Naomi Hokaiwado

Protective effects of citrus nobiletin and auraptene in transgenic rats developing adenocarcinoma of the prostate (TRAP) and human prostate carcinoma cells

Dietary phytochemicals, including nobiletin and auraptene, have been shown to exert inhibiting effects in several chemically induced carcinogenesis models. We here investigated the influence of nobiletin and auraptene on prostate carcinogenesis using transgenic rats developing adenocarcinoma of the prostate (TRAP) bearing the SV40 T antigen transgene under control of the probasin promoter and human prostate cancer cells. Starting at 5 weeks of age, male TRAP rats received powder diet containing 500 p.p.m. nobiletin or auraptene, or the basal diet for 15 weeks and then were sacrificed for analysis of serum testosterone levels and histological changes. The body and relative prostate weights and serum testosterone levels did not differ among the groups. Since all animals developed prostate carcinomas, these were semiquantitatively measured and expressed as relative areas of prostate epithelial cells. Nobiletin caused significant reduction in the ventral (P < 0.01), lateral (P < 0.001) and dorsal (P < 0.05) prostate lobes, while decreasing high grade lesions (P < 0.05) in the ventral and lateral lobes. Feeding of auraptene also effectively reduced the epithelial component (P < 0.05) and high grade lesions (P < 0.05), in the lateral prostate. A further experiment demonstrated that growth of androgen sensitive LNCaP and androgen insensitive DU145 and PC3 human prostate cancer cells, was suppressed by both nobiletin and to a lesser extent auraptene in a dose‐dependent manner, with significant increase in apoptosis. In conclusion, these compounds, particularly nobiletin, may be valuable for prostate cancer prevention. (Cancer Sci 2007; 98: 471–477)

Gpx2 Is an Overexpressed Gene in Rat Breast Cancers Induced by Three Different Chemical Carcinogens

Gene expression alterations are essential for the process of carcinogenesis. A carcinogen may have specific mechanisms for inducing tumors, which may involve inducing characteristic gene expression alterations. In this study, we attempted to identify genes crucial for mammary carcinogenesis. For this purpose, we used human c-Ha-ras proto-oncogene transgenic rats (Hras128), which are highly sensitive to mammary carcinogens including N-methyl-N-nitrosourea, 7,12-dimethyl benz[a]anthracene, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. DNA microarray analysis revealed that glutathione peroxidase 2 (Gpx2) was commonly up-regulated in the mammary carcinomas induced by the three different carcinogens, and its up-regulation was confirmed by quantitative reverse transcriptase-PCR and Western blotting analysis. In addition, expression of GPX2 was recognized in all 41 immunohistochemically examined cases of human breast cancer. Forced suppression of GPX2 expression by siRNA resulted in significant growth inhibition in both rat and human mammary carcinoma cell lines with wild-type p53 cells. Thus, these data suggested that GPX2 may be involved in mammary carcinogenesis and cell proliferation in both rats and humans, indicating that GPX2 may be a novel target for the prevention and therapy of breast cancer.

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), β‐cryptoxanthin‐ and hes‐peridine‐rich pulp (CHRP) and 1,1′‐acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium‐term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

Connexin 32 dominant‐negative mutant transgenic rats are resistant to hepatic damage by chemicals

Connexins are subunits of gap junction channels, which allow direct transfer of ions, secondary messenger molecules, and other metabolites between contacting cells. Gap junctions are believed to be involved in tissue homeostasis, embryonic development, and control of cell proliferation. Several studies have shown that cell damage signals are transmitted through gap junctions when cells are irradiated or when cells bearing the herpes simplex virus‐thymidine kinase (HSV‐TK) gene are treated with ganciclovir. We established 2 lines of transgenic rats with a dominant‐negative mutant of connexin 32 gene under control of the albumin promoter. In the livers of transgenic rats, membrane localization of normal endogenous connexin 32 protein is disturbed, and gap junction capacity measured by scrape dye‐transfer assay in vivo is markedly decreased when compared with wild‐type rats. The present investigation concerned susceptibility to the liver‐toxic substances D‐galactosamine and carbon tetrachloride. These toxicants induced massive liver cell death and elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the wild‐type rats; however, much fewer liver cells were damaged and serum enzyme elevation was much lower in the transgenic rats. In conclusion, gap junctional intercellular communication (GJIC) plays an important role in toxic effects of chemicals; damage or death signals may pass through gap junctions in the rat liver in vivo. (HEPATOLOGY 2004;40:205–210.)

Glutathione S-transferase Pi mediates proliferation of androgen-independent prostate cancer cells

Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the ‘transgenic rat adenocarcinoma in prostate’ (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription–polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA–atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells.

Prediction of carcinogenic potential by a toxicogenomic approach using rat hepatoma cells.

