BT-474 Human Breast Cancer Cells Research Articles

Synthetic progestins induce growth and metastasis of BT-474 human breast cancer xenografts in nude mice

Previous studies have shown that sequential exposure to estrogen and progesterone or medroxyprogesterone acetate (MPA) stimulates vascularization and promotes the progression of BT-474 and T47-D human breast cancer cell xenografts in nude mice (Liang et al, Cancer Res 2007, 67:9929). In this follow-up study, the effects of progesterone, MPA, norgestrel (N-EL), and norethindrone (N-ONE) on BT-474 xenograft tumors were compared in the context of several different hormonal environments. N-EL and N-ONE were included in the study because synthetic progestins vary considerably in their biological effects and the effects of these two progestins on the growth of human tumor xenografts are not known. Estradiol-supplemented intact and ovariectomized immunodeficient mice were implanted with BT-474 cells. Progestin pellets were implanted simultaneously with estradiol pellets either 2 days before tumor cell injection (ie, combined) or 5 days after tumor cell injections (ie, sequentially). Progestins stimulated the growth of BT-474 xenograft tumors independent of exposure timing and protocol, MPA stimulated the growth of BT-474 xenograft tumors in ovariectomized mice, and progestins stimulated vascular endothelial growth factor elaboration and increased tumor vascularity. Progestins also increased lymph node metastasis of BT-474 cells. Therefore, progestins, including N-EL and N-ONE, induce the progression of breast cancer xenografts in nude mice and promote tumor metastasis. These observations suggest that women who ingest progestins for hormone therapy or oral contraception could be more at risk for developing breast cancer because of proliferation of existing latent tumor cells. Such risks should be considered in the clinical setting.

Abstract 3805: Furanodienone downregulates EGFR and Her-2 signaling in BT-474 breast cancer cells

Abstract Epidermal growth factor receptor (EGFR) and Her-2 are members of the ErbB receptor family of type 1 tyrosine kinases. Also, ligand-induced EGFR /Her-2 heterodimerization triggers potent proliferative and survival signals. EGFR and Her2 frequently overexpress in breast cancer and their overexpression is correlated with a poor prognosis. Therefore dual EGFR/ Her-2 inhibition is an attractive therapeutic strategy for breast cancer treatment. Furanodienone is a compound isolated from Rhizoma Curcumae, which is commonly used in cancer treatment in China. In this study, we used the breast cancer cell BT-474 as the model to investigate the mechanism of furanodienone in breast cancer treatment. We found that furanodienone induced G1 arrest and effectively blocked EGF-stimulated cell cycle progression in BT-474 cells. Moreover, furanodienone decreased protein levels of EGFR, phosphorylation of EGFR and Her-2, so that it prevented the activation of downstream molecules like Akt, Gsk3β and c-Raf, which resulted in inhibiting cell cycle progression. These findings suggested that furanodienone induced growth inhibition in BT-474 human breast cancer cells through inhibiting EGFR /Her-2 signaling pathway. Furthermore, furanodienone and its related compounds provided insights into novel approaches for the prevention and treatment of human breast cancers, especially for those with EGFR /Her-2 overexpression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3805.

Abstract 611: Etanercept as a new tool for treatment of Herceptin-resistant breast cancer induced by tumor necrosis factor alpha

Abstract Breast cancer overexpressing ErbB2 is associated with high aggressiveness and elevated metastatic potential. Several therapies have been designed targeting receptor tyrosine kinase ErbB2, such as the low molecular weight inhibitor Lapatinib and the monoclonal antibody Herceptin. We have already demonstrated that tumor necrosis factor alpha (TNF) induces proliferation of BT-474 and SKBR-3 human and C4HD murine breast cancer cells which overexpress ErbB2, through the transactivation of ErbB2 and subsequent activation of transcription factor NF-κB, and through increase of Cyclin D1 protein expression. In the present study we evaluated the effectiveness of clinical drugs which block ErbB2, in order to inhibit NF-κB transcriptional activity and TNF-induced proliferation. We transfected BT-474 and SKBR-3 breast cancer cells with a luciferase reporter gene under control of a NF-κB response element and observed that the pharmacological ErbB2 inhibitor AG825 (100 μM)and the Lapatinib analog GW2974 (1 μM) blocked NF-κB activity induced by TNF. However, when we used 10 μg/ml Herceptin, TNF still activated NF-κB. We also monitored NF-κB activation through Western blot of phosphorylated IκBα, the protein which inhibits NF-κB traslocation to the nucleus, with same results. By reporter gene assays, we then explored activation of Cyclin D1 promoter, a target gene of NF-κB. While both AG825 and GW2974 blocked TNF-induced Cyclin D1 promoter activation, Herceptin did not. The endogenous protein Cyclin D1 mirrored the profile obtained with reporter gene assays. BT-474 proliferation increased in the presence of TNF (75 ± 12% vs control cells) measured by 3H-thimidine incorporation, cell count and flow cytometry of cells stained with propidium iodide. AG825 and GW2974 completely blocked TNF-induced proliferation, but Herceptin failed to do so. Bearing in mind that TNF is frequently expressed in invasive breast tumors, our results suggest that TNF may be one of the factors which confer Herceptin resistance. We therefore hypothesized that blocking TNF through the TNFR2-FcIgG fusion protein Etanercept may overcome Herceptin resistance. We observed that Etanercept (5 mg/kg, i.p. twice wk) inhibited TNF-induced in vitro proliferation of C4HD cells, a murine breast adenocarcinoma that produces TNF. When this tumor was growth in nude mice and treated with Etanercept the tumor size was reduced by 37.6 ± 1.1 % (P<0.05). Interestingly, histological examination of said tumors revealed that there were fewer mitotic figures and a lower percentage of necrotic and fibrotic areas in the tumors from mice treated with Etanercept, as compared to the group treated with an irrelevant IgG. Our results propose Etanercept as a new agent to overcome clinically observed Herceptin resistance. 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 611.

