NMU-induced Mammary Tumors Research Articles

Therapeutic effect of hydroethanolic extract of Trianthema portulacastrum L. against N-Nitroso-N-Methylurea-induced mammary tumors in Wistar rats

This study evaluated the therapeutic action of hydroethanolic extract of Trianthema portulacastrum L. (TPE) on N-nitroso-N-methylurea (NMU)-induced mammary tumors in Wistar rats. A hydroethanolic was prepared and subjected to qualitative and quantitative phytochemical screening. After acclimatization, Wistar rats were divided into 4 groups of 6 rats each: Group A (vehicle control), Group B (TPE control), Group C (TPE treatment) and group D (NMU control). NMU (50 mg/kg body weight) was injected intraperitoneally at 50, 80 and 110 days of age. After the induction of palpable tumors,the rats were administered 200 mg/kg bw of TPE by oral gavage for 2 months. The treatment with TPE significantly (p<0.05) decreased tumor incidence, frequency, size and malignancy in comparison to the tumor-bearing rats that were not administered TPE. Immunohistochemical analysis revealed that TPE treatment significantly reduced the expression of PCNA, VEGF, ER-α and ER-β, and caused non-significant reductions in matrix metallopeptidase-9 (MMP-9). Caspase-3 expression significantly increased in TPE-treated rats in comparison with NMU-treated controls. The qRT-PCR resultsshowed PCNA and ER-β expression was down regulated and caspase-3 expression was up regulated in the TPE-treated group. The present study showed the in vivo therapeutic action of TPE extract on NMU-induced mammary tumors. TPE exhibited antitumor activity through its antiproliferative, antiangiogenic, pro-apoptotic, and estrogen receptor-modulatory properties.

Metformin and melatonin improve histopathological outcome of NMU-induced mammary tumors in rats

ObjectiveNumerous reports showed inhibition of carcinogenesis after metformin (MF) and melatonin (MEL) administration. However, most in vivo studies used standard diet type, with relatively low fat content. As increase in fat intake may have a considerable impact on malignant transformation, we evaluated the effects of these two substances in a model of mammary carcinogenesis in rats fed a high-fat diet (10%). MethodsMammary tumors were induced by N-methyl-N-nitrosourea (NMU) in female rats of sensitive Sprague-Dawley strain. MF was administered in a diet (0.2%), MEL was administered in drinking water (20 mg/L). The chemoprevention was initiated 12 days prior to tumor initiation, both substances were administered through the termination of the experiment on 16th week after carcinogen application. Analysis of basic parameters of tumor growth, histopathological profile, and serum IGF-1 level were performed together with immunohistochemical detection of Ki67 (proliferation marker) and caspase-3 and BCL-2 (apoptosis markers) in mammary cancer cells. ResultsAlthough neither tumor incidence nor frequency were changed after MF and/or MEL administration, MF and MEL decreased high-grade/low-grade (HG/LG) tumor ratio. MEL decreased proliferation in mammary cancer cells; positive correlations between histological grade and Ki67 expressions were found after single administration of both MF and MEL. Serum IGF-1 levels were reduced to the level of intact rats in all groups receiving chemoprevention. ConclusionsMF and MEL administration did not inhibit growth of NMU-induced mammary tumors in rats in a significant manner but both substances ameliorated tumor histopathological profile. Surprisingly, combined treatment had no such effect.

Nelumbo nucifera Leaves Prevent NMU-Induced Mammary Tumor through Downregulation of Fatty Acid Synthase, Estrogen Receptor-α and Her2 Expression.

Diet polyphenol can reportedly prevent the formation of breast-cancer cells. Nelumbo nucifera leaf extract (NLE) is enriched with polyphenols and has several cellular functions, such as anti-atherosclerosis, anti-inflammation, and antitumor. In this study, we investigated the role of NLE in the prevention of N-methyl-N-nitrosourea (NMU)-induced mammary tumor formation. Cotreatment with NLE significantly reduced the NMU-induced tumor incidence, number, and volume. NLE administration significantly repressed the tumor growth and weight of nude mice upon inoculation with BT-474 cancer cells. Immunohistochemical staining indicated that fatty acid synthetase, estrogen receptor (ER)-, and phosphorylated ER- were obviously reduced in the cancer part of BT-474 inoculated nude mice upon administration of 2% NLE. Western blot analysis revealed that NLE and NLPE (polyphenol-rich NLE) repressed ER- expression and phosphorylation and decreased the phosphorylation of Her-2 without affecting their expression. Overall, NLE and NLPE exhibited more effective antitumor abilities in NMU-induced mammary cancer formation than with tamoxifen and Herceptin.

