Induced Rat Mammary Tumors Research Articles

Label-free multimodal nonlinear optical imaging of needle biopsy cores for intraoperative cancer diagnosis.

.Significance: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care.Aim: We aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment.Approach: A portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers.Results: The intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75.Conclusions: A label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

Gene Expression Changes by Diallyl Trisulfide Administration in Chemically-induced Mammary Tumors in Rats.

Diallyl trisulfide (DATS) was shown to be a potent inhibitor of luminal-type MCF-7 xenograft growth in vivo. The present study was conducted to determine the preventive effect of DATS administration using an N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model, which shares molecular resemblance to luminal-type human breast cancers. The DATS administration (50 mg/kg body weight, 5 times/week) was safe, but did not reduce mammary tumor latency, incidence, burden or multiplicity. Therefore, we conducted RNA-seq analysis using mammary tumors from control and DATS-treated rats (n = 3 for each group) to gain insights into lack of mammary tumor prevention by this phytochemical. The gene ontology and the Kyoto encyclopedia of genes and genomes pathway analyses of the RNA-seq data revealed upregulation of genes associated with ribosomes, translation, peptide biosynthetic/metabolic process, and oxidative phosphorylation but downregulation of genes associated with mitogen-activated protein kinases. A total of 33 genes associated with ribosomes were significantly upregulated by DATS treatment, including RPL11 and RPS14. Western blotting confirmed upregulation of RPL11 and neurofascin protein expression in mammary tumors from DATS-treated rats when compared to controls. A statistically significant increase in protein level of c-Jun N-terminal kinase 2 was also observed in tumors from DATS-treated rats when compared to controls. On the other hand, expression of complex I subunits NDUFV1 or NDUFS1 was not affected by DATS treatment. These results offer potential explanations for ineffectiveness of DATS in the chemically-induced rat mammary tumor model. Inhibitors of the proteins upregulated by DATS may be needed to improve chemopreventive efficacy of this phytochemical.

Abstract 5243: Averting tumor growth in rodent breast cancer models with a liquid ablation approach

Abstract In low-resource settings, surgery or radiation are inaccessible to 90% of cancer patients due to inadequate infrastructure and lack of trained personnel. Resource appropriate technologies must be developed to make cancer treatments accessible. Ablative techniques, such as radiofrequency ablation are often used as a minimally invasive alternative to surgery. However, energy-based ablation techniques require expensive machinery and a steady supply of electricity and expensive consumables. To address these shortcomings, we have developed a low-cost ablation technique that stabilizes tumor growth with a simple injection of ethanol in combination with a polymer, ethylcellulose (EC). The EC injected with ethanol creates a slow-release depot of ethanol at the injection site by undergoing a solvent induced phase-transition initiated by water in tissues. EC-ethanol improves upon traditional ethanol ablation by providing increased precision while remaining low-cost and simple to administer. We evaluated the precision of EC-ethanol as well as its vascular sclerosing capabilities as a strategy to avert tumor growth in rodent models of breast cancer. EC-ethanol injected at 1 mL/hr with 6% w/w EC was used as this demonstrated to be the optimal formulation and rate for causing tumor necrosis (Morhard et al., Scientific Reports, 2017) and enhanced the anti-tumor immunostimulatory response (Nief et al., 2019, Under Review). We demonstrate the precision delivery of EC-ethanol in orthotopic 67NR mammary tumor as with small-animal MRI. Further, ethanol devascularization is monitored with photoacoustic imaging 24 hours post ablation. Local tumor control was demonstrated both in 67NR mammary tumor and in 7,12-Dimethylbenz[a]anthracene (DMBA) induced rat mammary tumor models. Repeated ablations on significantly decrease primary tumor growth compared to untreated tumors in both the mouse and rat models (P<0.05). In rats weekly 200 µL EC-ethanol injections began when tumors reached 2 mL in volume (n=5), and in 30 days tumors decreased 20% ± 3% untreated controls grew an average of 721%. For mice (n=5), 50 µL was given daily for 6 days on day 3 after tumor implantation to account for small body weight and rapidly growing tumors. Mice treated with EC-ethanol lived significantly longer (P<0.05 log-rank test) than untreated mice and mice injected with the same volume of pure ethanol. EC-ethanol is a low-cost, non-invasive tumor destruction method that shows a clear control of tumor growth when applied as a fractionated dose. We utilized a mouse and rat tumor model to ensure that the EC-ethanol's success is not dependent on the model. Ongoing studies will examine the impact of varying injected volumes at well-defined fractionation schedules on ethanol distribution, necrosis, tumor growth, and survival. Citation Format: Corrine A. Nief, Erika Chelales, Robert Morhard, Maomao Chem, Jeffrey Everitt, Jenna Mueller, Junjie Yao, Mark W. Dewhirst, Nirmala Ramanujam. Averting tumor growth in rodent breast cancer models with a liquid ablation approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5243.