A New Application for the Drug Tranilast: Effects on Breast Cancer Cell Proliferation, Migration, and Invasion

BackgroundTranilast (Rizaben®) has been used to treat allergic and auto‐immune diseases for the last 26 years. The objective of this study was to investigate whether tranilast also has an effect on breast cancer cell biological features relevant to cancer progression, based on the its mechanism of action which involves TGFβsignaling.MethodsCultures of the BT‐474 human breast cancer cell line were used. Cell proliferation, clonogenicity, cell migration, endoglin and MMP‐9 expression were assessed by: MTT assay, soft agar colony formation, wound assay and immunocytochemistry respectively.ResultsTranilast was found to inhibit breast cancer cell proliferation by 70%. In addition, the drug decreased by 54% the number and size of cell colonies grown in soft agar, consistent with a decrease in clonogenicity. The closure of wounds made in cancer cell monolayers was slowed (55%) by Tranilast, consistent with an inhibitory effect on tumor cell migration. Endoglin, which plays a role in TGFβ signaling, was downregulated by the drug. MMP‐9, which is a metalloproteinase involved in tumor invasion and metastasis was also decreased by tranilast.ConclusionsThese results indicate that tranilast exerts inhibitory effects on key biological features of human breast cancer cells. The results are consistent with an anti‐tumor effect which can be exploited in preclinical and clinical therapeutic trials.

Auger-Mediated Cytotoxicity of Cancer Cells in Culture by an <SUP>125</SUP>I-Antisense Oligomer Delivered as a Three-Component Streptavidin Nanoparticle

We reported recently that a three-component nanoparticle, consisting of a targeting antibody, a transfecting peptide and an 111In-antiRIalpha MORF antisense oligomer, provided Auger electron-mediated, antisense-mediated, cytotoxicity of cells in culture. We have now measured the cytotoxicity of the nanoparticle in culture with the 111In replaced by 125I, another attractive Auger electron emitter. The nanoparticle consisted of streptavidin linking the 125I labeled antiRIalpha mRNA antisense MORF oligomer, the tat transfecting peptide and the anti-Her2 Trastuzumab antibody. Cytotoxicity was evaluated by a clonogenic survival assay in BT-474 (Her2+) human breast cancer cells. In a dose escalation study, as measured by the surviving fraction, the cytotoxicity of tumor cells to the 125I-labeled antisense nanoparticle was significantly higher than that for the identical sense control. When compared with our previous study with 111In as label, a similar level of cytotoxicity was achieved but the observed minimal therapeutic dose for the 125I-labeled nanoparticle in BT-474 cells was lower than that for 111In-labeled nanoparticle in SK-BR-3 cells. Thus, a radiolabeled antisense MORF oligomer delivered into cells by a three-component nanoparticle is an effective vehicle for Auger radiotherapy when radiolabeled with 111In or 125I.