Effects of the pesticide chlorpyrifos on breast cancer disease. Implication of epigenetic mechanisms

Chlorpyrifos (CPF) is an organophosphorus pesticide used for agricultural pest control all over the world. We have previously demonstrated that environmental concentrations of this pesticide alter mammary gland histological structure and hormonal balance in rats chronically exposed. In this work, we analyzed the effects of CPF on mammary tumors development. Our results demonstrated that CPF increases tumor incidence and reduces latency of NMU-induced mammary tumors. Although no changes were observed in tumor growth rate, we found a reduced steroid hormone receptor expression in the tumors of animals exposed to the pesticide. Moreover, we analyzed the role of epigenetic mechanisms in CPF effects. Our results indicated that CPF alters HDAC1 mRNA expression in mammary gland, although no changes were observed in DNA methylation. In summary, we demonstrate that the exposure to CPF promotes mammary tumors development with a reduced steroid receptors expression. It has also been found that CPF affects HDAC1 mRNA levels in mammary tissue pointing that CPF may act as a breast cancer risk factor.

Immunomodulatory effects of a bioactive fraction of Strobilanthes crispus in NMU-induced rat mammary tumor model

Ethnopharmacological relevanceStrobilanthes crispus Blume is traditionally consumed among local Malay and indigenous communities for the treatment of cancer and other ailments such as gastrointestinal disorders, inflammatory wounds of snake bite and immune system activation amongst others. We previously demonstrated that a bioactive fraction of S. crispus leaves (F3) was cytotoxic to breast cancer cells in vitro and inhibited tumor growth in N-methyl-N-nitrosourea (NMU)-induced breast cancer rat model. F3 also normalized the white blood cell count in the tumor-bearing animals, indicating its potential immuno-stimulatory effect. Aim of the studyTo evaluate the immune stimulatory effects of F3 from S. crispus in NMU-induced rat mammary tumor model. Materials and methodsImmunohistochemistry analysis of cellular immune parameters (CD4+ or CD8+ T cells, CIITA, MHC-II and CD68) was performed on NMU-induced rat mammary tumor nodules, followed by evaluation of the serum level of 34 cytokines using the cytokine antibody array. ResultsSignificant increase in MHC-II, CD4+ and CD8+ T cell and CIITA expression by tumor cells was observed in F3-treated rats compared to the tumor control group. F3-treated rats also displayed a significant decrease in the serum level of CCL2 and CD68+ infiltrating macrophages. Serum IFN-γ level in this group was increased by 1.7-fold suggesting enhanced infiltration of T cells, and upregulation of CIITA and MHC-II expression in the tumor cells might be triggered by F3-induced production of IFN-γ. ConclusionOur findings demonstrated for the first time that a subfraction from S. crispus, F3, is capable of activating the immune system in rats-bearing NMU-induced mammary tumor, which may contribute to the anticancer effects of F3, and additionally support the traditional use of S. crispus leaves to boost the immune system.

Anticancer and Cancer Prevention Effects of Piperine-Free Piper nigrum Extract on N-nitrosomethylurea-Induced Mammary Tumorigenesis in Rats.

