DMBA-induced Mammary Tumorigenesis Research Articles

Protective Effects of Pelargonidin against DMBA-Induced Mammary Tumorigenesis in BALB/c Mice through Reduced Oxidative Stress and Lipid Anomalies

Among luminal types of breast cancers, ER + breast cancer is the most frequently diagnosed cancer globally. ER + breast cancer is commonly treated with SERM drugs that block ER to prevent ER-mediated cancerous growth. Our previous computational screening found pelargonidin (PG) can inhibit ER-signaling with potent bioactivity and satisfactory toxicological features. The present study explored the anti-tumoral prospect of PG against DMBA-induced ER + murine mammary carcinogenesis. The female BALB/c mice were divided into control (A) and DMBA-exposed groups. Following tumor appearance, the DMBA-exposed group was divided into five groups: tumor control, PG-treated (Groups P25, P50, and P100), and tamoxifen-treated (TAM). The results indicated that PG-treatment dose-dependently reduced the mean tumor volume, reinstated body weight loss, and enhanced the percentage survival of tumor-bearing mice. In addition, we recorded a significant reduction in LPO, total cholesterol, and triglycerides and a surge in the activity of antioxidases and phase II detoxifying enzymes in PG-treated animals. PG also dose-dependently increased the serum level of unbound estradiol, an indicator of competitive ER binding by an ER agonist/antagonist. These data suggest that pelargonidin has potent anticancer potential against the animal model of ER + breast cancer that matches the efficiency of tamoxifen with conceivably fewer side effects.

Chemopreventive Effects of Pelargonidin Against DMBA-Induced Mammary Tumorigenesis in BALB/C Mice Through Reduced Oxidative Stress and Lipid Anomalies

Chemopreventive Effects of Pelargonidin Against DMBA-Induced Mammary Tumorigenesis in BALB/C Mice Through Reduced Oxidative Stress and Lipid Anomalies

Metformin and melatonin inhibit DMBA-induced mammary tumorigenesis in rats fed a high-fat diet.

The data from in-vitro and in-vivo studies show that both peroral antidiabetic metformin (MF) and pineal hormone melatonin (MT) inhibit the growth of many cancers, including breast cancer. However, most in-vivo studies used standard-type diet with low fat content. Therefore, in this study, we evaluated the chemopreventive effect of MF and MT in an in-vivo model of breast cancer in rats on a high-fat diet (10% of total fat). Mammary carcinogenesis was induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Chemoprevention with MF (administered in a diet, 0.2%) and MT (administered in tap water, 20 mg/l) was induced 20 days before the carcinogen administration through the termination of the experiment (14 weeks after carcinogen administration). Tumor growth parameters were analyzed together with histopathological examination and immunohistochemical detection of KI67 (proliferation marker), caspase-3, BAX, BCL-2 (apoptosis markers), and CD24 and CD44 (cancer stem cell markers) in mammary tumor samples. The combination of chemopreventive agents decreased tumor incidence by 29%. Cumulative tumor volume was lower in all groups treated with chemoprevention. Histopathology did not show significant changes in high-grade/low-grade tumor ratio. Immunohistochemistry showed increased expression of BAX in the combination group, and caspase-3 expression increased in both MT and combination groups. MT, and particularly the MF and MT combination, inhibited DMBA-induced mammary tumor growth in rats by apoptosis stimulation in cancer cells. Our results indicate that MT supplements in patients treated with MF may have a considerable effect on the incidence of breast cancer.

Anti-Inflammatory Mechanism Involved in Pomegranate-Mediated Prevention of Breast Cancer: the Role of NF-κB and Nrf2 Signaling Pathways.

