Masa-Aki Shibata

Promotion by ascorbic acid, sodium erythorbate and ethoxyquin of neoplastic lesions in rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine

The promoting effects of ascorbic acid, sodium erythorbate and ethoxyquin on two-stage urinary bladder carcinogenesis in F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at a dose of 0.05% in the drinking water were examined. Administration of 5% sodium erythorbate in the diet significantly increased the incidences of preneoplastic lesions, papilloma and cancer of the urinary bladder, whereas administration of 5% ascorbic acid in the diet did not. Administration of 0.8% ethoxyquin also increased the incidence of neoplastic lesions. Administrations of 5% sodium L-ascorbate and 5% sodium erythorbate caused increases in the pH, the sodium content and crystals of MgNH4PO4 in the urine. These results show that sodium erythorbate and ethoxyquin promote urinary bladder carcinogenesis, while ascorbic acid does not.

Induction of Renal Cell Tumors in Rats and Mice, and Enhancement of Hepatocellular Tumor Development in Mice after Long‐term Hydroquinone Treatment

Hydroquinone (HQ) was administered to F344 rats and B6C3F1 mice of both sexes at a level of 0.8% in the diet for two years. This treatment induced renal tubular hyperplasia as well as adenomas, predominantly in males of both species, and was associated with chronic nephropathy in rats. In addition, the occurrence of epithelial hyperplasia of the renal papilla was increased in male rats. Foci of cellular alteration of the liver were significantly reduced in number by HQ in rats, but in contrast, were increased in mice, where development of hepatocellular adenoma was also enhanced in males. The incidence of squamous cell hyperplasia of the forestomach epithelium was significantly higher in mice of both sexes given HQ than in the controls, but no corresponding increase in tumor development was observed. The present study strongly indicates potential renal carcinogenicity of HQ in male rats and hepatocarcinogenicity in male mice. Thus, it is possible that HQ, which is present in the human environment, may play a role in cancer development in man.

Lack of Carcinogenicity of Quercetin in F344/DuCrj Rats

Quercetin was administered at dietary levels of 0(control), 1.25 and 5.0% to groups of 50 male and 50 female rats for 104 weeks, and then all animals were maintained without quercetin supplement for a further 8 weeks. At 5.0% quercetin, both sexes showed growth retardation throughout the study. There were no treatment‐ascribed effects regarding clinical signs, mortality, urinalyses or hematology. Although serum glucose in 5.0% quercetin‐treated males was significantly decreased and some relative organ weights in 5.0% groups showed statistically significant increases, these latter changes seemed to be related to the growth retardation. An increased incidence of non‐neoplastic hyperplastic polyps in the cecum was noted in the 5.0% males. The incidences of cystic changes and fibroadenomas of the mammary gland, and foci (areas) of hepatocellular alteration in the 5.0% females, and liver bile duct proliferations in the 5.0% males were significantly decreased. No proliferative lesions of the urinary bladder related to treatment with quercetin were found in any rats. The incidences of several other nonneoplastic and neoplastic lesions which demonstrated statistically significant changes appeared to be related to the growth retardation or to be within the normal range, and therefore none was considered to be significant biologically. Thus, the investigation did not demonstrate any clear carcinogenic effect of quercetin on F344 rats at dietary levels of up to 5.0%.

Spontaneous Tumors in Aging (C57BL/6N X C3H/HeN)F1 (B6C3F1) Mice

Spontaneous tumors in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenicity tests were recorded. In both sexes, the development of spontaneous tumors was age-related. In 244 male mice, the most common tumors were hyperplastic nodules of the liver, hepatocellular carcinomas, malignant lymphomas/leukemias, lung adenomas, and adenocarcinomas. In 246 female mice, the most common tumors were malignant lymphomas/leukemias, pituitary adenomas, neoplastic nodules of the liver, hepatocellular carcinomas, and lung adenomas. Hepatocellular carcinomas metastasized in 20.3% of the animals with these tumors. Few other tumors except malignant lymphomas and leukemias metastasized. Various tumors of other organs and/or tissues were found at low incidences.

Changes in urine composition, bladder epithelial morphology, and DNA synthesis in male F344 rats in response to ingestion of bladder tumor promoters.

An investigation of changes in urine composition, morphology of bladder epithelium, and levels of DNA synthesis following 4 or 8 weeks oral administration of bladder tumor promoters or analogs without promotion potential was performed. The sodium salts of L-ascorbate, o-phenylphenate, and bicarbonate increased the pH value, sodium content, volume, and MgNH4PO4 crystalluria of the urine, while the parent compounds, L-ascorbic acid and o-phenylphenol, which in contrast are not tumor promoters, did not induce these changes. Sodium chloride ingestion caused natriuresis without increasing urinary pH. Diphenyl administration produced only microcalculi consisting of p-phenylphenol. Treatment with the antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and ethoxyquin was also not associated with any changes in urinary pH or Na ions. However, tert-butylhydroquinone did cause an increase in pH. Administration of the strong bladder carcinogens N-butyl-N-(4-hydroxybutyl)nitrosamine and N-ethyl-N-(4-hydroxybutyl)nitrosamine did not result in alteration of urine composition, with the exception of a decrease in phosphorus concentration. However, all the bladder promoters and carcinogens, without exception, brought about an elevation of DNA synthesis in the urothelium and produced morphologic surface alterations such as formation of pleomorphic or short, uniform microvilli and ropy or leafy microridges. Thus, an ability to induce proliferation and cell surface alteration was characteristic of the complete range of bladder promoters investigated. The results suggest that considerable variation in the mechanisms underlying these changes may be involved for different individuals or groups of agents.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

