Glibenclamide inhibits cell growth by inducing G0/G1 arrest in the human breast cancer cell line MDA-MB-231

Background: Glibenclamide (Gli) binds to the sulphonylurea receptor (SUR) that is a regulatory subunit of ATP-sensitive potassium channels (KATP channels). Binding of Gli to SUR produces the closure of KATP channels and the inhibition of their activity. This drug is widely used for treatment of type 2-diabetes and it has been signaled as antiproliferative in several tumor cell lines. In previous experiments we demonstrated the antitumoral effect of Gli in mammary tumors induced in rats. The aim of the present work was to investigate the effect of Gli on MDA-MB-231 breast cancer cell proliferation and to examine the possible pathways involved in this action. Results: The mRNA expression of the different subunits that compose the KATP channels was evaluated in MDA-MB-231 cells by reverse transcriptase-polymerase chain reaction. Results showed the expression of mRNA for both pore-forming isoforms Kir6.1 and Kir6.2 and for the regulatory isoform SUR2B in this cell line. Gli inhibited cell proliferation assessed by a clonogenic method in a dose dependent manner, with an increment in the population doubling time. The KATP channel opener minoxidil increased clonogenic proliferation, effect that was counteracted by Gli. When cell cycle analysis was performed by flow cytometry, Gli induced a significant cell-cycle arrest in G0/G1 phase, together with an up-regulation of p27 levels and a diminution in cyclin E expression, both evaluated by immunoblot. However, neither differentiation evaluated by neutral lipid accumulation nor apoptosis assessed by different methodologies were detected. The cytostatic, non toxic effect on cell proliferation was confirmed by removal of the drug. Combination treatment of Gli with tamoxifen or doxorubicin showed an increment in the antiproliferative effect only for doxorubicin. Conclusions: Our data clearly demonstrated a cytostatic effect of Gli in MDA-MB-231 cells that may be mediated through KATP channels, associated to the inhibition of the G1-S phase progression. In addition, an interesting observation about the effect of the combination of Gli with doxorubicin leads to future research for a potentialnovel role for Gli as an adjuvant in breast cancer treatment.Fil: Nuñez, Mariel Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Croci, Máximo. Instituto de Inmuno Oncología "Dr. Ernesto J. V. Crescenti"; ArgentinaFil: Cocca, Claudia Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rivera, Elena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Bergoc, Rosa Maria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Instituto Universidad de la Fundación "Héctor Barceló"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martin, Gabriela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Non-tumorigenic epithelial breast cells and ionizing radiation cooperate in the enhancement of mesenchymal traits in tumorigenic breast cancer cells

Aims: Radioresistance and recurrences are crucial hindrances in cancer radiotherapy. Fractionated irradiation can elicit a mesenchymal phenotype in irradiated surviving cells and a deep connection exists between epithelial mesenchymal transition, radioresistance, and metastasis. The aim of this study was to analyze the effect of the secretoma of irradiated non-tumorigenic mammary epithelial cells on surviving irradiated breast tumor cells regarding the gain of mesenchymal traits and migratory ability. Main methods: MDA-MB-231 and MCF-7 breast cancer cells, irradiated or not, were incubated with conditioned media from MCF-10A non-tumorigenic epithelial breast cells, irradiated or not. After five days, we evaluated the expression and localization of epithelial and mesenchymal markers (by western blot and indirect immunofluorescence), cell migration (using transwells) and metalloproteinases activity (by zymography). We also assessed TGF-β1 content in conditioned media by immunoblot, and the effect of A83-01 (a selective inhibitor of TGF-β receptor I) and PP2 (a Src-family tyrosine kinase inhibitor) on nuclear Slug and cell migration. Key findings: Conditioned media from MCF-10A cells caused phenotypic changes in breast tumor cells with attainment or enhancement of mesenchymal traits mediated at least in part by the activation of the TGF-β type I receptor and a signaling pathway involving Src activation/phosphorylation. The effects were more pronounced mostly in irradiated tumor cells treated with conditioned media from irradiated MCF-10A. Significance: Our results suggest that non-tumorigenic epithelial mammary cells included in the irradiation field could affect the response to irradiation of post-surgery residual cancer cells enhancing EMT progression and thus modifying radiotherapy efficacy.Fil: Vedoya, Guadalupe María. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; ArgentinaFil: Galarza, Tamara Ester. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Mohamad, Nora Alicia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; ArgentinaFil: Martin, Gabriela Adriana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Involvement of hydrogen peroxide in histamine-induced modulation of WM35 human malignant melanoma cell proliferation