Influence of paclitaxel therapy on expression of ßIII-Tubulin and Carbonic anhydrase IX proteins in chemically-induced rat mammary tumors

The manuscript is focused on analyze of βIII-Tubulin and Carbonic anhydrase IX proteins in experimental model of chemically induced rat mammary tumors. Overexpression of class III β-tubulin, a factor that confers dynamic properties to microtubules, is a candidate tumor biomarker for resistence to microtubule - targeting therapy in many solid tumors, including breast cancer. The expression of CAIX protein in malignant tumors plays an important role in progression of primary tumors and also contributes to increase risk of metastases. Nineteen rat females were used in this study. Mammary tumors were induced by 7,12 dimethylbenz(a)anthracene (DMBA). Rats were divided into two groups, group treated by paclitaxel and non-treated (control) group that was administered physiological solution. According to the tumor stage, they were subdivided into carcinoma in situ (CIS) and invasive carcinoma (IC). To detect the expression of both proteins, we used immunohistochemical staining (IHC). The ELISA method was used to detect the protein level in blood. The expression of CAIX and sIII-tubulin increased significantly not only in mammary tumors, but also in blood of treated groups examined by ELISA method (p < 0.05). Additionally, the results revealed significant increase in the expression of sIII-tubulin in IC samples and CAIX in CIS samples, when we compared treated to non-treated groups (p < 0.05). Paclitaxel-induced sIII-tubulin and CAIX overexpression in mammary tumors and their increase in tissue and serum should indicate drug-induced increase of resistance. The potential benefits of this finding is still uncertain, but our findings can contribute to newly developing diagnostic opportunities and therapy of the mammary tumors.

Chemopreventive Effects of Propolis in the MNU-Induced Rat Mammary Tumor Model.

Currently, one of the central problems in cancer management is the relapse of disease following conventional treatments, yet few therapeutic agents targeting resistance and tolerance exist. Propolis is known as a healing agent since ancient times. Therefore, over time, its curative properties have kept the interest of scientists, thus leading permanently to investigations of its other possible undiscovered effects. In this context, current experiments were performed to establish the chemopreventive potential of propolis extract (PE) (1.05 mg/kg BW/day) in N-methyl-N-nitrosourea- (MNU-) induced rat mammary tumors. MNU-inoculated/PE-treated rats had tumors of different physical attributes compared with control rats MNU-inoculated. The number of developed tumors (mean 49% versus 100%), incidence (mean 49% versus 100%), multiplicity (1.8 versus 3.7 (p < 0.001)), tumor volume (mean 10 cm3 versus 16 cm3 (p < 0.001)), and weight of the tumor mass (mean 7.42 g versus 9.00 g (p < 0.05)) were noted. The numbers of grade I tumors recorded for MNU-inoculated rats were 24 (Group 1) and 7 (Group 2) for MNU-induced/PE-treated rats. In the serum of rats MNU-inoculated/PE-treated were found higher levels of antioxidative enzymes (SOD, CAT, and GPx) than in MNU-induced. Taken together, these data indicate that propolis could be a chemopreventive agent against MNU-induced mammary carcinogenesis.