The long‐term rodent bioassay is the standard method to predict the carcinogenic hazard of chemicals for humans. However, this assay is costly, and the results take at least two years to produce. In the present study, we conducted gene expression profiling of cultured cells exposed to carcinogenic chemicals with the aim of providing a basis for rapid and reliable prediction of carcinogenicity using microarray technology. We selected 39 chemicals, including 17 rat hepatocarcinogens and eight compounds demonstrating carcinogenicity in organs other than the liver. The remaining 14 were non‐carcinogens. When rat hepatoma cells (MH1C1) were treated with the chemicals for 3 days at a non‐toxic dose, analysis of gene expression changes with our in‐house microarray allowed a set of genes to be identified differentiating hepatocarcinogens from non‐carcinogens, and all carcinogens from non‐carcinogens, by statistical methods. Moreover, optimization of the two gene sets for classification with an SVM and LOO‐CV resulted in selection of 39 genes. The highest predictivity was achieved with 207 genes for differentiation between non‐hepatocarcinogens and non‐carcinogens. The overlap between the two selected gene sets encompassed 26 genes. This gene set contained significant genes for prediction of carcinogenicity, with a concordance of 84.6% by LOO‐CV SVM. Using nine external samples, correct prediction of carcinogenicity by SVM was 88.9%. These results indicate that short‐term bioassay systems for carcinogenicity using gene expression profiling in hepatoma cells have great promise. (Cancer Sci 2006; 97: 1002–1010)

Rapid analysis of gene expression changes caused by liver carcinogens and chemopreventive agents using a newly developed three-dimensional microarray system

We investigated changes of gene expression in livers of rats treated with carcinogens and tumor promoters using a novel three‐dimensional microarray system developed by Olympus Optical Co., Ltd., to assess the feasibility of predicting modifying effects on hepatocarcinogenesis on the basis of changes in the patterns. For this purpose, two genotoxic carcinogens, two nongenotoxic carcinogens (promoters) and seven candidate chemopreventive agents were examined. Six‐week‐old male F344 rats were treated for 2 weeks with the 11 chemicals (0.05% phenobarbital, 0.3% clofibrate, 0.01% N‐diethylnitrosamine (DEN), 0.01% 2‐amino‐3, 8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 1% catechol, 1% caffeic acid, 0.05% nobiletin, 0.05% garcinol, 0.05% auraptene, 0.05% zermbone and 0.05% 1′‐acetoxychavicol acetate (ACA). Test chemicals were mixed in food with the exception of DEN, which was administered in drinking water. RNAs from liver were then analyzed using two kinds of customized microarrays (PamChip® microarray A spotted for 28 genes of drugmetabolizing enzymes in duplicate, and PamChip® microarray B spotted for 131 genes which are known to be up‐ or down‐regulated in hepatocarcinoma cells). Hybridization and subsequent analysis were usually completed within 2 h and the data obtained were highly reproducible. Carcinogens were classified into genotoxic and nongenotoxic substances by clustering analysis. We could also divide test chemicals into carcinogens and chemopreventive agents from their effects on gene expression. In this study, we have thus shown that it is feasible to predict the modifying effects of chemicals on the basis of changes of gene expression patterns after only 2 weeks of exposure, using our novel three‐dimensional microarrays.

Mammary Carcinomas Induced in Human c-Ha-ras Proto-oncogene Transgenic Rats Are Estrogen-independent, but Responsive to d-Limonene Treatment

We have previously shown that transgenic rats carrying three copies of the human c‐Ha‐ras proto‐ oncogene (Hras128) are highly susceptible to N‐methyl‐N‐nitrosourea (MNU) mammary carcino‐ genesis. All transgenic rats treated with 50 mg/kgMNU, i.v. at 50 days of age, were found to rapidly develop multiple, large mammary carcinomas within as short a period as 8 weeks. In the present study, the effects of ovariectomy and treatment with d‐limonene, known to inhibit mammary carcinogenesis in non‐transgenic female rats, were investigated in Hras128 animals treated withMNU to clarify the role of the human c‐Ha‐ras proto‐oncogene and to characterize the induced mammary carcinomas. Although ovariectomy completely inhibited development of mammary carcinomas in their wild‐type counterparts, it did not affect either the incidence or the multiplicity of the mammary carcinomas in the Hras128 rats. On the other hand, treatment with d‐limonene, an inhibitor of ras protein isoprenylation, inhibited the breast tumor development. These results indicate that aberrant c‐Ha‐ras gene expression is involved in ovarian hormone‐independent growth and c‐Ha‐ras protein isoprenylation plays an important role in mammary carcinogenesis

Age-dependent histopathological findings in the prostate of probasin/SV40 T antigen transgenic rats: Lack of influence of carcinogen or testosterone treatment

Sequential changes in the phenotype of prostatic lesions and the impact of additional carcinogen treatment or castration on development and progression of prostate cancers were examined in probasin/simian virus 40 (SV40) T antigen transgenic (TG) rats. Non‐invasive prostate adenocarcinomas were evident in all lobes at 15 weeks of age. Invasive tumors were limited to the anterior lobe at this time point and were found in all lobes in an age‐dependent manner thereafter. No metastasis was apparent at any age. Additional carcinogen treatment or castration did not enhance progression or generate selective growth of hormone‐independent prostate cancer cells. These results suggest that our TG rats are suitable for clarification of mechanisms in early stages of prostate carcinogenesis, that is, from prostatic intraepithelial neo‐plasia (PIN) to non‐invasive and then invasive lesions. (Cancer Sci 2003; 94: 153–157)

Frequent c-Ha-ras gene mutations in rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat and fish, is carcinogenic to the mammary glands of rats. Mutations in the H-ras gene were here examined in PhIP-induced mammary tumors of female F344 rats by the polymerase chain reaction followed by single strand conformation polymorphism analysis (PCR-SSCP) and restriction enzyme length polymorphism analysis (RFLP). Mutations in codon 12 of the H-ras gene were detected in four out of 13 tumors by PCR-SSCP. Three of them were GGA to GAA, and one was GGA to GTA. However, by RFLP analysis, four additional mutations in codon 13 were also detected in the same samples. Two had a GGC to CGC mutation, and the other shifts were GGC to GAC and GGC to GTC. Therefore, overall eight out of 13 cases had H-ras gene mutations. These results indicate that changes in H-ras function may play important roles in PhIP-induced mammary carcinogenesis.