Histamine is a recognized growth factor in melanoma, and exogenous histamine produces a dual effect on proliferation. We have previously reported that histamine at micromolar concentrations reduces the proliferation of melanoma cell lines. To investigate the mechanism by which histamine inhibits proliferation of WM35 human melanoma cells, we have studied the involvement of histamine in reactive oxygen species production and antioxidant enzyme regulation in these cells. Results indicate that histamine treatment (10 μM) significantly increased hydrogen peroxide levels, whereas it slightly decreased superoxide levels associated with an enhancement of superoxide dismutase and a reduction in catalase activity. Additionally, catalase treatment reversed the inhibitory effect of histamine on proliferation, and various treatments that reduce hydrogen peroxide formation increased proliferation of these cells. Furthermore, we demonstrate that the inhibition of proliferation produced by histamine was mediated at least in part by an induction of cell senescence. We conclude that hydrogen peroxide is involved in histamine-mediated modulation of proliferation in malignant melanoma cells.Fil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Massari, Noelia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Martín, Gabriela A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Bergoc, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Rivera, Elena Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Effect of histamine on the expression of metalloproteinases and cell adhesion in breast cancer cell lines

Changes in cell adhesion and matrix metalloproteinases production (MMPs) are pivotal for tumour progression to occur. MMPs degrade extracellular matrix, MMP2, MMP9 and MMP7 being associated with the malignant potential of cancer cells. MMPs proteolytic activity is precisely regulated by the endogenous tissue inhibitors of metalloproteinases (TIMPs). Histamine (HA) has been demonstrated to be an autocrine and paracrine growth factor in several neoplasms, modulating cell survival and invasiveness. Our aim was to study the effect of HA on cell adhesion, expression and activity of MMP2, MMP7 and MMP9 in human tumorigenic and non-tumorigenic mammary cell lines, MDA-MB-231 and HBL-100, respectively. MMPs protein expression was evaluated by immunocytochemistry, mRNA levels by RT-PCR and gelatinolytic activity by zimography. TIMP1 and TIMP2 mRNA levels were measured by RT-PCR. Cell adhesion was assessed by spectrophotometric measurement of methylene blue stained adherent cells. HBL-100 showed lower basal gelatinolitic levels than MDA-MB-231 cells. Basal activity and mRNA levels of MMP2 were higher than MMP9 in HBL-100, while MMP9 acitivity and RNA levels were predominant in MDA-MB-231. MMP7 protein and mRNA levels were very low in both cell lines. A significant basal expression of TIMP1 and TIMP2 mRNA levels was observed in these cell lines. 10 microM HA treatment reduced MMP9 and MMP2 activity and mRNA levels in MDAMB- 231 cells, while TIMP1 and TIMP2 mRNA levels were unaffected. In HBL-100, 10 microM HA slightly reduced MMP2 activity. Regarding cell adhesion, it was diminished by 10 microM HA in MDA-MB-231, but increased in HBL100 cells. These results disclose the ability of HA to modulate MMPs and cell adhesion in both cell lines, suggesting a potential role of HA in the events involved in mammary carcinoma progression.Fil: Genre, Fernanda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Valli, Eduardo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Medina, V.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gutiérrez, A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Sambuco, L.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Rivera, E.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Martin, Gabriela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Methodological approaches to investigate the effects of histamine receptor targeting compounds in preclinical models of breast cancer

Numerous studies demonstrated the pivotal role of histamine in breast cancer development and progression. This chapter aims to describe different preclinical breast cancer models, including in vitro soft agar clonogenic assay and in vivo chemically induced breast tumors in rats, human triple negative breast cancer (TNBC) xenograft, and murine TNBC syngeneic graft in mice. All these models could be useful and complementary to assess the role of histamine receptor ligands in breast cancer, which could originate advances in novel therapeutics to treat this disease. This chapter also describes the gold standard radiometric techniques to evaluate the activity of histamine metabolizing enzymes in breast cancer specimens.Fil: Martinel Lamas, Diego José. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nicoud, Melisa Beatriz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Sterle, Helena Andrea. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; ArgentinaFil: Martin, Gabriel Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cremaschi, Graciela Alicia. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Schwelberger, Hubert G.. Medical University Innsbruck; AustriaFil: Rivera, Elena Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; ArgentinaFil: Medina, Vanina Araceli. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Histamine prevents radiation-induced mesenchymal changes in breast cancer cells

