Agent For Treatment Of Breast Cancer Research Articles

Abstract 2110: Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7

Abstract mTOR inhibitor everolimus has been considered as a promising agent for breast cancer treatment especially among estrogen receptor (ER) positive postmenopausal advanced breast cancer. As expected, combination therapy of everolimus and steroidal aromatase inhibitor (AI) showed remarkable clinical efficacy for the first line nonsteroidal AI treatment failure patients in BOLERO-2 trial. However, the subsequent treatment after refractory to that therapy remains a great matter. We already reported several types of long term estrogen-depletion resistant (EDR) cell lines from MCF-7-E10, which has been constitutively ERE-GFP-transfected to monitor ER activity in living cells (Fujiki N et al., J Steroid Biochem Mol Biol. 2013 Oct 13;139C:61-72). They mimic AI resistance and these EDR cells have been characterized as the followings. 1) Type 1 as ER positive (ERE-GFP positive), constitutive ER overexpression without estrogen, and PI3K/Akt/mTOR pathway upregulated. 2) Type 2 as ER negative conversion (ERE-GFP negative) and IGF-1R/JNK signaling pathway upregulated. Then with those two types of sub-cell lines, we generated everolimus resistant cells after long term exposure to everolimus in estrogen depleted medium. Those everolimus resistant (EVR) cells are equivalent to resistance to the combination therapy of AI and everolimus as the second line endocrine treatment after the first line AI treatment failure. Though everolimus extremely inhibited cell proliferation of two EDR cell lines, everolimus resistant cells named as Type 1-EVR, Type 2-EVR no longer responded to everolimus. ER expression and estrogen sensitivity of two types of EVR cells were similar to those of the parental cells, respectively. Pure anti-estrogen fulvestrant inhibited cell proliferation of Type1-EVR cells. On the other hand, Type2-EVR did not respond to fulvestrant but JNK inhibitor inhibited the cell growth. Phosphorylation status of p70S6K and 4E-BP1 were different among individual parental EDR cells and EVR cells. Type1-EVR showed much higher expression of phosphorylation of MAP-kinase than the parental cells. In contrast, Type2-EVR expressed phosphorylation of MAP-kinase less than the parental cells. As above, the mechanism of acquired resistance to everolimus was likely to be different between those two sub-cell lines. This finding implies that different mechanisms of AI resistance follow different types of everolimus resistance. Citation Format: Mariko Kimura, Toru Hanamura, Toshifumi Niwa, Yuri Yamaguchi, Itaru Endo, Shin-ichi Hayashi. Molecular characterization of everolimus-resistant cell lines established from estrogen depletion-resistant MCF-7. [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 2110. doi:10.1158/1538-7445.AM2014-2110

Cytotoxicity Screening of Plants of Genus <i>Piper</i> in Breast Cancer Cell Lines

Purpose: To examine whether seven species of plants of genus Piper possess anti-cancer effects.Methods: One normal breast and three breast cancer cell lines were used to test cytotoxic effects over a period of 72 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The dried plants were extracted with methanol and dichloromethane, and the effective extract isolated by crystallization, acid/base extraction and column chromatography techniques. Fragmented DNA was purified by phenol/chloroform/isoamyl alcohol.Results: Methanol and dichloromethane extracts of Piper retrofractum, Piper betle, especially Piper nigrum, exhibited strong effect on MDA-MB-468. When the crude extract of P. nigrum was then separated by column chromatography, fraction D showed activity against both MCF-7 and MDA-MB-468 cells. Fraction DE that was isolated from D demonstrated a highly cytotoxic effect with IC50 values of 8.33 ± 1.27 and 7.48 ± 0.57 μg/ml on MCF-7 and MDA-MB-468 cells, respectively. Furthermore, fraction DF exhibited a strong cytotoxic effect only on MCF-7 with IC50 value of 6.51 ± 0.39 μg/ml. DNA smears of MCF-7 and MDA-MB-468 cells treated with fraction DE and DF were observed within 7 days.Conclusions: These results indicate that the compounds isolated from P. nigrum, viz, DE and DF, have cytotoxic effect on breast cancer cell lines. These fractions could be promising agent for breast cancer treatment. Further studies on the isolation, structural and mechanism elucidation of the active compound are still needed being carried out.Keywords: Cytotoxicity, Breast cancer, P. nigrum, DNA fragmentation

Synergistic anti-carcinogenic effect of interferon-β with cisplatin on human breast adenocarcinoma MDA MB231 cells.

