MDA-MB231 Cell Lines Research Articles

Olive secoiridoids and semisynthetic bioisostere analogues for the control of metastatic breast cancer

(−)-Oleocanthal (1) and ligstroside aglycone (2) are common bioactive olive oil secoiridoids. Secoiridoid 1 has been previously reported as a c-MET inhibitor. Chemically, (−)-oleocanthal is the elenolic acid ester of the common olive phenolic alcohol tyrosol. Therefore, several analogues (4–13) were synthesized by esterification and carbamoylation of tyrosol using diverse phenolic naturally occurring in olive and heterocyclic acids as elenolic acid bioisosteres to assess the effect of replacing the acid moiety of (−)-oleocanthal. Their c-MET inhibitory activity as well as their antiproliferative, antimigratory, and anti-invasive activities against the highly metastatic human breast cancer cell line MDA-MB231 has been assessed. Ligstroside aglycone (2) showed the best antimigratory activity. Generally, tyrosol esters showed better activities versus carbamate analogues. Tyrosol sinapate (5) showed the best c-MET phosphorylation inhibitory activity in Z′-LYTE™ kinase assay. Both 1 and 5 competitively inhibited the ATP binding into its pocket in the c-MET catalytic domain. Compound 5 showed selective activities against tumor cells without toxicity to the non-tumorigenic human breast MCF10A epithelial cell line. Tyrosol esters with a phenolic acid containing hydrogen bond donor and/or acceptor groups at the para-position have better anticancer and c-MET inhibitory activities. Olive oil secoiridoids are excellent scaffolds for the design of novel c-MET inhibitors.

Histone Deacetylase (HDAC) Inhibitors with a Novel Connecting Unit Linker Region Reveal a Selectivity Profile for HDAC4 and HDAC5 with Improved Activity against Chemoresistant Cancer Cells

The synthesis and biological evaluation of new potent hydroxamate-based HDAC inhibitors with a novel alkoxyamide connecting unit linker region are described. Biological evaluation includes MTT and cellular HDAC assays on sensitive and chemoresistant cancer cell lines as well as HDAC profiling of selected compounds. Compound 19i (LMK235) (N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide) showed similar effects compared to vorinostat on inhibition of cellular HDACs in a pan-HDAC assay but enhanced cytotoxic effects against the human cancer cell lines A2780, Cal27, Kyse510, and MDA-MB231. Subsequent HDAC profiling yielded a novel HDAC isoform selectivity profile of 19i in comparison to vorinostat or trichostatin A (TSA). 19i shows nanomolar inhibition of HDAC4 and HDAC5, whereas vorinostat and TSA inhibit HDAC4 and HDAC5 in the higher micromolar range.

Tumor associated fibroblasts are endowed with increased ability to support tumor development