Cancer cell-specific internalizing ligands from phage displayed -lactamase-peptide fusion libraries

The present study was focused on identifying cancer cell-specific internalizing ligands using a new kind of phage display library in which the linear or cysteine-constrained random peptides were at amino-terminus fusion to catalytically active P99 beta-lactamase molecules. The size and quality of libraries were comparable to other reported phage display systems. Several cancer cell-specific binding and internalizing beta-lactamase-peptide fusion ligands were isolated by selecting these libraries on the live BT-474 human breast cancer cells. The identified ligands shared several significant motifs, which showed their selectivity and possible binding to some common cancer cell targets. The beta-lactamase fusion made the whole process of clone screening efficient and simple. The ligands selected from such libraries do not require peptide synthesis and modifications, and can be used directly for applications that require ligand tracking. In addition, the selected beta-lactamase-peptide ligands have a potential for their direct use in targeted enzyme prodrug therapy. The cancer-specific peptides can also be adopted for other kinds of targeted delivery protocols requiring cell-specific affinity reagents. This is first report on the selection of cell-internalized enzyme conjugates using phage display technology, which opens the possibility for new fusion libraries with other relevant enzymes.

An Inhibitor of ADAMs (a Distintegrin and Metalloproteinase) Overcomes HER3-Mediated Resistance to Trastuzumab and Lapatinib.

Abstract Background: Inhibitors of HER-2/neu and EGF receptors such as trastuzumab, lapatinib, and erlotinib have demonstrated clinical efficacy but not all HER-2/neu or EGFR positive tumors respond and many that initially respond develop resistance. Ligand mediated HER-3 signaling results in tumor growth and survival and is a proposed mechanism of resistance to trastuzumab and lapatinib. Proteolytic cleavage of both ErbB ligands and receptors has been shown to be a critical event that results in ErbB pathway activation. Cleavage is necessary for the generation of soluble, functionally active forms of ErbB ligands and in the case of HER-2/neu, cleavage results in a shed extracellular domain (ECD) and a membrane bound fragment (p95) that contains a kinase domain with significant constitutive activity. In addition, it has been shown that the preferential association between HER3 and p95 can further activate the pathway. Both ErbB ligand and HER-2/neu cleavage are mediated by the ADAM family of proteases. Further, we have previously shown that the ADAM protease inhibitor, INCB7839, provides synergistic inhibition of HER2+ breast cancer cell line growth when combined with either trastuzumab or lapatinib. Materials and Methods: The HER-2 overexpressing BT-474 human breast cancer cell line was treated with either lapatinib or trastuzumab in the presence or absence of the HER-3 ligand, heregulin, and the effects on cell growth measured. The effects of the ADAM protease inhibitor INCB7839 in this system were also examined. Results: The addition of heregulin overcame the anti-proliferative effects of both lapatinib and trastuzumab on the growth of the BT-474 cell line in vitro. Addition of INCB7839 synergized with lapatinib or trastuzumab and importantly, restored the anti-proliferative effects of these agents in the presence of heregulin. Further, pretreatment of BT-474 cells with INCB7839 for 6 days, which we have previously shown completely eliminates the presence of the p95 form of HER2, amplified these effects, presumably by eliminating p95 before stimulating the cells with heregulin. Discussion: Together, these results confirm that heregulin can overcome the anti-proliferative effects of both trastuzumab and lapatinib as previously reported. Prevention of p95 formation by ADAM protease inhibitors appears to restore the anti-proliferative effects of both trastuzumab and lapatinib when heregulin/HER3 signaling occurs, negating a proposed mechanism of resistance to these agents in the clinic. These results suggest that combining an ADAM inhibitor with targeted inhibitors of the ErbB family can overcome HER-3-mediated resistance and enhance the clinical efficacy of approved HER2-targeted agents in the clinical setting. INCB7839 is currently under clinical investigation in combination with trastuzumab for women with metastatic breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3138.

Abstract C58: Causal Network™ Modeling identifies common and unique mechanisms for sensitivity to the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693

Abstract Background: Aberrant PI3K and AKT signaling play an important role in the development of some human tumors by promoting cell proliferation and survival. Inhibitors that target the kinase activity of PI3K or AKT may constitute an effective treatment for human malignancies that rely on these molecules for their growth. GlaxoSmithKline has developed a PI3K inhibitor, GSK1059615, and a pan-AKT inhibitor, GSK690693. Since AKT is activated by PI3K via generation of PIP3, and is a key effector of PI3K signaling, a high degree of overlap is expected in the effects of a PI3K and an AKT inhibitor. To determine whether the effects of the PI3K inhibitor are mediated solely through the AKT pathway, we compared the molecular response of BT474 human breast cancer cells to both GSK1059615 and GSK690693 as well as the sensitivity profile of 16 other human cancer cell lines. Methods: The mechanisms of action of the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693 were evaluated in BT474 human breast cancer cells using RNA expression and phosphoproteomic data. Causal Network™ Modeling was used to characterize these data sets to identify processes that contribute to the inhibition of proliferation observed in cells sensitive to these inhibitors, and processes that could explain differences in sensitivity to the two inhibitors. The sensitivity/resistance profile of 16 human cancer cell lines to GSK1059615 and GSK690693 was also determined. Results: Four common processes were found in BT474 cells treated with either GSK690693 or GSK1059615: RB1-Mediated Cell Cycle Arrest Increased FOXO Signaling Decreased MYC and TFRC Signaling Decreased Metabolism We identified one process specific to the PI3K inhibitor GSK1059615-treated cells: Decreased MAPK Signaling Conclusions: In BT474 cells, GSK690693 and GSK1059615 inhibit cell proliferation by increasing RB1-mediated cell cycle arrest, increasing FOXO signaling, decreasing MYC signaling, and decreasing cellular metabolism, as identified by Causal Network™ Modeling. In addition to these four shared mechanisms, the PI3K inhibitor GSK1059615 attenuates MAPK signaling. Five of 16 cell lines tested for sensitivity to the inhibitors were sensitive to GSK1059615 treatment only, and these five cell lines were the only ones that contained mutations activating MAPK signaling. Thus decreased MAPK signaling is likely an additional mechanism by which GSK1059615 inhibits cell proliferation, and may confer sensitivity to this PI3K inhibitor in cells resistant to the AKT inhibitor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C58.

