Dietary silymarin suppresses 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats

Yoshitame Yanaida1, Hiroyuki Kohno2, Koujiro Yoshida3, Yoshinobu Hirose3, Yasuhiro Yamada3, Hideki Mori3 and Takuji Tanaka2,4

1 Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942,
2 Department of Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293 and
3 Department of Pathology, Gifu University School of Medicine, 40 Tsuksa-machi, Gifu 500-8705, Japan

Abstract

The modifying effect of dietary administration of a polyphenolic antioxidant flavonoid silymarin isolated milk thistle [Silybum marianum (L.) Gaertneri] on 4-nitroquinoline 1-oxide (4-NQO)-induced tongue tumorigenesis was investigated in male F344 rats. Based on the results in pilot studies showing that silymarin treatment together with 4-NQO significantly reduced the occurrence of tongue dysplasia and gavaged with silymarin significantly elevated the phase II detoxifying enzymes' activities in the liver and tongue, the effects of dietary feeding of silymarin on tongue carcinogenesis were investigated in a long-term experiment, where rats were initiated with 4-NQO and fed silymarin containing diets during or after 4-NQO exposure. At 5 weeks of age, all animals except those treated with silymarin alone and untreated rats were given 20 p.p.m. 4-NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 1 week before 4-NQO administration, animals were fed the experimental diets containing silymarin (100 and 500 p.p.m.) for 10 weeks, and then maintained on a basal diet for 24 weeks. Starting 1 week after the cessation of 4-NQO exposure, the experimental groups given 4-NQO and a basal diet were fed the experimental diets containing 100 or 500 p.p.m. silymarin for 24 weeks. At week 34, feeding of 500 p.p.m. silymarin during the promotion phase significantly inhibited the incidence of tongue carcinoma, when compared with 4-NQO alone group (20% versus 64%, P = 0.019). Dietary silymarin decreased the cell proliferating activity and increased apoptotic index of tongue carcinoma. The treatment with silymarin decreased the polyamine content and prostaglandin (PG) E2 level in the tongue mucosa. Thus, the results indicate that feeding of silymarin (500 p.p.m.) during the promotion phase of 4-NQO-induced rat tumorigenesis exerts chemopreventive ability against tongue squamous cell carcinoma through modification of phase II enzymes activity, cell proliferation, and/or PGE2 content.

Abbreviations: AA, arachidonic acid; CDNB, 1-chloro-2,4-dinitrobenzene; COX, cyclooxygenase; DCNB, 1,2-dichloro-4-nitrobenzene; DMBA, 7,12-dimethylbenz[a]anthracene; GST, glutathione S-transferase; LOX, lipoxygenase; 4-NQO, 4-nitroquinoline 1-oxide; ODC, ornithine decarboxylase; PCNA, proliferating cell nuclear antigen; PG, prostaglandin; QR, quinone reductase; ROS, reactive oxygen species; ssDNA, single stranded DNA.

Introduction

Oral cancer is an important public health issue because its occurrence is strongly associated with cigarette smoking and alcohol drinking (1). Oral cancer is one of the ten most frequent cancers worldwide, with three quarters of all cases occurring in the developing countries (2). It varies in frequency very greatly among different countries and geographic regions (3). The incidence and the mortality of oral cancer have increased over the past decades in Europe (4) and in the United States (5). In particular, the incidence and mortality rate of tongue cancer, as compared with other types of oral cancer, have increased in younger adults (6–8). In Japan, oral cancer is relatively uncommon, but its incidence and mortality recently have increased. Nearly 5 000 new cases of oral cancer were diagnosed in 1999 and the age-standardized mortality rates among the males increased from 1.14 per 100 000 persons in 1957 to 1.84 in 1991 (9).

Active primary prevention such as chemoprevention against carcinoma development is now important. A number of studies are currently directed at identifying possible chemopreventive agents (10). We previously reported possible chemopreventive agents that are present in fruits and vegetables, against tongue carcinogenesis (11,12). Such chemopreventors possess anti-proliferation, anti-inflammatory and antioxidative effects.

