Receptor Negative Human Breast Cancer Research Articles

C/EBP\u03b2 isoform-specific regulation of migration and invasion in triple-negative breast cancer cells

The transcription factor C/EBPβ is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPβ-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPβ-isoforms in breast cancer development may reveal new avenues of treatment.

Sevoflurane modulates breast cancer cell survival via modulation of intracellular calcium homeostasis

BackgroundSome retrospective and in vitro studies suggest that general anesthetics influence breast cancer recurrence and metastasis. We compared the effects of general anesthetics sevoflurane versus propofol on breast cancer cell survival, proliferation and invasion in vitro. The investigation focused on effects in intracellular Ca2+ homeostasis as a mechanism for general anesthetic-mediated effects on breast cancer cell survival and metastasis.MethodsEstrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB-436) human breast cancer cell lines along with normal breast tissue (MCF10A) were used. Cells were exposed to sevoflurane or propofol at clinically relevant and extreme doses and durations for dose- and time-dependence studies. Cell survival, proliferation and migration following anesthetic exposure were assessed. Intracellular and extracellular Ca2+ concentrations were modulated using Ca2+ chelation and a TRPV1 Ca2+ channel antagonist to examine the role of Ca2+ in mediating anesthetic effects.ResultsSevoflurane affected breast cancer cell survival in dose-, time- and cell type-dependent manners. Sevoflurane, but not propofol, at equipotent and clinically relevant doses (2% vs. 2 μM) for 6 h significantly promoted breast cell survival in all three types of cells. Paradoxically, extreme exposure to sevoflurane (4%, 24 h) decreased survival in all three cell lines. Chelation of cytosolic Ca2+ dramatically decreased cell survival in both breast cancer lines but not control cells. Inhibition of TRPV1 receptors significantly reduced cell survival in all cell types, an effect that was partially reversed by equipotent sevoflurane but not propofol. Six-hour exposure to sevoflurane or propofol did not affect cell proliferation, metastasis or TRPV1 protein expression in any type of cell.ConclusionSevoflurane, but not propofol, at clinically relevant concentrations and durations, increased survival of breast cancer cells in vitro but had no effect on cell proliferation, migration or TRPV1 expression. Breast cancer cells require higher cytoplasmic Ca2+ levels for survival than normal breast tissue. Sevoflurane affects breast cancer cell survival via modulation of intracellular Ca2+ homeostasis.

PO-234 Transwell co-culture with human breast adipocytes alters the proteome expression profiles of MCF-7 and MDA-MB-231 human breast cancer cells

IntroductionBreast tumours grow within adipose tissue, and stromal adipocytes are likely the first cell type encountered by breast cancer cells as they metastasize. Adipocytes at the invasive edge of human breast tumours exhibit a modified phenotype, and consequently these adipocytes have been named Cancer Associated Adipocytes (CAA). Previous in vitro research has shown that co-culture with CAA promotes breast cancer cell resistance to radiation and some therapeutic agents, as well as breast cancer cell invasiveness both in vitro and in vivo. The aim of the current research was to identify cellular proteins that are differentially regulated in breast cancer cells co-cultured with CAA.Material and methodsThis study collected human breast adipose tissue samples, from which pre-adipocytes were isolated, differentiated into mature adipocytes, and co-cultured with hormone receptor positive (MCF-7) and negative (MDA-MB-231) human breast cancer cells for 3 days in a transwell co-culture system. The proteomes of co-cultured and control breast cancer cells were compared quantitatively using iTRAQ labelling and LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed by Western blotting.Results and discussionsThis study identified and quantified a total of 1126 and 1218 proteins expressed in MCF-7 and MDA-MB-231 cells, respectively. Of these, 85 in MCF-7 and 63 in MDA-MB-231 had a fold change >1.5 between co-culture and control breast cancer cell samples. Overall, MCF-7 cells had more proteins downregulated (n=53) than upregulated (n=32) after co-culture, whereas, more proteins were upregulated (n=51) than downregulated (n=12) in MDA-MB-231 cells. Enrichment analysis revealed an upregulation of proteins involved in metabolic pathways, namely TCA cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The metabolic enzyme PGK1 was upregulated in response to co-culture with CAA in both cell lines, and is associated with poorer overall survival and resistance to the chemotherapy agent paclitaxel in breast cancer. Western blotting results validated mass spectrometry ratios for three candidate proteins.ConclusionOverall, this study describes for the first time the local effect CAA have on the expression of proteins in breast cancer cells in vitro, and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA.

Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines.

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

NSG Mice Provide a Better Spontaneous Model of Breast Cancer Metastasis than Athymic (Nude) Mice.

Metastasis is the most common cause of mortality in breast cancer patients worldwide. To identify improved mouse models for breast cancer growth and spontaneous metastasis, we examined growth and metastasis of both estrogen receptor positive (T47D) and negative (MDA-MB-231, SUM1315, and CN34BrM) human breast cancer cells in nude and NSG mice. Both primary tumor growth and spontaneous metastases were increased in NSG mice compared to nude mice. In addition, a pattern of metastasis similar to that observed in human breast cancer patients (metastases to the lungs, liver, bones, brain, and lymph nodes) was found in NSG mice. Furthermore, there was an increase in the metastatic burden in NSG compared to nude mice that were injected with MDA-MB-231 breast cancer cells in an intracardiac experimental metastasis model. This data demonstrates that NSG mice provide a better model for studying human breast cancer metastasis compared to the current nude mouse model.

Sulforaphane inhibited tumor necrosis factor-α induced migration and invasion in estrogen receptor negative human breast cancer cells

Sulforaphane significantly (p<0.05) inhibited tumor necrosis factor (TNF)-α induced cellular migration and invasion in MCF10DCIS.com human breast cancer cells, compared with controls. mRNA and protein expressions of MMPs, including MMP-2, MMP-9, and MMP-13, and the enzymatic activities of MMP-2 and MMP-9 were suppressed by sulforaphane treatments at 1, 5, and 10 μM concentration in MCF10DCIS.com cells. Sulforaphane should be considered as a potent agent for retardation of mammary tumorigenesis.

Ethanolic extracts of California mugwort (Artemisia douglasiana Besser) are cytotoxic against normal and cancerous human cells

California mugwort (Artemisia douglasiana Besser) is used by many tribes throughout California to treat a variety of conditions, including colds, allergies, and pain. California mugwort is also utilized as women's medicine. Its use is on the rise outside of Native communities, often without the guidance of a traditional healer or experienced herbalist. Because it has been shown to have antiproliferative activity against plant and animal cells, we investigated whether California mugwort extracts have an effect on normal human cells as well as estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast cancer cells. Ethanolic and aqueous extracts of A. douglasiana leaves were tested for cytotoxicity against unstimulated normal human peripheral blood mononuclear cells (hPBMC), as well as against an ER+ human breast cancer cell line (BT-474) and an ER- human breast cancer cell line (MDA-MB-231). An ethanolic leaf extract killed hPBMC, BT-474, and MDA-MB-231 cells with IC50 values of 23.6 ± 0.3, 27 ± 5, and 37 ± 4 μg/ml, respectively. An aqueous extract killed hPBMC with an IC50 value of 60 ± 10 μg/ml, but had no effect on the two cancer cell lines at concentrations up to 100 μg/ml. The results of this study indicate that the cytotoxicity of California mugwort extends to normal human cells, as well as cancerous cells. Therefore, until further is known about the safety of this medicine, caution should be taken when consuming extracts of California mugwort, whether as a tincture or as a tea.

Anticancer Activity of Proapoptotic Peptides is Highly Improved by Thermal Targeting using Elastin-like Polypeptides

