Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide

Expression of cyclooxygenase (COX)-2 protein in 4-nitroquinoline-1-oxide (4-NQO)-induced rat tongue lesions and the postinitiation chemopreventive potential of a selective COX-2 inhibitor, nimesulide (NIM), were examined in Fischer 344 male rats. NIM was administered in the diet at doses of 150, 300, and 600 ppm for 14 weeks after treatment with 25-35 ppm 4-NQO in the drinking water for 12 weeks. Western blot analysis revealed COX-2 protein to be barely expressed in the normal tongue epithelia, whereas it was increased approximately 6-fold in squamous cell carcinomas (SCCs). Immunohistochemically, COX-2 protein was diffusely present in SCCs and dysplasia but expressed only in basal cells in hyperplasia and papillomas. In basal cells of normal epithelia, it was also occasionally weakly stained. NIM dose-dependently decreased at doses of 150 and 300 ppm, the incidences of SCCs to 4 of 12 (33.3%) and 1 of 13 (7.7%) and their multiplicity to 0.33+/-0.49 and 0.08+/-0.28 per rat, respectively, as compared with 4-NQO alone group values of 9 of 11 (81.8%) and 1.00+/-0.77. A lesser decrease was observed with 600 ppm, the values being 5 of 12 (41.7%) and 0.50+/-0.67. NIM did not significantly affect the development of hyperplasias, dysplasias, and papillomas. These results clearly indicate chemopreventive potential of a selective COX-2 inhibitor against the postinitiation development of SCCs in rat tongue carcinogenesis.     

An animal model for the rapid induction of tongue neoplasms in human c-Ha-ras proto-oncogene transgenic rats by 4-nitroquinoline 1-oxide: its potential use for preclinical chemoprevention studies

Oral squamous cell carcinoma is one of the most common human neoplasms, and prevention of this malignancy requires a better understanding of its carcinogenesis process. To this end, we tried to establish an animal model using the human c-Ha-ras proto-oncogene-carrying transgenic (Tg) rats and the carcinogen 4-nitroquinoline 1-oxide (4-NQO). 4-NQO (20 p.p.m.) was administered to Tg and non-Tg rats for 8 weeks in their drinking water, and then the occurrence of tongue carcinogenesis was compared during the experimental period of 22 weeks. In addition, we determined the DNA ploidy in tongue lesions and examined the immunohistochemical expression of five biomarkers such as cyclin D1, glutathione S-transferase placental form, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and beta-catenin. Next, the cancer chemopreventive effects of nimesulide, pioglitazone and a synthetic geranylated derivative, which have been reported to be inhibitors of tongue carcinogenesis, were examined in Tg rats treated with 4-NQO. Either during or after treatment with 4-NQO in the drinking water, tongue dysplasia and tumors were observed on the tongues of both Tg and non-Tg rats, with a greater incidence and multiplicity in Tg rats. Histopathologically, squamous cell dysplasia, papilloma and carcinoma with or without invasion were present in the tongue. Immunohistochemistry revealed that expression levels against five biomarkers increase with disease progression, and the changes correlated with those of the DNA ploidy pattern. Interestingly, a strong expression of COX-2, iNOS and beta-catenin was observed on the invasive front of squamous cell carcinomas. A subsequent chemoprevention study using Tg rats showed that the chemicals tested suppressed the occurrence of tongue carcinomas when they were administered after 4-NQO-exposure. These results may thus indicate that our 4-NQO-induced Tg rat tongue carcinogenesis model simulates many aspects of human oral carcinogenesis and it can be applied for an analysis of oral cancer development while also helping to identify potentially effective cancer chemopreventive agents against oral cancer.     

