MNU-induced Breast Cancer Research Articles

PP151 Preclinical evaluation of NK-1 receptor antagonist and COX-2 inhibitor against i.duc MNU-induced breast cancer in female Wistar rats

PP151 Preclinical evaluation of NK-1 receptor antagonist and COX-2 inhibitor against i.duc MNU-induced breast cancer in female Wistar rats

Histopathological Effect of Annona Muricata Fruit Extract on Methyl nitrosourea -Induced Mammary Gland Carcinoma in Female Albino Mice

The Annona muricata (Family-Annonaceae) fruit contain annonaceous acetogenins which have an effect on cancer cell as reported in many research studies. This study was aimed to evaluate the effect of methanolic fruit extract of Annona muricata (MFEAM) on breast cancer histology and invasive cell of MNU-induced breast cancer mice. Fifty female albino mice were divided into five groups (of ten each): control; MNU-induced breast cancer; MNU-induced breast cancer + (MFEAM) 200 mg/kg, MNU-induced breast cancer + (MFEAM) 300 mg/kg and MNU-induced breast cancer + (MFEAM) 500 mg/kg. The research indicates that breast cancer invasive ductal carcnoma (IDC) developed in the MNU-induced breast cancer group. The control group showed normal duct structure. The (MFEAM) -treated group showed lower invasive (DCIS) compared with the untreated cancer group. We concluded that (MFEAM), that improved the histological changes of breast cancer-induced MNU. Also (MFEAM) reduced proliferative epitheal cell of breast cancer-induced MNU and the most effective dose was 300 mg/kg. The present study concluded that the methanol extracts derived from Annona muricata fruit potentially exhibit antitumor activity. The control group showed normal duct structure. The (MFEAM) -treated group showed lower invasive (DCIS) compared with the untreated cancer group, We concluded (MFEAM) that improved the histological changes of breast cancer-induced MNU. Also (MFEAM) reduced proliferative epitheal cell of breast cancer-induced MNU and the most effective dose in 300 mg/kg.

Therapeutic Role of Annona muricata Fruit and Bee Venom Against MNU-Induced Breast Cancer in Pregnant Rats and its Complications on the Ovaries.

AimTo evaluate the potential therapeutic role of Annona muricata (graviola) fruit and bee venom (BV) against N-methylnitrosourea (MNU)-induced breast cancer in pregnant female rats and complications in the ovaries.MethodsA total of 24 female rats were induced with a single dose of MNU (50 mg/kg body weight). After confirmation of positive tumor marker, female rats were placed with the males for mating. The pregnant rats were randomly divided into four groups (n=6): MNU-induced only (group 1), MNU-induced rats and supplemented with A. muricata 200 mg/kg diet (group 2), MNU-induced and treated with two doses of BV 75 μg/kg (group 3), and MNU-induced and treated with both A. muricata and BV (group 4).ResultsIn group 1, the breast tissue of mothers revealed pronounced cellular hyperplasia and histopathological signs. Also, the ovarian tissue of mothers and their offspring displayed deleterious histological changes. In groups 2 and 4, histopathological signs and cellular hyperplasia markedly disappeared in breast tissue. However, the histopathological signs induced by MNU in the ovarian tissue reversed to normal in groups 2–4. Also in groups 2–4, levels of serum MMP1, NFκB, and TNFα significantly decreased, and serum caspase 3 significantly increased either in mother rats or their offspring compared to the MNU-alone group. Levels of serum MDA significantly decreased; however, levels of serum antioxidants (CAT and SOD) significantly increased in all groups 2–4 compared to MNU-alone group.ConclusionA. muricata has a more powerful therapeutic role than BV against MNU-induced breast cancer in rats; however, both have a powerful ameliorative role against ovarian histopathological alterations induced by MNU. Such ameliorative effects of A. muricata and BV are mainly attributed to their antioxidant, anti-inflammatory, and antiproliferative constituents.

Protective Effects of Antioxidant Chlorophyllin in Chemically Induced Breast Cancer Model In vivo.

