Human Mammary Tumor Cell Line Research Articles

α-tocopherol as a selective modulator of toxicogenic damage induced by antineoplastic agents cyclophosphamide and doxorubicin

ABSTRACT The aim of this study was to determine the oxidative/antioxidative effects, modulatory and selective potential of α-tocopherol (vitamin E) on antineoplastic drug-induced toxicogenetic damage. The toxicity, cytotoxicity and genotoxicity induced by antineoplastic agents cyclophosphamide (CPA) and doxorubicin (DOX) was examined utilizing as models Saccharomyces cerevisiae, Allium cepa, Artemia salina and human peripheral blood mononuclear cells (PBMCs) in the presence of α-tocopherol. For these tests, concentrations of α- tocopherol 100 IU/ml (67mg/ml), CPA 20 µg/ml, DOX 2 µg/ml were used. The selectivity of α-tocopherol was assessed by the MTT test using human mammary gland non-tumor (MCF10A) and tumor (MCF-7) cell lines. Data showed cytoplasmic and mitochondrial oxidative damage induced by CPA or DOX was significantly diminished by α-tocopherol in S. cerevisiae. In addition, the toxic effects on A. salina and cytotoxic and mutagenic effects on A. cepa were significantly reduced by α-tocopherol. In PBMCs, α-tocopherol alone did not markedly affect these cells, and when treated in conjunction with CPA or DOX, α-tocopherol reduced the toxicogenetic effects noted after antineoplastic drug administration as evidenced by decreased chromosomal alterations and lowered cell death rate. In human mammary gland non-tumor and tumor cell lines, α-tocopherol produced selective cytotoxicity with 2-fold higher effect in tumor cells. Evidence indicates that vitamin E (1) produced anti-cytotoxic and anti-mutagenic effects against CPA and DOX (2) increased higher selectivity toward tumor cells, and (3) presented chemoprotective activity in PBMCs.

Cytocompatibility of oleic acid modified iron oxide nanoparticles

Our long-term goal is to utilize oleic acid-modified iron oxide (OA-Fe3O4) nanoparticles embedded into a monoglyceride matrix for remote drug release utilizing an alternating magnetic field (AMF). However, the toxicological behavior of OA-Fe3O4 nanoparticles when in contact with biological tissues remains unknown. The size, shape, and surface morphology of OA-Fe3O4 nanoparticles prepared in our laboratory were evaluated using SEM and TEM. These data show that the OA-Fe3O4 nanoparticle preparations do not form aggregates and maintain the expected size and shape. We also used HTB-19, a human mammary tumor cell line, to assay the cytocompatibility of OA-Fe3O4 nanoparticles of different sizes (5, 10, 20, and 30 nm) at varying doses (6.25, 12.5, 25, and 50 µg/mL) using CytoTox-Glo™ cytotoxicity kit and phase-contrast inverted microscopy. These results indicate that there is no appreciable cytotoxicity at the lowest (6.25 µg/mL) concentration. Even the highest (50 µg/mL) concentration used in our study show > 75% cell viability. Microscopic morphological assessment performed using phase-contrast inverted microscopy revealed internalization of the nanoparticles and proliferation of cells in the presence of the OA-Fe3O4 nanoparticles. Taken together, our results show that OA-Fe3O4 nanoparticles are a safe and promising biocompatible excipient for drug delivery.

STAT5 is activated in macrophages by breast cancer cell-derived factors and regulates macrophage function in the tumor microenvironment

