Human Mammary Carcinoma Research Articles

Effect of xanthopterin and isoxanthopterin on nitric oxide production by a RAW264.7 cell line

The pteridines xanthopterin (2‐amino‐4,6(3H,5H)‐pteridinedione) and isoxanthopterin (2‐amino‐4,7(3H,8H)‐pteridinedione) are products of the degradation of tetrahydrobiopterin, a coenzyme used by aryl amino acid hydroxylases, glyceryl ether monooxidase and nitric oxide synthases. These pteridines have cytotoxic effects on a number of cell lines including human mammary carcinoma MCF‐7 cell lines (Lord et al 2005, Cancer Lett. 222, 119). We wished to test the possibility that the pteridines could affect the activity of a nitric acid synthase. Accordingly, we performed experiments in which xanthopterin and isoxanthopterin were tested for their effect on nitric oxide (NO) production by a RAW264.7 mouse macrophage cell line with the resultant nitrite detected by the Griess assay. Both pteridines showed a dose‐dependent reduction of nitric oxide production with respect to controls. Whether this lowering of nitric oxide concentrations is due to inhibition of the inducible nitric oxide synthase will have to be examined. Furthermore, this finding may be significant since nitric oxide affects apoptosis (Brüne 2003, Cell Death and Differentiation 10, 864) and because of the well‐recognized association between chronic inflammation, cell proliferation, cancer and other diseases (Korhonen et al 2005 Current Drug Targets – Inflammation and Allergy 4, 471).

Xanthopterin‐induced apoptosis in leukemic cell lines

The pteridines xanthopterin (2‐amino‐4,6(3H,5H)‐pteridinedione) and isoxanthopterin (2‐amino‐4,7(3H,8H)‐pteridinedione) exhibit anticancer effects in mice and cytotoxicity in confluent human mammary carcinoma MCF7 cells and other transformed cell lines (Lord et al, Cancer Lett. 2005, 222, 119). We wished to study the effects of these pteridines in additional cell lines in an attempt to understand the mechanism of their cytotoxicity. We now report that xanthopterin shows a dose‐dependent cytotoxic effect on Jurkat cells as shown by the MTT assay for cellular viability (Malich et al, Toxicology 1997, 124, 179). Xanthopterin and, to a much less extent, isoxanthopterin induce apoptotic and necrotic‐like morphology in Jurkat T, 769‐P and EL‐4, but not K‐562, cell lines. Dose response curves and cytofluorometric analysis show that the two pteridines in a time and concentration‐dependent manner decrease Jurkat cell viability by inducing caspase 6 activity and rapid cellular disorganization, nuclear condensation, and fragmentation. This suggests that xanthopterin may have some potential as an apoptosis‐inducing agent in leukemia cells (Korhonen et al 2005 Current Drug Targets – Inflammation and Allergy 4, 471).

Of mice and men: a comparative study of cancer-associated fibroblasts in mammary carcinoma

Introduction.The initial clinical experience from targeted therapy for breast cancer has been mixed. While important progress has been made in the care of a subset of patients characterized by amplification of HER2 through the use of trastuzumab, other targeted therapies have failed to improve the outcome for large, unselected groups of patients. Thus, efforts to find prognostic or predictive biomarkers to enable tailored therapy are highly warranted. Genetically engineered mouse models of human cancer provide a convenient setting in which to perform explorative studies. However, there is a paucity of comparative studies between mouse and human tumours in order to validate the use of mouse models as discovery tools.Materials and methods.Here, we have compared the localization of markers for cancer-associated fibroblasts in the MMTV-PyMT mouse model of mammary carcinoma with that of human breast cancer. The expression of α-smooth muscle actin, platelet-derived growth factor receptor-α, and fibroblast-specific protein-1 was assessed by immunostaining of sections from tumours of MMTV-PyMT mice. Information about the distribution of the same markers in human breast cancer was derived from the publicly available database the Human Protein Atlas.Results.Both mouse and human mammary carcinomas were infused by a rich fibrotic stroma. While no marker was capable of identifying all stromal fibroblasts, the expression pattern of each marker was remarkably similar in mouse and human.Discussion.We conclude that the MMTV-PyMT mouse model of breast cancer will have utility as a discovery tool for biomarkers of cancer-associated fibroblasts during malignant conversion.