A novel geranylated derivative,ethyl 3-(4'-geranyloxy-3'-methoxyphenyl)-2-propenoate,synthesized from ferulic acid suppresses carcinogenesis and inducible nitric oxide synthase in rat tongue

OBJECTIVES: We have previously observed the inhibitory effect of ferulic acid on rat tongue carcinogenesis. In this study, we investigated the modifying effects of a novel geranylated derivative, ethyl 3-(4'-geranyloxy-3'-methoxyphenyl)-2-propenoate (EGMP), synthesized from ferulic acid on tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO). METHODS: F344 male rats except those treated with EGMP alone and untreated rats were given 20 ppm 4-NQO in drinking water for 9 weeks to induce tongue neoplasms. Starting 1 week after the cessation of exposure to 4-NQO, the experimental groups given 4-NQO and a basal diet were fed the experimental diets containing EGMP (0.2% and 2%) for 20 weeks. The activities of glutathione S-transferase (GST) and quinone reductase (QR), the expression of proliferating cell nuclear antigen, polyamine content and ornithine decarboxylase activity in the tongue were examined for mechanistic analysis of modifying effects of EGMP on carcinogenesis. The expression of inducible nitric acid synthase (iNOS) was also assessed immunohistochemically. RESULTS: At week 30, the incidence and multiplicity of tongue squamous cell carcinoma were significantly reduced by feeding of a 2% EGMP diet. EGMP feeding (2% in diet) elevated tongue GST activity and lowered QR activity in the tongue. Dietary EGMP decreased the expression of cell proliferation biomarker expression. Interestingly, EGMP feeding altered iNOS expression in the invasive parts of carcinoma. CONCLUSIONS: These results suggest that EGMP, when given at the 2% dose level during the promotion phase, exerts a chemopreventive action against tongue tumorigenesis through modification of cell proliferation activity and/or detoxifying enzymatic activity.     

Inhibitory effects of the natural products indole-3-carbinol and sinigrin during initiation and promotion phases of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

The modifying effects of indole-3-carbinol (I3C) and sinigrin (SIN) on the initiation and post-initiation phases of tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) were investigated in male ACI/N rats. Rats were divided into eight groups: group 1 was given 4-NQO (10 ppm) in the drinking water for 12 weeks, starting at 7 weeks of age; groups 2 and 3 were given 4-NQO and fed the diets containing I3C (1,000 ppm) and SIN (1,200 ppm) for 14 weeks, respectively, starting at 6 weeks of age; groups 4 and 5 were given 4-NQO and then they were fed I3C and SIN containing diets for 23 weeks, respectively, starting one week after 4-NQO exposure; groups 6 and 7 were given I3C and SIN alone, respectively, during the experiment; group 8 served as an untreated control. At the termination of the experiment (week 37), the incidence of tongue neoplasms (squamous cell papilloma and carcinoma) in group 2 (1/15, 7%), group 3 (1/15, 7%), group 4 (3/15, 20%) or group 5 (2/15, 13%) was significantly smaller than that in group 1 (12/17, 71%) (P = 0.0003, P = 0.005 or P = 0.002). No tongue carcinomas developed in rats of groups 2, 3, and 5. Similarly, the incidence of preneoplastic lesions (hyperplasia and dysplasia) of the tongue in group 2 (11/15, 73%), group 3 (10/15, 67%), group 4 (11/15, 73%) or group 5 (10/15, 67%) was significantly lower than that in group 1 (17/17, 100%) (P = 0.04 or P = 0.02). There were no tongue neoplasms in rats of groups 6, 7, and 8. Administration of I3C and SIN also caused significant decreases in the number and area of silver-stained nucleolar organizer regions protein (AgNORs), a new cell proliferation index, of tongue squamous epithelium. Thus, I3C and SIN inhibited rat tongue carcinogenesis in both the initiation and post-initiation phases, when administered in these respective phases together with, or following treatment with, 4-NQO.     

A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies

We investigated the susceptibility of 4-nitroquinoline 1-oxide (4-NQO)-induced tongue carcinogenesis in male CB6F1-Tg-rasH2 @Jcl mice (Tg mice). The Tg mice were administered 4-NQO (20 p.p.m. in drinking water) for 2, 4, 6 or 8 weeks, and thereafter they were untreated up to week 24. At week 24, a higher incidence (80%) of tongue neoplasm with dysplasia was noted in the mice that received 4-NQO for 8 weeks in comparison with the other groups (20% incidence for each) treated with 4-NQO for 2, 4 and 6 weeks. Esophageal tumors also developed in the Tg mice were 4-NQO. Immunohistochemical observation revealed that the EP receptors, especially EP(1) and EP(2), expressed in the tongue and esophageal lesions induced by 4-NQO, thus suggesting the involvement of prostaglandin (PG) E(2) and EP(1,2) receptors in the tongue and esophageal carcinogenesis. Using this animal model, we investigated the potential chemopreventive ability of pitavastatin (1, 5 and 10 p.p.m. in diet for 15 weeks), starting 1 week after the cessation of 4-NQO-exposure (20 p.p.m. in drinking water for 8 weeks). Dietary pitavastatin at 10 p.p.m. significantly reduced the incidence and multiplicity of the tongue, but not esophageal neoplasms by the modulation of prostaglandin E2 biosynthesis, EP(1) and EP(2) expression and proliferation. Our results thus suggest that a rasH2 mouse model of 4-NQO-induced tongue and esophageal carcinogenesis can be utilized for investigating the pathogenesis of cancer development in these tissues and may well prove to be useful for identifying candidate cancer chemopreventive agents for the upper digestive organs.     