Pomegranate (Punica granatum L.), a nutrient-rich unique fruit, has been used for centuries for the prevention and treatment of various inflammation-driven diseases. Based on our previous study, a characterized pomegranate emulsion (PE) exhibited a striking inhibition of dimethylbenz(a)anthracene (DMBA)-initiated rat mammary tumorigenesis via antiproliferative and apoptosis-inducing mechanisms. The objective of the present work is to investigate the anti-inflammatory mechanism of action of PE during DMBA rat mammary carcinogenesis by evaluating the expression of cyclooxygenase-2 (COX-2), heat shock protein 90 (HSP90), nuclear factor-κB (NF-κB) and nuclear factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2). Mammary tumor samples were harvested from our previous chemopreventive study in which PE (0.2–5.0 g/kg) was found to reduce mammary tumorigenesis in a dose-dependent manner. The expressions of COX-2, HSP90, NF-κB, inhibitory κBα (IκBα) and Nrf2 were detected by immunohistochemical techniques. PE decreased the expression of COX-2 and HSP90, prevented the degradation of IκBα, hindered the translocation of NF-κB from cytosol to nucleus and increased the expression and nuclear translocation of Nrf2 during DMBA-induced mammary tumorigenesis. These findings, together with our previous results, indicate that PE-mediated prevention of DMBA-evoked mammary carcinogenesis may involve anti-inflammatory mechanisms through concurrent but differential regulation of two interrelated molecular pathways, namely NF-κB and Nrf2 signaling.

Lack of cortistatin or somatostatin differentially influences DMBA-induced mammary gland tumorigenesis in mice in an obesity-dependent mode.

BackgroundSomatostatin (SST) and cortistatin (CORT), two structurally and functionally related peptides, share a family of widespread receptors (sst1-5) to exert apparently similar biological actions, including endocrine/metabolic regulation and suppression of tumor cell proliferation. However, despite their therapeutic potential, attempts to apply SST-analogs to treat breast cancer have yielded unsatisfactory results. Actually, the specific roles of SST and CORT in mammary gland tumorigenesis (MGT), particularly in relation to metabolic dysregulation (i.e. obesity), remain unknown.MethodsThe role of endogenous SST and CORT in carcinogen-induced MGT was investigated under normal (lean) and obesity conditions. To that end, SST- and CORT-knockout (KO) mice and their respective littermate-controls, fed low-fat (LF) or high-fat (HF) diets, were treated with 7,12-dimethyl-benza-anthracene (DMBA) once a week (wk) for 3 wk, and MGT was monitored for 25 wk. Additionally, we examined the effect of SST or CORT removal in the development of the mammary gland.ResultsLack of SST did not alter DMBA-induced MGT incidence under lean conditions; conversely, lack of endogenous CORT severely aggravated DMBA-induced MGT in LF-fed mice. These differences were not attributable to altered mammary gland development. HF-diet modestly increased the sensitivity to DMBA-induced carcinogenesis in control mice, whereas, as observed in LF-fed CORT-KO, HF-fed CORT-KO mice exhibited aggravated tumor incidence, discarding a major influence of obesity on these CORT actions. In marked contrast, HF-fed SST-KO mice exhibited much higher tumor incidence than LF-fed SST-KO mice, which could be associated with higher mammary complexity.ConclusionsEndogenous SST and CORT distinctly impact on DMBA-induced MGT, in a manner that is strongly dependent on the metabolic/endocrine milieu (lean vs. obese status). Importantly, CORT, rather than SST, could represent a major inhibitor of MGT under normal/lean-conditions, whereas both neuropeptides would similarly influence MGT under obesity conditions. The mechanisms mediating these different effects likely involve mammary development and hormones, but the precise underlying factors are still to be fully elucidated. However, our findings comprise suggestive evidence that CORT-like molecules, rather than classic SST-analogs, may help to identify novel tools for the medical treatment of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0689-1) contains supplementary material, which is available to authorized users.

Abstract P4-07-14: The effects of calorie restriction on microRNA expression profiles and DMBA-induced mammary tumorigenesis in a model of luminal A breast cancer