Piper nigrum (P. nigrum) is commonly used in traditional medicine. This current study aimed to investigate the anticancer and cancer preventive activity of a piperine-free P. nigrum extract (PFPE) against breast cancer cells and N-nitrosomethylurea (NMU)-induced mammary tumorigenesis in rats. The cytotoxic effects and the mechanism of action were investigated in breast cancer cells using the MTT assay and Western blot analysis, respectively. An acute toxicity study was conducted according to the Organization for Economic Co-operation and Development guideline. Female Sprague-Dawley rats with NMU-induced mammary tumors were used in preventive and anticancer studies. The results showed that PFPE inhibited the growth of luminal-like breast cancer cells more so than the basal-like ones by induction of apoptosis. In addition, PFPE exhibited greater selectivity against breast cancer cells than colorectal cancer, lung cancer, and neuroblastoma cells. In an acute toxicity study, a single oral administration of PFPE at a dose of 5,000 mg/kg body weight resulted in no mortality and morbidity during a 14-day observation period. For the cancer preventive study, the incidence of tumor-bearing rats was 10% to 20% in rats treated with PFPE. For the anticancer activity study, the growth rate of tumors in the presence of PFPE-treated groups was much slower when compared with the control and vehicle groups. The extract itself caused no changes to the biochemical and hematologic parameters when compared with the control and vehicle groups. In conclusion, PFPE had a low toxicity and a potent antitumor effect on mammary tumorigenesis in rats.

Potentiation of the antitumor effect of tamoxifen by combination with the antiprogestin onapristone

The present study was undertaken to investigate the antitumor effect of a combination of the antiestrogen tamoxifen (TAM) and either the antiprogestin onapristone (ON) or the progestogen megestrol acetate (MEG) in experimental mammary tumor models. Rats bearing DMBA- or NMU-induced mammary tumors were treated with ON or MEG either alone or in combination with TAM for four weeks. In the DMBA-tumor model, treatment with ON or TAM alone caused tumor remissions, whereas the combination of ON and TAM was almost as effective as ovariectomy (100% remission) and led to a remission of 86–100%. The combination of TAM and ON was distinctly more effective than that of TAM and MEG. A similar potentiation of the antitumor effect of TAM and ON was observed in the NMU-tumor model. In DMBA-tumors, the concentration of progesterone receptors was found to increase after treatment for three days with TAM and ON. In hosts bearing DMBA-tumors, treatment with the combination of TAM and ON caused a reduction in ovarian and uterine weights. In the same animals, the basal level of progesterone was decreased in spite of a slight increase in the LH level. These findings suggest that the high antitumor effect of the combination of TAM and ON compared to the corresponding monotherapies can be related not only to the interaction of antihormones and receptors, but also to the up-regulation of PR and to a decrease in progesterone production. These data clearly suggest the sense of a combination of TAM with an antiprogestin in breast cancer treatment.

Tumor suppressor PTEN signaling is up-regulated in mammary epithelial cells by soy isoflavone genistein: implications for breast cancer protection.

Abstract Abstract #4069 Breast cancer is the second leading cause of death in the Western hemisphere affecting one of eight women in their lifetime. In addition to chromosomal and genetic alterations, nutrition is considered a risk determinant for breast cancer. Prevailing evidence suggests a negative correlation between intake of soy-rich foods and breast cancer incidence. Epidemiological and case control studies have shown a two-to eight fold lower occurrence of the disease in Asian women whose early intake of soy products is 20-25 times higher than their American counterparts. Previous work from our group has shown diet-induced protection against DMBA- and NMU-induced mammary tumors in vivo, with decreased tumor incidence and increased tumor latency in rats exposed to dietary soy protein isolate (SPI) or casein (CAS) supplemented with the major soy isoflavone genistein (GEN) when compared to those fed the control casein (CAS) diet. We also have shown that GEN enhanced apoptosis in mammary epithelial cells in vivo and in vitro (MCF-7 cells), attendant with increased expression of the tumor suppressor PTEN and several pro-apoptotic genes Bax, Bok, and p21. Here, we test the hypothesis that GEN induction of PTEN expression and nuclear localization in mammary epithelial cells result in decreased cellular proliferation and enhanced cellular differentiation, to confer protection from mammary tumorigenesis. Using the non-tumor human mammary epithelial cell line MCF-10A, we show that GEN at physiologically relevant concentrations (40 nM, 2 µM) increased PTEN mRNA and protein levels and enhanced nuclear accumulation of active (non-phosphorylated) PTEN. GEN induction of PTEN transcript levels was lost with the inclusion of the transcription inhibitor actinomycin D, suggesting PTEN is a direct GEN target. Further, cells treated with GEN for 6 days had lower cell viability when compared to control cells, concomitant with decreased expression of cyclin D1 and of the PTEN regulated gene pleiotrophin. Interestingly, GEN up-regulation of PTEN expression was accompanied by similar effects on another tumor suppressor p53, which co-localized with PTEN in the nuclei of GEN-treated cells. PTEN siRNA targeting, which decreased basal PTEN expression by 60%, significantly increased cyclin D1 but not p53 expression. Our findings provide support to the hypothesis that the protective effects of GEN in the mammary epithelium may be mediated by PTEN to enhance differentiation and inhibit cellular proliferation, and suggest the participation of another key tumor suppressor p53. Funding by USDA-CRIS 6251-5100002-06S. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4069.