Cisplatin is one of the most commonly used chemotherapeutic agents for breast cancer treatment. However, its efficacy is greatly limited by its toxic side effects. The present study investigated the synergistic effect of interferon β with cisplatin on MDA MB231 cells. The antiproliferative effect was measured by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) was calculated using the method of Chou and Talalay. Cytotoxicity was determined by trypan blue and clonogenic assay. Genotoxic and cytostatic effects were studied using micronucleus assay and nuclear division index (NDI). Protein expression was analyzed using immunoblotting. Interferon β (100-2500 IU/mL) and Cisplatin (0.01-100 μM) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 1500 IU/mL and 20 μM for interferon β and cisplatin, respectively. Western blot analysis revealed expression of interferon β binding receptor in MDA MB231 cells. More interestingly, synergistic, cytotoxic and genotoxic effects were observed after treatment with a combination of interferon β with reduced dosage of cisplatin. Decreased expression of Bcl-2 and increased expression of Bax stimulated the cytochrome c release, which triggers caspase-9 and -3 activation significantly increased in the combinational group. In conclusion the combination of interferon β with reduced dose of cisplatin results synergistically improved growth-inhibition and apoptosis-inducing effect on MDA MB231 cells.

Interplay between estrogen and retinoid signaling in breast cancer – Current and future perspectives

All-trans-retinoic acid (RA) is a promising agent for breast cancer treatment, but it induces several adverse effects and the few clinical trials performed up to now in breast cancer patients have provided disappointing results. The combination of RA and antiestrogenic compounds, such as tamoxifen, synergistically decreases the proliferation of breast cancer cells and an interplay between retinoid and estrogen signaling has begun to be unraveled, turning these combinations into an appealing strategy for breast cancer treatment. This review focus on the current knowledge regarding the interplay between retinoid and estrogen signaling in breast cancer and the combinations of RA with antiestrogens, aiming their future utilization in cancer therapy.

The pro-apoptotic role of autophagy in breast cancer.

Background:Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines.Methods:Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis.Results:LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells.Conclusions:Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa.

Identifying molecular features that distinguish fluvastatin-sensitive breast tumor cells

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatintriggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERα)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genesencoding regulators of mevalonate production, non-sterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug, Gemcitabine

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking or reversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategies to reduce cancer related death. One such promising chemopreventive agent which has attracted considerable attention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. The present study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent, gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the results by nuclear morphological examination. Further we analyzed the modulation of expression of Bcl-2 and COX-2 on treatment of these cells with SFN by RT-PCR. SFN showed cytotoxic effects on MCF-7 cells in a dose- and time-dependent manner via an apoptotic mode of cell death. In addition, a combinational treatment of SFN and gemcitabine on MCF-7 cells resulted in growth inhibition in a synergistic manner with a combination index (CI) <1. Notably, SFN was found to significantly downregulate the expression of Bcl-2, an anti-apoptotic gene, and COX-2, a gene involved in inflammation, in a time-dependent manner. These results indicate that SFN induces apoptosis and anti-inflammatory effects on MCF-7 cells via downregulation of Bcl-2 and COX-2 respectively. The combination of SFN and gemcitabine may potentiate the efficacy of gemcitabine and minimize the toxicity to normal cells. Taken together, SFN may be a potent anti-cancer agent for breast cancer treatment.