Abstract MicroRNA (miR) are small, post-transcriptional regulators that play an integral role in maintenance of cellular functions and whose dyregulation has been shown to promote many types of cancer, including breast cancer. While it has been shown that microRNA are dyregulated through the stages of breast cancer development, it was our goal to define the microRNA expression profiles of the individual stages in a model of luminal A breast cancer in order to identify which are specifically associated with progression. In addition, we aim to establish the effects of calorie restriction, which is known to inhibit breast cancer progression, on relevant microRNA. To address these questions, 100 female Sprague Dawley rats were administered either dimethylbenz(a)anthracene or vehicle control at 50 days of age, then randomized to receive either control (AIN-76A) diet ad-libitum (n = 40) or a 30% CR diet regiment (n = 60). Resultant mammary tumors were allowed to develop for 12 weeks. Calorie restriction significantly increased survival to study endpoint relative to control diet (75% vs 35%, respectively) (p = 0.0047). Furthermore, of the animals that developed tumors, CR significantly decreased median tumor area by 56% compared to control diet (109.4 mm2 vs 250.9 mm2, respectively) (p = 0.0286). To analyze the impact of microRNA modulation on tumor progression, mammary tumors from control animals were microdissected in a stage-dependent fashion, with areas of normal epithelium, intraductal proliferation, ductal carcinoma in-situ, and invasive ductal carcinoma collected and analyzed separately. We have identified 10 microRNAs that are associated with cancer progression in this model (miR-10a, -10b, -21, -92b, -124, -125b, -126, -145, -200a, -658). We are currently analyzing which of these microRNAs are CR-responsive and how their modulation affects tumorigenic indices. The results obtained will provide insights into the mechanisms of breast cancer progression and how calorie restriction inhibits progression through microRNA modulation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-14.

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice

BackgroundThe major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice.MethodsWe used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey’s post hoc procedure.ResultsAnalysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of “PAM50” genes in the SK-BR-3 cell line from an ER-/Her-2+ to that resembling a “normal-like” phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin.ConclusionsThe SK-BR-3 cells and DMBA-induced tumors, both with an ER- and Her-2+ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

Breast cancer prevention with Morinda citrifolia (noni) at the initiation stage

Background: It has been reported that noni has multiple health benefits for over 2000 years. In this study, the cancer preventive effects of Tahitian noni® juice (TNJ) at the initiation stage on DMBA-induced mammary tumorigenesis in female SD rats was investigated.Objective: We took advantage of the DMBA-induced mammary carcinogenic model to study the preventive effects of TNJ at the initiation stage of mammary carcinogenesis in female SD rats by using clinical observation, pathological examination, and 32P-postlabeling assay.Methods: One hundred and sixty female SD rats were divided into eight groups with 20 rats in each group. Three doses of TNJ or placebo was given to the animals at the age of 35 days until the end of the experiment. When the animals were 55 days old, 25 mg/kg DMBA was fed to the animals in the DMBA group, placebo, and TNJ groups. The 20 rats were kept at age-matched controls. Palpable tumors were examined twice a week after DMBA administration in each group by an experienced professional. The size of tumor was measured by a graduated caliper. A piece of tumor, vascularization area, and mammary glands in the thoracic and abdomen areas of each rat were dissected respectively and fixed in 10% neutral buffered formalin for light microscope examination. The DMBA-DNA adduct formation in mammary tissues was detected by 32P-postlabeling assay.Results: The tumor latency in TNJ groups was delayed about 60-90 days when compared with positive controls. The number of palpable tumors per group was significantly reduced by 73%, 72% and 80% in 3%, 5%, and 10% TNJ groups respectively when compared with positive controls at the end of 330 days after DMBA administration. The number of palpable tumors in the placebo groups was slightly reduced in the early stage, but much less than that in the TNJ groups. The multiplicity and malignancy of lesions were significantly reduced and the survival rate of animals in the TNJ groups was significantly increased compared with positive controls at different time points. Histological examination showed that the malignancy of lesions in TNJ groups did not show a significant change when compared with that in positive and placebo groups. Conclusion: In conclusion, this is the first study which indicates that TNJ possesses a cancer preventive effect at the initiation stage of chemical carcinogenesis induced by DMBA in female SD rates. Key words: Morinda citrifolia, Tahitian noni® juice, breast cancer, cancer prevention

Abstract B101: Breast cancer chemoprevention by a novel oleanane triterpenoid: Preclinical evidence