Study of a potential drug delivery system based on carbon nanoparticles: effects of fullerene derivatives in MCF7 mammary carcinoma cells

Fullerenes (C60) represent important carbon nanoparticles, widely investigated for diagnostic and therapeutic uses, mainly because they are characterized by a small size (between 7 and 10 A ˚ ) and a large surface area. The cytotoxicity of two fullerene derivatives, functionalized by 1,3-dipolar cycloaddition of azome- thine ylides to the C60 cage (1 and 2), the mechanism of cellular uptake (studied with a fluorescein-bearing derivative of 1, hereafter called derivative 3), and the intracellular distribution are the subject of this work. Cell cytotoxicity on human mammary carcinoma cell line (MCF7), evaluated with the MTT test and further confirmed by a flow cytometry approach with DiOC6 and PI probes, showed that derivative 1 was free of necrotic or apoptotic effects even after a long lasting cell exposure. Cell uptake and internalization of derivative 3 reach their zenith within 12 h after treatment, with a tendency to persist up to 72 h; this process was evaluated by flow cytometry and confirmed by confocal micros- copy. Thus, it appears that a compound such as derivative 1 may be unspecif ically taken up by MCF7 cells, in which it distributes throughout the cytoplasm, apparently avoiding any co-localization within the nucleus and secretory granules. These results suggest a strong interaction between the tested fullerene and mammalian cells and a significant ability of this compound to enter tumor cells, therefore resulting to be a suitable vector to deliver anticancer agents to tumor cells.

Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

Preliminary evidences on mitochondrial injury and impaired oxidative metabolism in breast cancer

Mitochondriopathy is emerging as a new cancer theory; however, the relevance of mitochondrial pathobiology in breast cancer has not yet been completely explored. Herein we report on altered expression levels of the oxidative phosphorylation system (OXPHOS) subunits, mitochondrial structural injury and impaired ATP content from a breast-infiltrating ductal carcinoma (IDC). With this purpose, a human mammary carcinoma (HMC-1) cell, referred to a human mammary epithelial cell (HMEC) line, was assayed for: a) OXPHOS levels by quantitative cryo-immunoelectron microscopy (CIEM) labeling; b) morphological characterization by a newly introduced damage grading (scale Mt-g1–3), calculated on the % of intact cristae carrying mitochondria; c) bioenergetic impairment by luminometric determinations of cellular ATP content and cytochemical visualization of COX activity. Drastic OXPHOS reduction was observed in HMC-1 cells for the succinate-dehydrogenase complex II SDH-B protein, while decreasing was reported for the NADH–ubiquinone oxidoreductase complex I NDUFS3 and the ubiquinol cytochrome c reductase complex III UQCRC2 subunits. A significant dropping was detected for the ATP-synthase complex V F1β protein. For the COX complex near-depletion of the mitochondrial-encoded COXI and no apparent variation of the COXIV subunits were observed. Injury grading was categorized assigning three levels of morphological damage in HMC-1 mitochondria: i) severe (4.6%), ii) moderate (23.1%), iii) slight (44.6%), corresponding to 0%, 1–50% and 51–75% of area occupied by intact cristae. ATP generation and COX activity appeared significantly reduced in HMC-1 cells. The structural damage grading here described could provide new insight on IDC mitochondrial impairment and represent hallmark in the breast cancer mitochondriopathy.

Synthesis, characterization and antitumor activity of novel amide derivatives containing ferrocenyl pyrazol-moiety

A series of novel 2-(5-ferrocenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl)-N-amide derivatives (1–38) were prepared. The compounds were characterized by 1H NMR, 13C NMR, IR, mass spectroscopy and element analysis. Compounds 19 and 30 were also gave crystals suitable for X-ray structural analysis. Biological evaluation of all the compounds were performed in the human prostatic carcinoma cell line (PC-3) and human mammary carcinoma cell line (Bcap-37) using the MTT method. Among them, compound 10 exhibited comparable in vitro antitumor activity to the positive control 5-Fluorouracil (5-FU) and cisplatin.

Bispecific antibody complex pre-targeted delivery of polymer-drug conjugates for cancer therapy.

