Inhibitory effects of soy isoflavones on rat prostate carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)
Atsuya Hikosaka1,3,
Makoto Asamoto1,
Naomi Hokaiwado1,
Koji Kato1,
Kazuya Kuzutani1,
Kenjiro Kohri2 and
Tomoyuki Shirai1
1 Department of Experimental Pathology and Tumor Biology and 2 Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
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Abstract
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Intake of isoflavones derived from soybean products may impact on prostate cancer risk. Here we evaluated the effects of Fujiflavone, a commercial isoflavone supplement, on rat prostate carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat. F344 male rats were given intragastric administrations of PhIP at the dose of 200 mg/kg twice weekly for 10 weeks. The rats subsequently fed a diet containing 0.25% Fujiflavone showed a significantly lower incidence of prostate carcinomas than those fed a soy-free diet. Interestingly fewer carcinomas but more foci of prostatic intra-epithelial neoplasia (PIN) were observed in the Fujiflavone group although the sum of the two lesions was not altered by Fujiflavone treatment. cDNA array analyses confirmed by semi-quantitative reverse transcription polymerase chain reactions (RTPCR) revealed Fujiflavone to alter gene expression of ornithine decarboxylase (ODC), prothymosin alpha (PTA) in the rat prostate. No modification of PhIP-induced colon carcinogenesis was evident, except for increased multiplicity of aberrant crypt foci >4 crypts in size. These results indicate that a commercial isoflavone supplement can inhibit PhIP-induced rat prostate carcinogenesis without any adverse effects, possibly by inhibiting progression of PIN to carcinoma, and that down-regulation of ODC and PTA could be related to the underlying mechanisms. Thus, intake of dietary isoflavones can be promising for prevention of human prostate cancer.
Abbreviations: ACF, aberrant crypt foci; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; ODC, ornithine decarboxylase; PIN, prostatic intra-epithelial neoplasia; PTA, prothymosin alpha
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Introduction
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Prostate cancer is one of the chief causes of male cancer death in North America and Europe, but is much less frequent in Asia (1). The reason might be partly explained by differences of dietary habits because Japanese emigrants to America who adopt a Western diet demonstrate higher risk of prostate cancer than native Asians (2).
Western people tend to consume a large amount of cooked meat (35), from which a number of heterocyclic amines (HCAs), known to be highly mutagenic carcinogens, have been isolated (6,7). Among them we have found previously 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant HCA (8), to be a carcinogen in the rat prostate (9). PhIP also causes mammary and colon cancers in the rat (10), both of which are common in Western countries as well as prostate cancer (1). Therefore, this HCA is a candidate for human carcinogen.
Asians consume a great deal of soy products containing abundant isoflavones (3). Many biological actions of isoflavones related to tumor growth suppression and cancer prevention have been reported (11) and epidemiological studies have revealed that a high intake of isoflavones is associated with low breast, prostate and colon cancer risk (12), suggesting that isoflavones might be promising candidates for chemoprevention of these cancers.
Many previous studies on the anticancer effects of isoflavones were focused mainly on genistein, a major isoflavone (11). However, single genistein for chemoprevention is not practical because of difficulties and expense in purification. In addition, promoting effects of pure genistein on colon carcinogenesis were found in a previous investigation (13). In the present study we evaluated the effects of Fujiflavone, a commercial isoflavone supplement containing relatively low genistein derivatives, on prostate carcinogenesis induced by PhIP. Alterations of gene expression in the rat prostate after the treatment with Fujiflavone were also examined.
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Materials and methods
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Animals
Five-week-old F344 male rats were purchased from Charles River Japan (Atsugi, Japan) and housed 2 or 3/plastic cage with wooden chips for bedding in an air-conditioned room at 22 ± 2 and 55 ± 5% humidity with a 12 h light/dark cycle. Tap water and food were available ad libitum.
Chemicals
PhIP hydrochloride with purity >99.9% was purchased from the NARD institute (Osaka, Japan). Fujiflavone P40, an enriched isoflavone extract from soy germ, was kindly provided by the manufacturer (Fujicco Co., Kobe, Japan). Manufacturing procedures and characteristics of Fujiflavone were described in a previous report (14) and are available on the website (http://www.fujicco.co.jp/english). Isoflavone components in Fujiflavone are listed in Table I.