Apoptosis induced by desmethyl-lasiodiplodin is associated with upregulation of apoptotic genes and downregulation of monocyte chemotactic protein-3

There is growing interest in the discovery of bioactive metabolites from endophytes as an alternative source of therapeutics. Identification of their therapeutic targets is essential in understanding the underlying mechanisms and enhancing the resultant therapeutic effects. As such, bioactive compounds produced by endophytic fungi from plants at the National Park, Pahang, Malaysia, were investigated. Five known compounds were identified using LC-UV-MS-NMR and they include trichodermol, 7-epi-brefeldin A, (3R,4S)-4-hydroxymellein, desmethyl-lasiodiplodin and cytochalasin D. The present study went on to investigate the potential anticancer effects of these compounds and the corresponding molecular mechanisms of the lead compound against human breast adenocarcinoma, MCF-7. For the preliminary screening, the cytotoxicity and apoptotic effects of these compounds against MCF-7 were examined. The compounds were also tested against noncarcinogenic hepatocytes (WRL68). The differential cytotoxicity was then determined using the MTT assay. Desmethyl-lasiodiplodin was found to suppress the growth of MCF-7, yielding an inhibitory concentration (IC50) that was seven-fold lower than that of the normal cells. The cytotoxic effect of desmethyl-lasiodiplodin was accompanied by apoptosis. Subsequent analysis demonstrated increased expression levels of caspase 3, c-myc and p53. Further, desmethyl-lasiodiplodin resulted in inhibition of monocyte chemotactic protein (MCP)-3, a cytokine involved in cell survival and metastasis. Hence, this study proposed that desmethyl-lasiodiplodin inhibited growth and survival of MCF-7 through the induction of apoptosis. This anticancer effect is mediated, in part, by upregulation of apoptotic genes and downregulation of MCP-3. As desmethyl-lasiodiplodin elicited minimal impact against normal hepatocytes, our findings also imply its potential use as a specific apoptotic agent in breast cancer treatment.

The Anti-Cancer Property of Proteins Extracted from Gynura procumbens (Lour.) Merr

Gynura procumbens (Lour.) Merr. belongs to the Asteraceae Family. The plant is a well-known traditional herb in South East Asia and it is widely used to treat inflammation, kidney discomfort, high cholesterol level, diabetic, cancer and high blood pressure. Our earlier study showed the presence of valuable plant defense proteins, such as peroxidase, thaumatin-like proteins and miraculin in the leaf of G. procumbens. However, the effects of these defense proteins on cancers have never been determined previously. In the present study, we investigated the bioactivity of gel filtration fractionated proteins of G. procumbens leaf extract. The active protein fraction, SN-F11/12, was found to inhibit the growth of a breast cancer cell line, MDA-MB-231, at an EC50 value of 3.8 µg/mL. The mRNA expressions of proliferation markers, Ki67 and PCNA, were reduced significantly in the MDA-MB-23 cells treated with SN-F11/12. The expression of invasion marker, CCL2, was also found reduced in the treated MDA-MB-231 cells. All these findings highlight the anti-cancer property of SN-F11/12, therefore, the proteins in this fraction can be a potential chemotherapeutic agent for breast cancer treatment.

Abstract 2087: A novel Btk tyrosine kinase inhibitor, CTN06, as a potential candidate agent for breast cancer treatment.

