Toshiya Kuno

The inhibitory effects of mangiferin, a naturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside, is one of xanthone derivatives and C-glucosylxanthones, is widely distributed in higher plants and is one of constituents of folk medicines. Recent studies showed that mangiferin has a potential as an anti-oxidant and an anti-viral agent. In this study, we examined the effects of mangiferin in rat colon carcinogenesis induced by chemical carcinogen, azoxymethane (AOM). We performed two experiments: a short-term assay to investigate the effects of mangiferin on the development of preneoplastic lesions by AOM, aberrant crypt foci (ACF), and the following long-term assay for the influence of mangiferin on tumorigenesis induced by AOM. In the short-term assay, 0.1% mangiferin in a diet significantly inhibited the ACF development in rats treated with AOM compared to rats treated with AOM alone (64.6+/-22.0 vs. 108.3+/-43.0). In the long-term assay, the group treated with 0.1% mangiferin in initiation phase of the experimental protocol had significantly lower incidence and multiplicity of intestinal neoplasms induced by AOM (47.3 and 41.8% reductions of the group treated with AOM alone for incidence and multiplicity, respectively). In addition, the cell proliferation in colonic mucosa was reduced in rats treated with mangiferin (65-85% reductions of the group treated with AOM alone). These results suggest that mangiferin has potential as a naturally-occurring chemopreventive agent.

Matrix metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation and localization of their activity

PurposeThe process of invasion and metastasis is closely related to the prognosis of oral squamous cell carcinoma (OSCC). Matrix metalloproteinases (MMPs) are a group of enzymes characterized by their ability to degrade extracellular matrix proteins and contribute to the tumor invasion and metastasis. Especially MMP-2 and MMP-9 are known to be related to destruction of basement membrane as collagenases. This study focused on protein expression of MMP-2 and MMP-9 and their extracellular matrix degradation activity in OSCCs.MethodsFreshly frozen samples from 31 OSCC patients were analyzed for the localization and activity of MMP-2 and MMP-9. Serial frozen sections were used by routine hematoxylin and eosin staining, immunohistochemistry for MMP-2 and MMP-9, and film in situ zymography (FIZ) for gelatinolytic activity. We also evaluated the activity of MMP-2 and MMP-9 by zymography using the same samples as frozen sections. The activated form/proform ratio of MMPs in zymography was evaluated using an image scanner.ResultsIn MMP-2 the proportion in T3 and T4 clinical stage groups was significantly higher than that in T1 and T2. The proportion in lymph node metastasis cases (N+) was also significantly higher than that in non-lymph node metastasis cases (N−). In contrast to MMP-2, the activated form/proform ratio of MMP-9 was very low, suggesting that MMP-9 is not activated in the matrix degradation of OSCC, although both MMP-2 and MMP-9 protein expression are presented in tumor cells. FIZ revealed MMP in both tumor cells and stromal cells of 70% of the N+ cases and of 47.6% of the N− cases.ConclusionsThese results indicate that two types of proform and activated form matrix metalloproteinases, MMP-2 and MMP-9, are present in human OSCC, and that the activated MMP-2 could be a main enzymatic activity of gelatinolysis in OSCC. Interaction of tumor cells and stromal cells seems to play an important role in the invasion and metastasis of human OSCC. Combination analysis of zymography and FIZ is a usuful method to detect activity and localization of MMPs in human OSCC.

Suppression of occurrence and advancement of β-catenin-accumulated crypts, possible premalignant lesions of colon cancer, by selective cyclooxygenase-2 inhibitor, celecoxib