Abstract Breast cancer is the most frequently diagnosed cancer in women in both developed and developing countries. Breast cancer remains highly resistant to chemotherapy and thus a major preventive approach, such as chemoprevention, is considered an important and viable strategy in reversing the morbidity and mortality of this disease. Several plant triterpenoids, including oleananes, have exhibited significant promise as chemopreventive agents against breast cancer. Amooranin (AMR), a triterpene acid (25-hydroxy-3-oxoolean-12-en-28-oic acid) isolated from Amoora rohituka (an Indian medicinal plant), has been shown to inhibit mammary tumorigenesis both in vitro and in vivo. Methyl-25-hydroxy-3-oxoolean-12-en-28-oate (AMR-Me) is a novel synthetic AMR analog with superior antitumor effects to AMR against a panel of human breast carcinoma cells. Nevertheless, mechanism-based chemopreventive potential of AMR-Me has not been tested in an experimentally validated preclinical model of breast cancer. The objective of the present study was to investigate the chemopreventive potential and underlying mechanisms of action of AMR-Me against 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinogenesis, an experimental rodent tumor model that closely resembles human mammary cancer. Rats were treated with three doses of AMR-Me (0.8, 1.2 and 1.6 mg/kg body weight) three times per week by oral gavage. Following two weeks of this treatment protocol, mammary tumorigenesis was initiated by DMBA (50 mg/kg body weight, per os). The treatment of rats with AMR-Me was continued. All animals were sacrificed 16 weeks following the DMBA exposure and the incidence and burden of mammary tumors were estimated. AMR-Me (at 1.2 or 1.6 mg/kg) displayed a striking attenuation of DMBA-induced mammary tumor incidence, total tumor burden, average tumor weight, and intratumor histopathological alterations without toxic effects. AMR-Me inhibited abnormal cell proliferation and induced apoptosis in mammary tumors. AMR-Me also upregulated the transcriptional levels of Bax, Bad, caspase-3, caspase-7 and poly (ADP-ribose) polymerase and downregulated Bcl-2. Based upon these results, we conclude that oral AMR-Me exerts a significant chemopreventive effect in DMBA-induced mammary tumorigenesis in rats through suppression of abnormal cell proliferation as well as induction of apoptosis in a dose-responsive fashion. The apoptosis-inducing effect of AMR-Me during mammary carcinogenesis may be mediated through promotion of mitochondrial pro-apoptotic mechanism. AMR-Me has considerable potential as a safe chemopreventive drug for human breast cancer that represents a dismal disease. This study was supported by a grant (R03CA136014) from the National Cancer Institute (NCI)/National Institutes of Health (NIH). The content of this abstract is solely the responsibility of the authors and does not necessarily represent the official views of NCI or NIH. Citation Format: Anupam Bishayee, Roslin J. Thoppil, Animesh Mandal, Altaf S. Darvesh, Deepak Bhatia. Breast cancer chemoprevention by a novel oleanane triterpenoid: Preclinical evidence. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B101.

The effect of dietary zinc - and polyphenols intake on DMBA-induced mammary tumorigenesis in rats

BackgroundThe aim of the study was to investigate the effect of dietary supplementation with zinc and polyphenol compounds, i.e. resveratrol and genistein, on the effectiveness of chemically induced mammary cancer and the changes in the content of selected elements (Zn, Cu, Mg, Fe, Ca) in tumors as compared with normal tissue of the mammary gland.MethodsFemale Sprague-Dawley rats were divided into study groups which, apart from the standard diet and DMBA (7,12-dimethyl-1,2- benz[a]anthracene), were treated with zinc ions (Zn) or zinc ions + resveratrol (Zn + resveratrol) or zinc ions + genistein (Zn + genistein) via gavage for a period from 40 days until 20 weeks of age. The ICP-OES (inductively coupled plasma optical emission spectrometry) technique was used to analyze the following elements: magnesium, iron, zinc and calcium. Copper content in samples was estimated in an atomic absorption spectrophotometer.ResultsRegardless of the diet (standard; Zn; Zn + resveratrol; Zn + genistein), DMBA-induced breast carcinogenesis was not inhibited. On the contrary, in the Zn + resveratrol supplemented group, tumorigenesis developed at a considerably faster rate. On the basis of quantitative analysis of selected elements we found - irrespectively of the diet applied - great accumulation of copper and iron, which are strongly prooxidative, with a simultaneous considerable decrease of the magnesium content in DMBA-induced mammary tumors. The combination of zinc supplementation with resveratrol resulted in particularly large differences in the amount of the investigated elements in tumors as compared with their content in normal tissue.ConclusionsDiet supplementation with zinc and polyphenol compounds, i.e. resveratrol and genistein had no effect on the decreased copper level in tumor tissue and inhibited mammary carcinogenesis in the rat. Irrespectively of the applied diet, the development of the neoplastic process in rats resulted in changes of the iron and magnesium content in the cancerous tissue in comparison with the healthy mammary tissue. The application of combined diet supplementation with zinc ions and resveratrol considerably promoted the rate of carcinogenesis and increased the number of DMBA-induced mammary tumors.