Regulation of adipocyte lipid homeostasis by genistein alters mammary epithelial cell differentiation: a paracrine mechanism for mammary tumor protection.

Abstract Abstract #5082 Epidemiological and animal studies have shown a negative correlation between breast cancer incidence and intake of soy-rich foods. Our laboratory has used soy protein isolate (SPI), the primary component of soy infant formula, as a paradigm to evaluate diet as a risk factor in mammary cancer. We previously demonstrated that lifetime exposure to dietary SPI reduced the incidence of NMU-induced mammary tumors in young adult rats relative to those fed the control Casein diet (CAS). This protection was associated with increased differentiation status of mammary epithelial cells prior to carcinogen insult, suggesting enhanced differentiation as a mechanism for mammary tumor protection. To identify early events contributing to diet-induced mammary differentiation, we used Affymetrix RAE230A GeneChips containing 14280 probes and GeneSpring Gx to analyze genomic profiles of mammary glands of prepubertal [postnatal day (PND) 21] rats which were lifetime exposed to dietary SPI or CAS. Of the 8 genes down-regulated by SPI, 4 are involved in lipid homeostasis: malic enzyme 1, fatty acid synthase (Fasn), stearoyl CoA desaturase-1 (Scd1), and insulin-induced gene 1. One of five SPI-induced genes is 11β-hydroxysteroid dehydrogenase 1 (Hsd1), which converts 11β-dehydrocorticosterone to corticosterone. Quantitative RT-PCR confirmed that Fasn and Scd1 transcript levels were down-regulated and Hsd1 was up-regulated, in PND21 mammary tissues of SPI vs CAS group. We evaluated potential paracrine effects of soy-mediated alterations in adipocyte lipid metabolism on mammary epithelial cell phenotype. In these studies, we used the mouse fibroblast 3T3-L1 cell line treated with the bioactive soy isoflavone genistein (GEN) and mouse mammary epithelial cell line HC11 as in vitro systems to recapitulate in vivo stromal/epithelial interactions. GEN (0.5 μM) regulated lipid homeostasis in fully differentiated 3T3-L1 adipocytes. GEN decreased Fasn and Scd1 and increased Hsd1 transcript levels in the absence of effects on leptin and PPARγ2 gene expression. To evaluate whether factors produced by GEN-treated adipocytes regulate mammary epithelial differentiation, mature adipocytes were incubated in medium with or without GEN (0.5 μM) for 48 h. Cells were refreshed with maintenance medium without GEN and 24 h later, conditioned medium (CM) was harvested, supplemented with prolactin (5 ng/ml), and added full-strength to HC11 cells. Cells incubated for 72 h in CM from GEN-exposed adipocytes had higher differentiation-associated β-casein transcript levels than those grown in non-GEN-treated CM. Our data support the hypothesis that GEN modulation of mammary fat pad metabolism and secretion constitutes one mechanism by which mammary epithelial differentiation is enhanced by diet to confer increased resistance to breast cancer. Studies are in progress to identify adipocyte-secreted factors regulated by this dietary isoflavone. USDA-CRIS-6251-5100-002-06S. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5082.

Gemini Vitamin D Analogues Inhibit Estrogen Receptor–Positive and Estrogen Receptor–Negative Mammary Tumorigenesis without Hypercalcemic Toxicity

Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1alpha,25(OH)(2)D(3), the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1alpha,25(OH)(2)D(3) in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using (a) an N-methyl-N-nitrosourea-induced estrogen receptor (ER)-positive mammary tumor model and (b) an MCF10DCIS.com xenograft model of ER-negative mammary tumors. Among vitamin D analogues we tested, Gemini 0072 [1alpha,25-dihydroxy-20S-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] and Gemini 0097 [1alpha,25-dihydroxy-20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMU-induced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.