Dietary garcinol inhibits 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in rats

The effects of dietary feeding with a polyisoprenylated benzophenone, garcinol, isolated from Garcinia indica fruit rind on the development of 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis were investigated in male F344 rats. At 7 weeks of age, animals were given 4-NQO at 20 ppm in the drinking water for 8 weeks to induce tongue neoplasms. They also received the diets containing 100 or 500 ppm garcinol either during (for 10 weeks) or after (for 22 weeks) the carcinogen exposure. The other rats were given tap water without 4-NQO throughout the experiment, and fed garcinol (500 ppm)-containing diet or basal diet alone. At the end of the study (week 32), incidences of tongue neoplasms and preneoplastic lesions, cell proliferation activity in the normal-like tongue epithelium estimated by 5-bromodeoxyurideine (BrdU)-labeling index and cyclin D1-positive cell ratio, and immunohistochemical expression of cyclooxygenase-2 (COX-2) in the tongue lesions were determined. Dietary garcinol significantly decreased the incidence and multiplicity of 4-NQO-induced tongue neoplasms and/or preneoplasms as compared to the control diet. Dietary administration of garcinol also significantly reduced the BrdU-labeling index and cyclin D1-positive cell ratio, suggesting reduction in cell proliferation activity in the tongue by garcinol. The COX-2 expression in the tongue lesions was also suppressed by feeding with garcinol. These results indicate that dietary administration of garcinol inhibited 4-NQO-induced tongue carcinogenesis through suppression of increased cell proliferation activity in the target tissues and/or COX-2 expression in the tongue lesions.     

Inhibitory Effects of the Natural Products Indole-3-carbinol and Sinigrin during Initiation and Promotion Phases of 4-Nitroquinoline 1-Oxide-induced Rat Tongue Carcinogenesis

The modifying effects of indole-3-carbinol (I3C) and sinigrin (SIN) on the initiation and post-initiation phases of tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) were investigated in male ACI/N rats. Rats were divided into eight groups: group 1 was given 4-NQO (10 ppm) in the drinking watar for 12 weeks, starting at 7 weeks of age; groups 2 and 3 were given 4-NQO and fed the diets containing I3C (1,000 ppm) and SIN (1,200 ppm) for 14 weeks, respectively, starting at 6 weeks of age; groups 4 and 5 were given 4-NQO and then they were fed I3C and SIN containing diets for 23 weeks, respectively, starting one week after 4-NQO exposure; groups 6 and 7 were given I3C and SIN alone, respectively, during the experiment; group 8 served as an untreated control. At the termination of the experiment (week 37), the incidence of tongue neoplasms (squamous cell papilloma and carcinoma) in group 2 (1/15,7%), group 3 (1/15, 7%), group 4 (3/15, 20%) or group 5 (2/15, 13%) was significantly smaller than that in group 1 (12/17,71%) ( P = 0.0003, P=0.005 or P=0.002). No tongue carcinomas developed in rats of groups 2, 3, and 5. Similarly, the incidence of preneoplastic lesions (hyperplasia and dysplasia) of the tougue in group 2 (11/15, 73%), group 3 (10/15, 67%), group 4 (11/15, 73%) or group 5 (10/15, 67%) was significantly lower than that in group 1 (17/17, 100%) (P=0.04 or P=0.02). There were no tongue neoplasms in rats of groups 6, 7, and 8. Administration of I3C and SIN also caused significant decreases in the number and area of silver-stained nucleolar organizer regions protein (AgNORs), a new cell proliferation index, of tongue squamous epithelium. Thus, I3C and SIN inhibited rat tongue carcinogenesis in both the initiation and post-initiation phases, when administered in these respective phases together with, or following treatment with, 4-NQO.     