Abstract B94: The roles of ERK1/2 and ERK5 in age-related breast cancer proliferation

Abstract The purpose of the current study is to examine the relative roles of the extracellular signal-regulated kinases 1, 2, and 5 (ERK1/2 and ERK5) and novel benzimidazole inhibitor compounds in cellular proliferation in breast tumors with aging. Breast cancer risk progressively increases with age, in part due to an accumulation of genetic mutations that occur during cellular division and/or exposure to environmental toxins. It has been estimated that over the next thirty years the number of Americans older than 65 years of age will double. With this increase in the aged population, the incidence of breast cancer will rise; therefore, it is important to examine the underlying mechanisms involved in breast tumor proliferation with aging. It has been well established that activation of the mitogen-activated protein kinase (MAPK) ERK1/2 leads to increased cellular proliferation and differentiation in cancer cells. Many of these studies rely on pharmacological inhibitors that were thought to be specific for the ERK1/2 pathways. Interestingly, recent studies demonstrate that these inhibitors also block the activation of a novel MAPK, ERK5. Indeed, an increase in ERK5 activity has been observed in mammary tumors in aged patients. However, the roles of ERK1/2 and ERK5 in breast cancer cell proliferation with aging have not been fully explored. In our studies, the transgenic MMTV-neu mouse model was utilized to examine ERK1/2 and ERK5 activity with age. MMTV-neu mice develop spontaneous mammary tumors which make them an appropriate model for the studies of human breast cancer. Tumors were extracted from young (3 mo), middle (9 mo), and old (15 mo) mice. Higher levels of ERK5 expression and activity were observed in middle- and old-aged animals as compared to their younger counterpart. Similarly, ERK1/2 expression and activation were increased with age. To further study the role of the ERK pathways on cell proliferation with aging, the BT-474 human breast cancer cell line and the MMTV-neu cell line (MMC) were utilized. Cell lysates were collected at passages 2, 12, 24 and 36 to mimic aging. At early passages ERK5 was constitutively active under basal conditions. At later passages, an age-dependent increase in ERK5 was observed. Breast cancer cell proliferation may be regulated by ERK5 exclusively or in conjunction with ERK1/2. We next investigated how novel benzimidazole compounds can act as specific inhibitors of ERK5 signaling in MMC and BT-474 cells. Overall, these studies will provide novel information about the cellular mechanisms involved in cancer cell proliferation and progression with aging. A better understanding of the ERK1/2 and ERK5 pathways in age-related mammary cell proliferation may lead to the development of preventative therapeutics or treatment possessing enhanced specificity for aging patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B94.

Darpp-32 and Its Truncated Variant t-Darpp Have Antagonistic Effects on Breast Cancer Cell Growth and Herceptin Resistance