Inhibitory effects of tea polyphenols by targeting cyclooxygenase-2 through regulation of nuclear factor kappa B, Akt and p53 in rat mammary tumors

Breast cancer has become the second leading cause of cancer-related deaths worldwide. The control of this disease can be achieved through chemoprevention, which refers to the consumption of synthetic or naturally occurring agents to block, reverse, or delay the process of tumor development. Tea (Camellia sinensis), the most widely consumed beverage, has shown promises in the field of cancer chemoprevention. Inhibition of tumorigenesis by green or black tea polyphenols has been demonstrated in various in vitro and in vivo models. Here, we examined the inhibitory effect of green tea polyphenol (GTP) and black tea polyphenol (BTP) on the development of mammary tumors- induced by 7, 12-dimethylbenz (a) anthracene (DMBA) in female, Wistar rats. 13% and 33% of animals developed tumors in GTP and BTP supplemented groups, respectively. Both GTP and BTP are effective in significantly inhibiting the cumulative number of mammary tumors (by ~92% and 77%, respectively) and in reducing their growth. Mechanistically, we investigated the effects of GTP and BTP on the components of cell signaling pathways, connecting biomolecules involved in cancer development. GTP and BTP supplementation as a sole source of drinking solution leads to scavenging of reactive oxygen species (ROS) (by ~72% and 69%, respectively) by inhibiting cyclooxygenase-2 (Cox-2) and inactivation of phosphorylated forms of nuclear factor-kappa B (NF-κB) and Akt. Altogether, the study suggests that both cultivars of tea, i.e. green and black, have anti-tumorigenic potential against DMBA-induced mammary tumorigenesis in Wistar rats. Further studies such as large and long term cohort studies and clinical trials are warranted.

Scaffold attachment factor B1 (SAFB1) heterozygosity does not influence Wnt-1 or DMBA-induced tumorigenesis

BackgroundScaffold Attachment Factor B1 (SAFB1) is a multifunctional protein which has been implicated in breast cancer previously. We recently generated SAFB1 knockout mice (SAFB1-/-), but pleiotropic phenotypes including high lethality, dwarfism associated with low IGF-I levels, and infertility and subfertility in male and female mice, respectively, do not allow for straightforward tumorigenesis studies in these mice. Therefore, we asked whether SAFB1 heterozygosity would influence tumor development and progression in MMTV-Wnt-1 oncomice or DMBA induced tumorigenicity, in a manner consistent with haploinsufficiency of the remaining allele.MethodsWe crossed female SAFB1+/- (C57B6/129) mice with male MMTV-Wnt-1 (C57B6/SJL) mice to obtain SAFB1+/+/Wnt-1, SAFB1+/-/Wnt-1, and SAFB1+/- mice. For the chemical induced tumorigenesis study we treated 8 weeks old SAFB1+/- and SAFB+/+ BALB/c mice with 1 mg DMBA once per week for 6 weeks. Animals were monitored for tumor incidence and tumor growth. Tumors were characterized by performing H&E, and by staining for markers of proliferation and apoptosis.ResultsWe did not detect significant differences in tumor incidence and growth between SAFB1+/+/Wnt-1 and SAFB1+/-/Wnt-1 mice, and between DMBA-treated SAFB1+/+ and SAFB1+/-mice. Histological evaluation of tumors showed that SAFB1 heterozygosity did not lead to changes in proliferation or apoptosis. There were, however, significant differences in the distribution of tumor histologies with an increase in papillary and cribriform tumors, and a decrease in squamous tumors in the SAFB1+/-/Wnt-1 compared to the SAFB1+/+/Wnt-1 tumors. Of note, DMBA treatment resulted in shortened survival of SAFB1+/- mice compared to their wildtype littermates, however this trend did not reach statistical significance.ConclusionOur data show that SAFB1 heterozygosity does not influence Wnt-1 or DMBA-induced mammary tumorigenesis.