Event Abstract Back to Event Tumor associated fibroblasts are endowed with increased ability to support tumor development Marius Toma1*, Radu-Adrian Lazarovicz1, Oana Gavriliuc1, Florina M. Bojin1, Calin Tatu1, Alexandra Gruia1, Carmen Tatu1, Gabriela Tanasie1, Carmen Panaitescu1 and Virgil Paunescu1 1 University of Medicine and Pharmacy Victor Babes Timisoara, Functional Sciences, Romania Tumor associated fibroblasts (TAFs) from solid human tumors provide structural and functional support to tumor development and progression. The main purpose of this study was to investigate TAF phenotype and functions comparative to bone marrow-derived mesencymal stem cells (MSCs) and to evaluate the role of these cells in tumorigenesis. TAFs were isolated using enzymatic digestion from surgical removed breast tumor pieces and were cultured for several passages in standard cultivation conditions, similar to MSCs. Cellular analysis included transmission electron microscopy (TEM), immunophenotyping (HLA-DR, CD29, CD44, CD73, CD90, CD106, and CD117), cytoskeleton and matrix proteins expression based on immunohistochemistry, and tri-lineage differentiation potential towards adipocytes, osteoblasts, and chondrocytes. ELISA assay was used for determining cells secretory profile, and we found that TAFs secrete significantly increased amounts of VEGF, TGF-β1, IL-4, IL-10, and TNF-α. Tumor cell lines SK-BR3, MDA-MB231 and MDA-MB468 were used for flowchamber adherence studies, under progressively increasing shear stress from 0.35 to 15 dyne/cm2, on MSCs or TAFs cellular substrate. The results showed that tumor cells have increased adherence to TAFs, for all values of shear stress, mainly because VEGF and IL-4 secretion, so that we may conclude that even though MSCs and TAFs share functional and structural characteristics, TAFs represent a more specialized cellular type, able to provide support for tumor growth and development, and can be a target for further anti-tumor therapies. Acknowledgements PN II IDEI 318/2011 Keywords: MSCs, TAFs, tumorigenesis, immunemodulation, Angiogenesis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Toma M, Lazarovicz R, Gavriliuc O, Bojin FM, Tatu C, Gruia A, Tatu C, Tanasie G, Panaitescu C and Paunescu V (2013). Tumor associated fibroblasts are endowed with increased ability to support tumor development. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00446 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Marius Toma, University of Medicine and Pharmacy Victor Babes Timisoara, Functional Sciences, Timisoara, 300041, Romania, tomamarius.forever@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marius Toma Radu-Adrian Lazarovicz Oana Gavriliuc Florina M Bojin Calin Tatu Alexandra Gruia Carmen Tatu Gabriela Tanasie Carmen Panaitescu Virgil Paunescu Google Marius Toma Radu-Adrian Lazarovicz Oana Gavriliuc Florina M Bojin Calin Tatu Alexandra Gruia Carmen Tatu Gabriela Tanasie Carmen Panaitescu Virgil Paunescu Google Scholar Marius Toma Radu-Adrian Lazarovicz Oana Gavriliuc Florina M Bojin Calin Tatu Alexandra Gruia Carmen Tatu Gabriela Tanasie Carmen Panaitescu Virgil Paunescu PubMed Marius Toma Radu-Adrian Lazarovicz Oana Gavriliuc Florina M Bojin Calin Tatu Alexandra Gruia Carmen Tatu Gabriela Tanasie Carmen Panaitescu Virgil Paunescu Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Hippuric acid nanocomposite enhances doxorubicin and oxaliplatin-induced cytotoxicity in MDA-MB231, MCF-7 and Caco2 cell lines

BackgroundThe aim of the current study is to design a new nanocomposite for inducing cytotoxicity of doxorubicin and oxaliplatin toward MDA-MB231, MCF-7, and Caco2 cell lines. A hippuric acid (HA) zinc layered hydroxide (ZLH) nanocomposite was synthesized under an aqueous environment using HA and zinc oxide (ZnO) as the precursors.MethodsThe hippuric acid nanocomposite (HAN) was prepared by the direct reaction of a HA solution with an aqueous suspension of ZnO.ResultsThe basal spacing of the nanocomposite was 21.3 Å, which is average of four harmonics at 2θ = 8.32°, 12.50°, 16.68°, and 20.84°. This result indicates that the hippurate anion was successfully intercalated into the interlayer space of ZLH. The combinations of HAN with chemotherapy (drugs) has inhibited the cell growth of the MDA-MB231, MCF-7, and Caco2 cancer cells when compared to drugs alone. An IC50 value for the combination of HAN with doxorubicin toward MCF-7 is 0.19 ± 0.15 μg/mL and toward MDA-MB231 is 0.13 ± 0.10 μg/mL. Similarly, the IC50 for the combination of HAN with oxaliplatin toward Caco2 is 0.24 ± 0.11 μg/mL. In the antiproliferative results, the equal combination of HAN (0.5 μg/mL) with doxorubicin (0.5 μg/mL) has reduced the cell proliferation in MCF-7 and MDA-MB-231 cells into 37.3% and 17.6%, respectively after 24 hours. Similarly, the antiproliferation percentage for equal combination HAN with oxaliplatin (5.00 μg/mL) toward Caco2 is 72.7% after 24 hours.ConclusionThe resulting combination HAN with drugs has exhibited higher inhibition in cells growth in all cancer cell lines.