BackgroundHerceptin (trastuzumab) is a humanized monoclonal antibody that is approved for the treatment of metastatic breast cancer patients whose tumors overexpress Her2 (erbB2/neu). Up to 70% of Her2-positive breast cancers demonstrate a response to Herceptin-based therapies, but resistance almost inevitably arises within a year of the initial response. To help understand the mechanism of Herceptin resistance, we isolated clonal variants of Her2-positive BT474 human breast cancer cells (BT/HerR) that are highly resistant to Herceptin. These cell lines exhibit sustained PI3K/Akt signaling as an essential component of Herceptin-resistant proliferation. Several genes in the protein kinase A (PKA) signaling network have altered expression in BT/HerR cells, including PPP1R1B, which encodes a 32 kDa protein known as Darpp-32 and its amino-terminal truncated variant, t-Darpp. The purpose of the current work was to determine the role of Darpp-32 and t-Darpp in Herceptin resistance.Methodology and ResultsWe determined expression of Darpp-32 and t-Darpp in BT/HerR cells selected for resistance to Herceptin. Subsequently, cDNAs encoding the two isoforms of Darpp-32 were transfected, separately and together, into Her2-positive SK-Br-3 breast cancer cells. Transfected cells were tested for resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. DNA binding activity by the cAMP response element binding protein (CREB) was also measured. We found that BT/HerR cells overexpressed t-Darpp but not Darpp-32. Moreover, t-Darpp overexpression in SK-Br-3 cells was sufficient for conferring resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. Darpp-32 co-expression reversed t-Darpp's effects on Herceptin resistance and Akt phosphorylation. t-Darpp overexpression led to increased CREB binding activity, which was also reversible by Darpp-32.Conclusionst-Darpp and Darpp-32 appear to have antagonistic effects on Herceptin resistance. We present a unified model by which these effects might be mediated via the PKA regulatory network.

In vivo examination of 188Re(I)-tricarbonyl-labeled trastuzumab to target HER2-overexpressing breast cancer

Trastuzumab (Herceptin), a humanized IgG1 monoclonal antibody directed against the extracellular domain of the HER2 protein, acts as an immunotherapeutic agent for HER2-overexpressing human breast cancers. Radiolabeled trastuzumab with beta- or alpha emitters can be used as radioimmunotherapeutic agent for the similar purpose but with additional radiation effect. In this study, trastuzumab was labeled with (188)Re for radioimmunotherapy of HER2/neu-positive breast cancer. (188)Re(I)-tricarbonyl ion, [(188)Re(OH(2))(3)(CO)(3)](+), was employed as a precursor for directly labeling the monoclonal antibody with (188)Re. The immunoreactivity of (188)Re(I)-trastuzumab was estimated by competition receptor-binding assay using HER2/neu-overexpressive BT-474 human breast cancer cells. The localization properties of (188)Re(I)-trastuzumab within both tumor and normal tissues of athymic mice bearing BT-474 human breast cancer xenografts (HER2/neu-overexpressive) and similar mice bearing MCF-7 human breast cancer xenografts (HER2/neu-low expressive) were investigated. When incubated with human serum albumin and histidine at 25 degrees C, (188)Re(I)-trastuzumab was found to be stable within 24 h. The IC(50) of (188)Re(I)-trastuzumab was found to be 22.63+/-4.57 nM. (188)Re(I)-trastuzumab was shown to accumulate specifically in BT-474 tumor tissue in in vivo biodistribution studies. By microSPECT/CT, the image of (188)Re localized BT-474 tumor was clearly visualized within 24 h. In contrast, (188)Re(I)-trastuzumab uptake in HER2-low-expressing MCF-7 tumor was minimal, and the (188)Re image at the localization of the tumor was dim. These results reveal that (188)Re(I)-trastuzumab could be an appropriate radioimmunotherapeutic agent for the treatment of HER2/neu-overexpressing cancers.

Effect of chronic exposure to tyrosine kinase inhibitors on chemotherapy-induced DNA damage in the SKBR3 breast cancer cell line.

Abstract Abstract #1039 Introduction: The HER family of receptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) is critical in the regulation of cell growth, proliferation and survival. Several studies have linked over-expression of HER1 and HER2 with resistance to chemotherapy and radiation. Clinical trials combining the HER tyrosine kinase inhibitors (TKI) gefitinib (GEF), an inhibitor of HER1, and lapatinib (LAP), a dual inhibitor of HER1 and HER2, with chemotherapeutic agents have not shown synergy comparable to that found in preclinical models. We investigated the effects of chronic treatment of breast cancer cells with TKI inhibitors on DNA damage and repair resulting from chemotherapy and ionising radiation (IR).
 Material and Methods: Western blotting was used to examine the expression of phosphorylated HER receptors, AKT and MAPK in the SKBR3 and BT474 human breast cancer cell lines. Etoposide (ET), doxorubicin (DOX) and IR induced DNA strand breaks (SB) were measured in the SKBR3 cell line using the single cell gel electrophoresis (comet) assay. Cisplatin-induced (PT) interstrand crosslinks (ICL) were measured with the modified alkaline comet assay. Cells were pre-treated with LAP or GEF for 1 hour or 48 hours, and TKI was replaced at 24-hourly intervals. Cell cycle analysis and intracellular DOX were measured by flow cytometry.
 Results: In the SKBR3 and BT474 cell lines pHER1, pHER2, pHER3, pAKT and pMAPK were inhibited within 1 hour of exposure to LAP or GEF. However, after 48 hours, pHER3 and pAKT signalling was detected in cells treated with GEF, but not LAP. pHER1, pHER2 and pMAPK were inhibited for at least 72 hours with both drugs. Cell cycle analysis on the SKBR3 cell line showed that 48 hours exposure to either TKI reduced the number of cells in S (12.4±1.1% to 3.2±0.3%), and G2 (16.0±0.55% to 7.1±1.0%) phases of the cell cycle. There was no alteration in intracellular DOX levels at 1 hour or following 48 hours exposure to either TKI. SB were induced by ET, DOX and IR, and ICL by PT, in cells exposed to GEF or LAP for 1 hour. Exposure for 48 hours did not alter the number of ICL or SB induced by PT or IR. In contrast there was a 30-90% reduction in SB following ET treatment (depending on drug concentration) in cells treated with TKI for 48 hours compared with 1 hour treatment. The effect of 48 hours exposure to TKI on DOX-induced SB was more marked with >90% reduction, even at high DOX concentrations. Higher concentrations of ET resulted in increased SB, but increasing concentration of DOX did not alter the level of detectable SB.
 Discussion: These results suggest why clinical trials of combinations of HER TKI with chemotherapy agents fail to show the magnitude of synergy predicted in preclinical studies. Whilst the cytostatic properties of both LAP and GEF may explain the reduction in DNA damage after 48 hours, this does explain why SB are absent in DOX-treated cells. Further studies investigating kinetics of DNA repair following chemotherapy or radiation in combination with TKI are on-going. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1039.