Animal experiment 1
PhIP suspended in corn oil was administered to 6-week-old F344 male rats intragastrically at the dose of 200 mg/kg twice a week for 10 weeks. The rats in one group were fed powdered soy-free diet (soybean-free modified NIH-07; Oriental yeast Co., Tokyo, Japan) and those in the other were given the same diet containing Fujiflavone at a concentration of 0.25% for the next 50 weeks (Figure 1). The animal weights were recorded once or twice a week until the end of the experiment. In addition, food consumption was monitored twice a month during the latter half of the experimental course. At the age of 66 weeks, all rats were killed under ether anesthesia and their prostates, kidneys, livers and colons were excised. Kidneys and livers were weighed and prostates and colons were processed for histological examination. Blood was collected from five rats of each group for determination of serum hormone levels.

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Fig. 1. Experimental protocols. Animals were F344 male rats 6 weeks old at commencement. Closed box, PhIP 200 mg/kg body weight intragastric administration twice weekly. Hatched box, 0.25% Fujiflavone administered in soy-free diet. Open box, Soy-free diet only. S, sacrifice.
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Tissue processing
Prostates and colons were fixed in 10% buffered formalin. Prostates were then divided into ventral lobes, dorso-lateral lobes and seminal vesicles with anterior lobes. Each lobe was weighed and the whole ventral lobe, two transverse slices of the dorso-lateral lobe and four transverse slices of the seminal vesicle with the anterior lobe from each side were embedded in paraffin for histological examination. Colons were stained with 0.5% methylene blue for counts of aberrant crypt foci (ACF), followed by paraffin embedding of macroscopically visible tumors for histological diagnoses.
Histological and histomorphometric examinations
Routine histological examination was performed with hematoxylin and eosin staining. Multiplicity of the prostate neoplastic lesions was determined as the number of neoplastic foci per cm2 of each section. Areas of each prostate lobe were measured with an image processor (Image Processor for Analytical Pathology; Sumika Technoservice, Osaka, Japan). ACF were counted with a stereoscopic microscope (SZH10; Olympus Optical, Tokyo, Japan).
Animal experiment 2
Six-week-old F344 male rats were divided into two groups. One group of five rats were fed the soy-free diet and the other of five rats were fed the same diet with 0.25% Fujiflavone for 2 weeks (Figure 1). At the age of 8 weeks, all rats were killed under ether anesthesia and their prostates were excised, frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Extraction of total RNA and cDNA array analysis
Total RNA extraction from the rats of experiment 2 followed by DNase treatment were performed with the Atlas Pure Total RNA Labeling System (Clontech, Palo Alto, CA) according to the manufacturer's instructions. Fifty micrograms of total RNA from rats in each group were pooled, respectively, and used for probe synthesis of cDNA arrays. Poly(A)+ RNA enrichment and 32P labeling were performed according to the manufacturer's instructions (Atlas Pure Total RNA Labeling System; Clontech). The Atlas Rat Stress/Toxicology Array (Clontech) was used to compare gene expression profiles of the rat prostate in the Fujiflavone group with those in the soy-free group. Signal spots for each gene were detected and analyzed with an image analyzer (FLA-3000G; Fujifilm, Tokyo, Japan) using Array Gauge software (Fujifilm). The expression ratio of each gene was calculated from the average signal intensity determined by duplicate analyses. More than 2-fold increase or decrease (ratio over 2 or under 0.5) was regarded as a significant change.
Semi-quantitative reverse transcriptionpolymerase chain reactions
One microgram of the RNA was converted to cDNA with avian myoblastosis virus reverse transcriptase (Takara, Otsu, Japan) in 20 µl of reaction mixture. Aliquots of 2 µl of cDNA samples were subjected to quantitative PCR in 20 µl reactions using FastStart DNA Master SYBR Green I and a Light Cycler apparatus (Roche Diagnostics, Mannheim, Germany). Primers are listed in Table II. Initial denaturation at 95°C for 10 min was followed by 40 cycles with denaturation at 95°C for 15 s, annealing at 5660°C for 5 s and elongation at 72°C for 30 s. The fluorescence intensity of the double-strand specific SYBR Green I, reflecting the amount of formed PCR product, was monitored at the end of each elongation step. mRNA levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used to normalize the sample cDNA content.
Statistics
MannWhitney's U-test was employed for statistical analyses of body and organ weight, tumor multiplicity and gene expression levels. Tumor incidence was analyzed with Fisher's exact probability test.
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Results
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Body and relative organ weights
Body weight gain of the rats did not differ between the Fujiflavone and soy-free diet groups through the experiment. Relative weights of the kidney and the liver were not affected by administration of Fujiflavone (Table III) but that of the prostate was smaller in the Fujiflavone group due to decrease in weight of the seminal vesicle with the anterior lobe (Table IV). The weight of the ventral lobe, where neoplastic lesions are usually caused by PhIP (9), demonstrated no significant difference between the two groups (Table IV).