Anti-inflammatory and anticancer activities of (−)-zeylenol from stems of Uvaria grandiflora

Uvaria grandiflora has stimulated rigorous phytochemical investigation aimed at determining chemical structures and biological activities. In recent years, many polyoxygenated cyclohexenes have been isolated from this genus. This study reports that zeylenol isolated from the stems of U. grandiflora possess potential anti-inflammatory, anticancer, and caspase-3 activities. The structure of this compound was elucidated from spectroscopic analysis, particularly 2D NMR techniques. The anti-inflammatory effect and toxicity of zeylenol were evaluated in animal models and compared to that of reference drugs. In addition, the cytotoxicity of the isolated compound against human breast cancer MDA-MB231 and hepatocellular carcinoma HepG2 cell lines were studied. The test ( )-zeylenol at the dose of 1 mg/ear significantly inhibited oedema formation by 90%, 69%, 52%, and 52% after 15, 30, 60, and 120 min, respectively. Zeylenol was found to be toxic to both MDA-MB231 and HepG2 cells in a dose response manner with IC50 values of 54 10 and > 80 µM, respectively. The compound induced MDA-MB231 cell apoptosis via caspase-3 activation. Zeylenol probably possesses anti-inflammatory activity by inhibiting the synthesis or release of various inflammatory mediators and might be used to induce human breast cancer MDA-MB231 cell apoptosis.

Abstract P6-04-20: Endocrine resistance in invasive lobular carcinoma cells parallels unique estrogen-mediated gene expression

Abstract Invasive lobular carcinoma (ILC) represents ∼10% of newly diagnosed breast tumors, accounting for ∼30,000 cases annually in the US. However, ILC has been understudied as a breast cancer subtype. ILC-specific signaling pathways and responses to endocrine therapies are not well characterized. Recent retrospective analyses of luminal-type breast cancers suggest that ILC patients treated with endocrine therapy have poorer disease-free survival and overall survival than invasive ductal carcinoma (IDC) patients with similar biomarkers. We hypothesize that unique transcriptional control of estrogen receptor-alpha (ERα) by estrogens and anti-estrogens in ILC cells drive a differential response of ILC tumors to endocrine therapies. The ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of ILC tumors to examine responses to estradiol (E2) and the anti-estrogens tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI). We examined cell growth and expression of canonical ERα-regulated genes in response to E2. Cells were also treated with anti-estrogens +/− E2 to examine their ability to inhibit E2-induced growth. To establish a genome-wide profile of ERα-mediated gene regulation in ILC cells, ILC cells were subjected to gene expression microarray analysis following 0–24hrs treatment with 1nM E2. In parallel to these in vitro studies, in vivo models of ILC are being assessed for endocrine responsiveness, including MDA MB 134VI xenografts and the primary human tumor xenograft HCI-013. We observed that both MDA MB 134VI and SUM44PE are growth-induced by E2 treatment. However, both cell lines also present de novo resistance to Tam, 4OHT, and Bx. While ICI treatment completely blocked E2-induced growth in MDA MB 134VI, Tam, 4OHT, and Bx acted as partial agonists. Treatment with these compounds alone induced ∼25% growth relative to E2, and E2-induced growth could only be inhibited ∼75%. Similarly, SUM44PE cells are strongly growth-inhibited by ICI treatment, whereas growth is not reduced by Tam, 4OHT, or Bx treatment. Consistent with these observations, novel patterns of gene expression are induced by E2 treatment in ILC cells. E2-treatment induced canonical targets (e.g. GREB1, IGFBP4) in both ILC cell lines. However, in MDA MB 134VI cells only ∼35% of genes regulated by E2 at 24hrs overlap with those regulated in the IDC cell line MCF-7. Further, a subset of the overlapping genes is differentially regulated between cell types, including ESR1 (1.25-fold and 0.67-fold versus control in MDA MB 134VI and MCF-7, respectively), HOXC6, PMP22, and L1CAM. Examination of these observations in in vivo models is currently ongoing. These data are consistent with the hypothesis that E2 and anti-estrogens differentially regulate ERα-mediated gene expression in ILC cells versus IDC cells. The de novo resistance to tamoxifen observed in ILC cells may correlate with the worse outcomes in ILC patients observed in retrospective analyses. We hypothesize that elucidation of the mechanisms responsible for differential ERα regulation may reveal novel therapeutic targets for treating ILC patients and overcoming endocrine resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-20.

