Rat Mammary Cancer Research Articles

Characterization of Molecular Subtypes of Rat Mammary Cancer and Their Association With Environmental Exposures.

Breast cancer is a heterogeneous disease with many subtypes, and the association between these subtypes and exposure to environmental factors such as radiation remains controversial. Although the rat is used widely for research into human breast cancer, the heterogeneity and subtype definitions are unclear. Here, we leveraged an archive of rat mammary cancer samples and gene expression microarray data to classify tumors and examine their association with exposures. Eighty-four mammary cancer and 12 normal mammary tissue samples were obtained from previous experiments in which rats were exposed to different types of radiation, chemical carcinogens, and diets. Tumors were then subjected to immunohistochemical (IHC) analysis of conventional biomarkers, as well as gene expression profiling; they were then classified by three approaches based on IHC results, the PAM50 classifier algorithm, and unsupervised clustering of gene expression profiles. IHC identified four subtypes (luminal A-like, luminal B-like 1, luminal B-like 2, and triple-negative), while PAM50 identified six (luminal A, luminal B, basal-like, HER2-enriched, normal-like, and claudin-low). Unsupervised clustering divided the tumors into three large, statistically significant, groups (named "luminal A", "luminal B", and "non-luminal" clusters). The results of the three approaches were significantly associated with each other. Exposure to radiation and chemical carcinogens during post-pubertal development was significantly associated with an increased risk of developing luminal A tumors, whereas exposure to a high corn-oil diet was associated with a higher likelihood of luminal B tumors. Rat mammary cancer subtypes resemble those in humans and are related to environmental factors.

Proteomics analysis of carcinogenesis in a rat model of mammary cancer induced by DMBA (7,12-dimethylbenz[a]anthracene)

Background: Mammary cancer, called breast cancer in humans, results from the abnormal growth of cells in the mammary glands that attack the surrounding tissue. The process of carcinogenesis at the molecular level can be monitored through the production of proteins as biomarkers for carcinogenesis. 7,12-Dimethylbenz[a]anthracene (DMBA) is a known carcinogenic compound. This study aimed to analyze the proteomic profile as critical data regarding DMBA-induced carcinogenesis in Sprague‒Dawley rats. Methods: Experimental animals were divided into two groups: a treatment group given DMBA at a dose of 10 mg/kg (intramammary) at intervals of 48 hours for a total of 10 doses, and a negative control group that was not given any treatment. Measurement of the total protein concentration was carried out using a spectrophotometer, and the data were analyzed using a t-test, while the characterization of protein profiles was carried out based on molecular weight data using SDS‒PAGE. Mammary gland histopathology was evaluated by hematoxylin and eosin (H&E) staining. Results: The results showed a significant (p<0.05) increase of 27% in the total protein concentration in the rat mammary cancer model. The results of proteomic characterization showed a protein profile containing proteins of 187, 169, 68, 64, 53, 41, 24, 18, and 14 kDa, which were suspected to be HER-2, Nischarin, COX-2, Albumine, Vimentin, ACTB, TNF, p16, and fatty acid binding protein 3 (FABP3), respectively. Histopathology of the mammary glands showed an irregular and indistinct arrangement of the alveoli and extensive epithelial cell proliferation from the surface to the lumen of the mammary ducts, and the mammary stroma showed the formation of new epithelial cells, which were cancer cells that spread to surrounding tissue. Conclusions: The proteomic profile was strongly associated with morphological alterations in mammary carcinogenesis in a rat model of DMBA-induced mammary cancer.

The role of natural compounds in rat mammary cancer: the beneficial effects of Santolina chamaecyparissus L. aqueous extract

Breast cancer is the most diagnosed cancer among women, and a leading cause of death worldwide. Santolina chamaecyparissus L. is a plant with multiple health benefits, including anticancer and anti-diabetic properties. This study aimed to assess the chemopreventive effects of S. chamaecyparissus aqueous extract (SCE) in an animal model of mammary cancer. A total of 28 four-week-old female Wistar rats were divided into four groups: control, MNU-induced (IND), SCE-supplemented (SCE), and SCE+IND. SCE was added to drinking water (12.72 mg/kg body weight) ad libitum. MNU was administered via the intraperitoneal route at 50 days of age. Weekly monitoring of body weight, food/drink intake, humane endpoints, and number of mammary tumours were recorded. Twenty weeks after MNU administration, animals were sacrificed by anaesthetic overdose and a necropsy was performed. Blood samples were used to determine blood count and serum biochemistry analysis, while kidney and liver samples were analysed for oxidative stress. Tumour samples were collected for gene expression and histology studies. SCE chemical composition was analysed by LC-MS and contained 19 phenolic compounds, with the most abundant being myricetin-O-glucuronide and 1,3-O-dicaffeoylquinic acid. Two animals in the IND group were sacrificed due to exceeding the humane endpoint limits. SCE supplementation delayed mammary tumour development, reducing its volume and weight. SCE had a positive impact on haematological parameters, particularly the neutrophil-lymphocyte ratio (P=0.026). No significant differences were observed in serum biochemistry, except for creatinine kinase MB, or in oxidative stress markers. Gene expression analysis showed significantly reduced VEGF expression levels (P=0.0158) in tumours from SCE+IND. These findings suggest that SCE is deserving of further study to identify the individual compounds and to understand its influence on animal models during cancer development.

Proteomics analysis of carcinogenesis in a rat model of mammary cancer induced by DMBA (7,12-dimethylbenz[a]anthracene).

Background: Mammary cancer, called breast cancer in humans, results from the abnormal growth of cells in the mammary glands that attack the surrounding tissue. The process of carcinogenesis at the molecular level can be monitored through the production of proteins as biomarkers for carcinogenesis. 7,12-Dimethylbenz[a]anthracene (DMBA) is a known carcinogenic compound. This study aimed to analyze the proteomic profile as critical data regarding DMBA-induced carcinogenesis in Sprague‒Dawley rats. Methods: Experimental animals were divided into two groups: a treatment group given DMBA at a dose of 10 mg/kg (intramammary) at intervals of 48 hours for a total of 10 doses, and a negative control group that was not given any treatment. Measurement of the total protein concentration was carried out using a spectrophotometer, and the data were analyzed using a t-test, while the characterization of protein profiles was carried out based on molecular weight data using SDS‒PAGE. Mammary gland histopathology was evaluated by hematoxylin and eosin (H&E) staining. Results: The results showed a significant (p<0.05) increase of 27% in the total protein concentration in the rat mammary cancer model. The results of proteomic characterization showed a protein profile containing proteins of 187, 169, 68, 64, 53, 41, 24, 18, and 14 kDa, which were suspected to be HER-2, Nischarin, COX-2, Albumine, Vimentin, ACTB, TNF, p16, and fatty acid binding protein 3 (FABP3), respectively. Histopathology of the mammary glands showed an irregular and indistinct arrangement of the alveoli and extensive epithelial cell proliferation from the surface to the lumen of the mammary ducts, and the mammary stroma showed the formation of new epithelial cells, which were cancer cells that spread to surrounding tissue. Conclusions: The proteomic profile was strongly associated with morphological alterations in mammary carcinogenesis in a rat model of DMBA-induced mammary cancer.

The transdermal cream of Formestane anti-breast cancer by controlling PI3K-Akt pathway and the tumor immune microenvironment.

Treatment of ER+ breast cancer with intramuscular formulation of Formestane (4-OHA) shrinks the tumor within weeks. Since the tedious way of intramuscular administration and side effects are not suited for adjuvant treatment, Formestane was withdrawn from the market. A new transdermal formulation of 4-OHA cream may overcome the defects and retain the effect of shrinking the breast cancer tumor. However, the effects of 4-OHA cream on breast cancer need further confirmatory studies. In this work, in vivo, the influence of 4-OHA cream on breast cancer was evaluated using the mode of 7,12-dimethylbenz(a)anthracene (DMBA) induced rat mammary cancer. We explored the common molecule mechanisms of action of 4-OHA cream and its injection formulation on breast cancer through RNA- sequencing-based transcriptome analysis and several biochemical experiments. The results showed that the cream substantially reduced the entire quantity, size, and volum of tumors in DMBA-treated rats consistent with 4-OHA injection, and indicated that there were comprehensive signals involved in 4-OHA antitumor activity, such as ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and proteoglycans in cancer. In addition, we observed that both 4-OHA formulations could enhance immune infiltration, especially CD8+ T cells, B cells, natural killer cells, and macrophages infiltration, in the DMBA-induced mammary tumor tissues. The antitumor effects of 4-OHA partly depended on these immune cells. 4-OHA cream could inhibit breast cancer growth as its injection formulation and may provide a new way for neoadjuvant treatment of ER+ breast cancer.