The pretargeting approach using bispecific affibody-antibody complex (BAAC) and targeting of chemotherapeutic drug loaded polymers have been used in breast cancer cell cultures to demonstrate targeted chemotherapy and reduce toxicity to non-pretargeted cancer and cardiac cells. HER2/neu-positive BT-474 and -negative BT-20 human mammary carcinoma cell lines were pretargeted with BAAC and targeted with multi-doxorubicin (Dox) loaded polyglutamic acid (PGA) site specifically modified with diethylene triamine pentaacetic acid (DTPA) (D-Dox-PGA) at the N-terminal of PGA. Toxicity to embryonic cardiocytes and human mammary carcinoma cells were assessed. BAAC was prepared by covalent conjugation of anti-HER2/neu affibody and anti-DTPA Fab via the thioether linkage. N-terminal DTPA modified polyglutamic acid was conjugated with doxorubicin via the amide bonds. Reduction in cardiotoxicity and IC50 of D-Dox-PGA and free Dox were determined in embryonic cardiocyte H9C2 cultures. Enhanced targeted tumor toxicity was demonstrated in BT-474 human mammary carcinoma cell line pretargeting with BAAC followed by targeting with D-Dox-PGA and compared to D-Dox-PGA alone with no pretargeting or free Dox. No enhanced targeted tumor toxicity was observed in HER2/neu negative BT-20 cells. IC50 of D-Dox-PGA and free Dox on embryonic cardiocytes was 15.75 and 1.20μg/ml, respectively. When BT-474 and BT-20 cells were pretargeted with BAAC followed by targeting with D-Dox-PGA, higher tumor cell-killing was seen only in BT-474 cells. Pretargeting with BAAC resulted in greater tumor cell death in BT-474 human breast cancer cells due to specific targeted delivery of D-Dox-PGA than cancer cells treated with D-Dox-PGA without pretargeting or treatment with free doxorubicin. In vitro targeted delivery of polymer drug conjugate resulted in highly specific, targeted HER2/neu positive BT-474 cancer cell death. Such a pretargeting and targeting approach using prodrug polymers may allow development of very efficient, lower non-target toxicity, and image-guided targeted therapy since these polymers can also be labeled with radioisotopes.

Application of a Nitric Oxide-Releasing Pro-drug for Halting Growth of Human Breast and Canine Mammary Carcinoma Cells

Breast carcinomas remain one of the most common forms of cancer among women. Likewise, mammary cancers are one of the leading causes of death in dogs. Among the principal challenges in tumor therapies, resistance to cytotoxic therapeutics represents a major bottleneck in the fight against cancer. The development of novel delivery methods for nitric oxide has fueled the re-emergence of nitric oxide as a potential adjuvant for enhancing sensitivity of cancer cells to traditional therapeutics. More importantly, when delivered under the right conditions, nitric oxide exhibits the capacity to reduce or altogether halt tumor cell proliferation. Here, we present a new class of nitric oxide-releasing pro-drugs using non-cytotoxic S-nitrosothiol nitric oxide donors to deliver nitric oxide in a controlled manner to halt the growth of both human breast and canine mammary carcinoma cells. Research Article Open Access

Preclinical Evaluation of Oncolytic Δγ134.5 Herpes Simplex Virus Expressing Interleukin-12 for Therapy of Breast Cancer Brain Metastases

The metastasis of breast cancer to the brain and central nervous system (CNS) is a problem of increasing importance. As improving treatments continue to extend patient survival, the incidence of CNS metastases from breast cancer is on the rise. New treatments are needed, as current treatments are limited by deleterious side effects and are generally palliative. We have previously described an oncolytic herpes simplex virus (HSV), designated M002, which lacks both copies of the γ 134.5 neurovirulence gene and carries a murine interleukin 12 (IL-12) expression cassette, and have validated its antitumor efficacy in a variety of preclinical models of primary brain tumors. However, M002 has not been yet evaluated for use against metastatic brain tumors. Here, we demonstrate the following: both human breast cancer and murine mammary carcinoma cells support viral replication and IL-12 expression from M002; M002 replicates in and destroys breast cancer cells from a variety of histological subtypes, including “triple-negative” and HER2 overexpressing; M002 improves survival in an immunocompetent model more effectively than does a non-cytokine control virus. Thus, we conclude from this proof-of-principle study that a γ 134.5-deleted IL-12 expressing oncolytic HSV may be a potential new therapy for breast cancer brain metastases.