Glutathione-related enzymes belong to the protection mechanism of the cells against harmful oxidative damage and chemicals. Glutathione S-transferase (GST) is frequently over-expressed in various cancer cells and is involved in drug resistance. Chlorophyllin is an antioxidant molecule interfering with the GST P1-1 activity. The purpose of this study is to evaluate the short- and long-term protective effects of chlorophyllin as an antioxidant molecule on DNA damage, antioxidant enzyme activities, trace elements, and minerals in chemically induced breast cancer model in vivo. In our study, N-methyl-N-nitrosourea (MNU) was used for inducing breast carcinogenesis in female Sprague-Dawley rats. A total of 36 rats were divided into groups as short term and long term. Each group was divided into four sub-groups as control group received physiological saline solution (n= 3), Chl group (n= 5) received chlorophyllin, MNU group (n= 5) was administered MNU, and Chl + MNU group (n= 5) was treated with both chlorophyllin and MNU. Results illustrated that chlorophyllin had a significant anti-genotoxic effect in the short term, and glutathione-related enzyme activities were protected by chlorophyllin treatment in MNU-induced breast cancer model. Additionally, MNU administration impaired mineral and trace element levels including Na, Mg, K, Fe, Zn, and Co in the liver, kidney, spleen, heart, and tumor tissues; however, adverse effects of MNU were recovered upon chlorophyllin treatment in the indicated tissues of the rats. In conclusion, chlorophyllin can be used as an antioxidant molecule to ameliorate adverse effects of MNU by enhancing antioxidant enzyme activities and regulating trace element and mineral balance in several organs and tumor tissue in the breast cancer model.

Evaluation of estrogenic potency of a standardized hops extract on mammary gland biology and on MNU-induced mammary tumor growth in rats

Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E2-benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms.

Abstract 5270: Disease subtype independent biomarkers of breast cancer prevention by withaferin a

Abstract Breast cancer is a rather complex and heterogeneous disease broadly grouped into four major subtypes, including luminal-type, basal-like, HER2 amplified, and normal-like, and each with a distinct molecular signature. A non-toxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study not only demonstrates chemoprevention of breast cancer in rats by the Ayurvedic medicine phytochemical withaferin A (WA) but also identifies its mechanistic biomarkers common to different subtypes of this disease. Chemopreventive efficacy of WA (4 and 8 mg per kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by western blotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat and/or mouse mammary tumor virus-neu (MMTV-neu) models. Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and MDA-MB-231 cells. Incidence, multiplicity, and burden of MNU-induced breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg per kg group was lower by 67% compared with controls (P = 0.004). Mitotic arrest and apoptosis induction were common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured MCF-7 and MDA-MB-231 cell lines. WA is a promising phytochemical with the ability to inhibit at least two different subtypes of breast cancer, including neu-driven estrogen receptor negative (ER-) breast cancer in MMTV-neu model and MNU-induced ER+ breast cancer in rats. This study was supported by the grant RO1 CA142604-07 awarded by the National Cancer Institute. Citation Format: Eun-Ryeong Hahm, Suman K. Samanta, Anuradha Sehrawat, Su-Hyeong Kim, Subrata K. Pore, Krishna B. Singh, Susan M. Christner, Yongli Shuai, Jan H. Beumer, Ruchi Roy, Nancy E. Davidson, Shivendra V. Singh. Disease subtype independent biomarkers of breast cancer prevention by withaferin a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2017-5270

Feature of Experimental Breast Cancer Induced by Intermammary Administration of N-Methyl-N-Nitrosourea in Wistar Rat

Abstract. Breast cancer is the leading cause of cancer death in women worldwide due to the complicated etiology involving both genetic and environmental factors. BC was induced by N-Methyl-N-Nitrosourea administration intermammary in Wistar female. Studied frequency of development of NMU-induced BC, phenotype of BC cells, phenotype of MSC from mammary gland tissue, quantity and quality of immunocompetent cells, expression of miRNAs, hormone levels and cytokines were done for estimate sensitivity to chemotherapy.We showed that MNU-induced BC developed in 90%, immunohistochemical similar to human luminal A type of BC with higher proliferation activity. We estimated significantly increased count of BC-MSC with CD45+CD90-, and CD45+CD90+, and CD45-CD90+ phenotype in animal with MNU-induced BC. Chemotherapy caused changes in quality and quantity of immunocompetent cells, decrease of prolactin and luteinizing hormone levels, and some changes in expression of pro-oncogenic or tumor-suppressing miRNAs. In MNU-induced BC animal were observed increased serum levels of pro-inflammatory cytokines. In regional lymph nodes found increased count of macrophages and presence of tumor cells. Intermammary administration of NMU possessed to breast cancer development with similarities to human luminal B phenotype. This MNU-induced breast cancer may serve as a model to study efficiency of chemotherapy.

Rapamycin and PF4 induce apoptosis by upregulating Bax and down-regulating survivin in MNU-induced breast cancer.