Design, development and evaluation of Resveratrol transdermal patches for breast cancer therapy

Resveratrol (RVT) is a polyphenolic phytoestrogen which has shown antiproliferative activity in breast cancer. However, its low bioavailability and short half-life have restricted its use. The current study aimed to develop transdermal patches of RVT and evaluate its site-specific delivery for breast cancer therapy. Different penetration enhancers were screened using a computational tool, quantitative structure propery relationship (QSPR). The best permeation of RVT was observed in a patch comprising hydroxypropyl methylcellulose (HPMC) E15LV: HPMC-K4M: polyvinyl pyrrolidone (PVP) K30 in the ratio of 3:1:2 as release controlling polymers with Glycerol:Capryol 90 (4:1) as penetration enhancer and plasticizer. To assess the localized delivery of RVT, the patch was applied to the breast of female rats. Higher breast tissue disposition with lower systemic concentration was observed compared to oral administration, demonstrated by increased AUC and MRT. Further, the optimized RVT patches were tested in 7,12-Dimethylbenz[a]anthracene (DMBA) induced rat mammary cancer. Compared to oral RVT, the application of RVT tansdermal patches significantly reduced the tumor volume and serum CA 15–3, a cancer biomarker. Thus, the RVT transdermal patch may be a viable approach for ensuring high local concentration of drug for site-specific delivery in breast cancer therapy.

Histopathological study of the protective effect of vitamin E against DMBA induced mammary cancer in rats

Histopathological study of the protective effect of vitamin E against DMBA induced mammary cancer in rats

INHIBITORY ACTIVITY OF SNAIL (Achatina fulica (Lam.) Bowdich) MUCUS ON GROWTH OF MAMMARY CANCER IN RAT INDUCED WITH DMBA (7,12-DIMETHYLBENZ(α)ANTHRACENE)

Back to nature as a medication concept has been accepted widely because it has fewer side effects than modern medicines. Researches on natural products as anticancer agent therapies are in progress. This present research was conducted to determine that the inhibitory activity of snail (Achatina fulica (Lam.) Bowdich) mucus inhibits the growth rate of mammary cancer in SpragueDawley rats. Five groups of female rats, with four individuals each, were induced with 7,12-dimethylbenz(α)anthracene (DMBA) for five weeks. Snail mucus was applied every seven days to the treated rats with three different dosages (15, 20, and 25 mg/kg BW). Observations were done on diameters and growth rate of the mammary cancer lump developed at the end of week 8, 10, 12, 14, and 16. Histopathological examination was carried out at the end of the 16th week. Inhibitory activity results showed a row of average diameter of the cancerous lumps on rats with the following details. The results obtained from the application of snail mucus at dosage of 15, 20, and 25 mg/kg BW were at 0.40, 0.60, and 0.09 cm respectively along with average growth rate of cancerous lumps at 1.50, 0.75, and 0.25. The histopathology results of the snail mucus treatment at dosage of 15, 20, and 25 mg/kg BW showed normal tissue depiction and similarly normal histopathological form indicated by the presence of their sub-clinic components. The results showed that a snail mucus dose of 25 mg/kg BW was able to perform inhibitory activity on growing mammary cancer in rats induced by DMBA.

CarcSeq Measurement of Rat Mammary Cancer Driver Mutations and Relation to Spontaneous Mammary Neoplasia.

The ability to deduce carcinogenic potential from subchronic, repeat dose rodent studies would constitute a major advance in chemical safety assessment and drug development. This study investigated an error-corrected NGS method (CarcSeq) for quantifying cancer driver mutations (CDMs) and deriving a metric of clonal expansion predictive of future neoplastic potential. CarcSeq was designed to interrogate subsets of amplicons encompassing hotspot CDMs applicable to a variety of cancers. Previously, normal human breast DNA was analyzed by CarcSeq and metrics based on mammary-specific CDMs were correlated with tissue donor age, a surrogate of breast cancer risk. Here we report development of parallel methodologies for rat. The utility of the rat CarcSeq method for predicting neoplastic potential was investigated by analyzing mammary tissue of 16-week-old untreated rats with known differences in spontaneous mammary neoplasia (Fischer 344, Wistar Han, and Sprague Dawley). Hundreds of mutants with mutant fractions ≥ 10-4 were quantified in each strain, most were recurrent mutations, and 42.5% of the nonsynonymous mutations have human homologs. Mutants in the mammary-specific target of the most tumor-sensitive strain (Sprague Dawley) showed the greatest nonsynonymous/synonymous mutation ratio, indicative of positive selection consistent with clonal expansion. For the mammary-specific target (Hras, Pik3ca, and Tp53 amplicons), median absolute deviation correlated with percentages of rats that develop spontaneous mammary neoplasia at 104 weeks (Pearson r = 1.0000, 1-tailed p = .0010). Therefore, this study produced evidence CarcSeq analysis of spontaneously occurring CDMs can be used to derive an early metric of clonal expansion relatable to long-term neoplastic outcome.

