Neu-induced retroviral rat mammary carcinogenesis: a novel chemoprevention model for both hormonally responsive and nonresponsive mammary carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.     

Suppression of mammary tumorigenesis in transgenic mice by the RXR-selective retinoid, LGD1069.

Retinoids have been used in the clinic for the prevention and treatment of human cancers. They regulate several cellular processes including growth, differentiation, and apoptosis. Previously, we demonstrated that a pan-agonist retinoid 9-cis retinoic acid was able to suppress mammary tumorigenesis in the C3(1)-SV40 T-antigen (Tag) transgenic mouse model. However, significant toxicity was seen with this naturally occurring retinoid. We hypothesized that the cancer preventive effects of retinoids could be dissected from the toxic effects by using receptor-selective retinoids. In this study, we used TTNPB, an retinoic acid receptor-selective retinoid, and LGD1069, an retinoid X receptor-selective retinoid, to preferentially activate retinoic acid receptors and retinoid X receptors. In vitro, both compounds were able to inhibit the growth of T47D breast cancer cells. We then determined whether these retinoids prevented mammary tumorigenesis. C3(1)-SV40 Tag mice were treated daily by gastric gavage with vehicle, two different doses of TTNPB (0.3 or 3.0 microg/kg), or two different doses of LGD1069 (10 or 100 mg/kg). Mice were treated from approximately 6-8 weeks to 7-8 months of age. Tumor size and number were measured twice each week, and toxicities were recorded daily. Our data show that LGD1069 suppresses mammary tumorigenesis in C3(1)-SV40 Tag transgenic mice with no observable toxicity, whereas TTNPB had a modest chemopreventive effect, yet was very toxic. Median time to tumor development was 129 days in vehicle-treated mice versus 156 days in mice treated with 100 mg/kg LGD1069 (P = 0.05). In addition, tumor multiplicity was reduced by approximately 50% in mice treated with LGD1069 (2.9 for vehicle, 2.4 for 10 mg/kg LGD1069, and 1.4 for 100 mg/kg, P < or = 0.03). TTNPB-treated mice showed a delayed median time to tumor development (131 days for vehicle versus 154 days for 3.0 microg/kg TTNPB; P < or = 0.05), but no changes were seen in tumor multiplicity. However, toxicity (skin erythema, hair loss) was seen in all of the mice treated with TTNPB. These data demonstrate that receptor-selective retinoids suppress mammary tumorigenesis in transgenic mice and that preventive effects of retinoids can be separated from their toxicity, demonstrating that receptor-selective retinoids are promising agents for the prevention of breast cancer.     

2-difluoromethylornithine and dehydroepiandrosterone inhibit mammary tumor progression but not mammary or prostate tumor initiation in C3(1)/SV40 T/t-antigen transgenic mice

Female transgenic mice that express SV40 T/t antigens under the regulatory control of the rat C3(1) gene spontaneously develop multifocal mammary lesions that predictably evolve into invasive, hormone-independent carcinomas, whereas male mice are prone to develop prostate cancer. Chemopreventive agents were administered to female C3(1)/SV40 large T-antigen mice from 7 to 19 weeks of age, during which time the mammary lesions developed and progressed to invasive carcinomas. No significant differences in the numbers of preinvasive mammary intraepithelial neoplasia lesions (histologically similar to human ductal carcinoma in situ) were observed after 2 or 8 weeks of treatment between mice receiving either vehicle alone, dehydroepiandrosterone (DHEA), or 2-difluoromethylornithine (DFMO). However, a dose-response reduction in invasive carcinoma growth was observed for both DFMO, an inhibitor of ornithine decarboxylase, and DHEA, the primary steroid precursor to both androgens and estrogens in primates. Despite unaltered expression of the transgene, tumor incidence was reduced approximately 20% by DFMO (8000 mg/kg) and 30% by DHEA (4000 mg/kg; P < 0.05). Tumor multiplicity was reduced by approximately 50% by both DFMO and DHEA (P < 0.05). DFMO had a dose-dependent effect on total tumor burden, which was reduced by 25% at low doses (4000 mg/kg) and 70% at high doses (8000 mg/kg). DHEA reduced tumor burden by 50% and 66% at low (2000 mg/kg) and high (4000 mg/kg) doses, respectively. Interestingly, despite its inhibitory effects on tumor development, DHEA caused a dose-dependent increase of serum estradiol levels that we have previously shown to increase mammary tumor formation in this model. No effect on the development of the prostate cancer precursor lesions (prostate intraepithelial neoplasia) was observed when mice were treated with DHEA, DFMO, tocopherol acetate, selenomethionine, or 9-cis-retinoic acid, although the effects on late-stage prostate cancer development were not determined. These results demonstrate that despite the expression of the highly transforming C3(1)/SV40 large T-antigen transgene, this transgenic model can be used to study the effects of chemopreventive agents on mammary cancer progression. The tumor-inhibitory effects of DHEA and DFMO on mammary cancer growth appear to occur after the development of preinvasive lesions, suggesting that these agents inhibit tumor progression but not initiation.     

