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Understanding of mechanisms of cancer progression is very important for reduction of cancer mortality. Of six rat hepatocellular carcinoma (HCC) cell lines, differing in their metastatic potential to the lung after inoculation into the tail vein of nude mice, the most metastatic featured particular overexpression of glutathione peroxidase 2 (GPX2). Therefore, we analyzed the influence of interference in highly metastatic L2 cells by siRNA transfection. Gpx2 siRNA significantly inhibited cell proliferation at 24 and 48 h time points with induction of apoptosis but not cell cycle arrest. High expression of mutated p53 was detected in all HCC cell lines, with reduction in Gpx2 siRNA-transfected cells. Migration and invasion in vitro were also suppressed as compared to control siRNA-transfected cells and secretion of matrix metalloproteinase 9 was reduced. In vivo, the numbers and areas of metastatic nodules per area in the lungs were significantly reduced in the mice inoculated with Gpx2 siRNA-transfected cells as compared to control siRNA-transfected cells. In conclusion, expression of GPX2 is associated with cancer metastasis from rat HCCs both in vitro and in vivo. Together with immunohistochemical findings of elevated expression in rat and also human liver lesions, the results point to important roles in hepatocarcinogenesis.

In this study, we focused on the in vitro effects of Kuguacin J (KuJ), a purified component of bitter melon (Momordica charantia) leaf extract (BMLE), on the androgen-independent human prostate cancer cell line PC3 and the in vivo effect of dietary BMLE on prostate carcinogenesis using a PC3-xenograph model. KuJ exerted a strong growth-inhibitory effect on PC3 cells. Growth inhibition was mainly through G1-arrest: KuJ markedly decreased the levels of cyclins (D1 and E), cyclin-dependent kinases (Cdk2 and Cdk4) and proliferating cell nuclear antigen. Interestingly, KuJ also dramatically decreased the levels of survivin expressed by PC3 cells. In addition, KuJ exerted anti-invasive effects on PC3 cells, significantly inhibiting migration and invasion: KuJ inhibited secretion of the active forms of MMP-2, MMP-9 and uPA by PC3 cells. In addition, KuJ treatment significantly decreased the expression of membrane type 1-MMP (MT1-MMP) by PC3 cells. In vivo, 1% and 5% BMLE in the diet resulted in 63% and 57% inhibition of PC3 xenograft growth without adverse effect on host body weight. Our results suggest that KuJ is a promising new candidate chemopreventive and chemotherapeutic agent for prostate cancer.

In this study, we focused on the effects of a bitter melon (Momordica charantia) leaf extract (BMLE) and a purified component, Kuguacin J (KuJ), on androgen-dependent LNCaP human prostate cancer cells. Both treatments exerted growth inhibition through G1 arrest and induction of apoptosis. In addition, KuJ markedly decreased the levels of cyclins (D1 and E), cyclin-dependent kinases (Cdk2 and Cdk4) and proliferating cell nuclear antigen, and caused an increase in p21 and p27 levels. Its induction of apoptosis was accompanied by an increase in cleavage of caspase-3 and poly (ADP-ribose) polymerase, attributable to augment of Bax/Bcl-2 and Bad/Bcl-xL and reduction of survivin levels. BMLE and KuJ also reduced the expression of androgen receptor (AR), prostate-specific antigen (PSA) while induced P53 protein level. Down-regulation of p53 by RNA interference indicated that BMLE and KuJ inhibited cell growth partly through p53-dependent cell cycle arrest and apoptotic pathways. Both BMLE and KuJ caused less toxicity in a normal prostate cell line, PNT1A. Our results suggest that BMLE and a purified component, KuJ, from its diethyl ether fraction could be promising candidate new antineoplastic and chemopreventive agents for androgen-dependent prostate cancer and carcinogenesis.

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. To test the hypothesis that it is metabolically activated by CYP1A2, we here investigated the effects of caffeine as a CYP1A2 inducer on MeIQx induced rat hepatocarcinogenesis in a medium-term liver bioassay system. Unexpectedly, no modifying effects of caffeine on MeIQx-induced hepatocarcinogenesis were evident, although up-regulation of CYP1A2 and NAT2 were detected. Therefore, mRNAs extracted from GST-P positive foci and the surrounding liver tissue in each group were analyzed to explore mechanisms in detail. The results suggest that suppression of syndecan-2 (Sdc2) and induction of cell cycle arrest through a p21-dependent pathway might have counter-acted any promotion effects of up-regulation of CYP1A2.