New half sandwich Ru(ii) coordination compounds for anticancer activity

With the aim of expanding the structure-activity relationship investigation, the series of Ru(II) half sandwich coordination compounds of the type [Ru([9]aneS3)(chel)(L)](n+) previously described by us (where [9]aneS3 is the neutral face-capping ligand 1,4,7-trithiacyclononane, chel is a neutral or anonic chelating ligand, L = Cl(-) or dmso-S, n = 0-2) was extended to 1,4,7-triazacyclononane ([9]aneN3). In addition, new neutral N-N, and anionic N-O and O-O chelating ligands, i.e. dach (trans-1,2-diaminocyclohexane), pic(-) (picolinate), and acac(-) (acetylacetonate), were investigated in combination with both [9]aneS3 and [9]aneN3. Overall, ten new half-sandwich complexes were prepared and fully characterized and their chemical behaviour in aqueous solution was established. The single-crystal X-ray structures of eight of them, including the versatile precursor [Ru([9]aneN3)(dmso-S)(2)Cl]Cl (9), were also determined. The results of in vitro antiproliferative tests performed on selected compounds against MDA-MB-231 human mammary carcinoma cells confirmed that, in this series, only compounds that hydrolyse the monodentate ligand at a reasonable rate show moderate activity, provided that the chelate ligand is a hydrogen bond donor.

Cytotoxic gold compounds: synthesis, biological characterization and investigation of their inhibition properties of the zinc finger protein PARP-1

The new gold(III) complexes: [Au{2-(2'-pyridyl)imidazolate}Cl(2)] and [Au{2,6-bis(2'-benzimidazolate)pyridine}(OCOCH(3))] and the mono- and binuclear gold(I) complexes: [Au{2-(2'-pyridyl)imidazole}(PPh(3))](PF(6)), [Au(2-phenylimidazolate)(DAPTA)] (DAPTA = 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane), [(PPh(3)Au)(2)(2-R-imidazolate)](PF(6)) (R = 2-C(5)H(4)N, Ph) have been synthesized and characterized. The structure of the [(PPh(3)Au)(2){2-(2'-pyridyl)imidazolate)](PF(6)) complex was also characterized by X-ray crystallography. The antiproliferative properties of the complexes were assayed against human ovarian carcinoma cell lines, either sensitive (A2780) or resistant to cisplatin (A2780cisR), human mammary carcinoma cells (MCF7) and non-tumorigenic human kidney (HEK293) cells. Most of the studied compounds showed important cytotoxic effects. Interestingly, the compounds containing the 2-(2'-pyridyl)imidazolate ligand showed selectivity towards cancer cells with respect to the non-tumorigenic ones, with the dinuclear compound [(PPh(3)Au)(2){2-(2'-pyridyl)imidazolate)](PF(6)) being the most active. Some compounds were also screened for their inhibitory effect of the zinc-finger protein PARP-1, essential for DNA repair and relevant to the mechanisms of cancer cell resistance to cisplatin. Interaction studies of the compounds with the model protein ubiquitin were undertaken by electrospray ionization mass spectrometry (ESI MS). The results are discussed in relation to the putative mechanisms of action of the cytotoxic gold compounds.

Synthesis, antiproliferative activity and estrogen receptor α affinity of novel estradiol-linked platinum(II) complex analogs to carboplatin and oxaliplatin. Potential vector complexes to target estrogen-dependent tissues

In the course of efforts to develop 17β-estradiol-linked to anticancer agents targeting estrogen-dependent tissue, we identified three estradiol-linked platinum(II) complex analogs to cisplatin (E-CDDP) derivatives namely: VP-128 ( 1), CD-38 ( 2) and JMP-39 ( 3) that exhibit potent in vitro and in vivo (for derivative VP-128) activity along with interaction with the estrogen receptor α (ERα). In this study, we prepared and biologically evaluated two novel classes of estradiol-linked platinum(II) complex analogs to carboplatin (E-CarboP, 1a– 3a) and oxaliplatin (E-OxaP, 1b– 3b). E-CarboP and E-OxaP were designed and based on the estradiol-linker scaffold of E-CDDP derivatives previously identified. Consequently, we assessed the importance of the nature of platinum(II) salt on the antiproliferative activity on MCF-7 and MDA-MB-231 human mammary carcinoma cell lines together with affinity for the ERα by replacing the dichloroplatinum(II) moiety by a cyclobutane-1,1-dicarboxylateplatinum(II) or an oxalateplatinum(II) moiety. Except for compound 3b which is inactive at the concentration tested, the antiproliferative activity of all compounds on both human mammary carcinomas cell lines are in micromolar range and are more active than carboplatin and oxaliplatin alone but less active that their E-CDDP counterparts ( 1– 3). In addition, E-CarboP derivatives 1a– 3a show very low affinity for ERα whereas E-OxaPs 1b and 2b show higher affinity for ERα than their parents E-CDDPs ( 1– 2), suggesting that the nature of the platinum(II) salt involved in the vector complexes is extremely important to both retain significant antiproliferative activity and selectivity for the ERα and possibility to target estrogen-dependent tissues. Finally, E-OxaPs 1b and 2b are potentially promising alternatives vector complexes to target estrogen-dependent tissues.