Prevention and treatment of experimental estrogen receptor-negative mammary carcinogenesis by the synthetic triterpenoid CDDO-methyl Ester and the rexinoid LG100268

PURPOSE: To test whether the triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) and the rexinoid LG100268 (268) prevent the formation of estrogen receptor (ER)-negative mammary tumors or either arrest the growth or cause regression of established tumors in MMTV-neu mice. EXPERIMENTAL DESIGN: For prevention, mice were fed control diet, CDDO-Me (60 mg/kg diet), 268 (20 mg/kg diet), or the combination for 45 weeks. For treatment, mice with established tumors at least 4 mm in diameter were fed control diet, CDDO-Me (100 mg/kg diet), 268 (60 mg/kg diet), or the combination for 4 weeks. RESULTS: CDDO-Me and 268 significantly delayed the development of ER-negative tumors, with a 14- and 24-week delay, respectively, compared with the control group for the time required to reach 50% tumor incidence. The combination of CDDO-Me and 268 was significantly more potent than the individual drugs, as only one tumor was found in the combination group, after 45 weeks on diet, at which time all control animals had tumors. Treating established tumors with CDDO-Me arrested the growth of 86% of the tumors, and 268 induced tumor regression in 85% of tumors. CDDO-Me and 268 target different signaling pathways and cell types. CDDO-Me inhibited constitutive STAT3 phosphorylation and the degradation of IKBalpha in ER-negative breast cancer cells, whereas 268 blocked IKBalpha degradation and the release of interleukin-6 in RAW264.7 macrophage-like cells, inhibited the ability of endothelial cells to organize into networks, and blocked angiogenesis in vivo. CONCLUSIONS: CDDO-Me and 268 are useful as individual drugs to prevent ER-negative mammary tumorigenesis and to treat established tumors. They synergize when used in combination for prevention.     

The combination of the rexinoid, LG100268, and a selective estrogen receptor modulator, either arzoxifene or acolbifene, synergizes in the prevention and treatment of mammary tumors in an estrogen receptor-negative model of breast cancer.

PURPOSE: We tested whether a selective estrogen receptor modulator (SERM) and a rexinoid are active for prevention and treatment in the mouse mammary tumor virus-neu mouse model of estrogen receptor-negative breast cancer. EXPERIMENTAL DESIGN: For prevention, mice were fed a powdered control diet, the SERM arzoxifene (Arz, 20 mg/kg diet), the rexinoid LG100268 (268, 30 mg/kg diet), or the combination for 60 weeks. In a second prevention study, mice were fed Arz (6 mg/kg diet), 268 (30 mg/kg diet), the combination of Arz and 268, the SERM acolbifene (Acol, 3 mg/kg diet), or the combination of Acol and 268 for 52 weeks. For the treatment studies, mice with tumors were fed combinations of a SERM and 268 for 4 weeks. RESULTS: The rexinoid 268 and the SERMs Arz and Acol, as individual drugs, delayed the development of estrogen receptor-negative tumors. Moreover, the combination of a SERM and 268 was strikingly synergistic, as no tumors developed in any mouse fed the combination of 268 and a SERM. Moreover, this drug combination also induced significant tumor regression when used therapeutically. These drugs did not inhibit transgene expression in vitro or in vivo, and the combination of Arz and 268 inhibited proliferation and induced apoptosis in the tumors. CONCLUSION: The combination of a rexinoid and SERM should be considered for future clinical trials.     

The rexinoid, bexarotene, prevents the development of premalignant lesions in MMTV-erbB2 mice

Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.     

9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice.

Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.     