Abstract 2097: Gemini vitamin D analogs inhibit estrogen receptor positive and estrogen receptor negative mammary tumorigenesis without hypercalcemic toxicity

Abstract Numerous preclinical, epidemiological and clinical studies with vitamin D and analogs have suggested the benefits of vitamin D and analogs for prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1α,25(OH)2D3, the hormonally active form of vitamin D, and many of its classical synthetic analogs as potent agents for cancer prevention and treatment. To identify vitamin D analogs with better efficacy and low toxicity, we have tested more than 60 novel Gemini vitamin D analogs, which have a unique structure of two six-carbon chains with a C-20-normal and a C-20-epi side chain. Our initial studies found that some Gemini analogs are many-fold more active than 1α,25(OH)2D3 in growth inhibition assay. We further examined the inhibitory effect of selected Gemini vitamin D analogs against mammary carcinogenesis in vivo by using N-methyl-N-nitrosourea (NMU)-induced mammary tumor model which is ER-positive. Among Gemini vitamin D analogs we tested, Gemini 0072 and Gemini 0097 treated groups showed approximately 60% suppressive activity in NMU-induced mammary tumor growth compared to the control group. In a dose dependent experiment, Gemini 0097 significantly reduced the average tumor burden per rat without affecting the body weight. At all doses tested, Gemini 0097 did not exert any calcemic toxicity. In addition, we determined the inhibitory effect of Gemini 0097 in MCF10DCIS xenograft model of ER-negative mammary tumors. Gemini 0097 significantly inhibited MCF10DCIS xenograft tumor growth without calcemic toxicity. We analyzed the tumor samples and found that the inhibitory effect of Gemini 0097 in vivo was through (a) increasing cyclin dependent kinase inhibitor, p21 and (b) inducing insulin-like growth factor binding protein 3 (IGFBP3) in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogs may be potent agents for the prevention and treatment of both ER positive and ER negative breast cancer without calcemic toxicity.

Hexachlorobenzene Is a Tumor Co-carcinogen and Induces Alterations in Insulin-Growth Factors Signaling Pathway in the Rat Mammary Gland

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Controversy still exists about the breast carcinogenic properties of organochlorines in humans. The ligands, receptors, and related signaling proteins of the insulin growth factor family are involved in the regulation of breast-cancer cell growth. The aims of this study were to determine: (1) whether HCB is co-carcinogenic in a medium term assay of N-nitroso N-methylurea (NMU)-induced mammary tumors in rats; (2) the effect of HCB on insulin receptor (IR), insulin-like growth factor-I receptor (IGF-IR) and insulin receptor substrate-1 (IRS-1) levels and on IRS-1 phosphorylation; (3) microsomal and cytosolic Protein Tyrosine Kinase (PTK) activities in mammary glands and NMU-induced tumors. Sprague Dawley rats were injected with 50 mg/kg body weight of NMU at 50, 80, and 110 days old. HCB (100 mg/kg body weight) was administered three times a week from 65 to 110 days of age. Rats were separated in four groups: control, NMU, HCB, and NMU-HCB. HCB alone did not induce tumor development. Parameters of tumor development were increased in NMU-HCB compared to NMU rats. A higher cellular undifferentiation was observed in NMU-HCB tumors. IR, IGF-IR, and IRS-1 levels were higher in HCB than in controls. Conversely IGF-IR levels decreased in NMU-HCB vs. NMU group. The IRS-1 phosphorylation increased in HCB rats; however, it decreased in NMU-HCB vs. NMU. HCB decreased microsomal PTK activity in tumors. This study showed for the first time that HCB is a co-carcinogenic agent in NMU-induced mammary tumors in rats. Our results suggest that the IR and/or IGF-IR signaling pathway may be involved in the mechanism of action of HCB.