Incidence and multiplicity of neoplastic lesions in the prostate
Prostatic intra-epithelial neoplasia (PIN), a putative pre- cancerous lesion of the rat prostate (15,16) as in the human prostate (1719) was found in anterior and ventral lobes of almost all rats in both groups. As reported previously (9), carcinomas were observed only in the ventral lobe, all of which were non-invasive. They were observed in 62.5% of rats in the soy-free group, while 21.4% of those in the Fujiflavone group, showing statistical difference with P < 0.05 (Table V). PIN and dysplasia were observed in the anterior lobe and the seminal vesicle of most rats in both groups, respectively. No neoplastic lesions were found in the dorsolateral lobe (data not shown).
Concerning multiplicity of PIN and carcinoma in the ventral lobe, more PIN foci and less carcinoma foci were observed in the Fujiflavone group than in the soy-free group with significant difference (P<0.05). However, the sums of the two lesions were about equal in each group (Table V).
Effects of Fujiflavone on PhIP-induced colon carcinogenesis
The incidence of colon carcinomas was 35.7% in the Fujiflavone group and 25% in the soy-free group showing no significant difference. All carcinomas were non-invasive and every tumor-bearing rat had only single lesions (Table VI). As to ACF, putative carcinoma-related lesions (20,21), no significant difference was shown on comparison of incidence. Multiplicity of ACF with
4 crypts was statistically higher in the Fujiflavone group (Table VI).
Change of gene expression profile by treatment with Fujiflavone
cDNA array analyses detected seven up-regulated genes and nine down-regulated genes with >2-fold expression change (Table VII). Confirmation of the data using semi-quantitative reverse transcriptionpolymerase chain reactions (RTPCRs) showed more considerable expression changes of the down-regulated genes by Fujiflavone treatment than those of the up-regulated genes, especially down-regulation of prothymosin alpha (PTA), ornithine decarboxylase (ODC) were evident with 0.36- and 0.52-fold expression, respectively (Figure 2).

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Fig. 2. Expression levels of genes detected by cDNA array analyses confirmed with semi-quantitative RTPCR. Expression levels are presented as ratios to GAPDH expression level. Values are means of triplicate experiments ±SD. GST mu2, glutathione S-transferase, mu type 2; mGST, microsomal glutathione S-transferase; CDK, cyclin-dependent kinase; GPx, glutathione peroxidase; BAD, Bcl-2 associated death agonist; ODC, ornithine decarboxylase; PTA, prothymosin alpha. *P < 0.05.
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Discussion
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In the present study significant inhibitory effects of Fujiflavone, a commercial isoflavone supplement, on rat prostate carcinogenesis were clearly shown, consistent with previous studies with various types of isoflavones (2227). We applied PhIP as a prostate carcinogen as it might be more relevant to the human situation than other carcinogens (2830), considering its target organs (9,10) and possible daily intake by people with Western-style diet (31). Anticancer effects of isoflavones have been explained by referring to various possible mechanisms including inhibition of tyrosine kinases (32,33), topoisomerase II (34), angiogenesis (35) and growth factor-induced c-fos expression (36). Antioxidant properties (37,38) and promotion of apoptosis (39) have also been reported. PhIP-induced rat prostate carcinomas are androgen receptor-positive and show androgen-dependent growth, like human prostate cancers (unpublished data). Although isoflavones have weak estrogenic properties (40) and a recent report has shown genistein is likely to inhibit 5-alpha reductase activity resulting in a reduction of dihydrotestosterone (DHT)active form of testosteronein several tissues including the prostate (25), serum luteinizing hormone, testosterone and DHT levels and DHT/TES ratio was not modified by Fujiflavone treatment (data not shown). Besides weights of each prostate lobe did not differ with the Fujiflavone treatment and microscopically prostate glands showed no involution (data not shown). These results suggest that anti-androgenic effects were not significantly involved in the mechanism of Fujiflavone to inhibit prostate carcinogenesis. Interestingly fewer carcinomas but more PIN foci were observed in the Fujiflavone group although the sum of the two lesions was not altered by Fujiflavone treatment. This might indicate Fujiflavone inhibits progression of PIN to carcinomas.