Abstract Backgrounds: Btk family kinases are non-receptor tyrosine kinases which are involved in the regulation of diverse cellular processes such as growth, survival and migration. Btk up-regulation has been shown to contribute to oncogenesis of myeloid malignacies. However its functions in solid tumors have not been explored due to its prominent expression in B cells. In this study, we present the first evidence that Btk overexpression contributes to breast carcinogenesis. Material and Methods: CTN06-treated and BTK silenced cells were subjected to an Affimetrix human Genome U133A 2.0 microarray analysis for a global examination of Btk's functions in epithelial carcinogenesis. Signaling cascades which were altered by CTN06 treatment include STAT3, mTOR, S6K and Wnt/β-catenin. In vitro proliferation, migration/invasion assays were employed to demonstrate CTN06-mediated anti-cancer effects. RNA interference experiments were performed to show Btk's contribution to breast carcinogenesis and metastasis. Finally, xenograft mouse model was used to demonstrate the effects of CTN06 and the down-regulation of Btk on breast tumorigenesis. Results: Our preliminary data indicates that Btk is aberrantly expressed in various breast cancer cell lines and tissues but particularly in cells of invasive phenotype. These findings prompted us to further explore the role of Btk in breast cancer carcinogenesis. Using available structural models, our team has developed a collection of small molecule inhibitors for Btk and shown that one of them, CTN06, prominently inhibited the growth and migration of different breast cancer cell lines. Interestingly, CTN06 also appeared to be a potent autophagy inducer in the highly invasive and triple-negative MDA-MB-231 cells. Based on these premises and Btk's participation in several signaling cascades such as Src, PI3K, PLCγ and PLC, we hypothesize that Btk overexpression confers a growth and/or survival advantage for breast cancer cells; Btk inhibitors (CTN06) should effectively suppress breast cancer development by negatively modulating Btk-related networks and be evaluated for clinical use. We have obtained promising results not only in vitro cell line models but also in vivo animal xenograft experiments to demonstrate its efficacy and safety profile. Conclusions: We demonstrated for the first time that Btk overexpression is detected in breast cancer cells particularly in metastatic MDA-MB-231 cells and Btk-specific inhibitor CTN06 effectively suppressed both tumor growth and metastasis via negatively modulating the aforementioned signaling cascades in vitro and in vivo. Citation Format: Chih-Ming Su, Chin-Nien Chuang, Alexander Wu, Liang-Shun Wang, Wei-Hwa Lee, Chih-Hsiung Wu, Kit Lam, Hsing-Jien Kung, Chi-Tai Yeh. A novel Btk tyrosine kinase inhibitor, CTN06, as a potential candidate agent for breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2087. doi:10.1158/1538-7445.AM2013-2087

Abstract 5579: Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

Abstract Breast cancer is a leading cause of death among women. Understanding breast cancer at the molecular level is imperative for finding more effective approaches to successfully treat these patients. Microtubule inhibitors are among the most frequently used agents for breast cancer treatment, with proven efficacy in both localized and metastatic disease. Paclitaxel (Taxol) is a member of the taxane class of anti-neoplastic microtubule damaging agents and exhibits activity against a wide range of human malignancies including breast cancer. Paclitaxel stabilizes microtubules, resulting in G2/M cell cycle arrest, and continuous treatment with paclitaxel ultimately leads to cell death. However, the precise mechanisms of how this mitotic arrest triggers cell death are still unclear. When cells undergo paclitaxel-induced cell death, the BCL-2 family-dependent mitochondrial apoptotic pathway is activated. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim−/− MEFs (mouse embryonic fibroblasts), the bim−/− mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak−/− MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that cdk activation at mitotic arrest could induce MCL-1 phosphorylation and subsequent degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance. Citation Format: Anna V. Miller, Mark A. Hicks, Amanda C. Richardson, Jolene J. Windle, Hisashi Harada. Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5579. doi:10.1158/1538-7445.AM2013-5579

Administration of the optimized β-Lapachone–poloxamer–cyclodextrin ternary system induces apoptosis, DNA damage and reduces tumor growth in a human breast adenocarcinoma xenograft mouse model

β-Lapachone (β-Lap) is a 1,2-orthonaphthoquinone that selectively induces cell death in human cancer cells through NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 is overexpressed in a variety of tumors, as compared to normal adjacent tissue. However, the low solubility and non-specific distribution of β-Lap limit its suitability for clinical assays. We formulated β-Lap in an optimal random methylated-β-cyclodextrin/poloxamer 407 mixture (i.e., β-Lap ternary system) and, using human breast adenocarcinoma MCF-7 cells and immunodeficient mice, performed in vitro and in vivo evaluation of its anti-tumor effects on proliferation, cell cycle, apoptosis, DNA damage, and tumor growth. This ternary system is fluid at room temperature, gels over 29°C, and provides a significant amount of drug, thus facilitating intratumoral delivery, in situ gelation, and the formation of a depot for time-release. Administration of β-Lap ternary system to MCF-7 cells induces an increase in apoptosis and DNA damage, while producing no changes in cell cycle. Moreover, in a mouse xenograft tumor model, intratumoral injection of the system significantly reduces tumor volume, while increasing apoptosis and DNA damage without visible toxicity to liver or kidney. These anti-tumoral effects and lack of visible toxicity make this system a promising new therapeutic agent for breast cancer treatment.