Betulin ameliorates 7,12-dimethylbenz(a)anthracene-induced rat mammary cancer by modulating MAPK and AhR/Nrf-2 signaling pathway.

The aim of the present study isto explore the preventive efficacy of betulin (BE) in 7,12-dimethylbenz(a)anthracene (DMBA)-administered mammary cancer by modulating Ahr/Nrf2 signaling in experimental models. The mammary cancer was stimulated by the addition of DMBA (25 mg/kg/b.Wt) mixed in 1 ml of vehicle solution (sunflower oil and saline 1:1) through subcutaneous injection. The DMBA-exposed mammary tumor models showed low bodyweight, elevated quantities of lipid peroxidation molecules (TBARS and LOOH), and low enzymatic (GPx, SOD, and CAT), and nonenzymatic (GSH, vitamin C, and vitamin E) antioxidant activities in plasma and mammary tissues. Moreover, histopathological studies confirmed that invasive ductal carcinoma was observed in DMBA-induced mammary tissue of the experimental model. Dietary oral supplementation of BE prevents the loss of bodyweight, overproduces lipid peroxidation, and restores the antioxidant activities in DMBA-exposed experimental animals. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial antioxidant protein that involves preventing numerous cancers. Therefore, Nrf2-associated signaling concern is a significant target for preventing mammary cancer. This study observed an increased expression of MAPKs, Keap1, ARNT, AhR, and CYP1A1, whereas decreased expression of HO-1 and Nrf2 in DMBA-induced cancer-bearing experimental animals. The oral supplementation of BE effectively modulates the expression of MAPKs, AhR/Nrf2-associated protein expressions in DMBA-exposed experimental animals. This current study concluded that BE is a strong antioxidant, which triggers the MAPKs-mediated oxidative stress and inhibits proliferative markers by restoring the activity of Nrf2 signaling.

Role of organophosphorous pesticides and acetylcholine in breast carcinogenesis.

Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.

Mangiferin Inhibits Inflammation and Cell Proliferation, and Activates Proapoptotic Events via NF-κB Inhibition in DMBA-Induced Mammary Carcinogenesis in Rats.

The purpose of this study was to investigate the anti-inflammatory, antiproliferatiive, and proapoptotic molecular mechanisms of mangiferin (MGN) against mammary carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA). Mammary cancer in rats was induced by single-dose subcutaneous injection of 0.5 ml DMBA (80 mg/kg in sesame oil) in the mammary gland. Increased tumor incidence and volume and other tumorigenic properties were observed. Further, we observed in these rats reduced antioxidant enzyme activity and elevated thiobarbituric acid reactive substance (TBARS) levels in plasma and tissues. DMBA-induced rats shows enhanced expression of the inflammatory markers NF-κBp65, COX-2, and iNOS and proliferation of PCNA and Cyclin D1, and overexpression of the antiapoptotic marker Bcl-2. Mangiferin (100 mg/kg body weight), administered orally once per day, significantly enhanced (p < 0.05) antioxidant levels and reduced TBARS levels. Moreover, MGN inhibited NF-κBp65 nucleus transcriptional activation, thereby suppressing inflammation and cell proliferation, and it increased proapoptotic proteins. Apoptosis was confirmed by TUNEL assay. In summary, MGN suppressed DMBA-induced mammary carcinogenesis through enhanced antioxidant levels, NF-κB inhibition, and positive regulation of apoptotic signals.