P2-04-02: Identification of a Novel Glycosyltransferase-Like Gene as an Autophagic Inducer in Human Mammary Carcinoma Cells Via Down Regulation of BCL-2.

Abstract Here we report the identification of a previously undescribed glycosyltransferase-like gene, GLTx, and its cellular effect on mammary carcinoma cells. GLTx codes for a novel human protein which is highly conserved in primates. Data obtained from the mRNA expression database Oncomine, demonstrate that GLTx is differentially expressed in a variety of cancers including breast cancer. Western blot analysis of a multiple human tissue blot using a rabbit polyclonal antibody against GLTx demonstrated that GLTx was highly expressed in liver, moderately in kidney, intestine and stomach and undetectable in any other tissues. To functionally characterize GLTx, we attempted to establish stable cell lines with forced expression. However, forced expression of GLTx was completely lethal and resulted in severe cell death in the mammary carcinoma cell lines, MCF-7 and BT549, as well as in any other cell lines tested. Staining of MCF-7 cells transiently transfected with a GLTx expression plasmid by either Hoechst 33258 or fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that the observed cell death was not caused by apoptosis. Further investigation confirmed that GLTx expressing cells exhibited cytoplasmic accumulation of autophagosomes, consistent with the induction of autophagy, as measured by the fluorescently labelled autophagosome marker LC3. In addition we have shown that GLTx downregulated BCL-2, an inhibitor of autophagy, as measured by BCL-2 gene promoter luciferase activity assay and western blot assay following transient transfection of a GLTx expressing plasmid. Thus, we have demonstrated that GLTx is a novel autophagic inducer that causes cell death by down regulating BCL-2. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-04-02.

P3-09-04: Comparative Preventive Efficacy of Aqueous Extracts from Lycium Barbarum Bark and Fruit on Estrogen Receptor Positive Human Mammary Carcinoma MCF-7 Cells.

Abstract Background: Chemotherapy, selective estrogen receptor modulators and aromatase inhibitors represent major treatment options for estrogen receptor positive (ER+) clinical breast cancer. These modalities frequently exhibit acquired tumor resistance and adverse systemic toxicity. Natural herbs are increasingly being used in the integrative support of breast cancer patients undergoing conventional treatment modalities and for prevention purposes. We previously reported favorable findings for the fruit of Lycium barbarum (LB), known commonly as goji berry, in the prevention for ER+ breast cancer (Nutrition & Cancer 61:408–414, 2009). The present study compares the preventive efficacy of non-fractionated aqueous extracts from the bark of LB (LBB) and the fruit of LB (LBF) on a cell culture model for ER + breast cancer. Material and Methods: The ER+ human mammary carcinoma derived MCF-7 cells represented the model. Anchorage dependent growth, anchorage independent colony formation and cellular metabolism of 17β-estradiol (E2) represented the mechanistic bases for efficacy. Results: MCF-7 cells grown in serum depleted medium retained their responsiveness to E2, exhibiting E2 stimulated promotion of anchorage dependent growth and anchorage independent colonies. LBB was much more potent than LBF as evidenced by a 95% reduction of IC50 from 0.38% for LBF to 0.02% for LBB, and a 95% reduction of maximum cytostatic concentration (MCC) from 1% for LBF to 0.05% for LBB. At their respective MCC, LBB treatment and LBF treatment produced a 93% and an 89% decrease, respectively, in the number of anchorage independent colonies. Also, LBB produced a 6.8 fold increase in 2-hydroxyestrone (2-OHE1), a 40% decrease in 16a-hydroxyestrone (16a-OHE1) and a 3.7 fold increase in estriol (E3) formation. The corresponding values for LBF were 3.9, 33 and 10.5. LBB treatment resulted in a 16.3 fold increase in 2-OHE1:16a-OHE1 ratio and a 2 fold increase in E3:16a-OHE1 ratio, while LBF treatment produced a 6.0 fold increase and a 2.9 fold increase, respectively, in these endocrine biomarkers. These data suggest that the preventive efficacy of LBB may predominantly be due to up-regulation of the anti-proliferative 2-OHE1 formation, while accelerated conversion of the pro-mitogenic 16α-OHE1 to the mitogenically inert E3 is the case with LBF. Discussion: Growth inhibitory profiles of LBB and LBF may in part be due to their distinct chemical composition and their complementary actions on E2 metabolism. The superior sensitivity of LBB relative to LBF, together with its non-toxic nature, suggests the feasibility of its use in the prevention of human breast cancer. A prevention trial to assess its efficacy and to establish relevant dose schedule is warranted. This study validates a mechanism based approach to identify and prioritize efficacious herbal extracts for the prevention of ER+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-04.