BackgroundIn breast cancer, complex interactions between tumor cells and cells within the surrounding stroma, such as macrophages, are critical for tumor growth, progression, and therapeutic response. Recent studies have highlighted the complex nature and heterogeneous populations of macrophages associated with both tumor-promoting and tumor-inhibiting phenotypes. Defining the pathways that drive macrophage function is important for understanding their complex phenotypes within the tumor microenvironment. Signal transducer and activator of transcription (STAT) transcription factors, such as STAT5, are key regulators of immune cell function. The studies described here investigate the functional contributions of STAT5 to tumor-associated macrophage function in breast cancer.MethodsInitial studies were performed using a panel of human breast cancer and mouse mammary tumor cell lines to determine the ability of tumor cell-derived factors to induce STAT5 activation in macrophages. Further studies used these models to identify soluble factors that activate STAT5 in macrophages. To delineate STAT5-specific contributions to macrophage function, a conditional model of myeloid STAT5 deletion was used for in vitro, RNA-sequencing, and in vivo studies. The effects of STAT5 deletion in macrophages on tumor cell migration and metastasis were evaluated using in vitro co-culture migration assays and an in vivo tumor cell-macrophage co-injection model.ResultsWe demonstrate here that STAT5 is robustly activated in macrophages by tumor cell-derived factors and that GM-CSF is a key cytokine stimulating this pathway. The analysis of RNA-seq studies reveals that STAT5 promotes expression of immune stimulatory genes in macrophages and that loss of STAT5 in macrophages results in increased expression of tissue remodeling factors. Finally, we demonstrate that loss of STAT5 in macrophages promotes tumor cell migration in vitro and mammary tumor metastasis in vivo.ConclusionsBreast cancer cells produce soluble factors, such as GM-CSF, that activate the STAT5 pathway in macrophages and drive expression of inflammatory factors. STAT5 deletion in myeloid cells enhances metastasis, suggesting that STAT5 activation in tumor-associated macrophages protects against tumor progression. Understanding mechanisms that drive macrophage function in the tumor microenvironment will ultimately lead to new approaches that suppress tumor-promoting functions while enhancing their anti-tumor functions.

Citronellol, an Acyclic Monoterpene Induces Mitochondrial-Mediated Apoptosis through Activation of Proapoptotic Factors in MCF-7 and MDA-MB-231 Human Mammary Tumor Cells

The present study investigated the anticancer activity of citronellol (CT) by analyzing the mitochondrial-mediated activation of apoptosis in MCF-7 and MDA-MB-231 human mammary tumor cell lines. Cytotoxicity, cell growth, and apoptosis were determined by measuring reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔΨm), DNA damage, and changes in morphology and expression of proteins involved in apoptosis in MCF-7 and MDA-MB-231 cells. Our results indicate that CT induces apoptosis as evidenced by the loss of cell viability, increase ROS generation, altered ΔΨm, and enhanced DNA damage. Further, CT inhibits Bcl-2 expression with the up-regulation of Bax, caspase-9, and -7 in both cancer cells. CT induces apoptosis in MCF-7 human mammary tumor cells by inducing oxidative damage and modulating the expression of various pro and anti-apoptotic proteins. Hence, CT might be a potential therapeutic agent for the treatment of breast cancer.

Chemo-treated 4T1 breast cancer cells radiation response measured by single and multiple cell ionization using infrared laser trap

We present a study that uses a laser trapping technique for measurement of radiation sensitivity of untreated and chemo-treated cancer cells. We used a human mammary tumor cell line (4T1) treated by an antitumor compound, 2-Dodecyl-6-methoxycyclohexa-2, 5-diene-1,4-dione (DMDD), which was extracted from the root of Averrhoa carambola L. The untreated control group, and both 2-hour and 24-hour treated groups of 4T1 cells were used in this study. The absorbed threshold ionization energy (TIE) and the threshold radiation dose (TRD) were determined using a high-power infrared laser (at 1064 nm) trap by single and multiple cells trapping and ionization. The results were analyzed using descriptive and t-statistics. The relation of the TIE and TRD to the mass of the individual cells were also analyzed for different hours of treatment in comparison with the control group. Both TIE and TRD decrease with increasing treatment periods. However, the TRD decreases with mass regardless of the treatment. Analyses of the TRD for single vs multiple cells ionizations within each group have also consistently showed this same behavior regardless of the treatment. The underlying factors for these observed relations are explained in terms of radiation, hyperthermia, and chemo effects.