Ursolic Acid Enhances Doxorubicin Cytotoxicity on MCF-7 Cells Mediated by G2/M Arrest

Ursolic acid has been widely known to possess biological activity against numerous tumor cell lines. Previous studies revealed its cytotoxicity on several cancer cells in vitro by either inducing apoptosis or cell cycle modulation. This study was conducted to investigate ursolic acid’s cytotoxicity solely and in combination with a chemotherapeutic agent, doxorubicin, on MCF-7 breast cancer cells, followed by observation on its mechanism. Cytotoxicity of single and combinational treatment of ursolic acid and doxorubicin on MCF-7 breast cancer cells were conducted by using MTT assay. Single treatment was then evaluated by determining IC50 value, while combinational treatment was evaluated by analyzing cell viability and evaluating combination index (CI). To explore the mechanism underlying cytotoxic effect on respected cells, further analysis on cell cycle profile of single and combinational treatment was conducted by flow cytometry. Twenty four hours treatment of ursolic acid inhibited MCF-7 cells’ growth with IC50 value of 37 µM, while combinational treatment showed that several concentration combinations of ursolic acid and doxorubicin exhibited synergism of cytotoxic activity on MCF-7 cells, giving optimum CI value of 0.54. Flow cytometric analysis showed that combinational treatment induced G2/M arrest in MCF-7 cells. These results show that ursolic acid is promising to be developed as either single chemopreventive agent, or as doxorubicin’s co-chemotherapeutic agent in breast cancer treatment. Observation on the selectivity as part of safety aspect together with in silico, in vitro, and in vivo study on its molecular mechanism should be conducted.Keywords: ursolic acid, doxorubicin,co-chemotherapeutic agent, breast cancer, cell cycle

The Role of Peroxisome Proliferator-Activated Receptor-&amp;gamma; in Breast Cancer

Peroxisome Proliferator-Activated Receptor-γ (PPARγ) is an extensively studied ligand-activated nuclear hormone receptor that functions as transcription factor and plays an important role in diverse biological processes, such as lipid metabolism and insulin sensitization. Recent studies have demonstrated that PPARγ is over-expressed in many tumor types, including breast cancer, suggesting a possible role in tumor development and/or progression and a putative prognostic value. Moreover, naturally-occurring and synthetic PPARγ agonists promote growth inhibition, apoptosis and differentiation of tumor cells. The present review summarizes the available information on PPARγ expression in breast tumors and the use of PPARγ ligands as anti-cancer agents for breast cancer treatment, both in vitro and in vivo. Considering the data so far, specific PPARγ agonists seem to exert beneficial effects against breast cancer and may therefore represent potential therapeutic agents.

Anti‐cancer agents for breast cancer treatment during pregnancy

Anti‐cancer agents for breast cancer treatment during pregnancy

Targeted therapy in breast cancer: what's new?

Breast cancer is the most commonly diagnosed malignancy and one of the major causes of death among women. Breast cancer is also one of the most investigated diseases but whose biological features are still not well understood, several effective treating strategies having been explored in dealing with different types of advanced breast cancer, such as endocrine therapy and molecular targeted therapy. Trastuzumab is the first approved targeted anti-cancer agent to show an attractive response rate and outcomes in treating HER-2 positive metastatic breast cancer patients. However, primary or acquired trastuzumab resistance usually occurs some time into the use of trastuzumab and leads to treatment resistance or tumour progression. The promising results with trastuzumab targeted therapy encouraged further investigations in this area exploring several novel targeted agents aiming to overcome the resistance drawback of trastuzumab. In this review we discuss the major newly developed targeted agents in breast cancer treatment, including the novel anti-HER-2 monoclonal antibody pertuzumab or ertumaxomab, small molecular tyrosine inhibitor lapatinib, selective PARP1 inhibitor olaparib, mTOR inhibitor rapamycin analogues, and sheddase inhibitors. Many of these novel targeted drugs or molecules showed additional or complementary effects to trastuzumab therapy that need further and wider investigation.