Abstract P2-06-05: Single-cell RNA sequencing of paclitaxol-treated breast cancer cell lines to find individual cell response

Abstract Cancer treatments act on a population of cells, each of which may experience different individual responses to treatment. Such differential response will result in resistance to treatment even if a majority of cancerous cells are eliminated. To examine differential cell response, we simultaneously profiled the gene expression and mutation spectrum of individual cells from the MDAMB231 cell line using next generation sequencing of isolated RNA. A total of 23 transcriptomes were characterized from paclitaxel-treated and paclitaxel-surviving cells. We found significant different changes in mutation rates between paclitaxel treated cells, with a dose-dependent increase in single nucleotide changes in RNA in paclitaxel-treated cells. Cells undergoing exposure to paclitaxel also showed higher pathway activity in SRC, as well as an integrin switch from ITGB1 to ITGB3. In contrast, cells that survived a high dose of paclitaxel showed an insignificant number of single nucleotide changes, suggesting that these cells either evaded initial paclitaxel exposure or were better able to repair the effects of paclitaxel exposure. Despite the RNA sequence similarity between surviving and untreated cells, there were changes in gene expression and pathway activities including higher PI3K activity. Paclitaxel-surviving cells also showed activation of pathways associated with higher proliferation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-05.

Cyclopalladated benzophenone imines: Synthesis, cytotoxicity against human breast adenocarcinoma cell lines and DNA interaction

Treatment of benzophenone imine with the stoichiometric amount of Pd(OAc)2 in acetic acid at 60 °C produced the corresponding acetato-bridged five-membered ortho-cyclopalladated dimer [Pd{C6H4CPhNH}(μ-OAc)]2 (1), which was isolated in pure form in a 79% yield. Reaction of 1 with an excess of LiCl in acetone gave rise to the corresponding chlorido-bridged cyclopalladated dimer [Pd{C6H4CPhNH}(μ-Cl)]2 (2) in a 78% yield. Compounds 1–2 reacted with an excess of py-d5 or the stoichiometric amount of PPh3 to give the mononuclear compounds trans-N,L-[Pd{C6H4CPhNH}(X)(L)] [3 (X = OAc, L = py-d5); 4 (X = Cl, L = py-d5); 5 (X = OAc, L = PPh3) and 6 (X = Cl, L = PPh3)]. Compounds 2–3 were prepared in CDCl3/py-d5 solution and were studied by 1H NMR, but were not isolated. In contrast, compounds 5–6 were prepared in acetone and were isolated in pure form in 43 and 79% yields, respectively. Compounds 1, 2, 5 and 6 were characterized by elemental analyses, mass spectrometry, IR, NMR and electronic spectroscopy. Compounds 1, 2, 5 and 6 showed high antiproliferative activity against MDA-MB231 and MCF7 human breast cancer cell lines, especially, compounds 5–6. These two latter compounds presented greater antiproliferative activity than cisplatin and produced IC50 values in the range 1–5 μM. The interaction of compounds 1, 2, 5 and 6 with DNA was also studied by the DNA electrophoretic migration, DNA-ethidium bromide fluorescence quenching and viscometry techniques.

Cytotoxic effect of ICD-85 (venom-derived peptides) on HeLa cancer cell line and normal LK cells using MTT Assay.