Honokiol inhibits breast cancer cell metastasis by blocking EMT through modulation of Snail/Slug protein translation

Honokiol (HNK), an active compound isolated from traditional Chinese medicine Magnolia officinalis, has shown potent anticancer activities. In the present study, we investigated the effects of HNK on breast cancer metastasis in vitro and in vivo, as well as the underlying molecular mechanisms. We showed that HNK (10-70 μmol/L) dose-dependently inhibited the viability of human mammary epithelial tumor cell lines MCF7, MDA-MB-231, and mouse mammary tumor cell line 4T1. In the transwell and scratch migration assays, HNK (10, 20, 30 μmol/L) dose-dependently suppressed the invasion and migration of the breast cancer cells. We demonstrated that HNK (10-50 μmol/L) dose-dependently upregulated the epithelial marker E-cadherin and downregulated the mesenchymal markers such as Snail, Slug, and vimentin at the protein level in breast cancer cells. Using a puromycin incorporation assay, we showed that HNK decreased the Snail translation efficiency in the breast cancer cells. In a mouse model of tumor metastasis, administration of HNK (50 mg/kgevery day, intraperitoneal (i.p.), 6 times per week for 30 days) significantly decreased the number of metastatic 4T1 cell-derived nodules and ameliorated the histological alterations in the lungs. In addition, HNK-treated mice showed decreased Snail expression and increased E-cadherin expression in metastatic nodules. In conclusion, HNK inhibits EMT in the breast cancer cells by downregulating Snail and Slug protein expression at the mRNA translation level. HNK has potential as an integrative medicine for combating breast cancer by targeting EMT.

Chemical composition and cytotoxicity of Philippine calamansi essential oil

The development of drug resistance is widespread in today's arsenal of chemotherapeutic agents and this has brought renewed interest in the use of natural products. Calamansi (Citrus microcarpa B.) is an essential oil-rich citrus from the Philippines popularly used in beverages and condiments. Calamansi wastes from processing plants are underutilized and may pose environmental concerns if not properly processed. This study aims to determine the volatile composition, d-limonene content, and cytotoxicity of calamansi essential oil. The calamansi essential oil was prepared by steam distillation and characterized using Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy and Gas Chromatography analysis. Cytotoxicity on human mammary tumor cell line MCF-7 and Chinese hamster ovary non-tumor cell line AA8 were assessed by MTT assay. Gas Chromatography-Mass Spectrometry qualitative analysis results revealed that d-limonene is the major constituent of calamansi essential oil comprising at least 92.67% of the total oil. Quantification of d-limonene in essential oil using a reference standard by Gas Chromatography-Flame Ionization Detector has shown 98.1% w/w. The IC50 of calamansi essential oil on MCF-7 and AA8 cells were identified to be 7.98 ± 1.77 and 16.15 ± 8.35 μg/mL respectively. Our findings suggest that calamansi essential oil has greater cytotoxic activity on MCF-7 than AA8 and this activity can be directly attributed to the high amount of d-limonene and perhaps the presence of other terpenes that may also have synergistic effects with d-limonene. Calamansi essential oil can be a good candidate for a natural low-cost chemopreventive and chemotherapeutic agent. These findings are of considerable economic interest to Philippine calamansi industry which produces large amount of calamansi pericarp waste.

Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization.