Toxicology studies of allyl bromide (CAS No. 106-95-6) in genetically modified (FVB Tg.AC hemizygous) mice and carcinogenicity studies of allyl bromide in genetically modified [B6.129-Trp53tm1Brd (N5) Haploinsufficient] mice (dermal and gavage studies)

Allyl bromide is primarily used as a starting material/chemical intermediate in organic synthesis and as an intermediate in the manufacture of polymers/resins, synthetic perfumes, pharmaceuticals, agricultural chemicals, and other allyl compounds. It has been described as an insecticidal fumigant used in crop protection. Male and female FVB/N and C57BL/6 mice received allyl bromide (greater than 99% pure) by gavage and dermal application, respectively, for 2 weeks, and FVB/N, C57BL/6, Tg.AC hemizygous, and p53 haploinsufficient mice received allyl bromide by gavage for 40 weeks. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. 2-WEEK STUDIES IN FVB/N MICE: Groups of five male and five female FVB/N mice were dermally administered 0, 7.5, 15, 30, 60, or 120 mg allyl bromide/kg body weight in acetone, 5 days a week for 2 weeks. The survival and mean body weights of all dosed groups of males and females were similar to those of the vehicle controls. There were no increases in the incidences of lesions in dosed mice. 2-WEEK STUDIES IN C57BL/6 MICE: Groups of five male and five female FVB/N mice were administered 0, 7.5, 15, 30, 60, or 120 mg allyl bromide/kg body weight in corn oil by gavage, 5 days a week for 2 weeks. Three 120 mg/kg male mice died prior to the end of the study. Mean body weights of all dosed groups of males and females were similar to those of the vehicle controls. Liver weights of 30 and 60 mg/kg males were significantly greater than those of the vehicle controls. Nonneoplastic lesions of the forestomach, including hyperplasia, inflammation, degeneration, and hyperkeratosis of the forestomach epithelium, were observed in dosed mice. 40-WEEK STUDIES IN FVB/N MICE: Groups of 15 male and 15 female FVB/N mice were administered 0 or 8 mg allyl bromide/kg body weight in corn oil by gavage, 5 days a week for 40 weeks. Survival of dosed mice was similar to that of the vehicle controls. Mean body weights of dosed mice were within 10% of those of the vehicle controls throughout most of the study. There were no chemical-related gross or microscopic findings in dosed mice. 40-WEEK STUDIES IN Tg.AC HEMIZYGOUS MICE: Groups of 15 male and 15 female Tg.AC hemizygous mice were administered 0, 0.5, 1, 2, 4, or 8 mg allyl bromide/kg body weight in corn oil by gavage, 5 days a week for 40 weeks. Survival of dosed mice was similar to that of the vehicle controls. Mean body weights were generally similar between dosed and vehicle control mice throughout the study. In female mice, there were increased numbers of cutaneous and mucocutaneous masses (gross observations) on the body, particularly the vaginal and vulvar area, and these papillomas were observed earlier in the dosed groups. There were positive trends in the incidences of squamous cell papilloma of the vulva and of all skin sites in females. 40-WEEK STUDIES IN C57BL/6 MICE: Groups of 15 male and 15 female C57BL/6 mice were administered 0 or 8 mg allyl bromide/kg body weight in corn oil by gavage, 5 days a week for 40 weeks. Survival of dosed mice was similar to that of the vehicle controls. Mean body weights and organ weights were similar between dosed and vehicle control mice throughout the study. There were no chemical-related gross or microscopic findings in dosed mice. 40-WEEK STUDIES IN p53 HAPLOINSUFFICIENT MICE: Groups of 15 male and 15 female p53 haploinsufficient mice were administered 0, 0.5, 1, 2, 4, or 8 mg allyl bromide/kg body weight in corn oil by gavage, 5 days a week for 40 weeks. Survival of dosed mice was similar to that of the vehicle controls. Mean body weights of dosed mice were within 10% of those of the vehicle controls throughout most of the study. Mean body weights of 8 mg/kg females were 11% to 15% greater than those of the vehicle controls from week 26 to week 33, and those of 4 mg/kg females were generally less after week 21. There were no chemical-related gross or microscopic findings. GENETIC TOXICOLOGY: Allyl bromide was mutagenic in S. typhimurium strain TA100, with and without exogenous metabolic activation (S9). No mutagenicity was detected in S. typhimurium strain TA98, with or without S9, over the same concentration range tested with TA100. The frequency of micronucleated erythrocytes was assessed in male and female mice for each of the four mouse strains administered allyl bromide by corn oil gavage for 40 weeks. Results in all four studies were concluded to be negative; in addition, no significant changes in the percentages of polychromatic erythrocytes (reticulocytes) among total erythrocytes were observed in any of the four strains of mice. CONCLUSIONS: Under the conditions of this study, there was no evidence of carcinogenic activity in male or female p53 haploinsufficient mice administered allyl bromide at 0, 0.5, 1, 2, 4, or 8 mg/kg per day by corn oil gavage, 5 days a week for 40 weeks. There was a marginal increase in the incidence of squamous cell papillomas, primarily of the vulva, in female Tg.AC mice administered allyl bromide by corn oil gavage for 40 weeks. No treatment-related neoplasms were seen in male Tg.AC hemizygous mice administered allyl bromide by gavage at 0.5, 1, 2, 4, or 8 mg/kg, 5 days per week for 40 weeks.     