The most frequently used animal models in oral cancer research studies have been the hamster buccal pouch model, rat, and less frequently, mouse (13–16). A fat-soluble 7,12-dimethylbenz[a]anthracene (DMBA) and a water-soluble 4-nitroquinoline 1-oxide (4-NQO) are the most frequently used carcinogens in these studies. One of the most important routes of oral exposure to carcinogens is through food and liquid containing different levels of water-soluble carcinogens. Since 4-NQO is water-soluble, it is well suited in examining the role of xenobiotics in experimental oral carcinogenesis. Topical application of 4-NQO and administration of 4-NQO in drinking water both induce premalignant and malignant lesions in the rat oral cavity. This results in development of dysplasia, papilloma, and invasive squamous cell carcinoma, resembling the histologic changes observed in these neoplasms in humans (17). Therefore we have used a rat model for tongue carcinogenesis induced by 4-NQO in drinking water to identify cancer chemopreventive agents, since 4-NQO exposure in drinking water could induce specifically tongue preneoplasms and neoplasms of squamous cell origin (11).

The collective name for an extract from the milk thistle [Silybum marianum (L.) Gaertneri] (18), silymarin is a naturally occurring polyphenolic flavonoid antioxidant (19). Silymarin is composed mainly ( ~80%, w/w) of silybin (also called silybinin, silibin or sibilinin) with smaller amounts of other stereoisomers such as isosilybin, dihydrosibilyn, silydianin, and silychristin (20). Silymarin protects experimental animals against a hepatotoxin (18). Silymarin has a strong antioxidant property and is able to scavenge both free-radicals and reactive oxygen species (ROS) (19,21). Silymarin and its components have other biological properties, such as inhibition of lipoxygenase (LOX) (22) and prostaglandin (PG) synthetase (23). There were no reports on the average of estimated dietary intake of silymarin in humans. However, for over 20 years, silymarin has been used clinically in Europe for the treatment of alcoholic liver disease and is used as an anti-hepatotoxic agent (24). More recently, silymarin has also been used in Asia as a therapeutic agent for treatment of liver diseases. As a therapeutic agent, silymarin is well tolerated and largely free of adverse effects (21). Recent studies suggested that silymarin acts as a potent anticarcinogenic agent against in vitro (25) and in vivo (26,27) carcinogenesis. However, animal chemopreventive studies with silymarin were limited to skin (27,28). We recently found the protective effect of silymarin on chemically induced colon tumorigenesis (Tanaka, T. et al., manuscript submitted). Silymarin inhibits tumor-promoter-caused induction of ornithine decarboxylase (ODC) activity and mRNA expression in mouse epidermis (29). Silymarin could inhibit mRNA expression of an endogenous tumor promoter TNF{alpha} (30). More recently, Zi et al. (31) reported that silymarin could inhibit activation of erbB1 signaling and induce cyclin-dependent kinase inhibitors, G1 arrest, and cause complete inhibition of growth of human prostate carcinoma DU145 cells. Also, silymarin, at lower non-toxic concentrations, inhibits transformation in cultured rat tracheal epithelial cells treated with benzo[a]pyrene (32). In addition, the silymarin group (silybin, silychristin, and silydianin) are xanthine oxidase inhibitors (33). We previously reported that a xanthine oxidase inhibitor 1'-acetoxychavicol acetate effectively inhibits 4-NQO-induced tongue carcinogenesis in rats (34). These findings led us to evaluate the possible suppressing effects of dietary silymarin on the occurrence of tongue neoplasms induced by 4-NQO.

In the present study, we investigated the modifying (possibly inhibiting) effects of dietary silymarin during the initiation or promotion phase on tongue carcinogenesis in rats initiated with 4-NQO. Before the long-term study, pilot studies were conducted. These included (i) a short-term dysplasia assay for predicting possible inhibitory action of silymarin in 4-NQO-induced dysplasia being a precursor lesion for tongue squamous cell carcinoma in rats and (ii) glutathione S-transferase (GST) and quinone reductase (QR) assay in rats gavaged with silymarin. The latter assay was done because certain inducers of the detoxifying enzymes, GST and QR, are candidates for cancer chemopreventive agents (35,36). The effects of dietary silymarin on the cell proliferation activity of tongue carcinoma were also assessed by measuring proliferating cell nuclear antigen (PCNA)-positive index and single stranded DNA (ssDNA) for apoptotic nuclei. Polyamine levels of tongue mucosa were assayed since polyamine metabolism is one of the targets for cancer chemoprevention (37). In addition, PGE2 level was determined in the tongue, since oral cancer including tongue carcinoma contains a higher level of PGE2 than surrounding tongue mucosa in humans (38).