Cancer is the fifth leading cause of death worldwide. There are considerable efforts to identify naturally occurring substances for use as new drugs in cancer therapy. Some components of animal venoms have been identified that possess substantial anticancer properties. In our previous studies, the cytotoxic effects of ICD-85 (venom-derived peptides) have been reported on HL-60 and MDA-MB231 cell lines. This has prompted us to investigate the comparative cytotoxic effects of ICD-85 on the HeLa cell line and normal lamb kidney (LK) cells. Cells were exposed to various concentrations (8 × 10-4 to 5.6 × 10 µg/ml) of ICD-85 at various incubation times (24, 48 and 72 hours). Cell viability was measured by the MTT assay. A morphological study was also carried out using an inverted microscope. Caspase-8 activity was assayed by the Caspase-8 Colorimetric Assay Kit in HeLa cells that were exposed to ICD-85 for 48 hours. Data analysis showed that ICD-85 has a dose-dependent cytotoxic effect on HeLa cells with an inhibitory concentration 50% (IC50) of 26.62 ± 2.13 µg/ml at 24 hours, 27.33 ± 2.35 µg/ml at 48 hours, and 28.13 ± 2.52 µg/ml at 72 hours. Results also indicated that the cytotoxic effect of ICD-85, at 48 and 72 hours incubation times did not show significant alteration compared to 24 hours of exposure. Interestingly, the minimum concentration of ICD-85 which showed a cytotoxic effect on LK cells was found to be 3500-fold less than the minimum concentration that showed a cytotoxic effect on the HeLa cancer cells. While morphological analysis revealed a significant difference that included the characteristic rounding of dying cells by treatment with ICD-85 compared with untreated HeLa cells, this difference was not observed in normal cells. ICD-85 increased caspase-8 activity in HeLa cells after 48 hours of exposure. ICD-85 has a dose-dependent cytotoxic effect on HeLa cancer cells in contrast with its negligible effect on normal LK cells.

WS9-6 - Analysis of Biological Features and Possibility of Chemotherapy of Breast Cancer Stem Cells Selected by Bone Marrow Microenvironment

ABSTRACT Background In breast cancer, especially in the relatively early stage cases, there have been emerging reports that bone marrow (BM) micrometastasis is seen or cancer stem cells (CSC) metastasize into BM. They suggest that breast CSCs are related with BM adhesion and general dissemination. Purpose We hypothesized that BM metastasis cells contain CSCs. To purify BM metastasis cells, we established the in vivo selection system by using immunodeficiency mice and analyzed the biological features of the selected cells in terms of the CSCs. Methods GFP-expressing breast cancer cell line MDA-MB231 were orthotopically transplanted in RAG2 gamma C (KO) mice, and GFP-positive tumor cells from BM cells were purified. Tumorigenicity, cell growth and metastasis, specific features of CSCs, between parent cells and BM meta cells were compared in vitro and in vivo. Next, gene-expression profiles of the two types of cells were also compared to analyze the differences of phenotypes by cDNA microarray. Results and discussion Tumorigenicity and tumor growth were convinced by orthotopical transplantation. BM meta was confirmed pathologically. Purified BM meta cells exhibited higher sphere formation ability and higher drug resistance in vitro. They showed higher serial transplantability, higher growth and metastasis abilities in vivo. From the result of gene-expression analyses, BM meta cells highly expressed Notch1, CD44 and CD49f. Besides that, BM cells growth was inhibited by Notch signaling inhibitor. Together, BM selects a cell population possessing characters of CSC, and control of BM meta may contribute to cancer cure and individualized therapy.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion

IntroductionTumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.MethodsA mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.ResultsWe found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.ConclusionsTogether these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Molecular docking and dynamics simulations of A.niger RNase from Aspergillus niger ATCC26550: for potential prevention of human cancer

The aim of the present research was to study the anticancer effects of Aspergillus niger (A.niger) RNase. We found that RNase (A.niger RNase) significantly and dose dependently inhibited invasiveness of breast cancer cell line MDA MB 231 by 55 % (P<0.01) at 1 μM concentration. At a concentration of 2 μM, the anti invasive effect of the enzyme increased to 90 % (P<0.002). Keeping the aim to determine molecular level interactions (molecular simulations and protein docking) of human actin with A.niger RNase we extended our work in-vitro to in-silico studies. To gain better relaxation and accurate arrangement of atoms, refinement was done on the human actin and A.niger RNase by energy minimization (EM) and molecular dynamics (MD) simulations using 43A(2) force field of Gromacs96 implemented in the Gromacs 4.0.5 package, finally the interaction energies were calculated by protein-protein docking using the HEX. These in vitro and in-silico structural studies prove the effective inhibition of actin activity by A.niger RNase in neoplastic cells and thereby provide new insights for the development of novel anti cancer drugs.