Reproductive effects have been observed in experimental animals treated with di(n-butyl)phthalate (DBP), one of phthalate esters used in soft plastics and a variety of consumer products. In this study, we investigated whether testicular toxicity of DBP is influenced by diminished renal function. To generate an experimental condition reflecting chronic renal disease in man, adult male F344 rats were given five consecutive weekly subcutaneous injections of folic acid at a dose of 300 mg/kg and then a diet containing 1200, 5000, and 20,000 ppm of DBP for 4 weeks. These concentrations roughly correspond to 60, 250, and 1000 mg/kg per day per rat, respectively. Folic acid clearly induced interstitial nephritis accompanied by impairment of renal function. Seminiferous degeneration, diminished spermatogenesis and increase in the number of morphologically abnormal sperm were more prominent in rats given folic acid and then 20,000 ppm DBP as compared to those exposed to DBP alone. These data suggest that DBP-induced male reproductive toxicity can be increased by folic acid-induced renal dysfunction.

p21/WAF1, a cyclin-dependent kinase inhibitory protein, functions as a tumor suppressor. Loss of p21/WAF1 expression has been reported to be an important prognostic predictor in several human malignancies, including bladder carcinomas. In this study, we induced p21/WAF1 using a Cre-loxP gene expression switching vector system in BC31 rat bladder carcinoma cells that feature p53 gene mutations. Cells in which p21/WAF1 was induced in nuclei were not labeled with BrdU and overall showed decreased cell proliferation, with increase in the G0-G1 population evident on flow cytometer analysis. The Cre treatment did not affect BC31 parental cells. These results show induction of p21/WAF1 can inhibit proliferation of tumor cells having p53 mutations, and could thus be applied as a potential therapy.

The importance of dietary factors for prostate carcinogenesis has been proven by epidemiological studies of immigrants from Asia into the USA. Intake of foodstuffs rich in fat, including meat, is suggested to be a risk factor. Experimentally, while some studies demonstrated high fat intake to promote rat prostate carcinogenesis, others did not. Charcoal-cooked red meat and fish have been demonstrated to contain heterocyclic amines that are carcinogenic in rodents and non-primates. Among them, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been shown to induce cancers in the mammary glands, colon and prostate of rats. Although there are epidemiological data showing that PhIP could contribute to the development of breast cancer, equivalent evidence for prostate cancer is lacking. However, as protective dietary factors, micronutrients such as selenium, zinc, isoflavones, carotenoids and lycopenes and vitamins E and D have been listed. Animal experimentation on prostate cancer has consistently supported preventive potential for carotenoids and isoflavones, in contrast to the inconsistent results with high fat diets. Although the diet has apparently an important influence on prostate carcinogenesis in man, further research is necessary for clarification of specific factors in man.

Fenbendazole (FBZ) is an anthelmintic drug known to be a potent CYP1A2 inducer. Combined effects of FBZ on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced hepatocarcinogenesis in rats were investigated using a medium-term liver bioassay system. No modifying influence was found in terms of glutathione S-transferase placental-form positive foci development although CYP1A2 protein expression in the livers of rats that were given MeIQx and FBZ was 2.3-fold higher than with MeIQx alone. NAT2 mRNA expression did not differ among the groups as revealed by quantitative reverse transcriptase-polymerase chain reaction analysis. These results suggest that elevated CYP1A2 expression is not sufficient to enhance MeIQx-induced hepatocarcinogenesis.

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.

Deletion of 9p21 has frequently been observed in human bladder carcinomas. A candidate target suppressor gene, p16, was recently identified within this deleted region. In this study, we therefore investigated the loss of heterozygosity (LOH) of the p16 gene which is located on mouse chromosome 4, as well as its expression in mouse bladder carcinomas. We also studied the effects of normal cell contamination on LOH analysis using xenografts in CD-1(ICR) nude mice from B6C3F1 bladder carcinomas. We could not detect any LOH at the p16 locus in the mouse primary bladder carcinomas and xenografts. Surprisingly, overexpression of p16 was found in all primary mouse bladder carcinomas. Using microsatellite polymorphisms, a distinction could be made between PCR products derived from B6C3F1 and CD-1(ICR) nude mice. It was thereby confirmed that effects of normal cell contamination on LOH analysis are negligible when only tumor tissue is carefully sampled. The results suggest that abnormalities of p16 expression may be involved in mouse bladder carcinogenesis, but that gene deletion is not involved.

An investigation was conducted to assess the chemopreventive potential of lycopene (LP), a naturally occurring hydrocarbon carotenoid found in tomatoes and their products, administered during the post-initiation stage in a multiorgan carcinogenesis model. One hundred eighteen B6C3F1 mice of both sexes were subjected to combined treatment with diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU) and 1,2-dimethylhydrazine (DMH) from day 11 after birth to week 9 (DMD treatment) (groups 1 and 2) or their vehicles (group 3). Then group 1 received LP (25 or 50 ppm in drinking water) for 21 weeks from weeks 11 to 32. Group 2 served as a carcinogen alone control and group 3 was given only LP (25 or 50 ppm). All surviving animals were sacrificed at week 32 and the major organs, including the liver, lung, kidney and colon, were histologically examined. The incidences and multiplicities of lung adenomas plus carcinomas combined in male mice in group 1 receiving 50 ppm LP were significantly decreased as compared to the DMD alone or DMD and 25 ppm LP groups (75.0 versus 18.8%, P < 0.02; 0.94 +/- 0.17 versus 0.25 +/- 0.14, P < 0.001). No such effect was observed for females. Although hepatocellular carcinomas were lacking in the DMD and LP groups while two cases were found in the DMD alone group, this difference was not statistically significant. The values for aberrant crypt foci (ACF) and tumors in the colon and kidney did not show any significant variation among the carcinogen-treated subgroups. The results suggest that LP exerts a chemopreventive effect limited to male lung carcinogenesis when given in the post-initiation stage to B6C3F1 mice.

Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[-4-(-5-nitro-2-furyl)-2- thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.

Prohibitin was isolated as a candidate antiproliferating gene in rat liver cells, and it has been suggested as a tumor suppressor gene in human breast cancer. We investigated the steady state level of prohibitin mRNA in rat bladder cell lines and in rat bladder carcinoma induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and sodium saccharin, as well as the protein level in rat bladder epithelial cells. We also examined the prohibitin gene for mutations using the polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) and direct sequencing methods. All rat bladder tumors investigated and several cell lines had unexpectedly increased steady state levels of prohibitin mRNA compared with that of normal rat bladder or liver. The prohibitin protein was easily detected by Western blotting in all cell lines regardless of their malignant status or growth rate. However, PCR-SSCP and direct sequencing analysis showed no mutations in the prohibitin gene. These results These results indicate that prohibitin overexpression, but not mutations, may be involved in the early stage of rat bladder carcinogenesis.

Combination effects of sequential treatment with uracil prior to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) with regard to carcinogenesis in the urinary bladder and renal pelvis were investigated in male F344 rats. Uracil was administered for 15, 10 or 5 weeks followed by BBN for 23 weeks, the total observation time being 40 weeks. Carcinoma(s) and papilloma(s) were induced in the urinary tract by the 15-week uracil treatment independent of subsequent BBN administration. It was concluded that uracil administration prior to BBN treatment is not associated with any synergistic effects, although both uracil and BBN alone exerted carcinogenicity.

The effects of various chemicals on the development of neoplastic lesions in the urinary bladder were investigated in male F344 rats given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) as an initiator in their drinking water for 4 weeks. The compounds tested, indomethacin, acemetacin, epsilon-aminocaproic acid (EACA), diphenyl, allopurinol and acetaminophen (AAP), were added to the diet or drinking water for 32 weeks, and all animals were killed at the end of week 36. Of the chemicals tested, only diphenyl significantly increased the incidences and average numbers (per 10 cm basement membrane) of papillary or nodular hyperplasias (PN hyperplasia), papillomas and carcinomas of the urinary bladder over those in animals treated with BBN alone. These findings show that diphenyl is a promoter of urinary bladder carcinogenesis in male F344 rats.

The mechanism of action of 2(3)-tert-butyl-4-hydroxyanisole (2-BHA or 3-BHA) on rat forestomach epithelium was studied by examining the metabolites of BHA in the stomach and the covalent binding of BHA to macromolecules in the forestomach epithelium. Male F344 rats 6 weeks old were given a single intragastric injection of 1 g/kg body wt of [tert-14C]-3-BHA (∗Bu-3-BHA) or [methyl-14C]-3-BHA (∗Me-3-BHA), and 6 h later BHA metabolites in the forestomach, glandular stomach and stomach contents were examined by thin-layer chromatography. No significant amounts of metabolites were detected in the forestomach or glandular stomach epithelium and almost all the radioactivity in these tissues was extracted with organic solvents. In in vitro experiments also, no significant amounts of metabolites were detected when the 9000 g supernatant of the forestomach or glandular stomach epithelium, or gastric juice was incubated with ∗Bu-3-BHA in the absence or presence of NADPH. In binding studies, rats were given ∗Bu-3-BHA, [tert-14C]-2-BHA (∗Bu-2-BHA), ∗Me-3-BHA or [methyl-14C]butylated hydroxytoluene (∗Me-BHT) intragastrically at a dose of 1 g/kg body wt with or without pretreatment with unlabelled 1% 3-BHA or BHT in the diet for 6 days. Six hours after treatment with a labelled compound, the rats were sacrificed and the DNA, RNA and protein of their forestomach, glandular stomach, liver and kidney were isolated. ∗Bu-3-BHA, ∗Bu-2-BHA and ∗Me-3-BHA did not bind covalently to forestomach DNA or RNA, and the amounts of radioactivity of these compounds bound to proteins in the 4 tissues were similar. These findings suggest that BHA acts on the forestomach epithelium directly without metabolic activation, and that its action is not related to its binding to DNA or RNA.

The effects of linolic acid hydroperoxides (product A) and secondary oxidative products of product A (product B), were examined in an in vivo mid-term test for hepatocarcinogens or hepatopromoters in rats. The number of placental type of glutathione S-transferase (GST-P)-positive foci of the liver was significantly reduced in rats given diethylnitrosamine (DEN) followed by products A (4.64 +/- 1.09, P less than 0.05) or B (3.62 +/- 1.65, P less than 0.01) as compared to the controls given carcinogen alone (6.31 +/- 2.82). The area of GST-P positive foci was also significantly reduced in rats given DEN followed by product B (0.30 +/- 0.21, P less than 0.05) as compared to the controls (0.47 +/- 0.23). These results suggest that linolic acid hydroperoxides or their secondary oxidative products are not hepatocarcinogens and rather may possess inhibitory potential for liver carcinogenesis.