Materials and methods

Animals, diets, and carcinogen
Male F344 rats (Charles River Japan, Kanagawa, Japan), 4 weeks old, were used. All animals were housed in wire cages (three or four rats/cage) with free access to drinking water and basal diet, powdered CE-2 (345.2 Cal, CLEA Japan Inc., Tokyo, Japan), under controlled conditions of humidity (50 ± 10%), light (12-h light/dark cycle) and temperature (23 ± 2°C). They were quarantined for 7 days and randomized into experimental and control groups. 4-NQO (CAS 56-57-5, 98% pure) was obtained from Wako Pure Chemical Ind. (Osaka, Japan). Experimental diets were prepared by mixing silymarin at a concentration of 100 or 500 p.p.m. on a weekly basis. The 4-NQO solution (20 p.p.m.) was prepared every week. The experimental diets and 4-NQO solution were stored in a cold room until used. They were freely available during the study.

Pilot studies
Twenty-four rats aged 5 weeks were used to test the modifying effect of silymarin on the development of tongue dysplasia induced by 4-NQO. They were divided into four groups of six rats each: group 1, 4-NQO alone; group 2, 4-NQO + 100 p.p.m. silymarin; group 3, 4-NQO + 500 p.p.m. silymarin; and group 4, untreated. Animals in groups 1–3 were given 4-NQO (20 p.p.m. in drinking water) with or without silymarin (100 p.p.m. or 500 p.p.m. in diet) for 10 weeks. All rats were killed 10 weeks after the start, and the incidence and multiplicity of tongue dysplasia was determined on hematoxylin and eosin-stained sections. In the next pilot study to determine the modifying effect of silymarin on GST and QR activities in liver and tongue, 26 rats (5 weeks of age) were daily gavaged with silymarin at different dose levels in 0.5 ml of 5% gum arabic (Sigma-Aldrich Japan, Tokyo, Japan) for 5 days: group 1 (six rats), 0 mg/kg body wt/rat; group 2 (six rats), 40 mg/kg body wt/rat; group 3 (seven rats), 200 mg/kg body wt/rat; group 4 (seven rats), 400 mg/kg body wt/rat. They were killed by cervical dislocation 30 min after the last gavage of silymarin. At death, the liver and tongue were excised immediately. The liver was minced into small pieces after blood was removed with phosphate-buffered saline (pH 7.4). After washing with phosphate-buffered saline (pH 7.4), the tongue mucosa was collected by scraping the mucosal surface using a microtome knife. Aliquots of minced liver and mucosal scraping of tongue were processed for cytosolic fraction as described (39,40). The activities of GST with 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates and QR with NADH and menadione as substrates were determined as described previously (41–43). All assays were performed by spectrophotometry at 340 nm and all samples were measured in quadruplicate. One unit of enzyme activity is the amount of enzyme catalyzing the conversion of 1 mmol substrate to product per min at 25°C. Cytosolic protein concentrations were determined by the Bradford (44) method using bovine serum albumin as the standard.

Experimental procedures of a long-term study
A total of 131 male F344 rats were divided into seven groups as shown in Figure 1Go. Groups 1 through 5 were given 20 p.p.m. 4-NQO in drinking water for 8 weeks. Groups 2 and 3 were fed diets mixed with 100 p.p.m. and 500 p.p.m. silymarin, respectively, for 10 weeks, starting 1 week before the start of 4-NQO exposure, and then maintained on the basal diet for 24 weeks. Groups 4 and 5 were fed the experimental diets containing 100 p.p.m. and 500 p.p.m. silymarin, respectively, for 24 weeks, starting 1 week after cessation of 4-NQO treatment. Group 6 was given 500 p.p.m. silymarin-containing diet alone for 34 weeks. Group 7 served as an untreated control. The experiment was terminated at 34 weeks after the start and all animals were killed to assess the incidences of tongue neoplastic and preneoplastic lesions. At the termination of the study, all organs including tongue in rats were carefully inspected for pathological lesions. The tongues of all rats were longitudinally cut into halves for histopathological, immunohistochemical, and biochemical examinations. The tongues without macroscopic lesions of randomly selected five rats each from all groups were for tissue polyamine content and PGE2 content. For histological examination, tissues and gross lesions were fixed in 10% buffered formalin, embedded in paraffin blocks and stained with hematoxylin and eosin. Tongue lesions (hyperplasia, dysplasia and neoplasms) were diagnosed according to the criteria described by Kramer et al. (45).




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Fig. 1. Experimental protocol.