Blood flow, volume and arterio-venous passages in induced mammary tumours of the rat

ObjectiveTo study blood flow, vascular volume and arterio-venous passages in induced mammary tumours of the rat to characterize parameters possibly responsible for tumour hyponutrition. MethodDimethylbenzanthracene-induced mammary tumours in Sprague-Dawley rats were studied. Regional blood flow was studied by use of the radioactive microsphere tracer technique using 141Cerium-labelled 15μm spheres coinjected into the left cardiac ventricle with 125Iodine-labelled 25μm spheres. Blood volume was studied by use of 125Iodine- or 99mTechnetium-labelled human serum albumin, the latter allowing autoradiography of tumour sections for visualization of flow and volume. ResultsTwenty-seven rats with 170 tumours had a mean tumour blood flow of 48 and 67mL×min−1×100g−1 using 15 and 25μm sphere data, respectively, indicating a significant passage through vessels between 15 and 25μm. The lungs showed a “nominal bronchial” blood flow of 260 and 135mL×min−1×100g−1 for the 15 and 25μm spheres, respectively, indicating pulmonary trapping, particularly of small spheres passing the systemic circulation in vessels larger than 15μm. There was a positive correlation between the total tumour blood flow within individual rats and trapped spheres of both dimensions in the lungs, indicating shunts also larger than 25μm.Normal tissues disclosed only small differences in regional blood flow as measured by the two spheres.Blood volume was studied in 20 rats with 120 tumours, with a vascular volume of 3.6mL×100g−1 representing a blood turnover >15 times/min. Blood volume co-localized with perfusion as seen in autoradiographs. ConclusionIn induced rat mammary tumours, a high fraction of blood, 28%, passes arterio-venous vessels between 15 and 25μm and there also exist passages >25μm. These findings indicate that the functional capacity of the tumour vascular bed might be impaired, adding to the abnormal microenvironment of tumours.

Anti-Tumor Action, Clinical Biochemistry Profile and Phytochemical Constituents of a Pharmacologically Active Fraction of S. crispus in NMU-Induced Rat Mammary Tumour Model.

Cancer patients seek alternative remedies such as traditional medicinal plants for safe and effective treatment and help overcome the side effects of conventional therapy. Current knowledge indicates that extracts of Strobilanthes crispus of the Acanthaceae family exhibit potent anticancer properties in vitro and are non-toxic in vivo. S. crispus was also reported to be protective against chemical hepatocarcinogenesis. We previously showed that a bioactive fraction of S. crispus leaves also synergized with tamoxifen to cause apoptosis of human breast cancer cell lines without damaging non-malignant epithelial cells. The present study aimed to evaluate the antitumor effect of S. crispus dichloromethane fraction (F3) using N-methyl-N-Nitrosourea (NMU)-induced rat mammary tumor model. Tumor regression was observed in 75% of the rats following 8-week oral administration of F3 with no secondary tumour formation and no signs of anemia or infection. However, no improvement in the liver and renal function profiles was observed. Major constituents of F3 were identified as lutein, 131-hydroxy-132-oxo-pheophytin a, campesterol, stigmasterol, β-sitosterol, pheophytin a and 132-hydroxy-pheophytin a. These compounds however, may not significantly contribute to the antitumor effect of F3.

