Autoantibodies against muscarinic receptors in breast cancer. Its role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size¡Ü2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10−8 M) and carbachol (10−9 M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.Fil: Lombardi, Maria Gabriela. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Negroni, María Pía.Fil: Pelegrina, Laura Tatiana. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Castro, Maria Ester. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina;Fil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Azar, María Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Cresta Morgado Carlos. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr.Ángel Roffo"; Argentina;Fil: Sales Maria Elena. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Centro de Estudios Farmacologicos y Botánicos; Argentina

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression

Evolución geológico-geomorfológica cuaternaria del tramo superior del valle del río Laja

La historia geológica del Cuaternario en el tramo superior del valle del río Laja corresponde a una compleja interrelación entre volcanismo, procesos de remoción en masa y sedimentarios aluvio-fluviales. El valle fue modelado inicialmente por la acción glaciaria del Pleistoceno inferior en rocas de las formaciones terciarias Cura-Mallin y Trapa-Trapa, ademas de rocas plutónicas, sobre las cuales se disponen en discordancia cuatro grandes unidades cuaternarias; el cono poligénico de Quilleco, una secuencia de rocas volcánicas pleistocenas, el volcán Antuco y el deposito de avalancha volcánica de Antuco. Los depósitos del cono poligénico de Quilleco representan a facies mixtas volcano-sedimentarias intermedias y distales de los estratovolcanes que originaron las secuencia de rocas volcánicas pleistocenas con las cuales engranan lateralmente. El volcán Antuco corresponde a un estratovolcán mixto y compuesto, de composición basáltica y andesitico-basáltica, cuya actividad se inicio ca. 130.000 a A.P. La primera etapa de su desarrollo (Antuco 1) culmino 9.700± 600 a A.P. con el colapso gravitacional lateral del edificio, que origino la gran avalancha volcánica de Antuco, cuyos materiales represaron el desagüe natural del lago del Laja y de sus quebradas afluentes. El colapso gravitacional fue el resultado de una actividad eruptiva freatomagmatica de tipo Bandai-San la que produjo, casi simultaneamente, flujos piroclasticos turbulentos, de tipo de oleadas de base húmeda, compuestos de cenizas basalticas negras cuyos depositos primarios se han denominado Arenas Negras de Trupan-Laja. El volcán actual (Antuco 2) incluye un cono principal de lavas y escorias y la emisión de, al menos, tres flujos piroclasticos importantes de poco espesor, localmente separados por depósitos de corrientes de barro y coluvios. Posteriormente, debido a la ruptura del represamiento del lago del Laja, las cenizas negras fueron removidas hasta la Depresión Central, donde formaron un gran abanico aluvial de aproximadamente 50 x 60 km2.The Quaternary geological history of the uppermost the Río Laja valley shows a complex interrelation between volcanic, mass wasting, and alluvial and fluvial sedimentary processes. The valley was initialiy carved by Lower Pleistocene glacial action on a Tertiary basement (Cura-Mallín and Trapa-Trapa Formations, intrusives), over which four major Quaternary units were deposited unconformably: the polygenic Quilleco alluvial cone, a Pleistocene volcanic sequence, and the products of the Antuco volcano and the Antuco volcanic avalancha. The Quilleco polygenic cone includes mixed intermediate and distal volcano-sedimentary facies which interfinger with the Pleistocene volcanic sequence derived from coeval stratovolcanos. The Antuco volcano is a mixed and composite andesitic to basaltic andesitic stratovolcano of basalt, which commenced its activity ca. 130,000 yr B.P. Its first constructive phase (Antuco 1) culminated at 9,700± 600 yr B.P. with the lateral gravitational collapse of the edifice; this event produced the major Antuco volcanic avalancha which dammed the natural outlet of Lago del Laja and its tributaries. The gravitational collapse was the final result of a Bandai-San type phreatomagmatic eruptive event associated with almost simultaneous wet turbulent pyroclastic base surges made up of black basaltic ash (Arenas Negras de Trupán-Laja). The present Antuco volcano (Antuco 2) includes the eruptive event that built the main cone with lavas and scoria falls and the eruption of, at least, three pyroclastic flows, locally separated by mud-flow and colluvial deposits. Later, due to the rupture of the Lago del Laja dam, the ash deposits were remobilized by debris flows which came down from the upper reaches of the river to the Central Depression where they formed a major alluvial fan of approximately 50 x 60 km2

Immunoglobulin G from breast cancer patients in stage I stimulates muscarinic acetylcholine receptors in MCF7 cells and induces proliferation: participation of nitric oxide synthase-derived nitric oxide