Mice expressing myrAKT1 in the mammary gland develop carcinogen-induced ER-positive mammary tumors that mimic human breast cancer

AKT1/PKB is a serine/threonine protein kinase that regulates biological processes such as proliferation, apoptosis and growth in a variety of cell types. To assess the oncogenic capability of an activated form of AKT in vivo we have generated several transgenic mouse lines that overexpress in the mammary epithelium the murine Akt1 gene modified with a myristoylation signal, which renders active this protein by localizing it to the plasma membrane. We demonstrate that expression of myristoylated AKT in the mammary glands increases the susceptibility of these mice to the induction of mammary tumors of epithelial origin by the carcinogen 9,10-dimethyl-1,2 benzanthracene (DMBA). We have found that while carcinogen-treated wild-type mice show mostly mammary tumors of sarcomatous origin, AKT transgenic mice treated with DMBA developed mainly adenocarcinoma or adenosquamous tumors, all of them displaying activated AKT. We analyzed other possible molecular alterations cooperating with AKT and found that neither Ras nor beta-catenin/Wnt pathways seemed altered nor p53 mutated. We have found that 100% of mammary DMBA-induced tumors and benign lesions in myrAKT mice are estrogen receptor (ERalpha)-positive and are more frequent than in wild-type littermates. These data show that AKT activation cooperates with deregulation of the estrogen receptor in the DMBA-induced mammary tumorigenesis model and recapitulate two characteristics of some human breast tumors. Thus, our model might provide a preclinical relevant model system to study the role of AKT and ERalpha in breast tumorigenesis and the response of mammary gland tumors to chemotherapeutics.

DMBA-Induced Mammary Pathologies are Angiogenic In Vivo and In Vitro

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.

Chemical carcinogen-induced tumorigenesis in parous, involuted mouse mammary glands.

Chemical carcinogen-induced tumorigenesis in parous, involuted mouse mammary glands.

Chemopreventive efficacy of curcumin-free aqueous turmeric extract in 7,12-dimethylbenz[ a]anthracene-induced rat mammary tumorigenesis

The modulating effects of turmeric (T), ethanolic turmeric extract (ETE) and curcumin-free aqueous turmeric extract (CFATE) on the initiation or post-initiation phases of DMBA-induced mammary tumorigenesis were investigated in female Sprague–Dawley rats. Dietary administration of 1% T/0.05% ETE 2 weeks before, on the day of DMBA treatment (day 55) and 2 weeks after the single dose (15 mg/animal) of DMBA (during the initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by a reduction in tumor multiplicity, tumor burden and tumor incidence. However, simultaneous administration of 1% T-derived CFATE as the sole source of drinking water during the initiation phase did not suppress DMBA-induced mammary tumorigenesis. Dietary administration of 1% T/0.05% ETE or 1% T-derived CFATE as the sole source of drinking water starting 48 h after DMBA treatment and continuing until the end of the experiment (during the post-initiation period) resulted in significant suppression of DMBA-induced mammary tumorigenesis as seen by reduction in the tumor multiplicity and/or tumor burden although tumor incidence was unaffected. The present data clearly indicate that dietary administration of T/ETE showed strong chemopreventive activity during initiation as well as post-initiation phases of DMBA-induced rat mammary tumorigenesis while CFATE was found to be weakly active only when it was administered during the post-initiation phase.

Chemopreventive efficacy of anethole trithione, N-acetyl-L-cysteine, miconazole and phenethylisothiocyanate in the DMBA-induced rat mammary cancer model

The chemopreventive efficacy of N-acetyl-L-cysteine (NAC), anethole trithione, miconazole and phenethylisothiocyanate (PEITC), each of which would be expected to alter carcinogen metabolism, was examined in the dimethylbenzanthracene (DMBA) mammary carcinogenesis model. In this protocol, animals were exposed to non-toxic doses of the chemopreventives in the diet beginning 7 days prior to DMBA administration and then continuously throughout the duration of the assay (100 days post carcinogen). Miconazole, an antifungal agent with relatively broad inhibitory activity toward a variety of cytochromes P450, increased mammary tumor latency, decreased tumor incidence at the highest dose and decreased tumor multiplicity up to 60%. Anethole trithione, a substituted dithiolthione and an analog of the relatively broad-spectrum chemopreventive oltipraz, was administered in the diet and significantly inhibited mammary cancer multiplicity but not cancer incidence. NAC, an antimucolytic agent, failed to inhibit DMBA-induced mammary tumorigenesis. Surprisingly, treatment with DMBA plus PEITC, a potent inhibitor of cytochrome P450 2E1, actually increased the multiplicity of tumors relative to that observed with DMBA alone.