Immunohistochemical evaluation of chemopreventive effect of hydro-alcoholic extract of garlic (Allium sativum) in N-methyl-N-nitrosourea (NMU) induced rat mammary tumours

Consumption of diets rich in vegetables and herbs is often associated with a reduced risk of developing cancer,particularly breast cancer.Many different cultures throughout the world have recognized the potential use of garlic for prevention and treatment of different diseases.The aim of the present study was to evaluate the chemopreventive effect ofhydro-ethanolic extract of Allium sativum(AS)on the expression pattern of biomolecular markers such as, proliferating cell nuclear antigen (PCNA),vascular endothelial growth factor (VEGF), and caspase-3 during N-methyl-N-nitrosourea (NMU) induced mammary carcinogenesis in Wistar rats.The NMU was injected at 50, 80, and 110days of age in both treatment and preventive group. In untreated control animals, 18 numbers of mammary tumours were successfully induced in 6 (50%) rats with average latency period of 85.84±2.47 days. In preventive group, the average latency period was 141.66±4.09 and total four tumours developed in 2 (16.67%) rats. Immunohistochemical analysis revealed over expression of PCNAand VEGF, and decreased expression of caspase-3 in the mammary tissues of NMU treated rats. Oral administration of hydro-ethanolic extract of AS in NMU treated rats significantly down regulated the expression of PCNA, VEGF and up regulated the expression of caspase-3. In conclusion, the hydro-ethanolic extract of AS treatment reduced cellular proliferation, angiogenesis, and induced more apoptosis in NMU induced mammary carcinogenesis in Wistar rats.

Evaluation of apoptosis using TUNEL assay and caspase-3 immunostaining in NMU induced rat mammary tumours

Avoidance of apoptosis is an important event of tumourigenesis. Tumour progression is dependent on balance between cell proliferation and apoptosis. The present study was carried out to evaluate apoptosis in N-methyl nitrosourea (NMU) induced rat mammary tumours by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL) assay in correlation with caspase-3 and PCNA immunostaining. A total of 22 female virgin Wistar rats of 28–30 days of age were selected for the experiment. NMU was injected at 50th, 80th and 110th days of age in 16 rats and 6 control group rats received the vehicle only. Six numbers of mammary tumours were successfully induced in 4 (25%) rats with average latency period of 87±2.63 days. The mean counts of PCNA and caspase-3 immunostaining were 57.67±9.45 and 1.82±0.21, respectively. The mean count of the TUNEL positive cells were 2.44±0.69. It was observed that apoptosis was evident by TUNEL assay and caspase-3 immunostaining in highly proliferative and more aggressive NMU induced rat mammary tumours, similar to that observed in human breast tumours.

Determination of cell proliferative activity by immnunohistochemical detection of proliferating cell nuclear antigen in N‐methyl nitrosoureainduced rat mammary tumours

Rodent experimental models are considered useful experimental systems to study mammary cancer, which occurs in rodents as a result of hormonal imbalance, much like the breast cancer in women. Rodent cells are easier to transform by chemical carcinogen due to their relatively poorer controls of genetic stability and DNA repair systems. Proliferating cell nuclear antigen (PCNA) is one of the most sensitive biomarkers to measure the cell proliferative activities in the neoplasms, especially when the otherwise undetectable PCNA protein over‐expresses to the immunohistochemically detectable levels. In the present study, mammary tumours were induced in Sprague Dawley rats by intraperitoneal administration of N‐methyl nitrosourea (MNU) chemical carcinogen. Palpable tumours were noticed after 60 days postinjection (dpi) with average latency period of 144 days, and 12 (46.15%) of 26 rats developed mammary tumours. A total of 18 tumours were diagnosed in 12 rats, including 2 cases (10%) of hyperplasia, 1 (5%) benign and 17 malignant tumours (85%). Malignant tumours included in situ ductal papillary carcinoma (11.11%), in situ ductal cribriform carcinoma (38.88%), invasive tubular adenocarcinoma (5.56%), invasive papillary carcinoma (16.67%) and invasive cribriform carcinoma (22.22%) were. PCNA immunohistochemistry revealed moderate to strong nuclear staining in neoplastic cells, with highest mean PCNA index of 55.53±10.13 in invasive cribriform carcinoma. The PCNA indices were significantly different (p&lt;0.01; one way ANOVA) between control mammary gland, hyperplasia, benign and malignant tumours. It was concluded that the measurement of PCNA expressing cells employing immunohistochemistry can help in the determination of the level of malignancy in MNU‐induced rat mammary tumours.