Chemopreventive effect of bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats.

The modifying effects of dietary feeding of bovine lactoferrin (bLF) on tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. The activities of phase II detoxifying enzymes, glutathione S-transferase (GST) and quinone reductase (QR), polyamine content and ornithine decarboxylase (ODC) activity in the tongue were also examined for mechanistic analysis of possible modifying effects of bLF on carcinogenesis. At 7 weeks of age, all animals except those treated with bLF alone and untreated rats were given 20 ppm 4-NQO in drinking water for 8 weeks to induce tongue neoplasms. Starting 7 days before 4-NQO exposure, experimental groups were fed experimental diets containing bLF (0.2% and 2%) for 10 weeks ("initiation feeding"). Starting 1 week after the cessation of exposure to 4-NQO, the other experimental groups given 4-NQO and a basal diet were fed the experimental diets for 22 weeks ("postinitiation feeding"). At week 32, the incidence and multiplicity of tongue neoplasms in the "initiation feeding" groups of 0.2% and 2% bLF and the "post-initiation feeding" group of 0.2% bLF were lower than those of the 4-NQO alone group, but without statistical significance. However, "post-initiation feeding" of 2% bLF caused a significant reduction in the incidence (20% vs. 55%, P=0.02418) and multiplicity (0.25+/-0.54 vs. 0.70+/-0.71, P<0.05) of tongue squamous cell carcinoma (by 64%, P=0.02418). bLF treatment elevated liver and tongue GST activities and liver QR activity. The "post-initiation feeding" with 2% bLF significantly decreased QR activity, proliferating cell nulcear antigen-positive index and ODC activity in the tongue. In addition, feeding with bLF decreased tongue polyamine content. These results suggest that bLF, when given at the 2% dose level during the post-initiation phase, exerts chemopreventive action against tongue tumorigenesis through modification of cell proliferation activity and/or the activities of detoxifying enzymes.     

Inhibitory effects of non-steroidal anti-inflammatory drugs, piroxicam and indomethacin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male ACI/N rats

The effects of the non-steroidal anti-inflammatory drugs (NSAIDs), piroxicam and indomethacin on 4-nitroquinoline 1-oxide-(4-NQO)-induced tongue carcinogenesis in male ACI/N rats were examined. Rats were given 4-NQO (10 ppm) in the drinking water for 12 weeks and followed by either diet containing 150 ppm piroxicam or the drinking water containing 10 ppm indomethacin for 24 weeks. The incidence of tongue neoplasms (squamous cell papilloma and carcinoma) in rats given 4-NQO plus piroxicam (4/13, 31%) and those given 4-NQO plus indomethacin (3/13, 23%) were significantly lower than that of animals given 4-NQO alone (12/17, 71%) (P less than 0.05 and P less than 0.005). Rats given piroxicam or indomethacin alone had no neoplasms in the tongue. Thus, piroxicam and indomethacin significantly inhibited the development of tongue neoplasms in male ACI/N rats.     

Dietary Protocatechuic Acid During the Progression Phase Exerts Chemopreventive Effects on Chemically Induced Rat Tongue Carcinogenesis

The modifying effects of dietary administration of protocatechuic acid (PCA) during the progression phase of ‍tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. For ‍tumor progression we developed a new animal model, where rats initiated by 4-week treatment of 20 ppm 4-NQO in ‍drinking water, received four cycles of 20 ppm 4-NQO to induce advanced tongue cancer (one cycle: 2 weeks of 4- ‍NQO followed by 2 weeks of tap water), starting at 14 weeks after the initiation. In this model, metastasis of tongue ‍cancer occurred in lungs. Starting two weeks before the cycle treatment with 4-NQO, animals were fed the 2000 ppm ‍PCA containing diet and continued on this diet until the end of the study. At the termination of the experiment (week ‍32), the incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell ‍proliferation activity estimated by morphometric analysis of silver-stained nucleolar organizer regions’ protein were ‍compared among the groups. Feeding with PCA containing diet during the progression phase significantly decreased ‍the occurrence of advanced tongue squamous cell carcinoma with metastasis (P<0.05) and preneoplasia (hyperplasia ‍and dysplasia) (P<0.001). In addition, PCA exposure decreased polyamine levels in the tongue tissue (P<0.001) during ‍progression phase. Our results suggest that dietary PCA inhibits progression of 4-NQO-induced oral carcinogenesis, ‍and such inhibition might be related to suppression of cell proliferation by PCA. ‍     