Extracellular vesicles (EVs) are membrane-bound vesicles produced by cells, known to play a key role in cell-to-cell communication. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (DNA, RNAs, proteins, and lipids) within the tumour microenvironment and throughout the body. In human cancer, EVs are known to interfere with pathways that lead to tumour progression and are used as novel cancer biomarkers. In veterinary medicine, very little is known on cancer-derived EVs. In this study, we preliminarily characterized EVs in mammary gland cancer of dogs and cats. EVs were isolated by ultracentrifugation from canine (CYPp), feline (FMCp) and human (MCF7) mammary tumour cell lines. EVs were visualized by transmission electron microscopy (TEM), counted using nanoparticle tracking analysis (NTA) and characterized by immunogold (CD63 and Alix) and western blot (Alix and TSG101). Additionally, EV production by "donor" cells (palmtdTomato+ ) and uptake by "recipient" cells (GFP+ ) were assessed. EVs were successfully isolated from all 3 cell lines by ultracentrifugation. Membrane-bound structures (50-400 nm) were identified by TEM and were positive for both CD63 and Alix at immunogold. Western blot showed positivity of EVs to Alix and TSG101. NTA analysis detected EVs from cell culture media ranging from 1.67 to 2.56 × 102 as number of EVs/cell and from 80 to 600 nm in size. Confocal microscopy identified the presence of palmtdTomato+ EVs into the cytoplasm of GFP+ cells. This preliminary study identified and characterized canine and feline mammary tumour cell-derived EVs, opening in veterinary medicine a new interesting unexplored field with several applications and limitless potential.

The transcription factor MAFK induces EMT and malignant progression of triple-negative breast cancer cells through its target GPNMB.

Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.

Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway

Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m2; normal weight (N): 18.5–24.9 kg/m2). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy.

Abstract 5203: Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression

Abstract PKC is a serine-threonine kinase family that controls malignant transformation and metastatic dissemination. ATRA is the main active metabolite of vitamin A. Some evidences indicate that PKCδ may regulate the expression of some retinoid acid (RA) dependent genes, and others indicate that retinoids could alter PKCα intracellular localization; both processes would lead to cell differentiation. In this work we have developed human (MDA-MB 231) and murine (LM3) cell models overexpressing PKCα or PKCδ, in order to determine whether PKC expression alters the sensitivity to retinoids treatment (ATRA). The effect of ATRA was studied in vitro, analyzing biological responses related to tumor growth. In LM3 cells, only PKCα overexpression was able to reduce in vitro population doubling time (PDT) as compared to control (PDT: 14,8±2,3 h vs 21,2±3,1 h for LM3-PKCα y LM3-Vector respectively). Moreover, these cells also responded to retinoid treatment with a significant delay in cell proliferation (PDT: 24,3±4,2 h vs. 14,8±2,3 h for LM3-PKCα ATRA treated or not respectively. In MDA-MB 231 derived cell lines, PKC overexpression as well as ATRA treatment have no effect on proliferative potential. No differences were observed on migratory and invasive capabilities either. Interestingly, in LM3, PKCδ overexpression induced an important increase in proteolytic enzymes secretion, which correlates with its major invasiveness, but this increase had no impact on in vivo metastatic dissemination. Only PKCα overexpression increased this parameter (lung nodes, median (range): 55 (20-75) vs 0 (0-10) for LM3-PKCα y LM3-Vector respectively). Contrary to LM3-PKCδ in MDA-PKCδ cells we could detect a decrease on proteolytic enzymes production and invasive capability. Moreover, colonies growing in Matrigel as 3D cultures showed a small and branched structures. Finally we studied whether the overexpression of α and δ PKC isoforms is able to alter the activity of retinoic acid responsive elements (RARE) through a reporter gene assay. On LM3 model, the constitutive expression of PKCδ highly increased RARE dependent activity. Surprisingly, in MDA-MB231 derived sublines, we could detect a significant increase on RARE activity when cells were treated with ATRA, Altogether, these results suggest that PKCα overexpression confers a more aggressive phenotype in LM3 model, but also makes these cells sensitive to ATRA effects. We could hypothesize, that the absence of response to ATRA treatment displayed by MDA-MB 231 sublines can be explained by their lack of RARβ, which is implied in AP-1 transrepression in response to ATRA. Among others phenomena, AP-1 is involved in cell proliferation and proteolytic enzymes production. Regarding the differences of response to PKCδ overexpression of both models, it has been reported that this PKC isoform has a differential role, pro or anti tumorigenic, depending on cellular context. Citation Format: Maria I. Diaz Bessone, D. E. Berardi, S. M. Cirigliano, C. Flumian, E. D. Bal de Kier Joffe, L. B. Todaro, A. J. Urtreger. Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2015-5203