Maternal handling during pregnancy reduces DMBA-induced mammary tumorigenesis among female offspring.

The present study investigated whether handling of pregnant rats would affect mammary tumorigenesis in their female offspring. Pregnant Sprague-Dawley rats were injected daily with 0.05 ml of vehicle between days 14 and 20 of gestation or were left undisturbed. Handling did not have any effects on pregnancy or early development of the offspring. The female offspring were administered 10 mg of 7,12-dimethylbenz(a)anthracene (DMBA) at the age of 55 days. The rats whose mothers were handled during pregnancy had a significantly reduced mammary tumour incidence when compared with the offspring of non-handled mothers. Thus, on week 18 after DMBA exposure, 15% of the handled offspring had developed mammary tumours, whereas 44% of the non-handled offspring had tumours. No significant differences in the latency to tumour appearance, in the size of the tumours or in their growth rates were noted. Daily handling performed during post-natal days 5 and 20 produced similar data to that obtained for prenatal handling; on week 18 after DMBA exposure, the mammary tumour incidence among the post-natally handled rats was 22% and among the non-handled rats 44%. Possible deviations in hormonal parameters were also studied in adult female rats exposed in utero to handling. The onset of puberty tended to occur later among the handled offspring, but no differences in the uterine wet weights or serum oestradiol levels between the groups were noted. In conclusion, maternal handling reduced the offspring's risk to develop mammary tumours, and this effect was independent of the oestrogenic environment at adulthood. We propose that handling of a pregnant rat reduces mammary tumorigenesis in her offspring by means of changing the morphology of the mammary gland, the pattern of expression of specific genes and/or immune functions.

A rice protein isolate alters 7,12-dimethylbenz[alpha]anthracene-induced mammary tumor development in female rats.

The effects of a rice protein isolate (RPI) on 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary tumor progression were investigated in female Sprague-Dawley rats. At 6 weeks of age, rats were fed a casein, RPI or soybean protein isolate (SPI) diet, respectively. After 1 week, DMBA was administered orally at the dose of 30 mg/kg body weight. The mean tumor number per tumor-bearing rat at autopsy was significantly lower only in rats fed RPI than in those fed casein. Palpable tumors at the mid point of the experiment were significantly lower in rats fed RPI and SPI than in those fed casein. Serum estradiol-17 beta concentrations were lower in rats fed the SPI (but not in those fed RPI) than in those fed casein. In a further experiment, no differences were found in hepatic microsomal DMBA-arylhydrocarbon hydroxylase activity after 7 days of feeding the respective diets. These results suggest that RPI exerts its inhibitory effect on DMBA-induced mammary tumorigenesis irrespective of changes in circulating estrogens or modulation of hepatic DMBA metabolism.

INHIBITION OF MAMMARY-GLAND TUMORIGENESIS IN THE RAT BY CARAWAY SEEDS AND DRIED LEAVES OF WATERCRESS

The effect of consumption of caraway seeds and dried leaves of watercress on 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced mammary gland tumorigenesis in female Sprague-Dawley rats was determined. At 6 weeks of age, animals were fed a basal (control) diet and experimental diets containing caraway or watercress (20%). Animals were maintained on their diets till termination of the experiment (25 weeks after DMBA). At 8 weeks of age, all rats were given DMBA 10 mg, one dose, p.o. in oily formulation). Neither caraway nor watercress affected the body weight or the rate of growth of animals. By week 25 after DMBA, 77% of the control rats developed mammary tumors, with a mean of 2.27 tumors per rat. Caraway decreased significantly (P<0.05) the percentage of rats with tumors (42.8% protection) and the mean number of tumors per rat (50.6% protection) and increased significantly (P<0.05) the mean latency period of tumor appearance. Watercress, though decreased the percentage of rats with tumors (28.5% protection) and the mean number of tumors per rat (31.7% protection), this decrease was significant (P < 0.05) during some week intervals only. The increase in the mean latency period of tumor appearance by watercress was not significant. The results of this study suggest that caraway and watercress possess chemopreventive effects against DMBA-induced mammary gland tumorigenesis in rats.