The C3(1)/SV40 T Antigen Transgenic Mouse Model of Prostate and Mammary Cancer*

BACKGROUND Prostate and mammary cancers are the most common malignant diseases in the Western world and have increased dramatically during the past decade (23). Both cancers are now the second leading cause of cancer deaths in men and women in the United States and their incidences continue to rise. According to recent estimates (23), approximately 317,000 men will be diagnosed with prostate cancer and 41,000 will die of the disease in the United States this year. For women, the cumulative lifetime risk of developing breast cancer is 12%, whereas the lifetime mortality risk has been estimated to be 3.5% (1 1). Despite this serious situation, the etiologies and risk factors for cancer in these organs have remained largely unknown. Some hereditary factors appear to be involved, but other unknown genetic alterations and environmental and life-style factors appear to be the most important causative factors. Although transgenic models for mammary cancer have been developed, no transgenic animal for prostate cancer has been available until recently. Several useful animal models for prostate cancer have been developed using chemical carcinogens (2, 30), but these models are labor intensive and generally require tumor promotion with pharmacologic doses of steroid hormone. Therefore, we developed a transgenic mouse model for prostate cancer by targeting the expression of the simian virus 40 (SV40) early region to the mouse prostate epithelium using the 5‘ flanking region of the rat C3(1) gene, a gene which is very highly expressed in the rat ventral prostate (20). This was the first reported transgenic model for prostate cancer. Other transgenic mouse models for prostate cancer have also been developed (Table I; see Ref. #7 for review). In addition to the development of prostate cancers, aged male C3( l)/TAg transgenic mice develop tumors of the urethral, bulbourethral, and salivary ‘glands, whereas 100% of female transgenic mice develop mammary adenocarcinomas.

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5′-flanking region display multistage mammary tumorigenesis. To delineate genetic changes associated with mammary tumor progression, comparative genomic hybridization (CGH) was performed. CGH revealed a consistent gain of the telomeric region of chromosome 6. This region contains the Ki-ras proto-oncogene. Analyses of genomic DNA by Southern blot demonstrated up to 40-fold amplification of the Ki-ras gene. Ki-ras amplification was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no amplifications were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene amplification exhibited high levels of Ki-ras RNA and protein. The over-expressed Ki-Ras protein in these tumors appeared functionally active as indicated by the elevated MAP kinase activity. These data demonstrate that while Ki-ras amplification might not be an early event, there is a strong association between Ki-ras amplification and over-expression and mammary tumor progression in this model. This study also shows that CGH is a powerful and useful technique for identifying chromosomal copy number changes during tumor progression, and that this model may provide a predictable in vivo system for studying gene amplification.

Alterations in Cell Cycle and Induction of Apoptotic Cell Death in Breast Cancer Cells Treated with α-Mangostin Extracted from Mangosteen Pericarp

The development of molecularly targeted drugs has greatly advanced cancer therapy, despite these drugs being associated with some serious problems. Recently, increasing attention has been paid to the anticancer effects of natural products. α-Mangostin, a xanthone isolated from the pericarp of mangosteen fruit, has been shown to induce apoptosis in various cancer cell lines and to exhibit antitumor activity in a mouse mammary cancer model. In this study, we investigated the influence of α-mangostin on apoptosis and cell cycle in the human breast cancer cell line MDA-MB231 (carrying a p53 mutation, and HER2, ER, and PgR negative) in order to elucidate its anticancer mechanisms. In α-mangostin-treated cells, induction of mitochondria-mediated apoptosis was observed. On cell-cycle analysis, G1-phase arrest, increased p21cip1 expression and decreases in cyclins, cdc(s), CDKs and PCNA were observed. In conclusion, α-mangostin may be useful as a therapeutic agent for breast cancer carrying a p53 mutation and having HER2- and hormone receptor-negative subtypes.

Promoting effect of sodium chloride in 2-stage urinary bladder carcinogenesis in rats initiated by N-Butyl-N-(4-hydroxybutyl)-nitrosamine

SummaryThe promoting effect of sodium chloride (NaCl) in 2-stage urinary bladder carcinogenesis in F344 rats initiated by 2 doses of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was investigated. The incidences of PN hyperplasia were significantly higher in rats initiated with 0.01 or 0.05% BBN when they were given diet containing 10% NaCl for 32 weeks than when they were given control diet. The incidence of papilloma in rats given 0.05% BBN followed by diet containing 10% or 5% NaCl tended to be higher than that in control rats. The urine of rats given diet containing NaCl was larger in volume and had lower osmolality than that of controls. The total urinary sodium and chloride contents were also increased, whereas those of potassium and phosphorus were decreased. No calculus formation or crystalluria was observed. These data suggest that excess intake of sodium as NaCl has a weak promoting effect in 2-stage urinary bladder carcinogenesis.