Gene expression profiling of NMU-induced rat mammary tumors: cross species comparison with human breast cancer

Breast cancer is a complex genetic disease characterized by the accumulation of multiple molecular alterations. The NMU breast cancer model induced in the rat is used for the study of mammary carcinogenesis because it closely mimics human breast disease. To assess the validity of this model from a more global molecular perspective, and also to devise a general technique to compare animal profiles with human microarray studies, we have characterized 25 NMU-induced mammary tumors and 11 normal glands using a combination of immunohistochemical and microarray analyses. The rat mammary carcinomas were classified as non-invasive, ER-positive ductal carcinomas with a composition of differentiated epithelial and myoepithelial cell lineages. Gene expression profiles generated using rat Affymetrix arrays containing 15,866 genes demonstrated that the rat mammary tumors are homogeneous and that H-ras mutations did not confer a unique molecular signature. We compared the resulting rat profiles with those obtained from a human dataset by merging the raw microarray data, using an approach that involves a combination of cross-species and cross-platform analysis. Using this novel strategy, we demonstrate the ability of 2305 rat orthologs to recapitulate the classification of human tumors derived from human Affymetrix arrays. The gene expression profiles of the NMU-induced primary tumors were most similar to ER-positive, low to intermediate grade breast cancer. Our technique provides a means to correlate gene expression data from animal models of cancer to human cancer and disease states.

Regression of NMU-induced mammary tumors with the combination of melatonin and 9- cis-retinoic acid

A significant increase in tumor regression was induced in N-nitroso- N-methylurea-induced mammary tumors in rats treated with the combination of melatonin and 9- cis-retinoic acid (9 cRA). Treatment groups included: control (ethanolic saline), 9 cRA (30 mg/kg chow/day), melatonin 500 μg/day, melatonin 1000 μg/day, melatonin 500 μg/day+9 cRA and melatonin 1000 μg/day+9 cRA. Rats treated with the lower dose of melatonin 500 μg+9 cRA show the greatest degree of tumor regression (78%), with 54% undergoing complete regression and a significant increase in apoptotic cells observed by TUNEL Assay. Furthermore, tumor multiplicity and burden were significantly decreased by the combination of melatonin and 9 cRA.

Synergistic effect of a retinoid X receptor-selective ligand bexarotene (LGD1069, Targretin) and paclitaxel (Taxol) in mammary carcinoma

We have previously shown that the retinoid X receptor (RXR) ligand bexarotene (LGD1069, Targretin) is efficacious as a chemopreventive and chemotherapeutic agent in rat N-nitroso-N-methylurea (NMU)-induced mammary carcinomas (Cancer Res 58: 479-484, 1998). To determine additional role for bexarotene in breast cancer treatment, we evaluated the effect of bexarotene on the efficacy of paclitaxel (Taxol) treatment in a rat NMU-derived mammary tumor cell line, NMU-417, in vitro and in rat NMU-induced mammary tumors in vivo. Our growth inhibition results showed that the bexarotene/paclitaxel combination produced a concentration-dependent synergy in NMU-417 tumor cell line. Synergistic growth inhibition by the combination was associated with an increase in cell death induced by both agents. In rat NMU-induced mammary tumor model in vivo, the benefit of combination therapy was observed as early as 1 week after treatment and increased as treatment continued. At the end of 6 weeks of treatment, the bexarotene/paclitaxel combination produced an overall objective response rate of 94% compared with a rate of 12% in paclitaxel-treated and 58% in bexarotene-treated animals, an effect that was more than the additive effects produced by single agents. Although both bexarotene alone and the bexarotene/paclitaxel combination reduced tumor multiplicity to similar extent, the combination regimen produced a statistically significant decrease in total tumor burden compared to single agents and untreated controls (two-tailed, p < 0.05). Combination therapy did not further alter body weight nor increase toxicity when compared to single agents. In summary, our results demonstrated the potential of using a RXR selective ligand in combination with chemotherapy for the treatment of breast cancer.

The effect of short intermittent light exposures on the melatonin circadian rhythm and NMU-induced breast cancer in female F344/N rats.