P4-01-11: Autocrine Human Growth Hormone (hGH) Reduces the Sensitivity of Breast Cancer Cells to Treatment with Tamoxifen and Fulvestrant.

Abstract Despite recent advances in breast cancer treatment regimes, intrinsic or development of resistance to conventional breast cancer therapies is still a significant clinical challenge. Expression of growth factors by tumour cells has been proposed as one mechanism for developing resistance to radiotherapy and chemotherapeutic agents. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development in human breast cancer. Recent studies have demonstrated that humans with growth hormone receptor deficiency are protected from developing cancer. Studies investigating human growth hormone (hGH) expression in human breast cancer have demonstrated that hGH expression is associated with specific histopathological features and survival outcomes for patients. Increased hGH expression is significantly associated with lymph node metastasis, tumour stage, proliferative index, worse relapse free survival and reduced overall survival in mammary carcinoma. In recent studies we have demonstrated that autocrine hGH promotes cell proliferation, cell survival and oncogenicity of human mammary carcinoma cells, enhancing anchorage independent cell growth, and supporting tumour formation in immunodeficient mice. Autocrine hGH also promotes mammary carcinoma cell migration/invasion and epithelial to mesenchymal transition and tumour vascularisation in xenograft studies. In this study we investigated whether autocrine hGH promotes resistance to anti-estrogen therapeutic drugs, sensitivity to tamoxifen and fulvestrent using the human breast cancer cell lines MCF-7, T47D and BT474. Stable transfection with an expression vector containing the hGH gene enhanced MCF-7, T47D and BT474 cell viability, total cell number and anchorage independent growth following treatment with tamoxifen or fulvestrant when compared to control transfected cells. Conversely, treatment of BT474 cells with an hGH receptor antagonist (B2036), increased BT474 cell sensitivity to treatment with tamoxifen or fulvestrant. Using an estrogen response element (ERE) luciferase assay, we observed that autocrine hGH enhanced basal estrogen receptor (ER)-mediated transcriptional activity. Our results demonstrate that forced expression of hGH in mammary carcinoma cells enhances ER-mediated signal tranduction and promotes resistance to tamoxifen and fulvestrant, while functional antagnonism of hGH increased cell sensitivity. Consequently, antagonism of the hGH receptor may help maintain sensitivity to anti-estrogen therapy and improve the prognosis of patients with hormone sensitive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-11.

P3-01-03: The Hominoid-Specific Gene SHON Is Oncogenic in Human Mammary Carcinoma.

Abstract Breast cancer is still the most common malignancy and the leading cause of cancer deaths in both developed and under developed countries. Molecular targeted therapy, in particular monoclonal antibody-based treatment, has emerged as a promising approach to treat this disease because of its high specificity and reduced toxicity. As a consequence, identification of novel molecular therapeutic targets is of crucial importance. In an effort to discover new targets for monoclonal antibody-based targeted therapy, we have identified a novel and secreted hominoid-specific molecule, SHON (secreted hominoid-specific oncogene), in the human mammary carcinoma cell line, MCF-7. Like other hominoid-specific genes, SHON has no known orthologs outside of the primate lineage. It is highly expressed in all cancer cell lines tested so far including breast, lung, liver, stomach, colon and prostate cancer as determined at the mRNA level by RT-PCR and at the protein level by western blotting. Forced expression of SHON in MCF-7 cells significantly increased cell proliferation and survival as demonstrated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and Annexin V/propidium iodide apoptosis assays, respectively. Forced expression of SHON also promoted MCF-7 cell anchorage independent growth in soft agar and enhanced cell migration and invasion. Moreover, forced expression of SHON in MCF-7 cells increased tumour volume in a xenograft model of human breast cancer in immunodeficient mice. Furthermore, depletion of endogenous SHON expression using small interfering RNAs, or functional inhibition using an inhibitory rabbit anti-SHON polyclonal antibody, decreased MCF-7 cell proliferation and survival, and reduced MCF-7 oncogenicity and invasiveness. Therefore, SHON is a novel oncogene for mammary carcinoma cells which may be useful as a therapeutic target for the treatment of breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-03.

