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

8-Chloro-cAMP-Related Changes in Mice Uteri TheScientificWorld

Histopathological effects of cAMP analog (8-Chloro-cAMP), tamoxifen, and medroxyprogesterone, alone or combined, upon BALB/c mice uteri are reported. 8-Chloro-cAMP diminished uterine weight, but did not modify its histopathology or estral cycle significantly. Tamoxifen diminished uterine weight showing cystic hyperplasia and an estral cycle arrested at diestrus. Medroxyprogesterone increased uterine weight, caused a swelling of the endometrium and a pseudopregnancy estrus. When combined with 8-Chloro-cAMP, tamoxifen or medroxyprogesterone always had a predominant effect. We concluded that the effects of 8-Chloro-cAMP on mice uteri did not cause significant changes on its histopathology, but diminished its weight. KEY WORDS: 8-Chloro-cAMP, tamoxifen, medroxyprogesterone, mice, uterus, histopathology DOMAINS: pharmaceutical sciences, cancer, endocrinology, pharmacology INTRODUCTION The study of 8-Chloro-cAMP pharmacological effects, alone or in combination with other compounds such as tamoxifen and medroxyprogesterone on a hormone-dependent tissue like the uterus, has two objectives: first, to explore how those pharmacological agents affect the cell development of a target tissue of estrogens and, second, to reveal the diverse protein-protein interactions by the different transductional pathways activated by each of them. In this work, we studied the effects of 8-Chloro-cAMP, alone or in combination with tamoxifen or medroxyprogesterone, on the histopa-1427 Actis et al.: 8-Chloro-cAMP-Related Changes in Mice Uteri TheScientificWorld (2002) 2, 1426-1432 thology of uterine mice. 8-Chloro-cAMP is a cAMP analog drug that binds to the regulatory subunit of cAMP-dependent protein kinase (PKA), as determined by Cho Chung EXPERIMENTAL MATERIALS AND PROCEDURES Animals Female BALB/c mice 50-60 days old were employed. The animals were kept under the conditions recommended by the Guide of Care and Use of Laboratory Animals, U.S. In Vivo Treatments Animals were randomly divided into six groups of five mice per group. One group was used as the control group. These were treated with vehicle. Mice of the other five groups received different treatments initiated simultaneously. The 8-Chloro-cAMP group received a subcutaneous pellet (15 mg/kg day); each pellet lasted 10 days and was changed successively. The medroxyprogesterone group received a subcutaneous drug depot (0.25 mg/kg day). The tamoxifen group was treated with a subcutaneous pellet (0.25 mg/kg day); drug delivering from the pellets of tamoxifen and medroxyprogesterone lasted 2 months with a linear-type releasing kinetic. In combined treatments, doses of the combination of drugs utilized were the same as previously described. The selection of dose employed was based in previous results obtained from murine mammary tumors Estrus Cycle and Uterine Weight Determination The estrus cycle was determined daily by vaginal smears. 1428 Actis et al.: 8-Chloro-cAMP-Related Changes in Mice Uteri TheScientificWorld (2002) 2, 1426-1432 Histopathology Representative samples of all uteri were harvested for histological examination. Reagents Gador S.A., Buenos Aires, Argentina, kindly provided tamoxifen and medroxyprogesterone. 8-chlorocAMP was a gift from Y.S. Cho Chung, M.D., National Cancer Institute, U.S. RESULTS Uterine Weight The drugs tested in this study acted diversely on this parameter as is shown in Estrus Cycle Histopathology DISCUSSION 8-Chloro-cAMP is a cyclic-AMP analog directly involved in cell proliferation and neoplastic transformation that causes growth inhibition in a variety of human cancer cell types. It is also known that in vitro it inhibits the expression of autocrine and paracrine growth factors 1430 Actis et al.: 8-Chloro-cAMP-Related Changes in Mice Uteri TheScientificWorld (2002) 2, 1426-1432 FIGURE 1. Endometrium slice of mouse uterus under different treatments. 1a: Vehicle; proliferative endometrial glands with normal number of mitoses and absence of secretory changes. Dense stroma. 1b: 8-Chloro-cAMP; proliferative endometrial glands with scarce number and scarce stroma. 1c: Tamoxifen; quistic endometrial hyperplasia with secretory apical mucosal sectors and multistratification; mitotic figures were seen. 1d: 8-Chloro-cAMP + tamoxifen; proliferative endometrial mucosae with multistratificated nucleous, mitoses and cystic glands. 1e: Medroxyprogesterone; highly indented endometrial glands with marked serrated appearance; secretory vacuole located in the basal portion of the cylindrical epithelium. Clear edematous and scarcely cellular stroma between glands. 1f: 8-Chloro-cAMP + medroxyprogesterone; important number of secretory glands with serrated appearance, scarce apical secretion, and edematous stroma. In all cases, HE 100X

