevated prostate-specific antigen levels, but did reduce serum lipids (39), an important issue for elderly men who are more likely to die from cardiovascular disease than from prostate cancer. ANALYSIS OF PHYTOCHEMICALS
There are many parallels between phytoestrogens and steroids regarding the development of suitable assays for clinical studies. Phytoestrogens, as well as steroids, were originally analyzed by gas chromatography-mass spectrometry (40, 41). However, the application of HPLC-mass spectrometry (42—44) greatly simplified the assay procedure since it eliminated most of the pre-analysis steps and the need for derivatization (45, 46). While this latter procedure offers high sensitivity and specificity, it does not have a high enough throughput for large scale epidemiologic studies. The introduction of immunoassay-based procedures (47—49) offers a partial solution to this problem if only measurement of one or two of the isoflavones or their metabolites is required. CONCLUSIONS AND FUTURE PERSPECTIVES
Future epidemiologic studies, particularly if carried out in the United States, will have to take into account the changing patterns of exposure to phytoestrogens. The US Food and Drug Administration approval of a health claim for prevention of heart disease by soy protein because of its lipid lowering effects, is leading to the introduction of new soy-based foods. For instance, the new generation soy milks are excellent from the consumers' taste considerations and could be consumed without realizing that they contain soy. This highlights the need for continuously updating the dietary intake databases (16, 50), and, where possible, questioning subjects carefully about what they eat. A more expensive but more accurate assessment is to measure the isoflavone content of fasting blood samples from the subjects. This could be done on randomized sub-
sets of the total population being studied. Even better are 24-hour urine collections—however, for large-scale studies these are impractical. The introduction of new soy foods also raises the issue of whether other components normally a part of traditional soy foods are retained by the processing methods that are used. As the effect of the phytoestrogens may depend on the matrix in which they are delivered, it is important for these new food materials to undergo careful examination in suitable pre-clinical models before making a health claim on their usefulness for prostate cancer. For similar reasons, soy supplement materials in which the isoflavones have been artificially concentrated may also lack critical nonisoflavone components. It should be noted that there is an on-going dose escalation, pharmacokinetics, and safety study at the University of North Carolina-Chapel Hill, funded by the US National Cancer Institute, on mixtures of substantially pure isoflavones. In future studies using chronic doses, this group will examine the effects of these isoflavones on prostate cancer biomarkers. Since Americans of African origin have the highest risk of prostate cancer, it would be of interest to determine thenintake of phytoestrogens (expected to be low, but perhaps now on the rise) with that of other ethnic groups that live in the same city, and compare these data with the associated risk of prostate cancer. Another interesting group to study is athletes who have used low-fat protein drinks containing large quantities of soy protein to sustain their sporting activities.
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