NTP Toxicity Studies of Dimethylaminopropyl Chloride, Hydrochloride (CAS No. 5407-04-5) Administered by Gavage to F344/N Rats and B6C3F1 Mice

Dimethylaminopropyl chloride, hydrochloride is used primarily as an industrial and research organic chemical intermediate acting as an alkylating reagent in Grignard and other types of reactions. It is also used as a pharmaceutical intermediate for the synthesis of many types of drugs, as an agricultural chemical intermediate, as a photographic chemical intermediate, and as a biochemical reagent for enzyme and other studies. Human occupational or other accidental exposure can occur by inhalation, ingestion, or skin absorption. Male and female F344/N rats and B6C3F1 mice received dimethylaminopropyl chloride, hydrochloride (greater than 99% pure) in water by gavage for 2 weeks or 3 months. Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes. In the 2-week toxicity studies, groups of five male and five female F344/N rats and B6C3F1 mice were administered doses of 0, 6.25, 12.5, 25, 50, or 100 mg dimethylaminopropyl chloride, hydrochloride/kg body weight in deionized water by gavage, 5 days per week for 16 days. All dosed male and female rats and mice survived until the end of the 2-week study; one vehicle control female mouse died early. Mean body weights of all dosed groups of rats and mice were similar to those of the vehicle control groups. No gross or microscopic lesions were considered related to dimethylaminopropyl chloride, hydrochloride administration. In the 3-month toxicity studies, groups of 10 male and 10 female F344/N rats and B6C3F1 mice were administered doses of 0, 6.25, 12.5, 25, 50, or 100 mg/kg in deionized water by gavage, 5 days per week for 3 months. One male rat in the 50 mg/kg group died during week 12 of the study, and one female mouse in the 100 mg/kg group died during week 9 and another during week 13. The final mean body weights of 50 mg/kg male rats and 50 mg/kg female mice were significantly less than those of the vehicle controls. Possible chemical-related clinical findings in rats included lethargy in one 50 mg/kg male and one 100 mg/kg male, tremors in one 100 mg/kg male, and ataxia in one 50 mg/kg male and two 100 mg/kg males. Absolute lung weights in the 25, 50, and 100 mg/kg groups of female mice were significantly less than those of the vehicle controls. Total serum bile acid concentrations were increased in 50 mg/kg male rats and 100 mg/kg male and female rats. The incidence of goblet cell hypertrophy of the nose was significantly increased in 100 mg/kg male rats compared to the vehicle controls. There were no significant histopathologic findings in mice. Dimethylaminopropyl chloride, hydrochloride was mutagenic in the Salmonella typhimurium base substitution strains TA100 and TA1535, with and without hamster or rat liver S9 activation enzymes; no mutagenic activity was seen in TA97 or TA98. No increase in the frequency of micronucleated erythrocytes was seen in peripheral blood of male or female mice administered dimethylaminopropyl chloride, hydrochloride for 3 months by gavage. In summary, dimethylaminopropyl chloride, hydrochloride caused increased incidences of goblet cell hypertrophy in the nose of male rats and increased serum bile acid concentrations in male and female rats. In mice, dimethylaminopropyl chloride, hydrochloride caused deaths in females administered 100 mg/kg. The estimated no-observed-effect levels were 50 mg/kg per day for male rats and female mice, 100 to 200 mg/kg per day for female rats, and greater than 100 mg/kg per day for male mice. Synonyms: 3-Chloropropyldimethyl-ammonium chloride; (3-chloropropyl)dimethylamine, hydrochloride; N-(3-chloropropyl)-N,N-dimethylammonium chloride; 3-dimethylamino-1-propyl chloride hydrochloride; 3-dimethylaminopropyl chloride hydrochloride; DMPC; 1-propylamine, 3-chloro-N,N-dimethyl-, hydrochloride.     