Abstract P4-05-04: Lack of the Effect of Mesenchymal Stem Cells on Breast Cancer Initiation and Growth in a Somatic Mouse Breast Cancer Model

Abstract Background: The impact of mesenchymal stem cells (MSCs) on breast cancer (BC) progression and growth remains controversial. Some studies using xenograft BC models in nude mice suggest that MSCs stimulate BC metastases, whereas other studies in syngeneic rat BC models suggest that MSCs suppress BC development and growth. In addition, whether MSCs have a role in BC initiation has not been tested. The objective of this study was to determine if bone marrow-derived MSCs affect BC initiation and progression in vitro and in clinically relevant somatic and synegeneic BC mouse models. Materials and Methods: MSCs were isolated from bone marrows of FVB wild-type mice, cultured, and characterized for their potential to differentiate into adipocytes and express flow-cytometric cell surface markers. MSC-conditioned medium (CM) was used to culture RCAS-Neu and RCAS-PyMT BC cell lines, derived from TVA-transgenic mice infected with an avian retroviral vector encoding Neu or polyoma middle T antigen (PyMT). Cell proliferation was analyzed with CellTiter 96 proliferation assay and 5-bromo-2-deoxyuridine labeling. The effect of RCAS-Neu and RCAS-PyMT cells and their CM on MSC migration was determined with Boyden chamber migration assay. In a somatic BC model, TVA-transgenic mice expressing the receptor for an avian retrovirus vector, RCAS, were infected with RCAS-PyMT vector by intraductal injection and treated with MSC (2x106cells/mouse) by i.v. injection. Mice were observed for BC development by palpation. In a syngeneic BC model, RCAS-Neu BC cells were co-implanted with MSCs (5x105/gland) at the ratios 1:0, 1:0.2, or 1:1 into the fat pad of FVB female mice. BC growth was monitored for 9 weeks. Results: CM from MSC did not significantly affect the proliferation of RCAS-Neu and RCAS-PyMT cells. However, RCAS-Neu and RCAS-PyMT cells and their CM induced morphologic changes in MSC and dramatically increased their motility. In somatic model, MSCs had no effect on BC initiation or growth with the mean tumor latency 27.5±7.5 days in MSC-treated mice and 29±5.5 days in control mice treated with PBS. In syngeneic BC model, there was no significant difference in the growth of RCAS-Neu alone or RCAS-Neu cells co-implanted with MSCs. Since BC in these models does not metastasize to distant organs, it could not be determined whether MSCs affect BC metastases. Discussion: Our results demonstrated that BC cell lines and their CM are able to induce MSCs migration and possibly differentiation. However, MSCs had no effect on BC initiation in a somatic BC model and on tumor growth in a syngeneic BC model. It is possible that the number of MSCs and schedule of MSC injection were not optimized or strong PyMT oncogene rapidly induced BC and overcame the effect of MSCs on BC formation. Experiments are ongoing to determine if multiple administrations of MSCs affect BC development induced by PyMT and Neu oncogenes. Lack of effect of MSCs in BC initiation and progression, if confirmed, will suggest that MSCs are safe for delivering novel antitumor agents for BC treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-04.

Dioscorealide B from the Traditional Thai Medicine Hua-Khao-Yen Induces Apoptosis in MCF-7 Human Breast Cancer Cells via Modulation of Bax, Bak and Bcl-2 Protein Expression