Nanotube–antibody biosensor arrays for the detection of circulating breast cancercells

Recent reports have shown that nanoscale electronic devices can be used to detect a changein electrical properties when receptor proteins bind to their corresponding antibodiesfunctionalized on the surface of the device, in extracts from as few as ten lysed tumor cells.We hypothesized that nanotube–antibody devices could sensitively and specifically detectentire live cancer cells. We report for the first time a single nanotube field effect transistorarray, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibitshighly sensitive and selective sensing of live, intact MCF7 and BT474 human breastcancer cells in human blood. Those two cell lines both overexpress IGF1R andHer2, at different levels. Single or small bundle of nanotube devices that werefunctionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases inconductivity upon interaction with BT474 or MCF7 breast cancer cells in twoµl drops of blood. Control experiments with non-specific antibodies or with MCF10A controlbreast cells produced a less than 5% decrease in electrical conductivity, illustrating the highsensitivity for whole cell binding by these single nanotube–antibody devices. We postulatethat the free energy change due to multiple simultaneous cell–antibody binding eventsexerted stress along the nanotube surface, decreasing its electrical conductivity due to anincrease in band gap. Because the free energy change upon cell–antibody binding, the stressexerted on the nanotube, and the change in conductivity are specific to a specificantigen–antibody interaction; these properties might be used as a fingerprintfor the molecular sensing of circulating cancer cells. From optical microscopyobservations during sensing, it appears that the binding of a single cell to a singlenanotube field effect transistor produced the change in electrical conductivity.Thus we report a nanoscale oncometer with single cell sensitivity with a diameter1000 times smaller than a cancer cell that functions in a drop of fresh blood.

Molecular profile of breast versus ovarian cancer cells in response to treatment with the anticancer drugs cisplatin, carboplatin, doxorubicin, etoposide and taxol

We assessed changes in the apoptosis-related genes BCL2, BAX, BCL2L12, FAS and CASPASE-3 in OVCAR-3 human ovarian cancer cells and BT-20 human breast cancer cells to provide an insight into the molecular mechanisms involved in the response of these cells to treatment with anticancer drugs and to assess their value as potential biomarkers of chemotherapy response in breast and ovarian cancer. Cells were treated with different chemotherapeutic drugs (cisplatin, carboplatin, doxorubicin, etoposide and taxol) and assessed for changes in the expression of apoptosis-related genes at the mRNA level. Total RNA was extracted, reverse-transcribed into cDNA and amplified by PCR using gene-specific primers. GAPDH was used as a housekeeping gene. Cytotoxicity was assessed by MTT assay. Both cancer cell lines responded differentially at the molecular level to the drug treatments. OVCAR-3 cells showed more pronounced sensitivity and changes compared to BT-20 cells at the mRNA level for different apoptosis-related genes, leading to cell and cancer type dependence in conjunction with drug dependence.