Iressa对肝癌细胞Hep-3B、HepG2裸鼠移植瘤的抑制作用

背景与目的：Iressa是一种新型分子靶向药物,在治疗晚期非小细胞肺癌中显示出较好疗效,对Iressa治疗肝癌的研究国内外报道较少。本研究旨在探讨Iressa对肝癌Hep-3B、HepG2细胞裸鼠移植瘤的抑制作用及其毒副作用。方法：肝癌Hep-3B、HepG2细胞移植瘤裸鼠随机分为对照组、低剂量组和高剂量组,分别用葡萄糖溶液、Iressa 100mg／kg和Iressa 200mg／kg每日一次灌胃,每周灌五天,连用3周,用药结束后2天处死动物。分别计算Iressa对两种肝癌的抑瘤率,比较各组裸鼠肿瘤大小、体重及脾指数。结果：给药前各组裸鼠体重和肿瘤体积无差异。低剂量组和高剂量组Iressa对Hep-3B肝癌移植瘤的抑瘤率分别为32．77％、46．99％;低剂量组和高剂量组Iressa对HepG2肝癌移植瘤的抑瘤率分别为68．57％、75．24％。两种肿瘤类型的高剂量组裸鼠的脾指数和体重下降与对照组比较均有显著性差异,低剂量组和对照组裸鼠的脾指数和体重比较无明显差异。结论：Iressa对肝癌Hep-3B、HepG2细胞移植瘤生长均有明显抑制作用。但高剂量可能引起裸鼠体重下降并影响免疫器官功能。     

Ineffectiveness of American ginseng in the prevention of dimethylbenzanthracene-induced mammary tumors in mice

The potential of American ginseng (AG) ( Panax quinquefolium), a commonly used herbal remedy believed to have anticarcinogenic effects, to prevent the development of mammary tumors was evaluated in a mouse model of dimethylbenzanthracene (DMBA)-induced mammary carcinoma. Ginsenosides, believed to be the active components of ginseng and that have a chemical structure similar to estradiol, have previously been shown to possess phytoestrogen-like qualities similar to the soy isoflavone genistein. The effects of AG, administered as powdered root, were compared to the selective estrogen receptor modulators tamoxifen and ospemifene. Eighty-three female SENCAR mice were divided into four treatment groups: control (N = 23), AG (N = 20), ospemifene (N = 20), and tamoxifen (N = 20). American ginseng, ospemifene, and tamoxifen were administered at a dose of 50 mg/kg/day orally by gavage, with the control mice receiving vehicle only. For the first 6 weeks, all mice received 20 microg/day DMBA in combination with their respective treatments. DMBA was then withdrawn, and daily treatments continued for a total of approximately 52 weeks. As expected, ospemifene (p = 0.01) and tamoxifen (p = 0.004) significantly reduced the incidence of mammary tumors compared to the control mice, which had a mammary tumor incidence of approximately 57%. The incidence of mammary carcinomas in the AG group was 40%, a reduction of approximately 29% compared to control. These results suggest that AG may still have the potential to prevent the development of mammary tumors in a chemically induced breast cancer mouse model, although the present study showed no significant difference between control and AG-treated mice.     

Prevention and treatment of experimental breast cancer with the combination of a new selective estrogen receptor modulator, arzoxifene, and a new rexinoid, LG 100268.

The selective estrogen receptor modulator arzoxifene and the rexinoid LG 100268 were active not only as single agents for prevention and treatment of breast cancer in the rat model that uses nitrosomethylurea as the carcinogen but also showed striking synergy, both preventively and therapeutically, in a series of six experiments with a total of 465 rats. Mechanistic studies in cell culture reported here suggest that enhancement of stromal-epithelial interactions may contribute to this synergy. The possible clinical use of the combination of arzoxifene and LG 100268 for prevention of breast cancer in women at high risk, for treatment of women in the adjuvant setting, or for treatment of end-stage disease should now be considered.     

Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (ZarnestraMT)

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female Strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at 20 weeks. At Week 20, the rank order for prevention of lung tumors was the combined treatment>budesonide>R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate endpoint biomarker for prevention.     

Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (Zarnestra MT)

Budesonide (an anti-inflammatory glucocorticoid), R115777 (afarnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinationsof them were evaluated for prevention of lung tumors and formodulation of DNA methylation in tumors. Lung tumors were inducedby vinyl carbamate in female strain A mice. One week later,mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week,0.8 or 1.6 mg/kg of budesonide in their diet, or their combinedtreatment until killed at 20, 28 and 36 weeks after administeringthe vinyl carbamate. Other mice were administered the drugsfor 2 weeks before killing at Week 20. At Week 20, the rankorder for prevention of lung tumors was the combined treatment> budesonide > R115777. At later killings, R115777 wasno longer effective, whereas budesonide and the combinationscontinued to prevent tumors, albeit at a reduced efficacy. DNAhypomethylation in lung tumors was prevented by treatment withR115777, budesonide and the combinations. When administeredstarting at Week 18 to tumor-bearing mice, the drugs reversedDNA hypomethylation in the tumors. In summary, combined treatmentwith budesonide and R115777 produced the following results:(i) it was more efficacious in preventing lung tumors than theindividual drugs; and (ii) it prevented and reversed DNA hypomethylationin lung tumors. These results support the combined use of budesonideand R115777 in prevention of lung tumors and suggest that reversalof DNA hypomethylation in lung tumors would be useful as a surrogateend-point biomarker for prevention.     

Toxicology studies of bromodichloromethane (CAS No. 75-27-4) in genetically modified (FVB Tg.AC Hemizygous) mice (dermal, drinking water, and gavage studies) and carcinogenicity studies of bromodichloromethane in genetically modified [B6.129-Trp53(tm1Brd) (N5) haploinsufficient] mice (drinking water and gavage studies)