Erufosine suppresses breast cancer in vitro and in vivo for its activity on PI3K, c-Raf and Akt proteins

This study investigated the antineoplastic effect of the membrane active alkylphosphocholine erufosine in breast carcinoma models in vitro and in vivo and determined its influence on the PI3K/Akt and Ras/Raf/MAPK signaling pathways. The antiproliferative effect of erufosine in vitro was determined by the MTT dye reduction assay, and the antineoplastic efficacy on tumor growth was investigated by relating the mean total tumor volumes of treated and control rats. Immunoblot analysis was used for detecting changes in the expression level of the signal molecules p-PI3K (p-p85), p-Akt at Thr 308 and p-cRaf. Based on their IC(50) (40 μM, respectively), the breast carcinoma cell lines MCF-7 and MDA-MB 231, which are estrogen receptor positive and negative, respectively, were equally sensitive to erufosine. In addition, erufosine caused dose-dependent decreases in the phosphorylation of PI3K (p85), Akt (PKB) at Thr 308 and cRaf in both cell lines. Moreover, administration of erufosine to rats bearing autochthonous methylnitrosourea-induced rat mammary carcinomas caused a significant dose-related tumor remission by more than 85 % (p < 0.05), which was well tolerated, as evidenced by a body weight loss of maximally 7 % and reduced tumor-related mortality (2 of 35 instead of 6 of 18 controls, p < 0.002). The results clearly indicate that erufosine possesses high antineoplastic activity not only in human breast cancer cell lines in vitro but also in rat mammary carcinoma in vivo. In addition, it can be derived that the mechanism of action of erufosine involves influence on both, PI3K/Akt and Ras/Raf/MAPK signaling pathways.

Synthesis, structural characterization and anticancer activity of some new complexes of 6-amino-4-hydroxy-2-thiopyrimidine

New complexes of 6-amino-4-hydroxy-2-thiopyrimidine (Hahtp), [Zn(ahtp)2(H2O)2], [Zn(ahtp)2(PPh3)(H2O)], [Zn(Hahtp)(bpy)Cl2], [Pd(phen)(ahtp]Cl, [Pd(Hahtp)(PPh3)2]Cl2, [Ag(ahtp)(H2O)2], [Ag(ahtp)(PPh3)(H2O)], [Ag(ahtp)L] (L=bpy, phen), have been synthesized and characterized on the basis of spectral (IR, 1H-NMR, ESI-mass and UV–visible), elemental analysis, thermal and molar conductivity measurements. Three modes of chelations have been observed for Hahtp; as a neutral bidentate ligand through cyclic nitrogen and thione sulfur atoms, mononegative bidentate ligand through either the deprotonated cyclic nitrogen and thione sulfur atoms or the deprotonated hydroxy and cyclic nitrogen atoms; all forming four-membered chelating rings. The free Hahtp and its complexes, [Zn(ahtp)2(H2O)2], [Zn(Hahtp)2(PPh3)(H2O)], [Zn(Hahtp)(bpy)Cl2], [Pd(phen)(Hahtp]Cl and [Ag(Hahtp)(PPh3)(H2O)] have been tested against the human breast cancer MDA-MB231 cell line. The [Ag(ahtp)(PPh3)(H2O)] complex exhibits the highest efficacy with a mean IC50 value of 4.7μM.

Dysregulated Recruitment of the Histone Methyltransferase EZH2 to the Class II Transactivator (CIITA) Promoter IV in Breast Cancer Cells

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types.