Abstract 2470: Cd177, a novel metastasis suppressor of breast cancer

Abstract Mammary glands contain luminal and basal epithelial cells. We recently found that both luminal and basal epithelial cells can initiate malignant mammary tumors with forced ErbB2/Her2/c-Neu expression (referred to as luminal and basal tumor-initiating cells, respectively). Basal tumor-initiating cell (TIC)-derived tumors had a greater incidence and larger volume than luminal TIC-derived tumors. Transcriptomic analysis was performed to evaluate the underlying transcriptional difference between ErbB2-driven luminal and basal TIC-derived tumors. CD177 was found to be downregulated within basal TIC-derived tumors. CD177 is an important membrane glycoprotein on neutrophils and serves as a marker for myeloproliferative diseases. The role of CD177 in solid tumors is unknown. Our initial analysis revealed that loss of CD177 expression is linked to higher metastasis incidence and shorter survival in breast cancer patients, but not other diagnostic markers like estrogen-, progesterone-, or HER2-receptors. Silencing CD177 in 4T1 cells, a mouse mammary tumor cell line, led to a significant increase for colonic growth in soft agar and pulmonary metastasis in mice. Overexpressing CD177 in MCF7 cells, a human luminal mammary tumor cell line, resulted in a significant decrease for colonic growth in soft agar. We also found that genetic deletion of CD177 in female mice results in significantly increased Lineage-CD24+CD49fhi basal epithelial cells, the major tumor-initiating cells for ErbB2-induced breast cancer. Collectively, we identified a novel potential metastasis-suppressor for human breast cancer with important prognostic value. Future studies will involve in the mechanistic understanding of how CD177 regulates metastasis of breast cancer. Citation Format: Qing Xie, Nicholas Borcherding, Ryan Kolb, Weizhou Zhang. Cd177, a novel metastasis suppressor of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2014-2470

Abstract P5-05-08: Obesity suppresses estrogen receptor beta expression in breast cancer cells via a HER2-mediated pathway

Abstract Purpose: Increased levels of estrogen receptor beta (ERb) are correlated with an improved breast cancer prognosis. Our preliminary data suggests that obesity may decrease ERβ expression in certain breast cancer subtypes and may contribute to the more aggressive disease associated with obesity. This study aims to elucidate a potential mechanism(s) mediating this suppression. Experimental Design: We utilized an in vitro model of obesity in which breast cancer cells were exposed to sera collected from postmenopausal breast cancer patients at the Cancer Therapy and Research Center at the University of Texas Health Science Center at San Antonio (UTHSCSA) and pooled by BMI category (obese (OB): ≥30 kg/m2; normal weight (NW): 18.5-24.9 kg/m2). Four human mammary tumor cell lines were used: SKBR3 (HER2 over-expressing, ERα negative), MCF-7 and ZR75 (ERα positive, HER2 negative) and MDA-MB-231 (triple negative), in addition to the mouse mammary tumor cell line, MMTV/neu (HER2 over-expressing). The effects of the sera on ERβ expression were assessed by qPCR. Protein expression levels were determined using Western blot analyses. Herceptin, a HER2 antagonist, was used to evaluate the role of HER2 in OB sera-induced ERβ modulation. Results: OB sera exposure led to lower ERβ expression in SKBR3 and MMTV/neu cells in comparison to NW. Treatment of the SKBR3 cells with Herceptin at the time of OB sera exposure resulted in a three-fold increase in ERβ expression. In contrast, there was only a small increase in SKBR3 cell ERβ expression when Herceptin was added to NW sera, suggesting that the effects of the OB sera may be mediated by HER2 activity. In support of this hypothesis, exposure to OB versus NW sera did not modulate ERβ expression in MDA-MB-231, MCF-7 and ZR75 cells, which have very low levels of HER2. Lapatinib, another HER2 antagonist, will be used to inhibit HER2 signaling in MMTV/neu cells. On-going studies utilizing siRNA to the HER2 receptor aim to elucidate the HER2-regulated pathways contributing to the ERβ suppressing effects of OB sera in SKBR3 cells. Conclusion: Based on our preliminary data, we hypothesize that obesity suppresses ERβ expression in breast cancer cells via a HER2-mediated pathway. Elucidation of the mechanism(s) mediating this effect could provide important insight into both the regulation of ERβ expression as well as how obesity promotes a more aggressive disease. Increased knowledge regarding the regulation of this effect may result in the identification of a new treatment target for the obese breast cancer patient population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-08.