Prevention of Doxorubicin Cardiopathic Changes by a Benzyl Styryl Sulfone in Mice

Cardiac toxicity is a major limitation in the use of doxorubicin (and related anthracyclins). ON 1910.Na (Estybon, Rogersitib, or 1910), a substituted benzyl styryl sulfone, is equally active as doxorubicin against MCF-7 human mammary carcinoma xenografted into nude mice. 1910 augments the antitumor activity of doxorubicin when given simultaneously. Furthermore, when given in combination, 1910 protects against cardiac weight loss and against morphological damage to cardiac tissues. Doxorubicin induces inactivation of glucose response protein 78 (GRP78), a principal chaperone that serves as the master regulator of the unfolded protein response (UPR). Inactivated GRP78 leads to an increase in misfolded proteins, endoplasmic reticulum (ER) stress, activation of UPR sensors, and increased CHOP expression. 1910 prevents the inactivation of GRP78 by doxorubicin, and the combination, while more active against the tumor, protects against cardiac weight loss.

Curcumin inhibits metastatic progression of breast cancer cell through suppression of urokinase-type plasminogen activator by NF-kappa B signaling pathways

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), is extracted from the plant Curcuma longa. It was recently reported for its anticancer effect on several types of cancer cells in vitro however, the molecular mechanisms of this anticancer effect are not fully understood. In the present study, we evaluated the effects of curcumin on human mammary epithelial carcinoma MCF-7 cells. Cells were treated with curcumin and examined for cell viability by MTT assay. The cells invasion was demonstrated by transwell assay. The binding activity of NF-κB to DNA was examined in nuclear extracts using Trans-AM NF-κB ELISA kit. Western blot was performed to detect the effect of curcumin on the expression of uPA. Our results showed that curcumin dose-dependently inhibited (P<0.05) the proliferation of MCF-7 cells. Meanwhile, the adhesion and invasion ability of MCF-7 cells were sharply inhibited when treated with different concentrations of curcumin. Curcumin also significantly decreased (P<0.05) the expression of uPA and NF-κB DNA binding activity, respectively. It is concluded that curcumin inhibits the adhesion and invasion of MCF-7 cells through down-regulating the protein expression of uPA via of NF-κB activation. Accordingly, the therapeutic potential of curcumin for breast cancer deserves further study.

Benzyl isothiocyanate inhibits basal and hepatocyte growth factor-stimulated migration of breast cancer cells

Benzyl isothiocyanate (BITC), which is found in cruciferous vegetables, has been shown to have anti-carcinogenic properties. Hepatocyte growth factor (HGF) has the ability to stimulate dissociation, migration, and invasion in various tumor cells, and abnormally increased expressions of HGF and its transmembrane tyrosine kinase receptor, c-Met, have previously been detected in human breast cancer, and are associated with high tumor grade and poor prognosis. In this study, in order to assess the mechanisms relevant to the BITC-induced regulation of breast cancer cell migration and invasion, MDA-MB-231 human breast cancer cells and 4T1 murine mammary carcinoma cells were cultured in the presence of 0-4μmol/l BITC with or without 10μg/l of HGF. BITC inhibited both the basal and HGF-induced migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner. In MDA-MB-231 cells, BITC reduced both basal and HGF-induced secretion and activity of urokinase-type plasminogen activator (uPA). In addition, BITC increased the protein levels of plasminogen activator inhibitor-1. HGF stimulated c-Met and Akt phosphorylation, but did not affect the phosphorylation of extracellular signal-regulated kinase-1/2 or stress-activated protein/c-jun N-terminal kinase. BITC suppressed NF-κB activity and reduced the HGF-induced phosphorylation of c-Met and Akt in a dose-dependent manner. LY294002, a specific Akt inhibitor, reduced both basal and HGF-induced uPA secretion and migration of MDA-MB-231 cells. In this study, we demonstrated that BITC profoundly inhibits the migration and invasion of MDA-MB-231 cells, which is associated with reduced uPA activity, and also that these phenomena are accompanied by the suppression of Akt signaling.