Inducing apoptosis in cancer cells is an effective strategy for cancer therapy. The cationic α-helix forming KLAKLAKKLAKLAK peptide (KLAK) has been known to induce apoptosis by disrupting the mitochondria. In the present study, we have designed a thermally targeted KLAK peptide by genetically engineering the KLAK sequence to the carboxy terminus of the heat responsive biopolymer elastin-like polypeptide (ELP). The cellular internalization of ELP-KLAK was made possible by engineering a cell penetrating peptide sequence (SynB1) to the amino terminus of ELP. The SynB1-ELP1-KLAK fusion polypeptide was cytotoxic against both estrogen receptor positive and negative human breast cancer cell lines. The potency of SynB1-ELP1-KLAK was further enhanced when mild hyperthermia was added to the treatment. In response to hyperthermia, SynB1-ELP1-KLAK selectively triggered apoptosis, which was associated with disruption of the mitochondria. The thermally responsive SynB1-ELP-KLAK polypeptide can have improved tumor targeting by the application of mild hyperthermia. Furthermore, the pharmacokinetic properties of ELP can prevent degradation of KLAK in vivo, and the use of SynB1 can mediate tumor cell uptake, thereby augmenting the effect of KLAK.

Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry

Breast cancer is the second most fatal cancer in American women. To increase the life expectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples.

Putative biomarkers and targets of estrogen receptor negative human breast cancer.

Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER), progesterone receptor (PR), and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self-renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.

Abstract 864: Benzyl isothiocyanate, a promising cancer chemopreventive constituent of cruciferous vegetables, inhibits epithelial to mesenchymal transition in human breast cancer cells in vitro and in vivo

Abstract Benzyl isothiocyanate (BITC), a constituent of cruciferous vegetables (e.g., garden cress), inhibits chemically-induced cancer in experimental rodents. We showed previously that BITC treatment inhibited growth of cultured (MDA-MB-231 and MCF-7) and xenografted (MDA-MB-231) human breast cancer cells by causing reactive oxygen species-dependent apoptosis, and suppressed mammary cancer development in a transgenic mouse model (MMTV-neu). We now demonstrate, for the first time, that BITC is a potent inhibitor of epithelial to mesenchymal transition (EMT) in cultured cells and tumor xenografts in vivo. Exposure of estrogen receptor negative (MDA-MB-231) and estrogen receptor positive human breast cancer cells (MCF-7) and a highly invasive pancreatic cancer cell line (PL-45) to pharmacologic concentrations of BITC (1 or 2.5 µmol/L) resulted in up-regulation of epithelial markers (e.g., E-cadherin and occludin) with a concomitant decline in protein levels of mesenchymal markers vimentin, fibronectin, snail, and/or c-Met. BITC-mediated induction of E-cadherin protein correlated with its transcriptional up-regulation. Experimental EMT induced by exposure to TGFβ and TNFα in a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was also largely reversible in the presence of BITC. Consistent with these results, the BITC treatment inhibited TGFβ/TNFα -induced migration by MCF-10A cells, which was partially but significantly attenuated by RNA interference of E-cadherin. Moreover, inhibition of MDA-MB-231 xenograft growth in vivo in female athymic mice by BITC administration correlated with a visible increase in protein level of E-cadherin and suppression of vimentin protein expression. In conclusion, the present study reports a novel anticancer effect of BITC involving inhibition of EMT, a process triggered during progression of carcinoma to invasive state. This investigation was supported by the USPHS grant RO1 CA129347-04, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 864. doi:10.1158/1538-7445.AM2011-864

Abstract 3556: New curcumin analogues are cytotoxic towards estrogen receptor negative human breast cancer cell lines

Abstract There is an urgent need for the development of a safe and efficacious drug therapy for the treatment of estrogen receptor negative breast cancers due to their high malignant nature and poor prognosis. In this study, a series of new heterocyclic monoketo curcumin analogues were synthesized and screened for anticancer activity. Among twenty eight analogues screened in estrogen receptor negative human breast cancer cell-lines MDA-MB-231 (ER-/HER2-), MDA-MB-468 (ER-/HER2-) and SKBr3(ER-/HER2+), the two most potent compounds were designated as RL-71 and RL-66. Cytotoxicity was assessed by sulforhodamine B (SRB) assay. Furthermore, the compounds were studied for their ability to modulate cell cycle progression and induce apoptosis. The mechanism of these drugs was also examined by determining protein expression by Western blot analysis. The results showed that RL-71 and RL-66 had sub micromolar EC50 values in all the cell lines and the cytotoxicity was concentration and time dependent. Mechanistic analyses demonstrated that RL-71 at 1µM and RL-66 at 2µM increased the proportion of MDA-MB-231 cells in G2/M-phase by 162.02 ± 1.53% and 134.12 ± 2.17% over control (P&amp;lt;0.001) respectively. In addition, the treatment with RL-71 (1µM) and RL-66 (2µM) increased the proportion of apoptotic MDA-MB-231 cells by 10-fold and 4-fold over control (p&amp;lt;0.001), respectively. Similar results were also produced in SKBr3 and MDA-MB-468 cells. Furthermore, Western blot analysis confirmed the effect of drugs on different cancer related genes involved in cell proliferation and apoptosis. Thus, these data suggest that the novel curcumin analogues RL-71 and RL-66 are potent antitumor drugs and have significant potential for further drug development for estrogen receptor negative breast cancer. 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 3556.