 
PCNA and ssDNA immunohistochemistry
For the determination of PCNA-incorporated nuclei, the PCNA-immunohistochemistry was performed according to the method described by Watanabe et al. (46). Apoptotic index was also evaluated by immunohistochemistry for ssDNA (46). The immunohistochemistry was done using a stain system kit (DAKO LSAB 2kit/HRP, DAKO Japan, Kyoto, Japan). A mouse monoclonal antibody against PCNA (1:50 dilution; PC10, DAKO Japan) and a rabbit polyclonal antibody against ssDNA (1:300 dilution; DAKO Japan) were applied to the sections according to the manufacturer's protocol. All incubation steps were carried out for 15 min at 37°C. The chromogen used was 3,3'-diaminobenzidine tetrahydrochloride. Slides were lightly counterstained with Meyer's hematoxylin for 1 min, dehydrated, and coverslipped. Negative controls were performed by substituting the primary antibodies with nonimmune mouse or rabbit serum. Slides were subsequently reviewed in a blinded fashion by the study pathologist (T.T.). The PCNA and apoptotic indices of squamous cell carcinomas in groups 1–5 and normal appearing squamous epithelium of groups 6 and 7 (three rats each) were determined by counting the number of positive cells among at least 200 cells in the lesion, and were indicated as percentages.

Measurement of polyamine level
Tongue mucosa from five rats each from all groups was scraped with a stainless steel disposable microtome bladed knife (S35, Feather Safety Razor, Osaka), pooled and homogenized in 1.5 ml of homogenizing buffer (250 mmol sucrose, 50 mmol Tris–HCl, pH 7.4, containing 1 mmol dithiothreitol, 1 mmol EDTA and 0.4 mmol pyridoxal 5'-phosphate) using a Polytron. The homogenates were centrifuged at 15 000 r.p.m. for 30 min at 4°C. The resulting cytosol fraction was used for determination of tissue polyamine contents and protein. Tissue polyamine contents were determined using the method described by Koide et al. (47).

Measurement of PGE 2 level in the tongue epithelium
For determination of PGE2 in the tongue mucosa, a commercially available PGE2 enzyme immunoassay system (Amersham Pharmacia Biotech, Tokyo, Japan) was employed. Cross-reactivity with other AA metabolites ranges from <0.1% to 7%. Results are expressed as pg PGE2/g tissue.

Statistical analysis
Where applicable, the data were analyzed using the Fisher's exact probability test or one-way ANOVA followed by a Newman-Keuls post-hoc test (software `JMP', SAS Institute, Cary, NC, USA), taking P < 0.05 as the level of significance.

Results

Incidence of tongue dysplasia and activities of GST and QR in pilot studies
The incidence of tongue dysplasia in a pilot study is given in Table IGo. The incidences of tongue dysplasia with various atypia in rats given 4-NQO and silymarin (100 p.p.m. or 500 p.p.m. in diet) were lower than those in rats treated with 4-NQO alone without statistical significance. However, no. of dysplasia/cm in these rats was significantly smaller than 4-NQO alone group (P < 0.02 and P < 0.005 for 100 p.p.m. and 500 p.p.m. silymarin, respectively). In addition, gavage with silymarin significantly elevated GST and QR activities in the liver and tongue, as shown in Table IIGo. In the liver of rats gavaged with silymarin (40, 200, and 400 mg/kg body weight), GST-CDNB or GST-DCNB increased by 1.23–1.69 (P < 0.001 for 40, 200, and 400 mg/kg body wt silymarin) or 1.08–1.29-fold over controls (P < 0.005 for 200 mg/kg body wt silymarin or P < 0.002 for 400 mg/kg body wt silymarin). There was a significant increase (1.84-fold) in the liver QR activity of rats given silymarin at 400 mg/kg body weight compared with controls (P < 0.002 for 400 mg/kg body wt silymarin). Similarly, in the tongue of rats gavaged with silymarin increased GST-CDNB by 1.20–1.36-fold (P < 0.02 for 40 mg/kg body wt silymarin; P < 0.05 for 200 mg/kg body wt silymarin; and P < 0.001 for 400 mg/kg body wt silymarin) and QR by 1.13–1.36-fold (P < 0.05 for 40 mg/kg body wt silymarin; P < 0.001 for 200 mg/kg body wt silymarin; and P < 0.002 for 400 mg/kg body wt silymarin) activities over the controls.