Abstract PR-10: PARP inhibition induces genomic instability in normal human cells.

Abstract Recent research has focused on poly(ADP-ribose) polymerases (PARPs) as potential chemotherapeutic targets. Tumors which harbor defects in DNA double-strand repair by homologous recombination (HR), including those with mutant BRCA1 and BRCA2, are exquisitely sensitive to PARP inhibitors, agents that block single-strand break repair. This approach to cancer therapy is known as synthetic lethality, given that PARP inhibitors are not toxic to normal cells at doses that kill HR-deficient cells. While initial trials with PARP inhibitors have demonstrated their promise, many questions remain regarding their clinical use. While PARP inhibition in HR-proficient cells is not cytotoxic, little data exists with regard to genotoxicity. To assess genotoxic effects resulting from PARP inhibition, we quantified frequencies of sister chromatid exchange (SCE), a type of HR with the potential to cause copy number variation at repeats, and chromosomal aberrations. Human non-tumorigenic cell lines (MCF-10A, HMEC-hTERT, EBV-transformed B cells), human mammary tumor cell lines (MCF-7, MDA-MB-468) and primary human T cells obtained from normal donors were tested. Short duration, non-cytotoxic dosing of three PARP inhibitors (olaparib, ABT-888 and BSI-201) currently in clinical trials was used. Olaparib was extensively studied and demonstrated profound induction of SCEs (4.4–9.6 fold) and chromatid-type aberrations (1.7–5.5 fold) in primary human T cells and non-tumorigenic and tumorigenic cell lines. Both olaparib and ABT-888 induced a dose-dependent increase in the frequency of SCEs at non-cytotoxic concentrations (typically greater than 90% cell viability). By contrast, even at cytotoxic doses, SCE frequencies were only slightly higher than the baseline with BSI-201. These results indicate that the SCE frequencies induced by PARP inhibitors are agent- and dose-dependent. A direct correlation between SCE induction and PAR activity inhibition was observed with the tested PARP inhibitors: whereas olaparib and ABT-888 markedly inhibited PARylation (97%), BSI-201 decreased PAR levels more modestly (60%). To assess these differences between agents in a cellular context, BRCA1 and BRCA2 mutant mouse embryonic stem (mES) cells along with wild-type mES cells were treated with olaparib and BSI-201. As expected, olaparib resulted in marked hypersensitivity in these HR-deficient cells as compared to wild type, with greater than a 200-fold increased sensitivity for Brca1-deficient cells. By contrast, BSI-201 resulted in minimally (1.5-fold) increased sensitivity. In conclusion, we have demonstrated genomic instability arising from short-term, low dose PARP inhibitors exposure of normal human cells. The genotoxic risk from clinical use of PARP inhibitors should be considered, especially for patients with early stage cancers and in prevention or non-oncologic indications. This work was supported by The Hecksher Foundation for Children (M.E.M.), National Institutes for Health grants P01CA94060 (M.E.M. and M.J.) and the Uehara Memorial Foundation and Sumitomo Life Social Welfare Services Foundation (S.I.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-10.