Effects of High-Fructose Diet vs. Teklad Diet in the MNUInduced Rat Mammary Cancer Model: Altered Tumorigenesis, Metabolomics and Tumor RNA Expression

Epidemiology, clinical and experimental animal studies suggest high fructose diets are detrimental to metabolic status and may contribute to tumor development. This due to increased obesity and metabolic syndrome, known risk factors for many types of cancer. We compared tumor development in N-methyl-N-nitrosourea (MNU)-treated rats fed either a high (60%)-fructose diet (HFD) or a standard diet (SD). Female Sprague-Dawley rats at 43 days of age (DOA) were fed a SD or HFD followed by administration of MNU at 50 DOA. Rats were palpated weekly and sacrificed at 190 DOA. MNU-treated rats on HFD exhibited decreased tumor latency and roughly a two-fold increase in tumor multiplicity. RNA-Seq on frozen tumors (SD vs. HFD rats) showed altered expression of approximately 10% of genes (P < 0.05). When examined by Ingenuity Pathway Analysis, multiple highly significant pathways were identified including A) mechanisms of cancer, B) Wnt pathway, C) immune response (e.g., "Th1 and Th2 activation" and "antigen presentation") and D) LXR/RXR nuclear receptor. These generalized pathways were indirectly confirmed by alterations of various interrelated disease pathways (epithelial cancers, T cell numbers and apoptosis). In a second study, serum was collected from rats on the HFD or SD pre-MNU and at the time of sacrifice. Metabolomics revealed that the HFD yielded: A) increased levels of fructose, B) increases of various monoglycerols, C) reduced levels of various diacylglycerols and oxygenated inflammatory lipids (9 and 13 HODE and 12,13 DHOME) and D) increased levels of secondary bile acids (hyodeoxycholate and 6-oxolithocholate), which may reflect microbiome changes. These metabolomic changes, which are distinct from those on a high-fat diet, may prove relevant when examining individuals who consume higher levels of fructose.

Abstract 1154: AFPep mitigates the risk of breast cancer

Abstract Women whose risk of breast cancer is elevated (based on Gail or Tyrer-Cuzik models) have only limited options to intervene in attempt to alter disease progression, and most options are of limited efficacy or have unwanted side effects. Options include increased surveillance, lifestyle changes, drugs such as tamoxifen or aromatase inhibitors, or removal of target tissue. Options that are more efficacious, better tolerated, and more empowering are needed for women in high-risk cohorts. For this purpose, we have developed AFPep, a homobiotic, selective anti-estrogenic/anti-breast cancer active site-analog of alpha-fetoprotein (AFP). AFPep stops growth of human breast cancers growing in xenograft models and prevents development of either estrogen-induced or MNU-induced mammary cancers in rats. In all models, AFPep is effective against tamoxifen-resistant breast cancer, is additive with tamoxifen (allowing dose reduction of tamoxifen), and reduces the uterine hyperplasia induced by tamoxifen. AFPep does not disrupt the estrous cycle of rats, nor does it alter fertility or fecundity and does not induce teratogenicity. The mechanism of action of AFPep differs from that of tamoxifen in that AFPep inhibits phosphorylation of the estrogen receptor leading to a downstream biomarker profile consistent with inhibition of tumor growth. AFPep is orally bioavailable and well tolerated in mice, rats, dogs and primates. The therapeutic index of AFPep in rats shows a substantially broader safety margin than that of currently used agents. AFPep may offer an effective, well tolerated option for women in high risk cohorts who wish to mitigate their risk of breast cancer. Citation Format: Samuel B. Fordyce, Thomas T. Andersen, James A. Bennett. AFPep mitigates the risk of breast cancer [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 1154.

The Effect of (E)-1-(4'-aminophenyl)-3-phenylprop-2-en-1-one on MicroRNA-18a, Dicer1, and MMP-9 Expressions against DMBA-Induced Breast Cancer.

Background:Most of breast cancer patients are estrogen receptor alpha-positive and have high resistance and side effect of chemotherapeutic drug. Therefore, discovering an effective anticancer agent is needed. This research explored the effect of (E)-1-(4’-aminophenyl)-3-phenylprop-2-en-1-one (APE) on miR-18a, Dicer1, and MMP-9 expressions. Methods:Twenty four female Sprague-Dawley rats were invetigated in this study. The rats were divided into 6 groups of 4. G1 was considered as normal rat. G2, G3, T1, T2, and T3 were given DMBA 20 mg/kgBW twice a week for 5 weeks to induce mammary cancer. After being affiliated with cancer, G2 was given vehicle and G3 was treated with tamoxifen. T1, T2, and T3 were treated with APE intraperitoneally everyday for 21 days at doses of 5, 15, and 45 mg/kgBW/day, respectively. Blood plasma was collected to measure miR-18a expression using qRT-PCR. Mammary tissues were also collected to determine Dicer1 and MMP-9 expressions by using immunohistochemistry. Results:The results showed significant down-regulation of miR-18a relative expression and up-regulation of Dicer1 expression in G3 and T1 compared to G2 (P<0.05). MMP-9 expression has significant decrease in T1 compared to G2 (P<0.05). Conclusion:APE can decrease miR-18a and MMP-9 expressions and increase Dicer1 expression in rat mammary cancer. Therefore, this compound could be a candidate of novel anticancer.

Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

The acquisition of MDR1-mediated chemoresistance poses a major obstacle to the success of conventional chemotherapeutic agents. HSF1 is also involved in chemoresistance, and several studies have demonstrated the relationship between HSF1 and MDR1 but without any consistent results. Paclitaxel- and doxorubicin-resistant cancer cells showed higher expression of MDR1 and HSF1. Depletion of HSF1 decreased mdr1 expression at mRNA level, and HSF1 directly interacted with the promoter site of mdr1, suggesting its role as a transcriptional regulator of MDR1. Phosphorylation of Ser303/307, which was involved in protein stability of HSF1 by FBXW7-mediated degradation, was found to be important for transcriptional activation of mdr1. Drug-resistant cells showed decreased expression of FBXW7, which was mediated by the activation of ERK1/2, thus indicating that over-activation of ERK1/2 in drug-resistant cells decreased FBXW7 protein stability, which finally inhibited protein degradation of pHSF1 at Ser303/307. There was a positive correlation between immunofluorescence data of pHSF1 at Ser303/307 and MDR1 in carcinogen-induced rat mammary tumors and human lung cancers. These findings identified the post-translational mechanisms of HSF1 transcription in MDR1 regulation of drug resistance development.

Effect of citronellol on NF-kB inflammatory signaling molecules in chemical carcinogen-induced mammary cancer in the rat model.

Inflammation plays a vital role in the process of carcinogenesis and anti-inflammatory properties of phytochemicals aregaining more attention in the chemoprevention of cancer. The present study was designed to evaluate the anti-inflammatory potential of citronellol (CT) on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats. The inflammation-associated gene and protein markers were analyzed by immunohistochemistry, reverse transcription polymerase chain reaction, and Western blot techniques. Markers such as nuclear factor-kB (NF-kB), tumor necrosis factor-α, interleukin-6 (IL-6), cyclooxygenase-2, macrophage inflammatory protein-1α, and inducible nitric oxide synthase are upregulated in DMBA-alone-treated mammary tumor tissues. The oral administration of CT (50 mg/kg BW) to DMBA-treated rats significantly downregulated the expression NF-kB and other inflammatory markers, and also increased the level of IL-10 in mammary tissues. The results suggested that the anti-inflammatory potential of CT prevented the incidence of chemical carcinogen-induced mammary cancer in rats.

Moderate hyperthermic heating encountered during thermal ablation increases tumor cell activity

Purpose The aim of this study was to determine whether moderate hyperthermic doses, routinely encountered in the periablational zone during thermal ablation, activate tumor cells sufficiently to secrete pro-tumorigenic factors that can induce increased proliferation. Material and methods R3230 rat mammary tumor cells and human cancer cell lines, MCF7 breast adenocarcinoma, HepG2 and Huh7 HCC, and HT-29 and SW480 colon adenocarcinoma, were heated in to 45 ± 1 °C or 43 ± 1 °C in vitro for 5-10 min and incubated thereafter at 37 °C for 1.5, 3 or 8 hr (n = 3 trials each; total N = 135). mRNA expression profiles of cytokines implicated in RF-induced tumorigenesis including IL-6, TNFα, STAT3, HGF, and VEGF, were evaluated by relative quantitative real-time PCR. HSP70 was used as control. c-Met and STAT3 levels were assessed by Western blot. Finally, naïve cancer cells were incubated with medium from R3230 and human cancer cells that were subjected to 43–45 °C for 5 or 10 min and incubated for 3 or 8 h at 37 °C in an xCELLigence or incuCyte detection system. Results Cell-line-specific dose and time-dependent elevations of at least a doubling in HSP70, IL-6, TNFα, STAT3, and HGF gene expression were observed in R3230 and human cancer cells subjected to moderate hyperthermia. R3230 and several human cell lines showed increased phosphorylation of STAT3 3 h post-heating and increased c-Met following heating. Medium of cancer cells subject to moderate hyperthermia induced statistically significant accelerated cell growth of all cell lines compared to non-heated media (p < 0.01, all comparisons). Conclusion Heat-damaged human tumor cells by themselves can induce proliferation of tumor by releasing pro-tumorigenic factors.