Radiotherapy is a prime option for treatment of solid tumors including breast cancer though side effects are usually present. Experimental evidence shows an increase in invasiveness of several neoplastic cell types through conventional tumor irradiation. The induction of epithelial to mesenchymal transition is proposed as an underlying cause of metastasis triggered by gamma irradiation. Experiments were conducted to investigate the role of histamine on the ionizing radiation-induced epithelial to mesenchymal transition events in breast cancer cells with different invasive phenotype. We also evaluated the potential involvement of Src phosphorylation in the migratory capability of irradiated cells upon histamine treatment. MCF-7 and MDA-MB-231 mammary tumor cells were exposed to a single dose of 2 Gy of gamma radiation and five days after irradiation mesenchymal-like phenotypic changes were observed by optical microscope. The expression and subcellular localization of E-cadherin, β-catenin, vimentin and Slug were determined by immunoblot and indirect immunofluorescence. There was a decrease in the epithelial marker E-cadherin expression and an increase in the mesenchymal marker vimentin after irradiation. E-cadherin and β-catenin were mainly localized in cytoplasm. Slug positive nuclei, matrix metalloproteinase-2 activity and cell migration and invasion were significantly increased. In addition, a significant enhancement in Src phosphorylation/activation could be determined by immunoblot in irradiated cells. MCF-7 and MDA-MB-231 cells also received 1 or 20 μM histamine during 24 h previous to be irradiated. Notably, pre-treatment of breast cancer cells with 20 μM histamine prevented the mesenchymal changes induced by ionizing radiation and also reduced the migratory behavior of irradiated cells decreasing phospho-Src levels. Collectively, our results suggest that histamine may block events related to epithelial to mesenchymal transition in irradiated mammary cancer cells and open a perspective for the potential use of histamine to improve radiotherapy efficacy.Fil: Galarza, Tamara Ester. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Mohamad, Nora Alicia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Táquez Delgado, Mónica Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vedoya, Guadalupe M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Crescenti, Ernesto J.. Instituto de Inmunooncología; ArgentinaFil: Bergoc, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martin, Gabriela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Antitumor activity of histamine and clozapine in a mouse experimental model of human melanoma

Functional presence of histamine H4 receptor (H4R) was demonstrated in human melanoma cell lines and biopsies. Objective: The purposes of this work were to investigate signal transduction pathways and biological responses triggered by the activation of H4R in human primary (WM35) and metastatic (M1/15) melanoma cell lines and to evaluate the in vivo antitumor activity of histamine (HA) and clozapine (CLZ) on human M1/15 melanoma xenografts. Methods: Clonogenic assay, incorporation of BrdU, cell cycle distribution, phosphorylation levels of ERK1/2 and cAMP production were evaluated in vitro. An experimental human melanoma model was developed into athymic nude mice. Tumor growth, survival and histochemical studies were performed in order to investigate the expression levels of H4R, HA, PCNA, mitotic index (MI), and angiogenesis. Results: The results indicate that H4R agonists inhibited forskolin-induced cAMP levels only in M1/15 cells while increased phosphorylation levels of ERK1/2 and decreased proliferation in both cell types. In vivo studies show that HA and CLZ (1 mg kg1, sc) significantly increased median survival and decreased tumor volume. These effects were associated to a reduction in MI, in the expression of proliferation marker and in intratumoral neovascularization. Conclusions: We conclude that HA and CLZ exhibit an antitumoral effect in vitro and in vivo on human melanoma, suggesting the therapeutic potential of these compounds for the treatment of malignant melanomaFil: Massari, Noelia Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Cricco, Graciela Patricia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Martinel Lamas, Diego José. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Sambuco, Lorena Andrea. Instituto de Inmunooncología; Argentina;Fil: Pagotto, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Ventura, Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Ciraolo, Pablo Juan. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Bergoc, Rosa Maria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Fisicomatemática. Cátedra de Física; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Rivera, Elena Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Fisico Matematica. Cat.de Fisica; Argentina