Antiproliferative effects of galectin-1 from Rana catesbeiana eggs on human leukemia cells and its binding proteins in human cells

Galectin-1 from American bullfrog, RCG1, was isolated to high purity, and its growth inhibitory properties against human cells were examined. The results demonstrated that highly purified RCG1 induced large cell aggregates and revealed cell-type-specific growth inhibition. It significantly inhibited all human leukemia cell lines tested such as HL-60, U937, and K562 cells but did not inhibit human colon cancer cell line, Colo 201, or mouse mammary tumor cell line FM3A cells. Although most of the galectin-induced growth inhibitions are known to be apoptic, RCG1 induced growth arrest and neither apoptosis nor necrosis. RCG1-mediated growth inhibition was specifically suppressed by the corresponding sugar, lactose, but not by sucrose or even the structurally similar sugar, melibiose. Several studies have reported that galectin-mediated biological functions were modulated by charge modification. Since the high purity of RCG1 was demonstrated but a moderate degree of growth inhibition occurred, it is possible protein charge modification was examined by isoelectric focusing, and it was found to be highly heterogeneous in charge. RCG1 binding proteins in human cells were analyzed by lectin blotting using biotinylated RCG1, and lectin blotting revealed that in human cell extracts the specific proteins at molecular weight 37 and 50kDa possessed the responsive features of RCG1 binding and lactose competition.

Abstract 609: Frondoside antagonizes the prostaglandin E receptor EP4 and inhibits breast tumor metastasis

Abstract Sea cucumber has been used as a folk medicine for many conditions and recently antitumor activities have been described. Frondoside A, a triterpenoid glycoside isolated from the sea cucumber Cucumaria frondosa, as well as the related compound Frondanol A5 inhibit growth of pancreatic, colon and breast tumor cell lines in vitro and show activity in a primary prevention model of colon cancer. The cyclooxygenase 2 pathway contributes to growth and progression of many solid tumors by multiple mechanisms. The COX-2 product, prostaglandin E2 (PGE2) contributes to malignancy by acting on a family of four G-protein-coupled receptors (EP1, EP2, EP3, EP4). We have shown that both human and murine breast tumor cell lines express all EP receptors and that EP4 contributes to metastatic potential in a syngeneic murine model of metastatic breast cancer. Other laboratories have also provided evidence that EP4 promotes malignancy but a paucity of effective EP4 antagonists has slowed the development of clinically evaluable EP-targeted compounds. Based on the reported anti-inflammatory properties of Frondoside A, we asked if this compound has EP antagonist properties. We now report the novel finding that Frondoside A is a potent antagonist of EP4 and, to a lesser degree, antagonizes EP2. Frondoside A inhibits the binding of radio-labeled PGE2 to Chem-1 cells expressing EP4 or HEK cells transduced to express EP2. Using EP4-expressing MDA-MD-231 human breast cancer or murine mammary tumor cell line 66.1, we show that EP4-mediated activation of adenylate cyclase is also blocked by Frondoside A indicating both pharmacologic and functional EP4 receptor antagonism. We determined the effect of Frondoside A on metastatic potential of line 66.1 tumor cells. Treatment of 66.1 cells with non-cytotoxic concentrations of Frondoside A prior to i.v. administration of tumor cells into syngeneic Balb/cByJ female mice reduced lung colonizing capacity by 58%. These studies describe a potent metastasis inhibitory activity of Frondoside A and show, for the first time, that this compound may be a potent novel EP4/EP2 antagonist with activity in a model of metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2011-609

Cell Biological Manifestations of Bisdioxopiperazines: Treatment of Human Tumor Cell Lines in Culture

British developed Bisdioxopiperazine compound (Biz compounds) {ICRF-159 or ICRF-187 (razoxane, Raz)} which was the first agent ever to be observed inhibiting and controlling the spontaneous pulmonary metastases of murine Lewis Lung Carcinoma (LLC) tumor model worldwide. Since 1980, two new Biz compounds {probimane (Pro, AT-2153, MST-2) and MST-16} have been synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, PR China, based on structural modifications from British developed Biz compounds. Despite some similarities and differences of structural and pharmacological activities observed between Raz, Pro and MST-16, the systematic comparisons of their pharmacological activities and mechanisms, especially on neoplasm metastases, are still much needed. This work demonstrates that Biz compounds may inhibit tumor cell migration in vitro through a Matrigel-Coated Transwell plate assay and a wound-healing assay by using three human mammary tumor cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468). Pro, ICRF-187 and MST-16 affect the network of actin assembly. We conclude that Biz compounds might inhibit neoplasm metastases via affecting a cascade of GTPases, cell skeleton polarizations and cell movements.