To evaluate the effects of Fujiflavone on gene expressions in the prostate tissue, cDNA array analysis of the rat prostate with Fujiflavone treatment was performed without PhIP pre- treatment in the present study. This protocol was used since PhIP is likely to alter gene expressions in the prostate by itself. For example, PhIP induces cytochromes P450, and is mutagenic and forms DNA adducts (41,42). Such changes could modify or mask the alterations that Fujiflavone might bring, and obscure which chemical is responsible for the observed effects. PTA, one of the down-regulated genes confirmed by semi-quantitative RTPCR in the present study, is a small acidic nuclear protein ubiquitously expressed in various types of cells. Its physiological role remains unclear but several studies have indicated its involvement with cell proliferation including processes leading to malignant transformation (43,44) and poor prognosis in some clinical cancers (45,46). ODC 1, another down-regulated gene in the Fujiflavone-treated prostate, codes the key enzyme for synthesis of polyamines known as cell proliferation factors. High activity of ODC in cancer cells or cancerous tissues including the prostate has been reported and over-expression of ODC can cause cellular transformation like a proto-oncogene product (47). Development of ODC inhibitors as anticancer agents is under way (48,49), also targeting prostate cancer (50,51). As described above, less PIN lesions progressed into carcinomas in the Fujiflavone-treated prostate in the present study. These findings indicated that down-regulation of PTA and ODC might be related to the mechanism of Fujiflavone to inhibit prostate carcinogenesis. It is probable that down-regulation of PTA and ODC by Fujiflavone is specific to the prostate because their expression changes were not detected at least on cDNA array analyses of the Fujiflavone-treated colon, forestomach and liver (data not shown). Therefore, further studies for involvement of these genes in prostate cancer might be necessary to reach unique preventive or therapeutic options against prostate cancer.
Most previous studies on anticancer effects of isoflavones focused mainly on genistein. Although soybean products generally contain genistein derivatives as major isoflavones, other isoflavone components such as daizein and glycitein cannot be negligible (52). A recent epidemiological study has shown a correlation between daizein-metabolizing ability and prostate cancer progression (53). Although Fujiflavone used in the present study contains more daizein and glycitein derivatives than other isoflavone mixtures used in previous reports (2325) and relatively low amounts of genistein derivatives, inhibitory effects on rat prostate carcinogenesis were apparent and not less than seen in previous studies using similar protocols. Some reports described adverse effects of pure genistein including the promotion of mammary and colon cancer (13,54). Here the incidence of ACF with
4 crypts was significantly elevated by Fujiflavone, whereas the final incidence and multiplicity of colon tumors were almost identical in the two groups, indicating no promotion of tumorigenesis. These findings do not accord with the reported correlation between ACF with
4 crypts and final colon tumor outcome (55). The present colon tumor results suggest that isoflavone mixture might have a less adverse effect than purified genistein and be more suitable as a cancer-preventive agent. And it would be important to study all critical aspects of each isoflavone component including their interactions.
In the present study each rat fed Fujiflavone consumed
80 mg/kg/day of total isoflavones, estimated by food consumption monitoring (data not shown). Such an isoflavone intake is very high compared with the Japanese daily isoflavone intake (52,56). As to toxicity, 868 mg/day has been found safe in a human trial (57) and a single administration of 5000 mg/kg demonstrated no toxicity in mice (T.Toda, personal communication). Previous studies with relatively high dose of isoflavones have not revealed any toxic findings (2226). Considering the data, together with body weight gain during the experimental course and the final organ weights in the present study, Fujiflavone seems to have no toxicity even at high dose.
In conclusion, Fujiflavone, a commercial isoflavone mixture supplement, showed inhibitory effects on rat prostate carcinogenesis induced by PhIP, an environmental human carcinogen candidate, possibly by inhibiting progression of PIN to carcinoma with no evident adverse effects. And the down- regulation of PTA and ODC could be related to the mechanism. These results suggest that intake of dietary isoflavones is promising for prevention of human prostate cancer.
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Notes
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3 To whom correspondence should be addressed Email: atsuhiko{at}med.nagoya-cu.ac.jp 
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Acknowledgments
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We thank Dr Toshiya Toda (Fujicco Co., Kobe, Japan) for providing Fujiflavone and helpful information. We also thank Dr Malcolm A.Moore for his kind linguistic advice during preparation of the manuscript. This research was supported partly by a Grant-in-aid from the Ministry of Health, Labour and Welfare and a grant from the Society for Promotion of Pathology of Nagoya, Japan.
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Received July 9, 2003;
revised November 13, 2003;
accepted November 15, 2003.