Chemopreventive effect of fermented brown rice and rice bran on 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats

The preventive effects of the dietary administration of brown rice and rice bran fermented with Aspergillus oryzae (FBRA) on oral carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. At 7 weeks of age, the animals were given 20 ppm 4-NQO in their drinking water for 8 weeks to induce tongue neoplasms. Groups of rats were fed diets containing 5 or 10% FBRA during the initiation or postinitiation phases of the 4-NQO-induced oral carcinogenesis. The other groups consisted of rats fed 10% FBRA or untreated rats. At the termination of the study (week 32), the incidences, multiplicities of tongue lesions (pre-neoplasms and neoplasms) and the cell proliferation activity estimated by the 5-bromodeoxyuridine (BrdU)-labeling index were compared among the groups. Feeding of 5% FBRA during the initiation phase significantly decreased the incidence (68.2 vs 36.8%; p<0.05) and multiplicity (1.05+/-0.84 vs 0.37+/-0.50; p<0.005) of the tongue carcinoma. When feeding of 10% FBRA occurred after the 4-NQO exposure, the multiplicity of tongue carcinoma was also reduced (1.05+/-0.84 vs 0.52+/-0.60; p<0.05). In addition, the dietary administration of FBRA at both doses significantly decreased the BrdU-labeling index in the oral squamous epithelium (p<0.05). Although a dose-dependent response was not observed, FBRA is effective in suppressing the development of 4-NQO-induced oral carcinogenesis by its concurrent exposure to the carcinogen. The inhibitory effect could be related to the suppression of the hyperproliferation of cells in the tongue epithelium and the radical scavenging activity of FBRA.     

A cyclooxygenase-2 inhibitor,nimesulide, inhibits postinitiation phase of N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters

The modification effects of nimesulide, a cyclooxygenase (COX)-2 inhibitor, administration during the postinitiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N-nitrosobis(2-oxopropyl)amine (BOP). Male Syrian hamsters were given 4 weekly s.c. injections of BOP at a dose of 10 mg/kg and thereafter administered 0, 100 or 400 ppm nimesulide in the diet for 36 weeks. Additional groups of hamsters were fed 400 ppm nimesulide without prior BOP initiation or nontreated. At week 40, all surviving animals were killed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidence of pancreatic adenocarcinomas was significantly (p < 0.05) decreased in the BOP/400 ppm nimesulide group compared to the BOP alone group. The multiplicity of total lesions of pancreatic adenocarcinoma plus atypical hyperplasia was also significantly (p < 0.05) lowered. Immunohistochemically, COX-2 was clearly expressed in pancreatic and lung tumor cells, whereas expression was not remarkably affected by the 400 ppm nimesulide treatment. Proliferating cell nuclear antigen labeling indices of pancreatic ducts were significantly (p < 0.01) reduced by nimesulide. The incidence and multiplicity of neoplastic lesions in other organs did not significantly differ among the BOP-treated groups, though only the multiplicity of lung tumors showed a tendency to decrease. No neoplastic lesions were detected in animals receiving nimesulide alone. Our results clearly indicate that nimesulide protects against BOP-induced pancreatic tumors in hamsters.     

Enhanced levels of glutathione and protein glutathiolation in rat tongue epithelium during 4-NQO-induced carcinogenesis