Bromodichloromethane is a by-product of the chlorination of drinking water. It is formed by the halogen substitution and oxidation reactions of chlorine and naturally occurring organic matter (e.g., humic or fluvic acids) in water containing bromide. Bromodichloromethane was nominated to the NTP by the United States Environmental Protection Agency for toxicology and carcinogenicity studies. Male and female Tg.AC hemizygous mice received bromodichloromethane (at least 98%pure) by dermal application for 26 or 39 weeks, in drinking water for 26 or 42 weeks, or by gavage for 26 or 41 weeks. p53 Haploinsufficient mice received bromodichloromethane in drinking water for 26 or 42 weeks or by gavage for 26 or 41 weeks. Genetic toxicology studies were conducted in mouse peripheral blood erythrocytes. 26- and 39-WEEK DERMAL STUDIES IN Tg.AC HEMIZYGOUS MICE: Groups of 15 male and 15 female Tg.AC hemizygous mice were dermally administered 0, 64, 128, or 256 mg bromodichloromethane/kg body weight in acetone, 5 days per week for 26 weeks, and groups of 10 male and 10 female Tg.AC hemizygous mice were dermally administered the same doses 5 days per week for 39 weeks. The survival and mean body and organ weights of all dosed groups of males and females were similar to those of the vehicle controls. There were no statistically or biologically significant increases in the incidences of neoplasms or nonneoplastic lesions. 26- AND 42-WEEK DRINKING WATER STUDIES IN TG.AC HEMIZYGOUS MICE: Groups of 15 male and 15 female Tg.AC hemizygous mice were exposed to drinking water containing 0, 175, 350, or 700 mg/L bromodichloromethane for 26 weeks (equivalent to average daily doses of approximately 20, 36, or 61 mg bromodichloromethane/kg body weight to males and 31, 61, or 130 mg/kg to females). The survival of exposed males and females was similar to that of the control groups. Mean body weights of males exposed to 350 or 700 mg/L were less than those of the controls during most of the study. Mean body weights of 175, 350, and 700 mg/L females were greater than those of the controls after weeks 10, 22, and 23, respectively. In exposed males, water consumption declined with increasing exposure concentration. Water consumption by exposed females was less at the beginning of the study, but was similar to that by controls at the end of the study. The decreased water consumption was related to poor palatability. Absolute heart and right kidney weights of exposed males were significantly less than those of the control group. The incidences of hepatocyte fatty change and hypertrophy in 350 and 700 mg/L females and cytoplasmic vacuolization in 700 mg/L females were significantly greater than those in the control group. Incidences of renal tubule dilatation in males exposed to 175 mg/L or greater, renal tubule hypertrophy in 350 and 700 mg/L males, and nephropathy and renal tubule degeneration in 700 mg/L males were also increased. Groups of 10 male and 10 female Tg.AC hemizygous mice were exposed to drinking water containing 0, 175, 350, or 700 mg/L bromodichloromethane for 42 weeks (equivalent to average daily doses of approximately 18, 33, or 64 mg/kg to males and 28, 49, or 111 mg/kg to females). The survival of exposed males and females was similar to that of the control groups. Mean body weights of 350 and 700 mg/L males were less than those of the controls at the end of the study. Due to poor palatability, water consumption decreased with increasing exposure concentration. Absolute right kidney weights of 350 and 700 mg/L males were significantly less than those of the control group. The incidences of hepatocyte fatty change in all exposed groups of females, renal tubule dilatation in all exposed groups of males, and nephropathy in 700 mg/L males were significantly increased. 26- AND 41-WEEK GAVAGE STUDIES IN TG.AC HEMIZYGOUS MICE: Groups of 15 male and 15 female Tg.AC hemizygous mice were administered 0, 25, 50, or 100 mg bromodichloromethane/kg body weight in corn oil by gavage, 5 days per week for 26 weeks. The survival of dosed males and females was similar to that of the vehicle control groups. Mean body weights of dosed females were generally greater than those of the vehicle controls at the end of the study. The incidence of multiple squamous cell papilloma of the forestomach in 100 mg/kg females was significantly greater than that in the vehicle controls. The incidences of hepatocyte fatty change in all dosed groups of females, hepatocyte cytoplasmic vacuolization in 25 and 50 mg/kg females, renal tubule hypertrophy in 100 mg/kg females, and renal tubule degeneration in 100 mg/kg males were significantly increased. Groups of 10 male and 10 female Tg.AC hemizygous mice were administered 0, 25, 50, or 100 mg/kg in corn oil by gavage, 5 days per week for 41 weeks. The survival of dosed males and females was similar to that of the control groups. Mean body weights of 25 mg/kg males and 100 mg/kg females were greater than those of the vehicle controls at the end of the study. The incidences of multiple squamous cell papilloma of the forestomach in 25 and 100 mg/kg females and of all squamous cell papillomas of the forestomach in 100 mg/kg females were significantly greater than those of the vehicle controls. The incidences of hepatocyte cytoplasmic vacuolization in 50 mg/kg females and hepatocyte fatty change in 50 and 100 mg/L females were significantly increased; the incidences of renal tubule degeneration in 100 mg/kg males was also significantly greater than that in the vehicle control group. 26- AND 42-WEEK DRINKING WATER STUDIES IN P53 HAPLOINSUFFICIENT MICE: Groups of 15 male and 15 female p53 haploinsufficient mice were exposed to drinking water containing 0, 175, 350, or 700 mg/L bromodichloromethane for 26 weeks (equivalent to average daily doses of approximately 16, 31, or 65 mg/kg to males and 26, 50, or 100 mg/kg to females). The survival of exposed males and females was similar to that of the control groups. Mean body weights of 350 and 700 mg/L males were less than those of the controls throughout most of the study. Mean body weights of 175, 350, and 700 mg/L females were less than control body weights after weeks 15, 23, and 18, respectively. In exposed males, water consumption declined with increasing exposure concentration. Water consumption by exposed females was similar to that by controls by the end of the study. The absolute heart weight of 700 mg/L males and absolute right kidney and liver weights of 350 and 700 mg/L males were significantly less than those of the control group. The incidences of renal tubule dilatation in all exposed groups of males, renal tubule degeneration in 350 and 700 mg/L males, and the incidence of fatty change in hepatocytes of 700 mg/L females were significantly greater than those in the control groups. Groups of 10 male and 10 female p53 haploinsufficient mice were exposed to drinking water containing 0, 175, 350, or 700 mg/L for 42 weeks (equivalent to approximately 14, 30, or 55 mg/kg to males and 22, 43, or 98 mg/kg to females). The survival of exposed males and females was similar to that in the control groups. Mean body weights of males exposed to 350 or 700 mg/L were less than those of the controls. Mean body weights in 700 mg/L females were less during the last three weeks of the study. Water consumption by exposed males was less than that by controls. The absolute right kidney weights in 350 and 700 mg/L males were significantly less than those of the control group. The incidences of renal tubule degeneration in 350 and 700 mg/L males were significantly greater than that in the control group. 26- AND 41-WEEK GAVAGE STUDIES IN P53 HAPLOINSUFFICIENT MICE: Groups of 15 male and 15 female p53 haploinsufficient mice were administered 0, 25, 50, or 100 mg bromodichloromethane/kg body weight in corn oil by gavage for 26 weeks. The survival of dosed males and females was similar to that of the vehicle control groups. The mean body weights of males administered 50 or 100 mg/kg and females administered 50 mg/kg were less than those of the vehicle controls during most of the study. The absolute heart, right kidney, and right testis weights in 100 mg/kg males were significantly less than those of the vehicle controls. The absolute liver weight of 100 mg/kg females was significantly greater. The incidences of fatty change in hepatocytes of 100 mg/kg females and renal tubule degeneration in 100 mg/kg males were significantly greater than those in the vehicle control groups. Groups of 10 male and 10 female p53 haploinsufficient mice were administered 0, 25, 50, or 100 mg/kg in corn oil by gavage for 41 weeks. The survival of dosed males and females was similar to that of the vehicle control groups. Mean body weights of 50 and 100 mg/kg males were less than those of the vehicle controls throughout the study and those of 25, 50, and 100 mg/kg females were less after weeks 9, 14, and 24, respectively. The absolute liver weight of 100 mg/kg females was increased with respect to the vehicle controls, and the absolute heart and right kidney weights of 100 mg/kg males were decreased. The incidences of hepatocyte fatty change in 100 mg/kg males and females and renal tubule degeneration and nephropathy in 100 mg/kg males were significantly greater than those in the vehicle controls. GENETIC TOXICOLOGY: Peripheral blood micronucleus tests on male and female Tg.AC hemizygous and p53 haploinsufficient mice exposed to bromodichloromethane in drinking water, by dermal application, and by gavage for 26 weeks yielded mixed results but no clearly positive responses. Results in Tg.AC hemizygous mice were judged to be equivocal for both males and females in the drinking water study, equivocal in males and negative in females treated by dermal application, and negative in males and females treated by gavage. (ABSTRACT TRUNCATED)     

