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

Plasma metalloproteinase activity is enhanced in the euglobulin fraction of breast and lung cancer patients

Matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis. We verified, by gelatin zymography, MMP activity in the euglobulin plasma fraction of 82 healthy controls, 66 patients with benign diseases and 149 patients with breast, lung, colon or brain cancer. The euglobulin fractions assayed showed 4 gelatinolytic bands of 62, 92, 120 and 200 kDa. The median (Md) value for 92 kDa-MMP activity was significantly increased in breast (Md 1.34 arbitrary units [AU]/ml plasma, range 0.0–7.2) and lung cancer patients (Md 1.43 AU/ml, range 0.0–3.6) compared with the controls (Md 0.48 AU/ml, range 0.0–1.8). Patients with colon cancer or gliomas presented values of MMP-9 similar to those of the healthy population. Multivariate analysis indicated that plasma MMP-9 activity was not predicted by the known clinicopathological parameters such as age, stage, tumor size, number of positive lymph nodes, histologic grade, histologic type, nuclear grade or mitotic index. Lung cancer patients also presented high values of MMP-9 (Md 1.43, range 0.0–3.6 [n = 26]), without association with tumor stage or histologic type. The levels of 92 kDa-MMP activity in the plasma euglobulin fraction could be a potentially useful tumor marker in breast and lung cancer.Fil: Farias, Eduardo Francisco. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ranuncolo, Stella Maris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cresta Morgado, Carlos. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Specterman, Sergio. Hospital Italiano; ArgentinaFil: Armanasco, Eduardo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; ArgentinaFil: Varela, Mirta. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lastiri, José. Hospital Italiano; ArgentinaFil: Pallotta, María Guadalupe. Hospital Italiano; ArgentinaFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Puricelli, Lydia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentin

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

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 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 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 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