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Table I. Incidence of tongue dysplasia in a pilot study (a dysplasia assay)
 

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Table II. GST and QR activities in (a) liver and (b) tongue of rats gavaged with silymarin
 
General observation in a long-term study
The rats tolerated well the oral administration of 4-NQO and/or silymarin. During the study, no clinical signs of toxicity were present in any groups. Histologically, there were no pathological alterations suggesting toxicity of silymarin in the liver, kidneys, heart, and lungs. The data on mean body, liver, and relative liver weights (g liver weight/100 g body weight) in all groups at sacrifice are given in Table IIIGo. The mean body weights of rats in groups 3 (4-NQO + 500 p.p.m. silymarin) and 5 (4-NQO -> 500 p.p.m. silymarin) were significantly lower than that of group 1 (4-NQO alone) (P < 0.01 and P < 0.001, respectively). The mean body weight of group 6 (500 p.p.m. silymarin alone) was significantly smaller than group 7 (P < 0.001). The mean liver weights did not significantly differ among the groups. The mean relative liver weight in group 6 was significantly greater than in group 7 (P < 0.02).


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Table III. Body, liver, relative liver weight, and incidence of tongue tumors in rats treated with 4-NQO and/or silymarin
 
Incidence of tongue neoplasms and preneoplastic lesions
Tongue tumors developed in the posterior tongue (dorsal region) of rats treated with 4-NQO (groups 1–5). Histologically, they were well differentiated squamous cell carcinoma or papilloma. Dysplastic and hyperplastic lesions also developed in the tongues of rats in these groups. No preneoplastic and neoplastic lesions in any organs, including tongue, were present in groups 6 and 7.

The incidences of tongue tumors (squamous cell papilloma and carcinoma) and preneoplasia (hyperplasia and dysplasia) in each group are given in Tables IV and VGoGo, respectively. In group 1 (4-NQO alone), the incidences of tongue squamous cell carcinoma and papilloma were 64% and 18%, respectively. On the other hand, the incidences of these tongue neoplasms in rats given silymarin either during or after 4-NQO exposure (groups 2–5) were lower than that of group 1 (Table IVGo). Statistical analysis revealed a significant reduction in the incidence of tongue carcinoma in rats fed the diet containing 500 p.p.m. silymarin during the post-initiation stage (group 5), when compared with that in group 1 (20% versus 64%, 69% reduction, P = 0.019). Also, feeding of 500 p.p.m. silymarin after 4-NQO administration (group 5) caused a significant reduction of combined incidence of tongue neoplasms (papilloma + carcinoma) (P = 0.019).


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Table IV. Incidence of tongue tumors in rats treated with 4-NQO and/or silymarin
 

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Table V. Incidence of tongue preneoplastic lesions in rats treated with 4-NQO and/or silymarin
 
As shown in Table VGo, various degrees of hyperplasia or dysplasia with or without neoplasms was observed in the tongue of rats given 4-NQO (groups 1–5). The incidences of tongue squamous hyperplasia in these groups were comparable. Among the various degrees of dysplasia, the incidence of severe dysplasia of rats in group 5 was significantly smaller than that of group 1 (P < 0.05).

PCNA-labeling index and apoptotic index
Data on PCNA-labeling index and apoptotic index in the tongue squamous cell carcinomas and normal squamous epithelium are shown in Table VIGo. The PCNA-labeling index of the tongue squamous cell carcinomas in groups 2–5 were smaller than that in group 1 without statistical significance. The apoptotic index of carcinoma in groups 2–5 were greater than that of group 1, but there was no statistical significance.


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Table VI. PCNA-labeling index and ssDNA-positive index in the tongue squamous cell carcinoma and squamous epithelium
 
Polyamine content and PGE2 level in the tongue mucosa
The results of PGE2 and tissue polyamine assays in the tongue are summarized in Table VIIGo. 4-NQO treatment significantly elevated tongue mucosal PGE2 content when compared with group 7 (P < 0.002). Silymarin feeding either during or after 4-NQO exposure significantly lowered the tongue PGE2 level, when compared with that of group 1 (P < 0.05 or P < 0.001). On the other hand, feeding of 500 p.p.m. silymarin during the experiment did not alter PGE2 content in the tongue. Tongue polyamine level (the sum of diamine, spermidine and spermine) of tongue mucosa in group 1 was significantly greater than group 7 (P < 0.05). Silymarin feeding (500 p.p.m. in diet) after 4-NQO exposure significantly lowered the tongue mucosal polyamine level when compared with group 1 (P < 0.05).