Upregulated Myc Expression in N-Methyl Nitrosourea (MNU)-induced Rat Mammary Tumours

The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75- 91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth

The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) such as docosahexaenoic acid (DHA, 22:6n−3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n−3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n−3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.

Molecular mechanisms of melatonin’s inhibitory actions on breast cancers

Melatonin is involved in many physiological functions and it plays an important role in many pathological processes as well. Melatonin has been shown to reduce the incidence of experimentally induced cancers and can significantly inhibit the growth of some human tumors, namely hormone-dependent cancers. The anticancer effects of melatonin have been observed in breast cancer, both in in vivo with models of chemically induced rat mammary tumors, and in vitro studies on human breast cancer cell lines. Melatonin acts at different physiological levels and its antitumoral properties are supported by a set of complex, different mechanisms of action, involving apoptosis activation, inhibition of proliferation, and cell differentiation.

Morphology of 1-methyl-1-nitrosourea induced rat mammary tumours after treatment with precursor of phytanic acid or its combination with vitamin D analogue

The proposed therapeutical effect of phytol (PHY), a precursor of the phytanic acid (PHYA), on mammary tumours induced with 1-methyl-1-nitrosourea (MNU), was investigated in Sprague-Dawley rats in combination with vitamin D analogue, Seocalcitol (SEO). Female Sprague-Dawley rats were administered intraperitoneally with MNU (50 mg/kg of body weight) at the 46th and 52th days of age. Controls and MNU animals received propyleneglycol appropriate to their body weight. PHY (MNU + PHY) (500 mg/kg) was administered after tumour detection (approximately in 100th day of the life) three times/week. Combination of PHY with SEO (7 μg/kg per week) was administered to rats after tumour detection (approximately in 100th day of the life) until the 181st day of age. Then the animals were sacrificed, the tumours removed, and fixed in 10% formalin. Haematoxylin and eosine stained sections were evaluated under microscope. Tumour invasiveness observed in all groups of animals was ranging from 80 to 90%. Treatment with PHY alone did not inhibit the progression of the MNU induced tumours in the rat breast but it decreased the tumour burden and volume in comparison with MNU treated controls. Decreased tumour burden and volume were induced by combined treatment of PHY with SEO. Malignity and invasivity of carcinomas were not affected. No redifferentiating effect on mammary tumour cells induced by NMU after treatment with PHY alone or in combination with SEO was observed in rats. SEO alone or in combination with PHY inhibited the progression of MNU induced mammary tumours and also inhibited the increase of tumour burden and volume in comparison with MNU treated control group. However, none of the compounds, either alone or in mutual combination, reduced the malignity or the number of invasive tumours in this experimental study.

Hypothalamus–pituitary–thyroid axis disruption in rats with breast cancer is related to an altered endogenous oxytocin/insulin-regulated aminopeptidase (IRAP) system

Associations of breast cancer with diseases of the thyroid have been repeatedly reported, but the mechanism underlying this association remains to be elucidated. It has been reported that oxytocin (OXT) attenuates the thyroid-stimulating hormone (TSH) release in response to thyrotrophin-releasing hormone (TRH) and decreased plasma levels of TSH as well as the thyroid hormones by an effect mediated by the central nervous system. Oxytocinase (IRAP) is the regulatory proteolytic enzyme reported to hydrolyze OXT. Changes in IRAP activity have been reported in both human breast cancer and N-methyl-nitrosourea (NMU)-induced rat mammary tumours. Here, we measure IRAP activity fluorometrically using cystyl-β-naphthylamide as the substrate, in the hypothalamus-pituitary-thyroid axis together with the circulating levels of OXT, and its relationship with circulating levels of TSH and free thyroxine (fT4), as markers of thyroid function in control rats and rats with breast cancer induced by NMU. We found decreased thyroid function in rats with breast cancer induced by NMU, supported by the existence of lower serum circulating levels of both TSH and fT4 than their corresponding controls. Concomitantly, we found a decrease of hypothalamic IRAP activity and an increase in circulating levels of OXT. We propose that breast cancer increases OXT pituitary release by decreasing its hypothalamic catabolism through IRAP activity, probably due to the alteration of the estrogenic endocrine status. Thus, high circulating levels of OXT decreased TSH release from the pituitary, and therefore, of thyroid hormones from the thyroid, supporting the association between breast cancer and thyroid function disruption.