We investigated the effects of altered endogenous nighttime melatonin concentrations on mammary tumor production in an N-nitroso-N-methylurea (NMU)-induced breast cancer model in female Fischer 344 (F344)/N rats. Experiments were designed 1) to evaluate whether short-duration intermittent exposures to light at night would affect the nocturnal rise of melatonin, resulting in a decrease in nighttime serum melatonin concentrations, 2) to evaluate whether any suppression of nighttime serum melatonin concentrations could be maintained for a period of weeks, and 3) to determine the effects of suppressed serum melatonin concentrations on the incidence and progression of NMU-induced breast cancer. In vivo studies were used to assess serum melatonin concentrations after 1 day and 2 and 10 weeks of nightly administration of short-duration intermittent light exposure at night and incidence of NMU-induced tumors. Five 1-minute exposures to incandescent light every 2 hours after the start of the dark phase of the light: dark cycle decreased the magnitude of the nocturnal rise of serum melatonin concentrations in rats by approximately 65%. After 2 weeks of nightly intermittent light exposures, an average decrease of the peak nighttime serum melatonin concentrations of approximately 35% occurred. The amelioration continued and, at 10 weeks, peak nighttime serum melatonin concentrations were still decreased, by approximately 25%. Because peak endogenous nighttime serum melatonin values could be moderately suppressed for at least 10 weeks, a 26-week NMU mammary tumor study was conducted. Serum melatonin concentrations and incidence, multiplicity, and weight of NMU-induced mammary tumors were assessed. A group of pinealectomized (Px) animals was also included in the tumor study. No effect on the development of mammary tumors in an NMU-induced tumor model in rats occurred when endogenous nighttime serum melatonin concentrations were moderately suppressed by short-duration intermittent light exposures at night. At necropsy, there were no alterations in mammary tumor incidence (28/40 NMU controls, 28/40 NMU + light, 31/40 NMU + Px), multiplicity (2.18 tumors/tumor-bearing NMU control, 1.89 NMU + light, 2.39 NMU + Px), or average tumor weight (1.20 g NMU control, 1.19 g NMU + light, 0.74 g NMU + Px). Tumor burden had no effect on the serum melatonin cycle. At 26 weeks, however, animals exposed to intermittent light at night exhibited approximately 3-fold higher serum melatonin concentrations as compared with controls. Additionally, rats that had been pinealectomized at 4 weeks of age had serum melatonin concentrations that were markedly higher than the expected baseline concentrations for pinealectomized rats (<15 pg/ml), suggesting the reestablishment of a melatonin cycle. This finding was unexpected and suggests that melatonin can be produced by an organ or tissue other than the pineal gland.

Inhibition by dietary menhaden oil of cyclooxygenase-1 and -2 in N-nitrosomethylurea-induced rat mammary tumors.

Studies in laboratory animals and epidemiological surveys suggest a relationship between the type and amount of dietary fat and mammary cancer. One mechanism proposed to explain this relationship is modulation by dietary fat, of mammary tumor eicosanoid levels through action at the rate limiting enzyme in eicosanoid synthesis, cyclooxygenase (COX). Until recently there have been no studies which have examined COX gene expression in human breast or rodent mammary tissues. In this study we have demonstrated the presence of two immunoreactive isoforms of cyclooxygenase (COX-1 and -2), and the modulating effects of n-3 fatty acids on their expression, in N-nitrosomethylurea (NMU)-induced rat mammary tumors. Three different high fat diets were compared namely, corn oil (CO) 23%; CO 18% menhaden oil (MO) 5%; CO, 5%/MO 18%; low fat corn oil (5%) served as a control. It was found that immunoreactive COX-2 protein levels were approximately 3x higher than COX-1 levels in NMU-induced mammary tumors. Moreover, the high menhaden oil diet (rich in n-3 fatty acids) significantly suppressed both COX-1 (-28%) and COX-2 (-36%) protein levels when compared to the high corn oil diet. No differences were found among the other treatment groups when compared pair-wise or with low-fat control. The mechanism(s) by which n-3 fatty acids suppress COX-1 and COX-2 remain to be determined.

A comparative analysis of allelic imbalance events in chemically induced rat mammary, colon, and bladder tumors.