The effect of trastuzumab alone and combined with α‐linolenic acid, γ‐tocopherol, sesame lignan and its metabolites on the growth of BT‐474 and MCF‐7 human breast cancer cells

About 25% of breast cancers overexpress epidermal growth factor receptor‐2 (HER2), are aggressive and mainly treated with trastuzumab (TRAS). Our objective was to determine the effect of some food components on the proliferation of breast cancer cells which overexpress HER2 (BT‐474) and those which have physiological HER2 levels (MCF‐7). Using a 4‐day in‐vitro incubation method, we examined the effect of sesame lignan (sesamin, SES, 1 uM), its metabolites (enterodiol (ED) and enterolactone (EL), 1 uM), α‐linolenic acid (ALA, 50 uM), and γ‐tocopherol (GTOC, 20 uM), alone and combined with TRAS (10 μg/ml). Compared with control, in MCF‐7 cells, tumor reduction was 33% (P<0.001) with ALA alone, 9% (P<0.05) with ALA combined with TRAS, and none with the others. In BT‐474 cells, there were 53% and 35% tumor reductions with TRAS and ALA alone (P<0.001), respectively, 20% tumor increase with GTOC (P<0.01) and no effect with the other treatments. Tumor reductions were observed with TRAS co‐treatment with SES (59%), ED (62%), EL (57%), ALA (61%), and GTOC (64%) (all P<0.001), however they did not differ from TRAS treatment alone in BT‐474 cells. In conclusion, ALA is the most effective anti‐cancer food component in both cell lines. Although other treatments are less effective, they and ALA do not antagonize the effectiveness of TRAS in HER2 overexpressing tumors.

A Cathepsin K Inhibitor Reduces Breast Cancer–Induced Osteolysis and Skeletal Tumor Burden

Osteoclasts mediate bone destruction in breast cancer skeletal metastases. Cathepsin K is a proteinase that is secreted by osteoclasts and degrades bone. Here, immunohistochemistry revealed that cathepsin K was expressed not only by osteoclasts but also by breast cancer cells that metastasize to bone. Following intratibial injection with cathepsin K-expressing human BT474 breast cancer cells, tumor-bearing mice treated with a clinical dosing regimen of cathepsin K inhibitor (CKI; 50 mg/kg, twice daily) had osteolytic lesions that were 79% smaller than those of tumor-bearing mice treated with the vehicle. The effect of CKI was also studied in a mouse model in which the i.v. inoculation of human B02 breast cancer cells expressing cathepsin K leads to bone metastasis formation. Drug administration was started before (preventive protocol) or after (treatment protocol) the occurrence of osteolytic lesions. In treatment protocols, CKI (50 mg/kg, twice daily) or a single clinical dose of 100 microg/kg zoledronic acid (osteoclast inhibitor) reduced the progression of osteolytic lesions by 59% to 66%. CKI therapy also reduced skeletal tumor burden by 62% compared with vehicle, whereas zoledronic acid did not decrease the tumor burden. The efficacy of CKI at inhibiting skeletal tumor burden was similar in the treatment and preventive protocols. By contrast, CKI did not block the growth of s.c. B02 tumor xenografts in animals. Thus, CKI may render the bone a less favorable microenvironment for tumor growth by inhibiting bone resorption. These findings raise the possibility that cathepsin K could be a therapeutic target for the treatment of bone metastases.

Progestin-Dependent Progression of Human Breast Tumor Xenografts: A Novel Model for Evaluating Antitumor Therapeutics

Recent clinical trials indicate that synthetic progestins may stimulate progression of breast cancer in postmenopausal women, a result that is consistent with studies in chemically-induced breast cancer models in rodents. However, progestin-dependent progression of breast cancer tumor xenografts has not been shown. This study shows that xenografts obtained from BT-474 and T47-D human breast cancer cells without Matrigel in estrogen-supplemented nude mice begin to regress within days after tumor cell inoculation. However, their growth is resumed if animals are supplemented with progesterone. The antiprogestin RU-486 blocks progestin stimulation of growth, indicating involvement of progesterone receptors. Exposure of xenografts to medroxyprogesterone acetate, a synthetic progestin used in postmenopausal hormone replacement therapy and oral contraception, also stimulates growth of regressing xenograft tumors. Tumor progression is dependent on expression of vascular endothelial growth factor (VEGF); growth of progestin-dependent tumors is blocked by inhibiting synthesis of VEGF or VEGF activity using a monoclonal anti-VEGF antibody (2C3) or by treatment with PRIMA-1, a small-molecule compound that reactivates mutant p53 into a functional protein and blocks VEGF production. These results suggest a possible model system for screening potential therapeutic agents for their ability to prevent or inhibit progestin-dependent human breast tumors. Such a model could potentially be used to screen for safer antiprogestins, antiangiogenic agents, or for compounds that reactivate mutant p53 and prevent progestin-dependent progression of breast disease.