Abstract 648: Effects of bazedoxifene and lapatinib in the prevention of estrogen receptor positive (ER+) and negative (ER-) mammary cancers

Abstract Bazedoxifene is a third-generation selective estrogen receptor modulator (SERM) that has been clinically approved in Europe for the prevention and treatment of postmenopausal osteoporosis. Our research previously showed that this agent (at a dose of 5 mg/kg diet) would decrease ER+ mammary cancers in rats by 73%. Lapatinib, an epidermal growth factor receptor (EGFR) inhibitor, is commonly used to treat patients with HER2+ breast cancer. We have also shown that the compound inhibits the development of ER+ mammary cancers in rats and ER- mammary cancers in mice at a dose of 75 mg/kg BW/day. The present studies were done to determine if lower doses and different treatment regimens designed to reduce possible toxicity would also be effective in preventing mammary cancers. Bazeodxifene was added directly to the diet (Teklad, 4% fat), while lapatinib was given by oral gavage. When evaluated in rats receiving methylnitrosourea (MNU), lapatinib given daily at doses of 75, 50 and 25 mg/kg BW/day reduced mammary cancer multiplicity by 93, 84 and 67%, respectively. Equivalent doses given 1x/week caused reductions of 87, 56, and 40%. When lapatinib was given to dimethylbenzanthracene (DMBA) treated MMTV/Neu mice at dose levels of 250, 125, and 63 mg/kg BW, 5x/week, mammary cancer multiplicity was reduced by 89, 60 and 80%, respectively. When equivalent doses were given 1x/week, the number of cancers were reduced by 82, 61, and 70%. Bazedoxifene given at 5.0 and 2.5 mg/kg diet to female MMTV/Neu mice also receiving DMBA caused reduction of mammary cancers by 73 and 51%. In a biomarker study lasting two weeks, bazedoxifene at the 5.0 mg/kg BW/day dose decreased the proliferation rate of normal mammary epithelial cells in MMTV/Neu mice by 60%. Additional studies to determine why this SERM is having a preventive effect against ER- mammary cancers are warranted. Studies to evaluate the efficacy of the combination of low doses of bazedoxifene and lapatinib in prevention of ER+ and ER- mammary cancers are in progress and will be presented. Supported by the NCI contract number HHSN261201500036I, Task Order HHSN26100005. Citation Format: Clinton J. Grubbs, Altaf Mohammed, Shizuko Sei, Robert H. Shoemaker. Effects of bazedoxifene and lapatinib in the prevention of estrogen receptor positive (ER+) and negative (ER-) mammary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 648.

Screening of Chemopreventive Agents in Animal Models: Results on Reproducibility, Agents of a Given Class, and Agents Tested During Tumor Progression.

Because of the importance of testing reproducibility of results, we present our findings regarding screening agents in preclinical chemoprevention studies in rodent models performed by the Chemopreventive Agent Development Research Group (CADRG) of the Division of Cancer Prevention of the NCI. These studies were performed via contracts to various commercial and academic laboratories. Primarily, results with positive agents are reported because positive agents may progress to the clinics. In testing reproducibility, a limited number of direct repeats of our standard screening assays were performed; which entailed initiating treatment shortly after carcinogen administration or in young transgenic mice and continuing treatment until the end of the study. However, three additional protocols were employed relating to reproducibility: (i) testing agents at lower doses to determine efficacy and reduced toxicity; (ii) testing agents later in tumor progression when microscopic lesions existed and, (iii) testing multiple agents of the same mechanistic class. Data with six models that were routinely employed are presented: MNU-induced ER-positive mammary cancer in rats; MMTV-Neu ER-negative mammary cancers in transgenic mice; AOM-induced colon tumors in rats; intestinal adenomas in Min mice; OH-BBN-induced invasive rat urinary bladder cancers in rats; and UV-induced skin squamous carcinomas in mice. It was found that strongly positive results were highly reproducible in the preclinical models evaluated. Cancer Prev Res; 11(10); 595-606. ©2018 AACR.