In this paper, patterns of allelic imbalances (Als) in chemically induced rat mammary, colon, and bladder tumors from (Wistar Furth x Fischer 344)F1 rats are described and compared. Male F1 rats were administered azoxymethane (AOM), and colon tumors were collected at 58 wk after treatment. Female F1 rats were given either N-nitroso-N-methylurea (NMU) or N-butyl-(hydroxybutyl)-nitrosoamine (BBN), and mammary and bladder tumors were collected at 15 and 52 wk after treatment, respectively. DNA was extracted from a subset of 18 of the largest tumors from each group, and a genome scan was performed by using polymerase chain reaction and 90 polymorphic microsatellite markers. Als, such as loss of heterozygosity, gene duplication, and microsatellite instability, were observed at low frequencies in all of the tumor models. Thirty random Als were observed in the AOM-induced colon tumors but only four in the NMU-induced mammary tumors. In both these models, all the tumors were classified as adenocarcinomas, and most of the Als observed were confined to single tumors with atypical histopathology. In contrast, 27 random Als were identified in the BBN-induced bladder tumors. Als were observed in both transitional-cell carcinomas and papillomas, although most were in the carcinomas. Statistical analysis of the Al data revealed no significant nonrandom Als within or among the tumor models, although several of the infrequently observed Al events identified in the rat tumors may also be observed in the corresponding human tumor type.

Radiochemical and Radiopharmacological Properties of Pirocarbotrat and Other Labeled Charcoal Dispersions: Comparative Studies in Rats with NMU-Induced Mammary Tumors

The purpose of this work is to study the physicochemical properties of Pirocarbotrat to explain its radiopharmacological behavior. We also studied a mixture of charcoal plus chromic [ 32P]phosphate and charcoal plus sodium [ 32P]orthophosphate only for comparative purposes. The results show that the mean diameter of the Pirocarbotrat particles was 2.5 μm with an homogeneous distribution, while the other products show an heterogeneous distribution of the particle sizes, with a mean size diameter between 0.5 and 0.9 μm. Hydrolysis studies with a solution of 0.1 N HCl and with sulfochromic mixture revealed that in Pirocarbotrat the 32P is strongly bound to the charcoal particles. Bioelimination studies of Pirocarbotrat show that the total eliminated activity was 12.70 ± 3.90%, with a higher amount in urine (8.30 ± 1.80%) than in feces (4.40 ± 3.50%). When biodistribution studies of Pirocarbotrat were carried out, we found that the 84.50 ± 2.60% of the activity remained in the tumor with almost null irradiation of the other organs under study. When therapeutic action was evaluated, we observed that the percentage of tumor regression was 78.3% for the tumors injected with Pirocarbotrat. The other dispersions under study showed different behaviors with high activity percentages distributed throughout the organism. These studies demonstrate that Pirocarbotrat has the best radiopharmacological properties to ensure irradiation of the tumor with the least concomitant irradiation of surroundings or other organs or tissues.

Prolactin is a local growth factor in rat mammary tumors.

PRL is a mitogenic hormone that shares many characteristics with growth factors. The recent demonstration that rat mammary tissue expresses PRL messenger RNA (mRNA) led us to hypothesize that PRL may act as an autocrine/paracrine growth factor in the mammary gland and may be a determinant in mammary carcinogenesis. To examine this, mammary tumors were induced in rats by injection of the carcinogen nitrosomethylurea (NMU). In vitro studies used a cell line derived from NMU-induced mammary tumors. Expression of PRL and PRL receptor was assessed by reverse transcriptase-polymerase chain reaction. The NMU-induced mammary tumors and the cell line express mRNA for both PRL and PRL receptor (the long and short isoforms); additional hybridizing polymerase chain reaction products were seen in the tumors, but not in lactating mammary tissue. Immunoreactive PRL was detected in the NMU-induced tumors. The effect of PRL on cell proliferation was assessed by culturing NMU cells with PRL antiserum. The PRL antiserum inhibited cell proliferation by up to 70% compared to the effect of normal rabbit serum or GH antiserum. In summary, we showed that NMU-induced mammary tumors express mRNA for PRL and PRL receptor. Addition of PRL antiserum to cultured NMU cells significantly inhibited their growth. We propose that PRL may be acting as a local growth factor that stimulates the proliferation of mammary tumors.