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Table VII. PGE2 level and polyamine content in the tongue mucosa
 
Discussion

Our results clearly indicate that dietary administration of 500 p.p.m. silymarin during the promotion phase of 4-NQO-induced tongue carcinogenesis significantly lowered the incidence of squamous cell carcinoma. Feeding of silymarin during the initiation phase also reduced the tongue cancer development, but the reduction was not statistically significant. Silymarin is known to inhibit chemically induced carcinogenesis in other organs, such as skin (27) and colon (26). We also found the protective effect of silymarin on colon carcinogenesis in rats (Tanaka,T., manuscript submitted). Also, feeding of silymarin could inhibit the development of preneoplastic lesions of these organs. These results reported indicate that silymarin might be a candidate chemopreventive agent against carcinogenesis in multiple organ including tongue.

It is known the defense against carcinogenic events is provided by phase II enzymes such as GST and QR (36). These enzymes are of importance because they can conjugate electrophiles and protect the host from the carcinogenic effects of chemical carcinogens. In a pilot study, gavage with silymarin significantly elevated GST and QR activities in the liver and tongue. Subsequent long-term study revealed that the protective effect of silymarin feeding during the initiation phase of 4-NQO-induced tongue tumorigenesis was not strong when compared with that in rats fed during the promotion phase. This may suggest that the enzyme alteration is not enough for significant inhibition of silymarin in initiating events of 4-NQO, and other mechanism(s) including antioxidant ability and/or alteration of phase I enzymes' activity.

Cell proliferation plays an important role in multistage carcinogenesis and involves multiple genetic alterations (48,49). Silymarin is a strong antioxidant (21) and inhibits increased cell proliferation activity caused by a radical-generating promoter (29). Polyamines and polyamine synthetic enzyme activities are associated with cell proliferation. A decrease in the numbers of PCNA-positive cells reflects a decrease in S phase cells and thus a reduced proliferative activity. Our previous study demonstrated that a specific ODC inhibitor {alpha}-difluoromethylornithine suppressed 4-NQO-induced tongue carcinogenesis in rats (50). In the current study, feeding of silymarin (500 p.p.m. in diet) during the promotion phase significantly lowered the PCNA-labeling index and the polyamine levels in the tongue, suggesting that silymarin in diet could suppress the high-proliferative activity of cells initiated with a water-soluble carcinogen 4-NQO and inhibit carcinogenesis. In a mouse skin photocarcinogenesis model, application of silymarin inhibits ODC activity and suppresses skin tumor formation (27,29). Thus, one of the mechanisms by which silymarin exerts chemopreventive ability might be related to suppression of cell proliferation in the target tissue.

In the present study, we measured PGE2 content in the tongue mucosa, since alteration in arachidonic acid (AA) metabolism plays a role in oral carcinogenesis. In humans, PGE2 level in oral cancer tissues is higher than that of the surrounding normal oral tissue (38). We and others found that non-steroidal anti-inflammatory drugs can effectively inhibit oral carcinogenesis (51,52). In the current study, 4-NQO administration significantly elevated PGE2 level in the oral mucosa and dietary feeding of silymarin during either the initiation or promotion phase of carcinogenesis reduced this increased level of PGE2. The reduction was prominent when silymarin was fed to rats during the promotion phase. The findings support that silymarin could affect the AA metabolic pathway (22,23). Recently, we (Yoshida,K. et al., manuscript submitted) and others (53) found that a specific cyclooxygenase (COX)-2 inhibitor inhibits 4-NQO-induced tongue carcinogenesis. Although we did not examine the effect of silymarin on COX-2 expression in this study, silymarin can inhibit COX-2 expression induced by a tumor promoter in SENCAR mouse epidermis and can suppress iNOS and COX-2 expression in the macrophage cell line J774A.1 treated with lipopolysaccharide (54).

In conclusion, our results suggest that dietary silymarin exerts a chemopreventive effect on 4-NQO-induced rat tongue carcinogenesis, when fed during the promotion phase. This cancer protective effect of silymarin might relate to the control of carcinogen-induced hyper-cell proliferation, and/or alteration of the AA metabolic pathway. Additional works to investigate the modifying effect of silymarin in other organs, especially hormone-related organs, need to be done, since silymarin inhibits the function of androgen receptor in the human prostate cancer cell line LNCaP (55).

Notes

4 To whom correspondence should be addressed. Email: takutt{at}kanazawa-med.ac.jp Back

Acknowledgments

This research was supported in part by a Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare in Japan; by a Grant-in-Aid for Cancer Research (11-18 and 13-15) from the Ministry of Health, Labour and Welfare in Japan; by a Grant-in-Aid (no. 13671986) from the Ministry of Education, Science, Sports and Culture in Japan; and by a grant (H2001-4) for Project Research from High-Technology Center of Kanazawa Medical University.

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Received November 20, 2001; accepted January 29, 2002.