Abstract 3024: Elucidation of WNT signaling pathway players “sFRP-1” and “TCF7L2” as novel potential biomarkers of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by an unfavorable prognosis. While patients do not benefit from endocrine or Her2 targeted therapies except chemotherapy, there is an increased propability of response to taxane-/anthracycline-containing therapy compared to other breast cancers. However, molecular determinants of chemotherapy sensitivity for patients with TNBC specifically remain largely unknown. The WNT signaling cascade has been shown to play an important role in breast cancer pathogenesis. Material and Methods: Differentially expressed genes in triple negative (TN) vs. non-TN breast cancers of patients treated with neoadjuvant taxane-/anthracycline-containing chemotherapy were identified and validated in an independent AffymetrixU133A gene chip dataset. Genes were tested for correlation with relapse-free survival, response to neoadjuvant chemotherapy and Ki67 expression. The effects of siRNA mediated knockdown in the TNBC and non-TNBC cell lines on proliferation, chemo sensitivity invasion competency and gene expression of target genes via quantitative real-time PCR were analyzed. Results: sFRP-1 (secreted frizzled related protein 1) and TCF7L2 (transcription factor-7 like 2) were identified as significantly over expressed genes in TNBC vs. non-TNBC and validated in an independent dataset. While sFRP-1 expression was not associated with relapse-free survival in TNBC, it was significantly correlated with a pCR. Importantly, no correlation to Ki67 expression could be demonstrated. While mRNA knockdown of sFRP-1 in MDA-MB 468 was associated with an increased resistance to paclitaxel, doxorubicin and cisplatin, target gene expression analysis suggest sFRP-1 as a biphasic modulator in breast tissue. TCF7L2 knockdown analysis in the TN cell line MDA MB 231 as well as the non-TN lines MCF7 and SKBR-3 shows an increased cell invasiveness of up to 200 %. Conclusion: We suggest sFRP-1 as predictive biomarker and potential mediator of sensitivity to taxane-, anthracycline and platinum-containing chemotherapy in TNBC in vitro referring to its biphasic WNT modulation. Furthermore TCF7L2 is suggested as a prognostic biomarker in vitro breast cancer cell lines including TN and non-TN cell lines. Our results provide proof of principle that high-throughput gene expression analysis may be exploited as a means to derive at a biological / translational hypothesis that may be supported by basic science. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3024. doi:1538-7445.AM2012-3024

Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Abstract MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 463. doi:1538-7445.AM2012-463

Abstract 2250: Combination of PARP inhibitor, Olaparib with pathway-targeted drug, vandetanib (Caprelsa™) in TNBC: A proof of principle study to determine the role of PI3K-AKT-mTOR or RAS-MAPK pathways in targeted drug-response

Abstract Introduction: Identification of the driving genetic alteration(s) (e.g. BRCA, PTEN) and deregulated signaling pathway(s) are critical for the successful discovery of molecular targets in Triple Negative Breast Cancer (TNBC). Understanding of the biology of TN tumor cells has resulted in the recognition of a few molecular targets for the development of novel therapeutics including, (1) cell surface receptor tyrosine kinase(s) (RTKs, e.g. EGFR), (2) signaling pathway(s) (PI3K-mTOR, RAS-MAPK), and (3) DNA-damage (chemo- or radiotherapy) repair pathway. We hypothesize that the genetic background (expression of RTKs, BRCA mutations, absence of PTEN or activating mutations of PIK3CA / RAF) influences the response of TNBC cells to a combination treatment of drugs. Methods: We tested the effects of combination of EGFR/VEGFR inhibitor (vandetanib) with PARPi (Olaparib) in presence of DNA-damaging agent (carboplatin) on, (a) cell survival/proliferation and their, signaling marker(s), (b) integrin-directed migration, (c) fibronectin-directed matrigel-invasion, (d) adhesion-dependent survival, (e) vascular mimicry (VM), and (f) clonogenic survival in TNBC cell lines with high expression of EGFR (MDA-MB468, SUM149), BRCA mutations (HCC1937, SUM149), no expression of PTEN protein (HCC70, HCC1937, MDA-MB468, SUM149), PIK3CA mutation (BT20), and RAS/RAF mutation (MDA-MB231). Results: Data showed that, (1) although EC50s ranged from 5-15 µM for vandetanib in cells, the PTEN-null cells exhibited comparatively favorable EC50s, (2) vandetanib (10 µM) inhibited phosphorylation of AKT (S473 &amp; T308), S6RP, 4EBP1 and ERK by 1 hour, (3) MDA-MB468 cell line was the most sensitive to vandetanib (∼ 0.05 µM) when combined with 10 µM fixed dose of Olaparib, and (4) a combination of vandetanib with Olaparib plus carboplatin time dependently increased caspase-3 and PARP cleavage, inhibited VM (6, 24 h), blocked fibronectin-directed migration, and suppressed clonogenic growth to a greater extent in PTEN-null cells lines. Conclusion: Anti-proliferative/pro-apoptotic, and anti-migratory/invasive effects of vandetanib (alone or in combination with carboplatin plus Olaparib) were most prominent in PTEN-null and high EGFR-expressing cells. However, given that the inhibitory activity of olaparib occurred at relatively high concentrations we are currently pursuing studies to refine the dose response relationship. In addition we are examining (1) the mechanistic relationship between the anti-proliferative/pro-apoptotic effects and the effectors status of EGFR/ PI3K pathway following treatment with vandetanib (in combination with PARPi plus temozolomide), and (2) effect of vandetanib on the hypoxic-response of tumor and endothelial cells, the results of which will be presented in the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2250. doi:1538-7445.AM2012-2250