To elucidate the role of rapamycin and PF4 on apoptosis regulation via Bax (pro-apoptosis), Bcl-2 (anti-apoptosis) and survivin activation on the growth in the 1-methyl-1-nitrosourea -induced invasive breast carcinoma model. Thirty five female Sprague Dawley rats at age 21-day old were divided into 4 groups; Group 1 (control, n=10), Group 2 (PF4, n=5), Group 3 (rapamycin, n=10) and Group 4 (rapamycin+PF4, n=10). MNU was administered intraperitionally, dosed at 70 mg/kg body weight. The rats were treated when the tumors reached the size of 14.5 ± 0.5 mm and subsequently sacrificed after 5 days. Rapamycin and PF4 were administered as focal intralesional injections at the dose of 20 μg/lesion. The tumor tissue was then subjected to histopathological examinations for morphological appraisal and immunohistochemical assessment of the pro-apoptotic marker, Bax and anti-apoptotic markers, Bcl-2 and survivin. The histopathological pattern of the untreated control cohort showed that the severity of the malignancy augments with mammary tumor growth. Tumors developing in untreated groups were more aggressive whilst those in treated groups demonstrated a transformation to a less aggressive subtype. Combined treatment resulted in a significant reduction of tumor size without phenotypic changes. Bax, the pro-apoptotic marker, was significantly expressed at higher levels in the rapamycin-treated and rapamycin+PF4-treated groups compared to controls (p<0.05). Consequently, survivin was also significantly downregulated in the rapamycin-treated and rapamycin+PF4-treated group and this was significantly different when compared to controls (p). In our rat model, it could be clearly shown that rapamycin specifically affects Bax and survivin signaling pathways in activation of apoptosis. We conclude that rapamycin plays a critical role in the induction of apoptosis in MNU-induced mammary carcinoma.

Abstract P5-05-09: Extract ERr731® does not mimic estrogen stimulated growth of MNU-induced breast cancer in ovariectomized Sprague-Dawley rats

Abstract Background: ERr731® (trade names Phyto-Strol® or femi-loges®), an extract isolated from the roots of the Siberian rhubarb (Rheum rhaponticum), is used as an alternative treatment option for menopausal complaints. In addition, it is regularly used for the treatment of oligomenorrhoea or amenorrhea. The efficacy of the extract on treatment of menopausal complaints was shown in clinical trials and follow-up studies. Although clinical trials did not show changes of mammographic density, we addressed the question whether ERr731 is capable to stimulate experimental breast cancer in an estrogen dependent manner. Objective: The aim of the study was to assess whether or not the special extract ERr731® in a dosage relevant to human applications is capable to stimulate the growth of methyl-nitroso-urea (MNU) induced experimental breast cancer in ovariectomized Sprague-Dawley (SD) rats. Experimentals: 32 female SD rats were obtained at the age of postnatal day (PND) 40. On PND 49 and 56 all animals received an intra peritoneal MNU injection of 50 mg/kg body weight (BW). 28 days after the second injection 24 animals were ovariectomized (OvEx) and randomly allocated to 3 experimental groups, 8 animals were SHAM operated and served as positive control. SHAM operated animals (first positive control) and one group of OvEX animals (negative control) were fed a (phyto)estrogen-free diet. A second positive control group of OvEx animals received the phytoestrogen-free diet supplemented with 10 mg/kg diet of estradiol-benzoate. To mimic human exposure, ERr731®, in a dose of 0.067 mg/kg BW/d was chosen which is equivalent to 2.5 mg of the extract/kg chow. The experiment was terminated 25 weeks after MNU injection. Tumor-specific parameters, as well as uterine wet weights (UWW) were assessed. Results: Ovariectomy reduced the UWW &amp;gt; 6fold. Estradiol-benzoate restored UWW almost completely, whereas ERr731® did not lead to an increase in UWW. Tumor incidence in estrogenized animals was 62.5% (5/8 animals) in the SHAM group and 50% (4/8 animals) in the estradiol-benzoate treated group. No tumors were detected in OvEx negative controls and OvEx animals supplemented with the human relevant dose of ERr731®. The total numbers of tumors were 17 in the group of SHAM operated animals and 5 in the estradiol-benzoate treatment group. Conclusions: The human dose of ERr731® did neither exhibit estrogenic properties in the uterotrophic assay nor did it stimulate growth of MNU-induced breast cancers in OvEx SD rats, whereas estradiol supplementation at a low dose as well as estradiol levels in animals with a regular estrus cycle significantly promoted tumor growth. In this experimental model the used human dosage of ERr731® therefore appears safe regarding estrogen dependent growth stimulation of experimental MNU breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-09.