Combination of a Novel Growth Hormone Releasing Hormone Antagonist JMR 132 and Tamoxifen Enhances the Inhibitory Effects on Growth of ZR 75 and T47D Estrogen Dependent Human Breast Cancer Cells In Vitro and In Vivo.

Abstract Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs.Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2.5µM, 5µM and 10µM. The cell viability after a period of 72 h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132 at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks.Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p&amp;lt; 0.05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells.Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5073.

Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines

Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3β transiently decreased, while β-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

Leptin enhances growth inhibition by cAMP elevating agents through apoptosis of MDA-MB-231 breast cancer cells

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

Combination of a novel growth hormone releasing hormone Antagonist JMR 132 and Tamoxifen enhances the inhibitory effects on growth of ZR 75 and T47D estrogen dependent human breast cancer cells in vitro and in vivo

Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs. Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2,5µM, 5µM and 10µM. The cell viability after a period of 72h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks. Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p<0,05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells. Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects

Effective tumor growth inhibition of MX1 and MDA-MB-231 estrogen receptor negative human breast cancer xenografts by treatment with LHRH antagonist cetrorelix.

Abstract Abstract #2137 Introduction&amp;#x2028; Agonists of LHRH are a treatment option in premenopausal women with estrogen receptor positive breast cancer. The treatment with those agonists decreases the levels of FSH and LH in the blood and thus reduces the levels of sex hormones. Patients with estrogen receptor negative breast cancers do not benefit from therapy with LHRH agonists. Antagonists of LHRH lower LH, FSH and sex steroid levels but without the initial flare up of FSH and LH. In addition to the endocrine effects, Cetrorelix also shows a direct inhibition of cell growth in LHRH receptor (LHRH-R) positive gynecological cancer in vitro where alterations of FSH, LH and sex hormone levels are clearly excluded. In view of these findings we decided to investigate Cetrorelix on the tumor growth in two estrogen receptor negative but LHRH-R positive breast cancer cell lines.&amp;#x2028; Materials and Methods&amp;#x2028; The mRNA expression of LHRH-R was detected by PCR. For proliferation assays cells were treated with increasing doses of Cetrorelix acetate in 0.5% FBS supplemented RPMI 1640 media. MTS assays were performed after 72h. Fluorescence microscopy using ethidium bromide staining was performed to show cell death. In the in vivo study, nude mice were treated with Cetrorelix pamoate preparation at a dose of 3mg s.c. releasing an estimated 100mg Cetrorelix/day.&amp;#x2028; Results&amp;#x2028; MX1 and MDA-MB-231 estrogen receptor negative human breast cancer cell lines were positive for the expression of mRNA for LHRH-R. The cell proliferation in vitro of MX1 and MDA-MB-231 lines treated with different concentrations of Cetrorelix was significantly inhibited in a dose dependent manner. Time and dose dependent cell death was observed by fluorescence staining. Experimental treatment in vivo with Cetrorelix pamoate preparation reduced a significant inhibition of tumor growth in MX1 xenografts as compared to the Controls after 14 days and the suppression remained significant until the end of the study on day 28. MDA-MB-231 xenografts treated with Cetrorelix also showed a significant inhibition of tumor growth compared to the Control as early as day 7 and the inhibition remained significant until the end of the study on day 28.&amp;#x2028; Conclusion&amp;#x2028; Our results indicate a direct inhibitory effect of Cetrorelix on LHRH-R positive breast cancers. Since LHRH-R are expressed in ER negative breast cancers, these findings suggest a new approach to the treatment of LHRH-R positive breast cancer patients independent of the estrogen receptor status. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2137.