Suppression of occurrence and advancement of premalignant lesions is important for cancer prevention. Our previous studies clarified that β‐catenin‐accumulated crypts, independent of aberrant crypt foci (ACF), are probably direct precursors of colon cancers in rats. Here we investigated the effects of a selective cyclooxygenase‐2 inhibitor, celecoxib, on the development of β‐catenin‐accumulated crypts in comparison with those on ACF. Male F344 rats were divided into 4 groups. Groups 1‐3 were administered azoxymethane (AOM) s.c. at a dose of 15 mg/kg body weight, once weekly for 3 weeks to induce β‐catenin‐accumulated crypts. Groups 2 and 3 also received experimental diet containing celecoxib (500 and 1500 ppm, respectively) for 8 weeks, starting a week before the first dosing of AOM. At termination, the frequency and crypt multiplicity (number of crypts/lesion) of β‐catenin‐accumulated crypts of groups 2 and 3 were significantly less than that of group 1. Furthermore, numbers of silver‐stained nucleolar organizer regions (AgNORs)/nucleus in β‐catenin‐accumulated crypts were also decreased by exposure to celecoxib. In this study, celecoxib had greater effects on the frequency and growth of β‐catenin‐accumulated crypts than on those of ACF. These findings represent additional evidence that β‐catenin‐accumulated crypts are premalignant lesions of colon cancer. The results also suggest that β‐catenin‐accumulated crypts could be a novel target for evaluation of possible chemopreventive agents against colon carcino‐genesis, and indicate that possible chemopreventive effects of celecoxib on the initial stage of colon carcinogenesis may be related to modulation of cell proliferation activity in such early lesions.

Inhibitory effects of troglitazone, a peroxisome proliferator-activated receptor γ 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)

Lung Toxicity of 16 Fine Particles on Intratracheal Instillation in a Bioassay Model Using F344 Male Rats

We have developed a bioassay model to estimate toxicity of fine particles in the lungs at an early stage after intratracheal instillation (Yokohira et al. 2005; Yokohira et al. 2007). The present experiment was conducted to improve the model by estimating appropriate doses based on dose-dependent toxicity of instilled quartz (4 mg to 0 mg) as a positive control and assessing the impact of powdered particles without suspension (Experiment 1). In addition, examination of the toxicity of a series of particles was performed with the developed bioassay (Experiments 2A, 2B, and 2C). The materials chosen were sixteen particles, including nanoparticles and diesel powder. Histopathological and immunohistochemical analysis of bromodeoxyuridine (BrdU) incorporation and inducible nitric oxide synthase (iNOS) were performed after exposure of the lungs. A dose of 2 mg quartz suspended in 0.2 mL saline was suggested to be most appropriate for sensitive detection of acute and subchronic inflammatory changes. Although some materials, including nanoparticles, demonstrated toxicity that was too strong for sensitive assessment, the ranking order could be given as follows: CuO > quartz > neutralized Na2PdCl4 > NiO > hydrotalcite > MnO2 > diesel > titanium dioxide (in Experiment 2B) > β-cyclodextrin > diesel standard > titanium dioxide (in Experiment 2A) > CaCO3.

Dietary supplementation of the citrus antioxidant auraptene inhibits N,N-diethylnitrosamine-induced rat hepatocarcinogenesis.

Objectives: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemopreventive action in other organs. Methods: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm2) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-α were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage (‘initiation’ feeding for 7 weeks) or post-initiation phase (‘post-initiation’ feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. Results: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-α-positive EAF/cm2, and the reduction in the number of TGF-α-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the ‘initiation’ feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 ± 1.09 vs. 1.96 ± 1.85, p < 0.005) of liver cell carcinoma. Also, the ‘post-initiation’ feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 ± 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 ± 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. Conclusions: The results suggest that the citrus antioxidant AUR is a potential chemopreventive agent against DEN-induced hepatocarcinogenesis in rats.

Tumor formation is correlated with expression of β‐catenin‐accumulated crypts in azoxymethane‐induced colon carcinogenesis in mice

We have reported that β‐catenin‐accumulated crypts (BCAC) are independent of aberrant crypt foci (ACF) in the colonic mucosa of rats exposed to colorectal carcinogens, and we suggested that they may be premalignant lesions. In the present study, we performed a comparative study on the formation of the two types of early‐appearing lesions (BCAC and ACF), and tumors of the colon in two mouse strains with different susceptibility to azoxymethane (AOM). SWR/J mice are known to be relatively susceptible to AOM, whereas AKR/J mice are reported to be virtually resistant. Both AKR/J and SWR/J mice, 6 weeks old, received subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks, and were sacrificed at 16 and 41 weeks of age. Colons of the animals sacrificed at 16 and 41 weeks of age were processed to examine expression of the early‐appearing lesions and neoplasms. Although AKR/J mice had a lower incidence of colonic tumors than SWR/J mice did, AKR/J mice showed a similar frequency of ACF to that in SWR/J mice. In both strains, ACF were detected at high frequency in the proximal colon, whereas tumors developed mainly in the distal colon. Importantly, the incidence of BCAC in SWR/J mice was significantly higher than that in AKR/J mice, and the highest frequency was observed in the distal segments of the colon. These results support the idea that BCAC are a reliable surrogate endpoint for colon carcinogenesis in mice.