Intrinsic Susceptibility MR Imaging of Chemically Induced Rat Mammary Tumors: Relationship to Histologic Assessment of Hypoxia and Fibrosis

To investigate relationships between magnetic resonance (MR) imaging measurements of R2* and carbogen-induced DeltaR2* in vivo with subsequent histologic assessment of grade, hypoxia, fibrosis, and necrosis in a chemically induced rat mammary tumor model. All experiments were performed in accordance with the local ethics review panel, the UK Home Office Animals Scientific Procedures Act of 1986, and the UK Co-ordinating Committee on Cancer Research guidelines. Of 30 rats injected with N-methyl-N-nitrosourea, 17 developed mammary tumors. Prior to MR imaging, rats were administered pimonidazole. Tumor R2* was then quantified while the host first breathed air and then carbogen (95% O(2), 5% CO(2); n = 16). Tumor sections were subsequently stained for pimonidazole, sirius red, cytokeratin 14, and hematoxylin-eosin for quantitative assessment of hypoxia, fibrosis, malignancy, and necrosis, respectively, and graded according to the Scarff-Bloom-Richardson scale. Linear regression analysis was used to identify any correlates of the MR imaging data with histologic data. Tumors exhibited wide heterogeneity in the magnitude of carbogen-induced reduction in R2*, an emerging imaging biomarker of fractional blood volume. Significant correlations were found between pimonidazole adduct formation and both baseline tumor R2* (r = -0.54, P = .03) and carbogen-induced DeltaR2* (r = 0.56, P = .02), demonstrating that tumors with a larger fractional blood volume were less hypoxic. There was also a significant correlation between pimonidazole and sirius red staining (r = 0.76, P < .01), indicating that more fibrotic tumors were also more hypoxic. There were no correlations of R2* with grade. In this model of breast cancer, baseline tumor R2* and carbogen-induced DeltaR2* are predictive imaging biomarkers for hypoxia and primarily determined by blood volume.

Antitumor activity of edotecarin in breast carcinoma models

Edotecarin (J-107088, formerly ED-749) is a potent indolocarbazole topoisomerase-I inhibitor that has the potential to treat solid tumors. The current studies evaluated the potency and antitumor activity of edotecarin, as a single agent and in combination with capecitabine or docetaxel. Antiproliferative activity was tested in vitro in a panel of 13 mammary cell lines and antitumor efficacy was tested in vivo in various breast cancer models. Edotecarin inhibited cellular proliferation in breast carcinoma cell lines: 50% inhibitory concentrations ranged from 8 nmol/L in SKBR-3 cells to approximately 30 micromol/L in BT20 cells. Single dose and weekly intravenous treatments with edotecarin 30 and 150 mg/kg produced significant antitumor activity in the SKBR-3 human breast carcinoma xenograft model, with no major toxicities, compared with vehicle solvent treatment. Daily administration of edotecarin 15 mg/kg for 10 days was not well tolerated, whereas the total dose of 150 mg/kg was safe when administered in a single injection. Edotecarin 3 and 30 mg/kg given after docetaxel in the nude mouse SKBR-3 xenograft model produced tumor growth delays that were greater than those observed with either agent alone and with no toxicity as evaluated on the basis of body weight reduction (<20%). Furthermore, edotecarin 3 mg/kg in combination with capecitabine produced more than additive effects and the combination was well tolerated. However, edotecarin at a dose of 30 mg/kg in combination with capecitabine was lethal. Edotecarin also exhibited potent antitumor activity against xenografted human MX-1 cells, MMTV-v-Ha-ras oncogene-driven mouse breast tumors, and chemically induced rat mammary tumors. The data suggest that edotecarin may be useful as a single agent or a component of combination chemotherapy regimens for treating human breast cancer.