Abstract LB-236: Differential regulation of STAT3 in mammary gland involution as well as in mammary tumorigeneis

Abstract STAT3 (Signal Transducer and Activator of Transcription 3) is persistently activated in many human primary tumors as well as in cancer cell lines, including breast cancer. This activated STAT3 is associated with cell survival and proliferation, inhibition of apoptosis, activation of angiogenesis and suppression of immune surveillance. Thus, STAT3 is generally considered as a growth-promoting factor. However, STAT3 activation also plays an important role in initiation of apoptotic signaling, especially, in mammary epithelial cells during mammary gland involution. Conditional deletion of STAT3 is associated with delayed involution mainly due to inadequate induction of apoptosis in mammary epithelial cells. Additionally, STAT3 activation is associated with induction of growth arrest and apoptosis in mouse mammary epithelial cells, myeloid cells and granulocyte. Thus, STAT3 plays dual role as a growth promoter as well as a growth inhibitor. However, the molecular switch that is responsible for switching the STAT3- associated pro-oncogenic signal to pro-apoptotic signal is not known. In order to identify this molecular switch we screened for signaling molecules that regulate STAT3 as well as those that are regulated by STAT3 during mammary gland involution, in human primary breast tumor tissues, and in human breast cancer cell lines. Our results show that early in the onset of mammary gland involution there is a significant up regulation of STAT3 and SLC5A8, a Na+-coupled transporter for monocarboxylates, including butyrate an inhibitor of histone deacetylases (HDACs). Further, STAT3 expression during mammary gland involution was found to be predominantly acetylated and phosphorylated. However, in human breast cancer cell lines we found an increase in phosphorylated STAT3 with almost undetectable expression of SLC5A8 and significantly decreased expression of acetylated STAT3 expression. Heterologous expression of SLC5A8 in human mammary tumor cell lines resulted in a butyrate-dependent induction of apoptosis with an increase in acetylated forms of STAT3. These results suggest that SLC5A8 is the molecular switch that converts the pro-oncogenic STAT3 signal to a pro-apoptotic signal by mediating the butyrate entry into mammary epithelial cells and thus it activates death receptor signaling by inhibiting HDACs and activation of STAT3 acetylation during mammary gland involution. However, in cancer cells the expression of SLC5A8 is silenced and thus it no longer able to inhibits HDACs and activates Ac-Stat3 and death receptor signaling. Thus, our studies provide a molecular link between the STAT3-associated pro-apoptotic and pro-oncogenic signaling that observed in normal mammary epithelium and in human breast cancer, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-236.

Construction of Targeting Vector for Expressing Human GDNF in Cattle Mammary Gland

Glial cell line-derived neurotrophic factor (GDNF) is a type of neurotrophic factor with significant potential in treatment of Parkinson's disease. Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The aim of this study was to construct a gene-targeting vector for the human gdnf gene knockin at the bovine beta-casein gene locus so that human GDNF protein can be produced in the mammary gland of the gene-targeted bovine. The constructed vector contains the 2.2 kb 5' homologous arm and the 5.7 kb 3' homologous arm. The human gdnf cDNA was located at the downstream of the 5' homologous arm. The neo gene placed between the 5' and 3' homologous arms as positive selection marker gene. The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes, respectively. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the plasmid DNA was transfected into human mammary tumor cell line Bcap-37 by lipofectamine. Reverse transcription polymerase chain reaction and Western-blotting analysis showed that the transfected cells produced human GDNF mRNA and protein. The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells. At the same time, it is first time to confirm that human mammary tumor cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.