INTRODUCTION: Muscarinic acetylcholine receptors (mAChR) belong to the G-protein-coupled receptor family and are extensively expressed in most cells in mammals. We had reported the expression of mAChR in murine and human breast tumors. METHODS: The presence of antibodies in the sera of patients with different tumors directed against self-proteins has been recently described. In this work, we investigated the presence of autoantibodies against mAChR in the sera of breast cancer patients in stage I (T1N0Mx-IgG). IgG purification was performed by affinity chromatography in protein G-agarose. We also studied the ability of these antibodies to modulate the proliferation of MCF-7 breast tumor cells by the MTS colorimetric assay. The ability of T1N0Mx-IgG to stimulate muscarinic signaling pathway via nitric oxide synthase was tested by Griess reaction. RESULTS: We demonstrated M(3) and M(4) receptors expression in MCF-7 cells. T1N0Mx-IgG promotes cell proliferation, mimicking the action of the muscarinic agonist carbachol. This effect was preferentially due to M(3) receptor activation in tumor cells via phospholipase C-induced nitric oxide liberation by calcium-dependent nitric oxide synthases. IgG from control patients was unable to produce this effect. DISCUSSION: IgG from patients with breast cancer in early stages could be promoting tumor progression by muscarinic activation, and its presence could be determining the prognosis of this illness.Fil: Negroni, María Pía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Fiszman, Gabriel Leon. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Azar, María Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Cresta Morgado, Carlos. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Español, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Pelegrina, Laura Tatiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: de la Torre, Eulalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Sales, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentin

Effect of BFA-IgG and normal IgG on VEGF-A expression in MCF-7 cells.

<p>Tumor cells were treated with 10⁻⁸ M benign fibroadenoma (BFA) or normal-IgG during 1 h and Western blot assays were performed in MCF-7 cell A) supernatants (Sn) and B) lysates (Lys). Densitometric analysis of the bands is shown. Values were relativized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and to VEGF-A in MCF-7 cells without treatment considered as 1, and is represented by a dotted line. Molecular weights are indicated on the right. Values are mean±S.E.M. of three experiments.</p

Effect of T1N0Mx-IgG or carbachol on MCF-7 cells-induced angiogenesis.

<p>MCF-7 cells were adjusted to 2×10⁶ cells/ml and treated during 1 h with A) T1N0Mx-IgG (10⁻⁸ M) or B) carbachol (CARB) (10⁻⁹ M) in the absence or presence of 10⁻⁸ M atropine (AT), 4-diphenylacetoxi-N-methylpiperidine (4-DAMP), or tropicamide (TROP). Cell suspensions (0.1 ml) were inoculated (i.d.) in both flanks of NUDE mice. Values are means±S.E. of three experiments performed in triplicate. Basal: animals without treatment. <i>p</i><0.001 <i>vs.</i>T1N0Mx-IgG or CARB. Photographs of the angiogenic sites from each experimental group stated in A) and B) are shown in upper panels. Magnification 6.4×.</p

Effect of carbachol on VEGF-A expression in MCF-7 cells.

<p>Western blot assays were performed in A) supernatants (Sn) and B) lysates (Lys) from MCF-7 and MCF-10A cells. Optical density (O.D.) of the bands was calculated by densitometric analysis, and values were relativized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). N. D. not detectable. One representative experiment of three is shown. Values are mean ± S.E.M. of three experiments. Molecular weights are indicated on the right. C) MCF-7 cells were treated for 1 h with 10⁻⁹ M carbachol (CARB) in the absence or presence of 10⁻⁸ M atropine (AT), 4-diphenylacetoxi-N-methylpiperidine (4-DAMP), or tropicamide (TROP) and VEGF-A expression was measured in Lys. Densitometric analysis of the bands is shown in lower panel. Values were relativized to the expression of VEGF-A in MCF-7 cells without treatment, considered as 1, and is represented by a dotted line. Molecular weights are indicated on the right. Values are mean±S.E.M of three experiments. *<i>p</i><0.1; **<i>p</i><0.01 <i>vs</i>. CARB.</p

Effect of T1N0Mx-IgG on VEGF-A expression in murine tumor cells.

<p>A) LMM3 and NMuMG cell lysates (Lys). Optical density (O.D.) of the bands was calculated by densitometric analysis, and values were relativized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). N. D. not detectable. One representative experiment of three is shown. Values are mean±S.E.M. Molecular weights are indicated on the right. LMM3 cells were treated with B) T1N0Mx-IgG or C) carbachol (CARB) in the absence or presence of 10⁻⁸ M atropine (AT). Densitometric analysis of the bands is shown in the right pannel. Values were relativized to the expression of VEGF-A in LMM3 cells without treatment considered as 1, and is represented by a dotted line. Values are mean±S.E.M. of three experiments. *<i>p</i><0.01 <i>vs.</i> T1N0Mx-IgG or CARB. Molecular weights are indicated on the right.</p

Effect of T1N0Mx-IgG on VEGF-A expression in MCF-7 cells.

<p>Western blot assays were performed in cell lysates (Lys) obtained from A) untreated MCF-7 cells or treated with IgG from breast cancer patients in stage I (T1N0Mx-IgG) (10⁻⁸ M) during 1 h in the absence or B) presence of 10⁻⁸ M atropine (AT) 4-diphenylacetoxi-N-methylpiperidine (4-DAMP), or tropicamide (TROP). Densitometric analysis of the bands is shown. Values were relativized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and to the expression of VEGF-A in MCF-7 cells without treatment considered as 1, and is represented by a dotted line. Molecular weights are indicated on the right. Values are mean±S.E.M. of three experiments. *<i>p</i><0.01; **<i>p</i><0.001 <i>vs.</i>T1N0Mx-IgG.</p