High glutathione (GSH) levels are commonly found in oral tumors and are thought to play an important role in tumorigenesis. While posttranslational binding of GSH to cellular proteins (protein glutathiolation) has recently been recognized as an important redox-sensitive regulatory mechanism, no data currently exist on this process during carcinogenesis. Our goal was to determine the effects of 4-nitroquinoline-N-oxide (4-NQO)-induced carcinogenesis on tongue levels of protein-bound and free GSH and related thiols in the rat. Male F-344 rats (6 weeks of age) were administered either 4-NQO (20 ppm) in drinking water or tap water alone (controls) for 8 weeks. Twenty-four weeks after cessation of 4-NQO, squamous cell carcinomas of the tongue were observed in all rats. The levels of both free and bound GSH in tumors, as well as in adjacent tissues, were 2- to 3-fold greater than in tongue epithelium from control rats (p < 0.05). Prior to tumor formation, at 8 weeks after cessation of 4-NQO, hyperplasia, dysplasia and carcinoma in situ were observed in 100%, 25% and 12.5% of 4-NQO-treated rats, respectively. At this early stage of carcinogenesis, levels of free and bound GSH were increased 50% compared with tongue tissues from control rats (p<0.05). Glutathione disulfide (GSSG) levels were also 2-fold greater in tongue tissues from 4-NQO treated vs. control rats (p<0.05). Altogether, these results suggest that protein glutathiolation, together with GSH and GSSG levels, are induced during oral carcinogenesis in the rat possibly as a result of enhanced levels of oxidative stress.     

Chemopreventive Effect of a Xanthine Oxidase Inhibitor, 1'-Acetoxychavicol Acetate, on Rat Oral Carcinogenesis

The effect of a xanthine oxidase inhibitor, 1'-acetoxychavicol acetate (ACA), on 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis was investigated in male F344 rats. All rats except those in the ACA-alone and untreated groups were given 4-NQO (20 ppm) in the drinking water for 8 weeks to induce oral cancer. Starting 1 week before the 4-NQO exposure, animals were fed diet containing 100 ppm or 500 ppm ACA for 10 weeks, followed by the basal diet without ACA for 22 weeks. Other groups were fed the diet containing ACA at 100 ppm or 500 ppm for 22 weeks, starting 1 week after the cessation of 4-NQO exposure. The remaining groups consisted of rats given 500 ppm ACA alone or untreated rats. At the termination of the experiment (32 weeks), the incidences of tongue neoplasms and preneoplastic lesions, polyamine levels in the tongue tissue, and cell proliferation activity estimated in terms of 5-bromodeoxyuridine (BrdU)-labeling index and by morphometric analysis of silver-stained nucleolar organizer regions' protein (AgNORs) were compared among the groups. Feeding of ACA at the two doses during initiation or postinitiation significantly decreased the development of tongue carcinoma (93–100% reduction, P< 0.001) and preneoplasia (43–50% reduction for hyperplasia and 34–48% reduction for dysplasia, P<0.05). There were no such lesions in rats fed ACA alone or those in the untreated control group. The number of AgNORs per cell nucleus was significantly decreased by feeding of ACA at a high dose (500 ppm) (29% inhibition, P<0.05). The BrdU-labeling index was also reduced by dietary administration of ACA (23–32% inhibition, P<0.01). In addition, ACA feeding reduced tongue polyamine levels (35–40% inhibition, P<0.05). These results indicate that ACA inhibited rat oral carcinogenesis, and such inhibition might be related to suppression of cell proliferation in the oral mucosa by the xanthine oxidase inhibitor.     

A prostaglandin E2 receptor subtype EP1-selective antagonist, ONO-8711, suppresses 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

We previously reported that certain cyclooxygenase (COX) inhibitors could inhibit chemically induced tongue carcinogenesis. In the present study, we investigated the effects of prostaglandin E(2) (PGE(2)) receptor EP(1)-selective antagonist ONO-8711 on 4-nitroquinoline 1-oxide (4-NQO)-induced oral carcinogenesis to know whether an EP(1) receptor involves in oral carcinogenesis. Male Fischer 344 rats were given drinking water containing 4-NQO for 8 weeks (20 p.p.m. for the initial 2 weeks, 25 p.p.m. for 2 weeks, and then 30 p.p.m. for 4 weeks). After 4-NQO treatment, animals were given 400 or 800 p.p.m. ONO-8711 containing diets for 23 weeks. The incidence of tongue squamous cell carcinomas (SCC) in the 4-NQO-treated rats was 64%, while that in the rats given ONO-8711 after 4-NQO exposure was 29 (P<0.05) and 29% (P<0.05) in the 400 and 800 p.p.m. of ONO-8711, respectively. The multiplicity of tongue cancer was also smaller in the 4-NQO + ONO-8711 (400 p.p.m. ONO-8711, 0.35 +/- 0.61; and 800 p.p.m. ONO-8711, 0.29 +/- 0.47; P<0.05), when compared with the 4-NQO alone group (0.88 +/- 0.88). Feeding with ONO-8711 significantly reduced PGE(2) level and cell proliferation activity in the non-tumorous epithelium of the tongue. Also, treatment with ONO-8711 resulted in the decrease in EP(1) immunohistochemical expression in the tongue lesions induced by 4-NQO. The results suggest that EP(1) receptor involves in oral carcinogenesis, and that an EP1-selective antagonist ONO-8711 exerts the cancer chemopreventive effects through the suppression of EP(1) expression, PGE(2) biosynthesis and cell proliferation.     

Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis by citrus auraptene in rats

The modifying effects of citrus auraptene given during the initiation and post-initiation phases of oral carcinogenesis initiated with 4- nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. At 6 weeks of age, animals were divided into experimental and control groups, and fed the diets containing 100 ppm or 500 ppm auraptene. At 7 weeks of age, all animals except those treated with auraptene alone and control groups were given 4-NQO (20 ppm) in the drinking water for 8 weeks to induce tongue carcinoma. Starting 7 days before the 4-NQO exposure, groups of animals were fed the diets containing auraptene (100 and 500 ppm) for 10 weeks and then switched to the basal diet. Starting 1 week after the cessation of 4-NQO exposure, the groups given 4-NQO and a basal diet were switched to the diets mixed with auraptene (100 and 500 ppm), and maintained on these diets for 22 weeks. The other groups consisted of rats fed auraptene alone (500 ppm) or untreated rats. All rats were necropsied at the termination of the study (week 32). The incidences of tongue lesions (neoplasms and preneoplasms), polyamine levels in the tongue tissue and cell proliferation activity estimated by 5-bromodeoxyuridine (BrdU)- labelling index were compared among the groups. In addition, the activities of gluthathione S-transferase (GST) and quinone reductase (QR) in liver and tongue of rats gavaged various doses of auraptene (0, 200, 400 and 800 mg/kg body wt) for 5 days were assayed. Feeding of auraptene at both doses during the initiation phase caused a significant reduction in the frequency of tongue carcinoma (100 ppm auraptene, 91% reduction, P < 0.001; 500 ppm auraptene, 63% reduction, P < 0.05). When fed auraptene after 4-NQO exposure, the frequency of tongue carcinoma was also decreased (100 ppm auraptene, 100% reduction, P < 0.001; 500 ppm auraptene, 74% reduction, P < 0.01). The incidences of tongue severe dysplasia in these groups were significantly smaller than those in carcinogen controls (P < 0.05). There were no pathological alterations in rats treated with 500 ppm auraptene alone or those in an untreated control group. Dietary administration of auraptene significantly decreased BrdU-labelling index and polyamine concentrations in the oral mucosa (P < 0.05). In addition, auraptene administration significantly increased the activities of GST and QR in the liver and tongue. Although dose-dependent effect was not found, citrus auraptene is effective in inhibiting the development of oral neoplasms induced by 4-NQO. Thus, suppression by the initiation-feeding of auraptene might relate to elevation in the phase II enzymes GST and QR of the liver and tongue, and inhibition occurring during the post- initiation might be related to suppression of increased cell proliferation caused by 4-NQO in the oral mucosa.      

Inhibitory effects of troglitazone,a peroxisome proliferator-activated receptor gamma ligand,in rat tongue carcinogenesis initiated with 4-nitroquinoline 1-oxide