Abstract 3367: Breast cancer stem cell-like cells are sensitive to ionizing radiation: Role of ATM

Abstract The aim of this study was to determine the radiosensitivity of cancer stem (tumor-initiating) cells (CSC), which are known to be responsible for tumorogenicity and metastasis. We used breast cancer stem cell (CSC)-like MDA-MB231 and MDA-MB453 cell lines which were established by Dr. Edward V. Prochownik (Sajithlal et al., Stem Cells, 28:1008, 2010). Interestingly, these cells can proliferate without differentiation and have characteristics of tumor-initiating cells. CSC-like cells and their alternate subset non-CSC cells were exposed to α-rays (1.25-8.75Gy) and survival curves were determined by colony formation. A final slope, D0, of the survival curve for each cell line was determined to measure radiosensitivity. D0 of CSC-like MDA-MB453 cells and that of non-CSC MDA-MB453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB231 cells. We further examined whether CSC-like cells are relatively radiosensitive owing to different intrinsic factors including cell cycle distribution, free-radical scavengers and DNA repair. We observed that, unlike non-CSC cells, CSC-like cells have little/no sublethal damage repair and a low intracellular level of ATM. These results suggest that low DNA repair capability is responsible for the high radiosensitivity of these CSC-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3367. doi:1538-7445.AM2012-3367

Abstract 500: DMP1 activates osteolytic cycle in a tumor environment

Abstract Bone metastasis is one of the skeletal malignancies that could be caused by breast cancer. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein localized specifically in the mineralized matrix of bone and dentin. It is a multifunctional protein, involved in gene regulation (within the cell) and HAP nucleation (in the ECM). DMP1 is endocytosed by preosteoblasts, triggering the calcium dependent and calcium independent signaling pathways resulting in a series of downstream events leading to osteoblast differentiation. These signaling events facilitate the activation of a calcium dependent stress-induced p38 MAP kinase and a calcium independent integrin mediated ERK1/2 MAP kinase pathway resulting in the expression of downstream target genes such as Runx2 and osteocalcin. Results from this study have shown that the osteoblast cells upon differentiation secrete receptor activator of nuclear factor kappa B ligand (RANKL) that stimulates the differentiation of precursor monocytes to osteoclasts. The study also focuses on identifying the role of DMP1 in breast cancer cells that could enhance osteoclast formation thereby accelerating bone resportion. Thus, the study reveals a new mechanism by which DMP1 can activate the vicious cycle leading to the aggressive growth and behavior of the cancer cells. To address the mechanism by which DMP1 might contribute to the osteolytic process, DMP1 was overexpressed in the metastatic cancer cell line MDA MB231. Upregulation of markers like Runx2, MMP2, MMP9, RANKL, OPN, BSP, VEGF and the activation of Smad2/3 and MAP kinase pathways (p38 and ERK1/2 pathways) were observed in overexpressed cells. Results from this study have shown that DMP1 secreted by the breast cancer cells into the extracellular environment could stimulate the differentiation of osteoblasts, leading to the secretion of factors that signal monocytes to differentiate to osteoclast thus resulting in bone metastasis. Overall, the results obtained from this study identify a new role for DMP1 in the differentiation of breast cancer tumor cells that may be directly related to their metastatic potential. Supported by NIH DE 115657 and the Brodie Endowment Fund Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 500. doi:1538-7445.AM2012-500