N-methylnitrosourea Induced Breast Cancer in Rat, the Histopathology of the Resulting Tumours and its Drawbacks as a Model

Several animal models of breast cancer have been developed to study various aspects of breast cancer biology. Substantial evidence suggests that the N-methylnitrosourea (MNU) animal model mimics human breast cancer in many respects. It has therefore been used extensively to evaluate preventive and therapeutic agents for human breast cancer. Chemically induced rodent models are also suitable for studying malignant progression. Recently, Liska et al. [7] established two protocols of MNU administration depending on the animal's age and number of applications of carcinogen, with the aim of investigating the advanced stages of mammary gland tumours. We used the same protocol as Liska but have obtained substantially different results. These results are presented and discussed in the frame of suggested key drawbacks of the MNU induced breast cancer rat model, as a contribution to the debate about the suitability of that model for evaluating preventive and therapeutic agents.

Effects of different dietary fatty acid on expression of nuclear receptor genes in breast cancer of rats

To study the effects of different dietary fatty acid on the expression of nuclear receptor genes in the breast cancer of rats. Fifty-day-old female Sprague-Dawley rats were fed on eight different diets containing following fatty acids: saturated fatty acid (SFA); monounsaturated fatty acid (MUFA); n-6 polyunsaturated fatty acid (PUFA); n-3 PUFA; 1:1 n-6/n-3; 5:1 n-6/n-3; 10:1 n-6/n-3; 1:2:1 S/M/P (n-6/n-3 at 1:1). The rats were given a single intraperitoneal injection of methyl-nitrosourea (MNU) at 50 mg/kg body weight to establish the rat model of mammary carcinogenesis, the ultrastructure changes of mammary gland cells in rats were observed by transmission electron microscope, the cell proliferation activity was detected by BrdU-labeled immunocytochemistry, and the expression of PPARbeta and PPARgamma mRNA were assayed by RT-PCR. There was no breast cancer occurring in control groups and the MNU-treated n-3 PUFA group, and the ultrastructure and proliferation activity of mammary gland cells in these groups were normal. In contrast, there appeared obvious marker of adenocarcinomas in mammary gland cells of MNU-induced breast cancer, and a high cell proliferation activity was found in tumor growth-enhancing groups (SFA, MUFA, n-6 PUFA, 5:1 n-6/n-3, 10:1 n-6/n-3 and S/M/P, 21% - 22% of BrdU-labeled cells), while a low cell proliferation activity was detected in rats fed with 1:1 n-6/n-3 diet (13% of BrdU-labeled cells, P < 0.05). Moreover, peroxisome proliferator-activated receptors (PPARs), as important nuclear receptor genes of relating lipid metabolism, the expressions of PPARbeta and PPARgamma mRNA were significantly up-regulated in mammary adipose tissues of MNU-induced breast cancer as compared with the control groups, but the expression levels of peroxisome proliferator-activated receptors (PPARs) in rats fed with 1:1 n-6/n-3 group were lowest (P < 0.05). The different dietary fatty acid compositions should diversely adjust the expression of PPARs gene in rats, which maybe have an important role in affecting incidence of breast cancer.

The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate

Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2-20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus 'deprotect' rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.

Detection of VIP receptors in MNU-induced breast cancer in rats: implications for breast cancer targeting.

Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with a wide range of biological activities. Receptors for VIP (VIP-R) are overexpressed in breast cancer, where they may have diagnostic and therapeutic implications. Although N-methyl nitrosourea (MNU)-induced breast cancer in rats is used extensively as a model to study mammary carcinogenesis, there is no information about the expression of VIP-R in this model. Therefore, the purpose of this study was to investigate the presence of VIP-R in MNU-induced breast cancer in rats so that this model can be used to perform studies involving VIP-R. Breast cancer was induced in 36-day-old virgin female Sprague-Dawley rats, by a single intravenous injection of MNU (50 mg/kg body weight). The breast tumors were detected 100-150 days after injection. The normal and cancerous rat breast tissue were excised and 20 micro sections were incubated with 40 nM fluorescein-labeled VIP (Fluo-VIP(TM)), in the presence and absence of 1000-fold excess unlabeled VIP, pituitary adenylate cyclase activating polypeptide (PACAP) or secretin. The sections were visualized under a fluorescence microscope and photographed. Fluo-VIP(TM) stained rat breast cancer tissue homogeneously and to a much greater extent than normal rat breast tissue (p < 0.05). This staining was specific as indicated by displacement of Fluo-VIP(TM) by excess unlabeled VIP and PACAP. Displacement of Fluo-VIP(TM) by secretin indicated the probable presence of VIP receptors of type VPAC1 (VIP receptor subtype 1) in the rat breast. These data suggest that, as in human breast cancer, VIP-R, predominantly of type VPAC1, are overexpressed in MNU-induced rat breast cancer tissue as compared to the normal rat breast tissue. Thus, MNU-induced rat breast cancer model can be used as a tool to study the functional role of VIP-R in human mammary carcinogenesis and VIP-R mediated active breast cancer targeting. This could have implications in the diagnosis, prognosis and therapy of human breast cancer.