Genistein affects HER2 protein concentration, activation, and promoter regulation in BT-474 human breast cancer cells

The HER2 proto-oncogene, a member of the epidermal growth factor receptor family, is overexpressed in 20-30% of breast cancers. Genistein, the main soy isoflavone, interacts with estrogen receptors (ER) and it is also a potent tyrosine kinase inhibitor. Previously, our laboratory found that genistein delayed mammary tumor onset in transgenic mice that overexpress HER2 gene. Our goal was to define the mechanism through which genistein affects mammary tumorigenesis in HER2 overexpressing mice. We hypothesized that genistein inhibits HER2 activation and expression through ER-dependent and ER-independent mechanisms. Genistein inhibited total HER2 protein expression and tyrosine phosphorylation in BT-474, an ERalpha (-) and ERbeta (+) human breast cancer cell line, however, E2 had no effect. Taken together, these data suggest that genistein has an ER-independent inhibitory effect, presumably, through tyrosine kinase inhibition activity. Genistein at 1.0 microM mimicked E2 and down-regulated HER2 protein phosphorylation when BT-474 was co-transfected with ERalpha, but not ERbeta. Although E2 and overexpression of HER2 can promote mammary tumorigenesis, an inverse relationship between ER expression and HER2 overexpression has been found in human breast cancer. We cloned a 500-bp promoter region upstream of the HER2 transcription initiation site. Co-transfection with ERalpha, but not with ERbeta, down-regulated HER2 promoter reporter in BT-474. At concentrations > or =1 microM, genistein inhibited HER2 promoter reporter in the absence of ERalpha. In conclusion, genistein at > or =1 microM inhibited HER2 protein expression, phosphorylation, and promoter activity through an ER-independent mechanism. In the presence of ERalpha, genistein mimicked E2 and inhibited HER2 protein phosphorylation. These data support genistein's chemo-prevention and potential chemo-therapeutic roles in breast cancer.

Trastuzumab-Resistant HER2-Driven Breast Cancer Cells Are Sensitive to Epigallocatechin-3 Gallate

Overexpression of the epidermal growth factor receptor family member HER2 is found in approximately 30% of breast cancers and is a target for immunotherapy. Trastuzumab, a humanized monoclonal antibody against HER2, is cytostatic when added alone and highly successful in clinical settings when used in combination with other chemotherapeutic agents. Unfortunately, HER2 tumors in patients develop resistance to trastuzumab or metastasize to the brain, which is inaccessible to antibody therapy. Previously, we showed that the green tea polyphenol epigallocatechin-3 gallate (EGCG) inhibits growth and transformed phenotype of Her-2/neu-driven mouse mammary tumor cells. The different modes of action of EGCG and trastuzumab led us to hypothesize that EGCG will inhibit HER2-driven breast cancer cells resistant to trastuzumab. We studied trastuzumab-resistant BT474 human breast cancer cells, isolated by chronic trastuzumab exposure, and JIMT-1 breast cancer cells, derived from a pleural effusion in a patient who displayed clinical resistance to trastuzumab therapy. EGCG treatment caused a dose-dependent decrease in growth and cellular ATP production, and apoptosis at high concentrations. Akt activity was suppressed by EGCG leading to the induction of FOXO3a and target cyclin-dependent kinase inhibitor p27Kip1 levels. Thus, EGCG in combination with trastuzumab may provide a novel strategy for treatment of HER2-overexpressing breast cancers, given that EGCG can cross the blood-brain barrier.

Estrogenic constituents of the heartwood of Dalbergia parviflora

From the heartwood of Dalbergia parviflora, five compounds, dalparvin A ( 1), B ( 2), C ( 3), dalparvinol C ( 4), and neokhriol A ( 5), along with 11 known compounds, kenusanone G ( 6), cajanin ( 7), sophorol ( 8), alpinetin ( 9), hesperetin ( 10), 3′- O-methylorobol, odoratin, (2 R)(3 R)-2,3- trans 7-hydroxy-5-methoxydihydroflavonol, (6a R, 11a R)-3,8-dihydroxy-9-methoxypterocarpan, (6a R, 11a R)- vesticarpan, and methyl-3,4-dihydroxy-2-methoxybenzoate were isolated and characterized. Isolates were evaluated for their cell proliferation stimulatory activity against MCF-7, T-47D, and BT20 human breast cancer cell lines. Along with 7– 10, two compounds 2 and 3 stimulated not only MCF-7, but also T-47D human breast cancer cell proliferation. Compound 6 had activity only against MCF-7 cells, and the activity of 7 was more than equivalent to that of daidzein. On the other hand, none of the isolates had any significant effects on BT20 cell proliferation, and these results indicated that the stimulative activity of these compounds was not general to any cell proliferations. Furthermore, these compounds were tested in the estrogen-responsive transient luciferase reporter assay.