Role of Polyamines in Breast Cancer Growth, Development and Progression

The polyamines putrescine, spermidine, and spermine are small, aliphatic amines that play an important role in multiple cellular functions. Activation of the polyamine pathway is involved in carcinogenesis and other aspects of tumor biology including in breast cancer. Levels of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis as well as the levels of polyamines, are higher in human breast cancer specimens than in normal and benign breast tissue. Inhibition of ODC with -difluromethylornithine exerts a protective effect in the promotion of chemically induced rat mammary tumors as well as in the development of breast cancers in a transgenic model system. Inhibition of polyamine biosynthesis has also been shown to inhibit the growth of hormone-dependent and -independent mammary tumors, both in vitro and in vivo, thus indicating a major role for polyamines in breast cancer cell proliferation. Several lines of evidence indicate that increased ODC activity may contribute to breast cancer progression. Increased tumor ODC activity has been shown to be an independent, adverse prognostic factor for overall survival in women with localized breast cancer. This finding suggests that polyamines may be critically involved in the development of breast cancer metastasis. We have, indeed, observed that administration of DFMO markedly reduced in vitro invasiveness of the hormone-independent MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. More importantly, we have observed that DFMO administration significantly reduced the development of pulmonary metastasis from orthotopically implanted MDA-MB-435 breast cancer xenografts in nude mice. The mechanism of antitumor action of DFMO is currently under active investigation. Several laboratories have shown that inhibition of polyamine biosynthesis results in activation of the MAPK pathway and STAT signaling. We have recently shown that DFMO induced activation of the MAPK pathway in MDA-MB-435 human breast cancer cells is directly involved in the production of the anti-invasive protein, thrombospondin-1. Inhibition of MAPK phosphorylation suppressed DFMO-induced stimulation of thrombospondin-1 and reversed its anti-invasive effect. Collectively, these data indicate that the polyamine pathway may be an attractive target for breast cancer prevention and treatment. Keywords: antipolyamines, breast cancer, cell proliferation, tumor progression

Characteristic Patterns of VEGF, Integrins, ER.ALPHA. and HER2 Immunoreactivity Suggest Two Tumor Cell Populations in DMBA-Induced Rat Mammary Tumor

Recent evidence has shown that certain tumors have characteristic cell populations related to tumor progression. Therefore, we investigated tumor cell populations in 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumor, an established estrogen-sensitive angiogenic tumor model. The tumors pathologically identified as invasive ductal carcinoma were immunostained using antibodies against the following various molecules: vascular endothelial growth factor (VEGF), integrins, estrogen receptor alpha (ERα), human epidermal growth factor receptor-2 (HER2), p27, and laminin, all of which are known to be related to tumor progression, proliferation and/or prognosis. The basal layer, the outermost layer of tumor lobules facing the basement membrane, was mainly VEGF and integrin (α3, α6, β1) positive. In contrast, the inner layer, the internal cell layers of the tumor lobules, was mainly ERα and HER2 positive. The p27-positive rate in the inner layer was similarly low to that in the basal layer, suggesting a similar high proliferative potency between these layers. Laminin was positive in the basement membrane surrounding the tumor lobules. These results suggest that the tumor lobules consist of at least two cell populations, the basal and inner layers, with respect to the expression of VEGF, integrins, ERα and HER2; and that these cell populations may have distinct functions in the pathophysiology of this mammary tumor.

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.