Potential role of Renin–Angiotensin-system for tumor angiogenesis in receptor negative breast cancer

Objective This study examined the potential role of Angiotensin II for the regulation of angiogenesis associated genes in receptor positive and negative human breast cancer. Methods Expression of different Renin–Angiotensin system (RAS) components in human breast cancer tissue was investigated using immunofluorescence, and in a receptor positive (MCF-7) and receptor negative (MDA-MB 468) breast cancer cell line by performing immunocytochemistry and RT-PCR. Both cell lines were stimulated with Angiotensin II and Angiotensin II receptor type 1 (At 1R) blocker Candesartan, and gene expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 and 2 (Ang-1 and Ang-2), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), and hypoxia inducible transcription factor 2α (HIF-2α) were quantified by TaqMan-Real-Time PCR analysis. Results RAS components, Angiotensinogen, Renin, Angiotensin I-converting enzyme (ACE), and At 1R and At 2R were expressed in hormone-receptor negative and positive human breast cancer tissue as well as in MDA-MB 468 and in MCF-7 human breast cancer cells. In addition, we found expression of VEGF, Ang-1, TIMP-1, and HIF-2α in both cell lines. However, only in receptor negative MDA-MB 468 cells, did Angiotensin II significantly increase gene expression of VEGF, HIF-2α, and TIMP-1. This effect was completely inhibited by Candesartan. Conclusion In conclusion, it is hypothesized that Angiotensin II may be involved in regulation of tumor angiogenesis especially in receptor negative breast cancer by regulation of angiogenesis associated genes via At 1R. These findings are the first evidence for targeting tumor angiogenesis by inhibition of At 1R in receptor negative human breast cancer cells and may lead to new therapeutical anticancer strategies based upon inhibition of At 1R.

Synergistic tumoricidal effect between celecoxib and gabexate mesilate on the human breast cancer cells

21179 Background: Selective cyclooxygenase 2 (COX2) inhibitors, including celecoxib, has shown its antitumor effects against various cancers including breast cancer. Gabexate mesilate (GM) can be found to have anti-cancer activities against tumors such as colon cancer. We tried to determine whether synergism can exist between GM and celecoxib against human breast cancer cells. Methods: MCF7/Her18 (estrogen receptor positive) and MDA-MB-436 (estrogen receptor negative) human breast cancer cells were used. Celecoxib and GM were added into the culture media at a dose of 30 and 50 μM for estrogen receptor (ER) (+) cells and 50 and 75 μM for ER (-) cells. Cellular proliferation assays wer performed and trypan blue exclusion method was adopted to count the viable cells after culture. Cell cycle analysis and fluorescein-assorted cell sortings were done to check the cell cycle changes after treatment. Immunoblotting using Akt, phosphorylated Akt (p- Akt), β-catenin, COX2 and VEGF was done to evaluate changes in expression of these proteins. Results: Combination treatment group showed significantly enhanced tumoricidal effect than either mono-treatment group on cellular proliferation study. Combined treatment yielded more G1 phase population and G2-M phase cells. (p&lt;0.05) The level of Akt, p-Akt, VEGF, COX2 expression were decreased more significantly than either mono-treatment group. (p&lt;0.05) Conclusions: We think that there could be synergistic effect between GM and celecoxib on the human breast cancer cells. Either drug has been used for many cases without significant problem, and can be tried for further ex vivo studies to determine synergistic effects between them. Possible antiangiogenentic actions after the combination should be evaluated thoroughly in other cell lines to get more preclinical data. No significant financial relationships to disclose.

Novel retinoic acid metabolism blocking agents have potent inhibitory activities on human breast cancer cells and tumour growth

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER −ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumuor xenografts. The ER +ve cell lines were more sensitive (IC50 values between 3.0 and 609 nM) to the RAMBAs than the ER −ve MDA-MB-231 cell line (IC50=5.6–24.0 μM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-α (ER-α). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.