Histamine protects bone marrow against cellular damage induced by ionising radiation

Purpose: Based on our previous data on the histamine radioprotective effect on small intestine, in the present work we aimed to determine whether histamine is able to protect bone marrow cells against ionising radiation damage. Materials and methods: 56 mice and 40 rats were divided into four groups. Histamine and histamine-irradiated groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24 h before irradiation. Irradiated groups received a single dose on whole-body using Cesium-137 source and were sacrificed three days after irradiation. We evaluated the number of medullar components, bone marrow trophism, oedema, vascular damage, and other histological characteristics and also proliferation markers by immunohistochemistry. Results: Histamine treatment substantially reduced the grade of aplasia, the oedema and vascular damage induced by ionising radiation on bone marrow of mice and rats. Additionally, histamine preserved medullar components increasing the number of megakaryocytes (14.0 ± 1.0 vs. 7.3 ± 1.0 in mice; and 9.9 ± 1.3 vs. 4.1 ± 1.0 in rats, P < 0.01) and also myeloid (253.4 ± 37.6 vs. 7.8 ± 1.5 in mice; and 52.0 ± 3.7 vs. 31.8 ± 3.1 in rats, P < 0.01), lymphoid (97.4 ± 6.5 vs. 19.8 ± 1.6 in mice; and 23.4 ± 0.9 vs. 11.7 ± 2.5 in rats, P < 0.01) and erythroid cells (165.0 ± 9.1 vs. 8.8 ± 2.8 in mice; and 27.3 ± 2.3 vs. 15.6 ± 3.5 in rats, P < 0.01) per mm2. This effect was associated with an increased proliferation rate of bone marrow cells. Conclusions: Histamine reduces ionising radiation toxicity on bone marrow cells being a suitable candidate for use as radioprotector, especially for patients undergoing radiotherapy who are at the risk of bone marrow or small intestine damage.Fil: Medina, Vanina Araceli. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Croci, Máximo. Instituto de Inmuno Oncología Dr. Ernesto J. V. Crescenti; ArgentinaFil: Carabajal, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Bergoc, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Rivera, Elena S.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Radioprotection of Sensitive Rat Tissues by Oligoelements Se, Zn, Mn Plus Lachesis Muta Venom

In this study we first evaluated the general radioprotective efficacy of Se, Zn and Mn (4 μg/ml each) plus Lachesis muta venom (4 ng/ml) combination (O-LM) by determining survival on rats irradiated with lethal doses of gamma-rays. The aim of the second part of the study was to investigate the O-LM ability to prevent ionizing radiation-induced damage on small intestine, bone marrow and submandibular glands. Hence, histological characteristics and functional studies, together with proliferation and apoptotic marker levels on whole body irradiated rats with a 5 Gy dose were evaluated. Results show that all animals of the untreated group died after whole body irradiation with 8 and 10 Gy while 60 day-survival was more than 80% and 40% in O-LM-treated animals, respectively. Histopathological examinations revealed a high degree of small intestine and submandibular gland radioprotection 3 days post-irradiation. O-LM inhibited histological damage on small intestine, restoring the radiation-induced reduction in villous height and crypt number. O-LM prevented radiation-induced loss of salivary gland function and morphological alterations. These effects were associated to a complete inhibition of radiation-induced apoptosis. Furthermore, studies performed 30 days post-irradiation revealed that O-LM significantly improved bone marrow repopulation, increasing all medullar progenies to the extent of the non-irradiated animals, and completely prevented permanent submandibular gland alterations. Based on the present results and taking into account that O-LM is being safely administered in phase I clinical trial as an immunomodulator, we conclude that O-LM is a non-toxic promising approach to achieve radioprotection for patients undergoing radiotherapy.Fil: Crescenti, Ernesto J. V.. Instituto de Inmuno Oncología "Dr. Ernesto J. V. Crescenti"; ArgentinaFil: 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: Croci, Máximo. Instituto de Inmuno Oncología "Dr. Ernesto J. V. Crescenti"; ArgentinaFil: Sambuco, Lorena Andrea. Instituto de Inmuno Oncología "Dr. Ernesto J. V. Crescenti"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Prestifilippo, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Elverdín, Juan Carlos. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Bergoc, Rosa Maria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Rivera, Elena Susana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Role of H4 receptor in histamine-mediated responses in human melanoma