Ligands for peroxisome proliferator-activated receptor (PPAR) γ have been implicated in growth inhibition and cell differentiation in certain malignancies. In this study, the effects of troglitazone, a PPARy ligand, given during the postinitiation phase of oral carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. Rats aged 6 weeks were given 4-NQO at 20 ppm for 8 weeks to induce tongue neoplasms. Starting 1 week after the cessation of 4-NQO exposure, animals were fed diets containing 0, 30 or 100 ppm troglitazone for 22 weeks. At the end of the study (week 32), the incidences of 4-NQO-induced tongue neoplasms and preneoplasms were determined histo-pathologically and cell proliferation activity was estimated by counting bromodeoxyuridine (BrdU)-labeling indices and cyclin D1-positive cell ratios. In addition, immunohistochemical expression of cyclooxygenase (COX)-2 and PPARγ was assessed in the tongue lesions. Feeding with 100 ppm troglitazone significantly decreased the incidence of squamous cell carcinoma when compared to the group without troglitazone treatment (5.0% vs. 45.8%, P <0.005). Interestingly, the BrdU-labeling index and cyclin D1-positive cell ratio assessed in the non-lesional tongue squamous epithelium were reduced by dietary administration of troglitazone ( P <0.0001–0.005). Additionally, the immunoreactivity of COX-2 in the tongue lesions was also decreased by the treatment ( P <0.01–0.05). These results clearly showed that dietary troglitazone inhibits 4-NQO-induced tongue carcinogenesis and such inhibition is related to suppression of increased cell proliferation and/or COX-2 expression. This study warrants further investigation on the use of PPARy ligands as a novel preventive approach for oral malignancy. (Cancer Sci 2003; 94: 365–371)     

Chemopreventive effect of dietary flavonoid morin on chemically induced rat tongue carcinogenesis.

The modifying effects of dietary exposure of the flavonoid morin on 4-nitroquinoline 1-oxide (4-NQO)-induced tongue tumorigenesis, the activities of phase II detoxifying enzymes glutathione S-transferase (GST) and quinone reductase (QR) in liver and tongue, and cell proliferation activity in tongue were investigated in male F344 rats. At 7 weeks of age, all animals except those treated with morin alone and control group were given 4-NQO (20 ppm) in drinking water for 8 weeks to induce oral neoplasms. Starting 7 days before 4-NQO exposure, experimental groups were fed experimental diets containing morin (100 and 500 ppm) for 10 weeks ("initiation feeding"). Starting 1 week after the cessation of exposure to 4-NQO, other experimental groups given 4-NQO and a basal diet were given experimental diets for 22 weeks ("post-initiation feeding"). At week 32 week, "initiation feeding" of morin caused a significant reduction in the incidence of tongue carcinoma (by 44-100%). "Post-initiation feeding" with morin also significantly decreased the frequency of tongue carcinoma (by 44%). Morin feeding elevated liver GST and QR activities and GST activity in the anterior portion of tongue. Feeding with morin significantly lowered QR activity of the posterior part of the tongue. Dietary exposure to morin significantly decreased the proliferating cell nuclear antigen-positive index in the posterior portion. Also, morin feeding lowered tongue polyamine levels, especially in the "post-initiation feeding" group. Our results indicate that morin acts as a chemopreventive agent against tongue carcinogenesis induced by 4-NQO through modification of detoxifying enzyme activities and/or cell proliferation activities.     

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

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) E(2) 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 PGE(2) content.     

Inhibition of 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis by the naturally occurring plant phenolics caffeic, ellagic, chlorogenic and ferulic acids

The modifying effects of dietary administration of the plantphenolic antioxidants caffeic acid (CA), ellagic acid (EA),chlorogenic acid (CGA) and ferulic acid (FA) during the initiationphase on 4-nitroquinoline-1-oxide (4-NQO)-induced tongue carcinogenesisand on the number and area of silver-stained nucleolar organizerregion proteins (AgNORs), a new cell proliferation marker, ofthe tongue squamous epithelium were investigated in male F344rats. Rats were fed the diet containing 500 p.p.m. CA, 400 p.p.m.EA, 250 p.p.m. CGA or 500 p.p.m. FA for 7 weeks. One week afterthe commencement of the diets, 4-NQO (20 p.p.m.) was administeredin the drinking water for 5 weeks. Feeding of four phenoliccompounds significantly reduced the incidences of tongue neoplasms(squamous cell papilloma and carcinoma) and preneoplastic lesions(hyperplasia and dysplasia) by 32 weeks, and rats fed CA orEA had no tongue neoplasms. The number and area of AgNORs pernucleus were decreased significantly by dietary treatment withthese four phenolics. Thus, CA, EA, CGA and FA inhibited thetongue carcinogenesis induced by 4-NQO when they were administeredconcurrently with the carcinogen. These results might suggestpossible application of these natural substances for cancerchemoprevention in tongue in addition to other tissues (skin,lung, liver and esophagus).