Fatty acid synthase inhibitors are chemopreventive for mammary cancer in neu-N transgenic mice

High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions of the colon, stomach, esophagus, oral cavity, prostate, and breast. Inhibition of FAS with C75 has led to a significant antitumor effect in both human breast and prostate cancer xenografts. Recently, HER2/neu, which has also been identified in preneoplastic breast lesions, has been shown to regulate FAS expression through the PI3K/Akt signal transduction pathway rendering them susceptible to FAS inhibition. Utilizing the neu-N transgenic mouse model of mammary cancer, weekly treatment of the neu-N mice with C75 (30 mg/kg) for 10 weeks significantly delayed tumor progression. Only 20% of the C75-treated transgenic mice developed mammary carcinoma by 220 days, compared to 50% in the vehicle control animals. Two C75-treated animals never developed mammary cancer. Analysis of mammary tissue following 10 weeks of C75 treatment revealed a significant delay in mammary maturation as manifested by a reduction of the number and caliber of mammary ducts and budding epithelial structures. Apoptotic changes were increased, DNA synthesis was decreased, and the expressions of FAS, neu, Akt, phospho-Akt, and p21(waf1) were all decreased when compared to vehicle controls and FVB/N mice. Importantly, these effects were restricted to the breast epithelial cells that overexpressed neu, not involving other normal duct structures in the skin, liver, or kidney. C247, an FAS inhibitor chemically distinct from C75, significantly delayed mammary maturation similar to C75. Thus, pharmacological inhibition of FAS affects the expression of key oncogenes involved in both cancer development and maintenance of the malignant phenotype. Moreover, these data identify FAS as a potential novel drug target for breast cancer chemoprevention.     

Assessing the therapeutic and toxicological effects of cesium chloride following administration to nude mice bearing PC-3 or LNCaP prostate cancer xenografts

Purpose The purpose of this study was to assess the therapeutic and toxicological effects of cesium chloride (CsCl) administration in mice bearing prostate cancer tumors. Methods Three CsCl dose titration studies were completed in tumor-bearing and non-tumor-bearing athymic nude mice. All mice were administered either vehicle (controls), 150, 300, 600, 800, 1,000, or 1,200 mg/kg of CsCl once daily by oral gavage for 30 consecutive days. Body mass was measured daily, food and water consumption were measured every 2 days, and tumor volume was measured twice weekly. Histopathological analysis was conducted on tissues collected from each of the studies. Serum AST/ALT and creatinine were also measured. Results Administration of 800–1,200 mg/kg CsCl reduced PC-3 tumor growth but had no effect on LNCaP tumors. Administration of 800–1,200 mg/kg CsCl also resulted in increased water consumption, bladder crystal development, and higher prevalence of cardiac fibrin clots. An observed loss in body mass was dependent on the xenograft type and concentration of CsCl administered. CsCl did not affect serum AST/ALT and creatinine levels. Conclusions CsCl may have a therapeutic effect against prostate cancer, but one cannot overlook the acute toxicities also described.