We have previously reported that histamine at micromolar concentrations reduces the proliferation of melanoma cell lines. It is also known that melanoma cells express histamine H1, H2 and H3 receptors. The aim of this work was to investigate the presence of histamine H4 receptor (H4R) in human melanoma cells and its associated biological processes. In order to better understand the importance of histamine in tumor development, we explored the expression of H4R in human melanoma tissue biopsies. The expression of H4R in WM35 and M1/15 cells was analyzed by RT-PCR, Western blot and immunocytochemistry. To characterize the biological responses we evaluated cell proliferation by clonogenic assay and BrdU incorporation. In addition, cell senescence and differentiation were determined by β-galactosidase enzyme assay and dopa oxidase activity, respectively. The expression levels of H4R were determined by immunohistochemistry in 19 samples of human malignant lesions. Results indicate that melanoma cells express H4R at the mRNA and protein level. By using histamine agonists, antagonists and H4 siRNA we showed that the inhibitory effect of histamine on proliferation was in part mediated through the stimulation of the H4R. The decrease in proliferation was associated with an induction of cell senescence and an increase in melanogenesis that is a differentiation marker of these cells. Furthermore, H4R was expressed in 42% of human melanoma biopses. To our knowledge this is the first report that describes the presence of the H4R in melanoma cells and tissue suggesting a potential therapeutic application of H4R ligands.Fil: Massari, Noelia Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: 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: Martinel Lamas, Diego José. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Cricco, Graciela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Croci, Máximo. Instituto de Inmunooncología; ArgentinaFil: Sambuco, Lorena Andrea. Instituto de Inmunooncología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Bergoc, Rosa Maria. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Rivera, Elena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Bioevaluation of 32P patch designed for the treatment of skin diseases

Objective: The objective of this study was to design and evaluate a 32P patch for the treatment of skin diseases. Materials and Methods: The patch was prepared from chromic phosphate 32P and silicone. Bioelimination and biodistribution in healthy and treated animals, and the therapeutic efficacy of two treatment schemes (single dose and fractionated dose) in an animal model of skin cancer were studied. Results: Based on the bioelimination and biodistribution studies, no leakage of 32P from the patch was observed. The treated tumors reduced their mean diameter compared to controls. The single-dose therapeutic scheme showed a higher number of complete and partial remissions compared to the fractionated scheme. These results were confirmed by histopathological analysis of the samples. Conclusion: The 32P patch was designed and produced according to specifications for the treatment of superficial lesions of the skin. Although the 32P patch is an open source, it behaves like a sealed one for use in brachytherapy treatments.Fil: Salgueiro, María Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Duran, Hebe Alicia. Comisión Nacional de Energía Atómica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palmieri, Mónica Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Pirchio, Rosana. Comisión Nacional de Energía Atómica; ArgentinaFil: Medina, Vanina Araceli. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ughetti, Ricardo. No especifíca;Fil: Croci, Máximo. No especifíca;Fil: Nicolini, Jorge. No especifíca;Fil: Zubillaga, Marcela Beatriz. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Aminoguanidine impedes human pancreatic tumor growth and metastasis development in nude mice

AIM: To study the action of aminoguanidine on pancreatic cancer xenografts in relation to cell proliferation, apoptosis, redox status and vascularization