IDO1 plays an immunosuppressive role in 2,4,6-trinitrobenzene sulfate-induced colitis in mice.

IDO, an enzyme that degrades the essential amino acid l-tryptophan to N-formylkynurenine, is known to exert immunomodulatory effects in a number of diseases and disorders. IDO expression is increased in tumors, where it is thought to be involved in tumor evasion by suppressing the immune response. A competitive inhibitor of IDO is currently being tested in clinical trials for relapsed or refractory solid tumors; however, there remains a concern that attenuation of the immunosuppressive function of IDO might exacerbate inflammatory responses. In this study, we investigated the role of IDO in 2,4,6-trinitrobenzene sulfate (TNBS)–induced colitis in mice by gene deletion and pharmacological inhibition. TNBS treatment induced significantly more severe colitis in Ido1 gene–deficient (Ido1−/−) mice than in Ido1 wild-type (Ido1+/+) mice, indicating a role for IDO1 in suppression of acute colitis. Consistent with this, the expression of Ido1 was increased in the colonic interstitial tissues of TNBS-treated Ido1+/+ mice. Furthermore, transplantation of Ido1+/+ bone marrow cells into Ido1−/− mice reduced the pathological damage associated with colitis, altered the expression of cytokines, including IFN-γ, TNF-α, and IL-10, and increased the number of CD4+ Foxp3+ regulatory T cells in the colon. Pharmacological inhibition of IDO enzymatic activity by oral administration of 1-methyltryptophan (1-methyl-l-tryptophan or 1-methyl-d-tryptophan) significantly increased the severity of TNBS-induced colitis in mice, demonstrating that both stereoisomers can promote colitis. Collectively, our data indicate that IDO1 plays an important immunoregulatory role in the colon.

Antioxidant Effects of Flavonoids Used as Food Additives (Purple Corn Color, Enzymatically Modified Isoquercitrin, and Isoquercitrin) on Liver Carcinogenesis in a Rat Medium-Term Bioassay

To clarify the effects of purple corn color, enzymatically modified isoquercitrin (EMIQ), and isoquercitrin (IQ), registered as natural food additives in Japan, on liver carcinogenesis in vivo, a medium-term bioassay was employed. A total of 100 male F344 rats were divided into 5 groups; groups 1 to 4 were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) on day 1. From weeks 2 to 8, they were administered basal diet purple corn color, EMIQ, or IQ as containing test chemicals at doses of 1.0% (groups 1 and 5), 0.1% (group 2), 0.01% (group 3), or 0% (group 4) (experiments 1, 4, and 5). All rats were subjected to two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Purple corn color exerted no significant modifying effects on GST-P positive foci, preneoplastic foci, development in the liver. However, serum of rats treated with purple corn color provided evidence of antioxidant power significantly by potential antioxidant (PAO) test in vivo (experiment 2). And microarray analyses showed purple corn color to induce RNA expression such as P450 (cytochrome) oxidoreductase, phosphatidylinositol 3-kinase, and phospholipase A2 (experiment 3). Higher doses of EMIQ or IQ with strong antioxidant power in vivo by PAO test treated groups were correlated with smaller numbers of GST-P positive foci, with Spearmans rank correlation coefficients of P= 0.002 and P= 0.049, respectively (experiments 4 and 5). Therefore, the tested food additives may be effective as antioxidants in vivo and have chemopreventive potential against liver preneoplastic lesion development.

Ellagic acid, a component of pomegranate fruit juice, suppresses androgen-dependent prostate carcinogenesis via induction of apoptosis

Ellagic acid (EA), a component of pomegranate fruit juice (PFJ), is a plant‐derived polyphenol and has antioxidant properties. PFJ and EA have been reported to suppress various cancers, including prostate cancer. However, their chemopreventive effects on development and progression of prostate cancer using in vivo models have not been established yet.