Dioscorealide B is a pharmacologically active compound from the rhizome of the Thai medicinal plant Dioscorea membranacea. Here, we demonstrated that in vitro treatment of dioscorealide B resulted in a cytotoxic effect on MCF-7 human breast cancer cells (IC50 = 2.82 microM). To determine whether this compound induces apoptosis in MCF-7, the Annexin V assay was performed. The data showed that the number of apoptotic cells were increased 7-12 folds over that of the control cells after treatment with various concentrations of dioscorealide B (3, 6 and 12 microM) for 24 hours. Dioscorealide B-induced apoptosis was associated with modulation of the multidomain Bcl-2 family members Bax, Bak and Bcl-2. After treatment with 3 microM dioscorealide B, acceleration of the level of proapoptotic proteins Bax and Bak were observed at 6 hours and 12 hours, respectively, while the decrease in the expression of antiapoptotic protein Bcl-2 was observed 3 hours after the treatment. These effects of dioscorealide B might result in the activation of caspase-8, -9 and -7, which lead to apoptosis in MCF-7 cells. Taken together, the results of this study provide evidence that dioscorealide B possesses an antitumor property against human breast cancer cells and thus provide the molecular basis for the further development of dioscorealide B as a novel chemotherapeutic agent for breast cancer treatment.

Abstract PR-05: Effects of presurgical administration of tea polyphenols in women with operable breast cancer

Abstract Background: Various epidemiologic studies have demonstrated an inverse association between green tea consumption and breast cancer risk. The primary polyphenol of green tea, epigallocatechin gallate (EGCG), is a potent antioxidant that acts on multiple stages of carcinogenesis, including proliferation and angiogenesis. The hepatocyte growth factor (HGF)/c-Met pathway is deregulated in breast cancer, with high levels of serum HGF and its tissue receptor c-Met being associated with a poorer prognosis in the clinical setting. Polyphenols have been shown to modulate these pathways in human breast cancer cell lines and mouse xenograft models, however, studies in humans are notably lacking. The purpose of this study was to determine the effects of short-term presurgical administration of an oral green tea extract, Polyphenon E (Poly E), on serum and tissue-based biomarkers in patients with operable breast cancer. Methods: This is a phase II single-arm open-label trial of oral Poly E 800 mg daily for 2-4 weeks in women with histologically confirmed breast cancer on core biopsy who were scheduled for surgical resection. Poly E is a purified tea fraction containing 800 mg of EGCG and lesser amounts of other green tea catechins. Safety was assessed by monitoring clinical and laboratory parameters at baseline and the end of treatment. Serum was collected at baseline and the day prior to surgery and analyzed for HGF and VEGF levels by ELISA. All pretreatment and posttreatment samples were run in the same batch and analyzed in a blinded fashion. Formalin-fixed paraffin-embedded tumor tissue from the diagnostic core biopsy (pretreatment) and surgical specimen (posttreatment) were analyzed for expression of the Ki-67 proliferation index by immunohistochemistry. Pre- and posttreatment values for each biomarker were compared using paired t-test or nonparametric analog. All statistical analyses were two-sided and performed using SAS version 9.1. Results: From February 2008 to September 2009, 25 women were enrolled of which 21 are evaluable. Median age: 49 (33-71); White/Hispanic/Black: 7/13/1; Stage 0/I/II/III: 3/11/4/2; Tumor hormone receptor +/−: 18/3. The study drug was well tolerated with no grade 2 or higher toxicities. Poly E administration was associated with a trend toward decreasing serum HGF levels (mean absolute change of −240 pg/ml, mean % change of −13.5%, p=0.077). No significant change in serum VEGF levels was observed. Tissue-based biomarker analyses are currently ongoing. Conclusions: Our results show a nonsignificant trend towards a decrease in serum HGF levels in women with newly diagnosed early stage breast cancer after short-term administration of Poly E. The treatment was well tolerated, with no significant elevation of liver or pancreatic enzymes. This presurgical study design is a useful and feasible model for testing the biologic effects of potential agents for breast cancer treatment and prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):PR-05.

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF-7 and MDA-MB-231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI-Annexin-V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner; however, MDA-MB-231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell-cycle arrest, p53 accumulation and activation selectively in MCF-7 cells. Inspiringly, the PI-Annexin-V staining assay and western blot analysis confirmed cell apoptosis executed by caspase-3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down-regulation of Bcl-2 and up-regulation of BCL2-associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier-induced apoptosis was confirmed by pan-caspase inhibitor, Z-VAD-fmk. As expected, the inhibitor decreased Huaier-induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER-positive and ER-negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment.