RESUMEN
HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.
Asunto(s)
Neoplasias de la Mama , Resistencia a Antineoplásicos , Complejo I de Transporte de Electrón , Proteómica , Receptor ErbB-2 , Trastuzumab , Humanos , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Complejo I de Transporte de Electrón/metabolismo , Proteómica/métodos , Receptor ErbB-2/metabolismo , Línea Celular Tumoral , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/efectos de los fármacos , Proteoma/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.
Asunto(s)
Neoplasias de la Mama/genética , Técnicas de Cultivo de Célula/métodos , Resistencia a Antineoplásicos , Células Madre Neoplásicas/citología , Receptor ErbB-2/genética , Trastuzumab/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cobalto/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Receptor ErbB-2/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/metabolismoRESUMEN
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate diverse cellular functions including cell death, proliferation, and survival. Recent studies have reported that PKCδ, are involved in apoptosis or autophagy induction. In the present study we focused on how PKCδ regulates proliferation and cancer stem cell (CSC) properties of the hormone-independent mammary cancer cell line LM38-LP, using pharmacological and genetic approaches. We found that pharmacological inhibition of PKCδ, by Rottlerin treatment, impairs in vitro LM38-LP proliferation through cell cycle arrest, inducing the formation of cytoplasmic-vacuoles. Using immunofluorescence we confirmed that Rottlerin treatment induced the apparition of LC3 dots in cell cytoplasm, and increased autophagy flux. On the other side, the same treatment increased CSC growth rate and self-renewal. Furthermore, Rottlerin pre-treatment induced in CSC the development of a "grape-like" morphology when they are growing in 3D cultures (Matrigel), usually associated with a malignant phenotype, as well as an increase in the number of experimental lung metastasis when these cells were inoculated in vivo. The PKCδ knockdown, by RNA interference, induced autophagy and increased CSC number, indicating that these effects are indeed exerted through a PKCδ dependent pathway. Finally, the increase in the number of mammospheres could be reversed by a 3MA treatment, suggesting that autophagy mechanism is necessary for the increased of CSC self-renewal induced by PKCδ inhibition. Here we demonstrated that PKCδ activity exerts a dual role through the autophagy mechanism, decreasing proliferative capacity of mammary tumor cells but also regulating tumor stem cell self-renewal.
Asunto(s)
Autofagia , Neoplasias Pulmonares/enzimología , Neoplasias Mamarias Experimentales/enzimología , Células Madre Neoplásicas/fisiología , Proteína Quinasa C-delta/metabolismo , Acetofenonas/farmacología , Animales , Antineoplásicos/farmacología , Benzopiranos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Trasplante de Neoplasias , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/genética , Neoplasias de la Mama/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Femenino , Expresión Génica , Humanos , Fenotipo , Receptor ErbB-2/genética , Esferoides Celulares , Trastuzumab/toxicidad , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Autoanticuerpos/inmunología , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/inmunología , Neovascularización Patológica/inmunología , Receptores Muscarínicos/inmunología , Animales , Neoplasias de la Mama/patología , Carbacol/farmacología , Femenino , Fibroadenoma/irrigación sanguínea , Fibroadenoma/inmunología , Fibroadenoma/patología , Humanos , Inmunoglobulina G , Células MCF-7 , Ratones , Ratones Desnudos , Estadificación de Neoplasias , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: We have previously reported the expression of muscarinic acetylcholine receptors (mAChR) in human breast tumors. The activation of these receptors triggered tumor cell proliferation. Considering that invasion and metastasis is the major cause of death in cancer, we investigated the action of autoantibodies against mAChR derived from breast cancer patients in stage I (T1N0Mx-IgG) on MCF-7 cells migration and metalloproteinase-9 (MMP-9) activity. We also analyzed the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway. METHODS: Immunoglobulin G (IgG) was purified by chromatography in protein G-agarose from blood samples of breast cancer patients obtained under informed consent. Migration was assayed by an in vitro wound assay. MMP-9 activity was quantified by zymography. RESULTS: T1N0Mx-IgG promoted tumor cell migration and increased MMP9 activity mimicking the action of the muscarinic agonist carbachol. This effect was reduced not only by the presence of atropine but also by 4-DAMP or tropicamide, antagonists for M(3) and M(4) mAChR subtypes respectively. The actions of T1N0Mx-IgG and carbachol on MCF-7 cells, involved the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway. CONCLUSIONS: IgG from breast cancer patients in stage I could be promoting tumor progression by regulating migration and MMP-9 activity in tumor cells via mAChR activation. The presence of these autoantibodies could be determining the prognosis of breast cancer in these patients.
Asunto(s)
Autoanticuerpos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Movimiento Celular , Inmunoglobulina G/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores Muscarínicos/inmunología , Autoanticuerpos/farmacología , Carbacol/farmacología , Movimiento Celular/efectos de los fármacos , Agonistas Colinérgicos/farmacología , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Inmunoglobulina G/farmacología , Células MCF-7 , Óxido Nítrico Sintasa/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de SeñalRESUMEN
The epidermal growth factor receptor 2 (HER2) is a tyrosine kinase overexpressed in nearly 20% to 25% of invasive breast cancers. Trastuzumab is a humanized monoclonal antibody that targets HER2. The majority of patients with metastatic breast cancer initially respond to trastuzumab, however, within 1 year of treatment disease progresses. Several molecular mechanisms have been described as contributing to the development of trastuzumab resistance. They could be grouped as impaired access of trastuzumab to HER2, upregulation of HER2 downstream signaling pathways, signaling of alternative pathways, and impaired immune antitumor mechanisms. However, since many of them have overlapping effects, it would be of great clinical impact to identify the principal signaling pathways involved in drug resistance. Significant efforts are being applied to find other therapeutic modalities besides trastuzumab treatment to be used alone or in combination with current modalities.
RESUMEN
Low O(2) levels in solid tumors are associated with increase in hypoxia-inducible factor 1alpha (HIF-1alpha). The present study examines functional changes involved in adaptation to hypoxia of the LMM3 mammary tumor cell line, using CoCl(2) as hypoxic mimetic. Our results showed that LMM3 cells were not only tolerant to 150 microM CoCl(2) but they can overgrowth in vitro respect to untreated cells. Hypoxia inhibited cell invasion, migration, MMP-9 activity and NO levels. Macrophage cytotoxicity augmented under hypoxia but was blunted by conditioned media from tumor cells. In vivo tumorigenicity of CoCl(2)-treated cells was greater than controls. Our results show stabilization of HIF-1alpha in LMM3 cells under CoCl(2) and functional changes associated with enhanced cell survival and growth but not with tumor dissemination.
Asunto(s)
Cobalto/farmacología , Hipoxia , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesis , Relación Estructura-Actividad , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Autoanticuerpos/farmacología , Neoplasias de la Mama/inmunología , Carcinoma/inmunología , Inmunoglobulina G/farmacología , Óxido Nítrico/metabolismo , Autoanticuerpos/aislamiento & purificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Carbacol/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Carcinoma/fisiopatología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Agonistas Colinérgicos/farmacología , Cromatografía de Afinidad , Progresión de la Enfermedad , Femenino , Humanos , Inmunoglobulina G/aislamiento & purificación , Estadificación de Neoplasias , Óxido Nítrico Sintasa/metabolismo , Receptor Muscarínico M3/inmunología , Receptor Muscarínico M4/inmunología , Transducción de Señal/efectos de los fármacosRESUMEN
Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for alpha-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.
Asunto(s)
Línea Celular Tumoral/citología , Células Epiteliales/citología , Fibroblastos/citología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Animales , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral/metabolismo , Resistencia a Antineoplásicos/fisiología , Células Epiteliales/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Femenino , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Neoplasias Mamarias Experimentales/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Translocación GenéticaRESUMEN
Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10(-9) M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that nonneuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.
Asunto(s)
Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Neovascularización Patológica/etiología , Óxido Nítrico Sintasa/fisiología , Receptores Muscarínicos/fisiología , Neoplasias de la Mama/enzimología , Carbacol/farmacología , Línea Celular Tumoral , Proliferación Celular , Humanos , Receptores Muscarínicos/clasificación , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
We have previously reported the expression of functional muscarinic acetylcholine receptors (mAChR) in two different murine mammary adenocarcinoma cell lines LM2 and LM3. Activation of mAChR with carbachol (CARB) increased proliferation in both tumor cell lines in a concentration-dependent manner. In LM3 cells CARB promoted proliferation via M(3) receptor activation by inositol 1,4,5-triphosphate and nitric oxide (NO) production. CARB-induced LM2 cells proliferation needed both M(2) and M(1) receptor activation increasing prostaglandin E(2) liberation and arginase catabolism respectively. Our present results indicate that CARB stimulates LM2 and LM3-induced angiogenesis and tumor growth. This activation follows different patterns. In LM2 tumor, M(1) and M(2) receptors activation stimulates neovascularization by arginase II and cyclooxygenase-2 (COX-2)-derived products while M(1) and M(3) receptors mediate CARB-induced tumor growth by the same effector enzymes. In LM3 tumor, we observe that M(1) and M(2) receptors are involved in agonist-stimulated angiogenesis by COX and NOS1-derived products while tumor growth is stimulated by M(3) and M(2) receptors activation and COX-2-derived prostanoids. Taken together these data present, at least in part, a picture of the regulation that different mAChR subtypes activation exerts on angiogenesis and growth of two different murine mammary adenocarcinomas.
Asunto(s)
Neoplasias Mamarias Experimentales/patología , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Animales , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Carbacol/farmacología , Línea Celular Tumoral , Agonistas Colinérgicos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Receptores Colinérgicos/metabolismo , Sulfonamidas/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
The ability of tumor cells to stimulate adaptive immunity, particularly by inducing anti-tumor antibodies (Abs), has been extensively reviewed. LM3 is a tumorigenic cell line derived from a murine mammary metastatic adenocarcinoma that spontaneously overexpressed mAchR. Here we investigate the ability of Abs purified from the sera of LM3 tumor-bearing mice, directed against muscarinic acetylcholine receptors (mAchR) to modulate tumor cells' proliferation and angiogenesis. We observed that IgG from early tumor bearers (ETB), 14-day LM3 tumor, and from late tumor bearers (LTB), 28-day LM3 tumor, displaced tritiated quinuclidinyl benzilate binding to LM3 tumor cells, confirming Abs interaction with cholinoceptors, while IgG from normal mice did not modify the antagonist binding to mAchR at any concentration tested. In addition, Abs from ETB and LTB immunoblotted a protein of 70 kDa on murine tumor cells and on heart homogenates that was also recognized by a specific anti-M(2) receptor monoclonal antibody. We also observed that IgG purified from ETB-stimulated LM3 cells' proliferation in a more effective manner than the muscarinic agonist carbachol (CARB) did. IgG from LTB-potentiated LM3 cells induced angiogenesis by increasing the number of blood vessels and VEGF-A production in peritumoral skin "via" mAchR, in an agonist similar manner. All effects were blocked by preincubating cells with the non-selective antagonist atropine. In conclusion, autoAbs purified from LM3 tumor-bearing mice sera exert different pro-tumor actions depending on the stage of tumor development: in ETB, they stimulate tumor cells' proliferation, while in LTB they potentiate tumor neovascularization.
Asunto(s)
Adenocarcinoma/sangre , Autoanticuerpos/sangre , Neoplasias Mamarias Experimentales/sangre , Receptores Muscarínicos/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Atropina/farmacología , Autoanticuerpos/aislamiento & purificación , Autoanticuerpos/farmacología , Western Blotting , Carbacol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/farmacología , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Antagonistas Muscarínicos/farmacología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Quinuclidinil Bencilato/farmacología , Receptores Muscarínicos/metabolismo , Timidina/metabolismo , Tritio , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have developed multicellular spheroids (MCS) established from LM05e and LM3 spontaneous Balb/c-murine mammary adenocarcinoma and B16 C57-murine melanoma derived cell lines as an in vitro model to study the efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide system. We demonstrated for the first time that HSVtk-expressing cells assembled as MCS manifested a GCV resistance phenotype compared to the same cells grown as sparse monolayers. HSVtk-expressing LM05e, LM3 and B16 spheroids were 16-, three- and nine-fold less sensitive to GCV than their respective monolayers, even though they could express transgenes 10-, eight- and five-fold more efficiently. Mixed populations of HSVtk- and their respective beta gal-expressing cells displayed a cell-type specific bystander effect that was higher in monolayers than in MCS. However, HSVtk-expressing cells in two- or three-dimensional cultures were always significantly more sensitive to GCV than the beta gal-expressing counterparts, supporting the feasibility of this suicide approach in vivo. We present evidence showing that HSVtk-expressing tumor cells, when transferred from monolayers to MCS, displayed: (i) lower GCV cytotoxic activity and bystander effect; (ii) higher and efficient expression of genes transferred as lipoplexes; (iii) lower cell proliferation rates; and (iv) changes in intracellular Bax/Bcl-xL rheostat of mitochondria-mediated apoptosis.
Asunto(s)
Adenocarcinoma/terapia , Antivirales/farmacología , Ganciclovir/farmacología , Neoplasias Mamarias Animales/terapia , Timidina Quinasa/genética , Proteínas Virales/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/virología , Animales , Efecto Espectador/efectos de los fármacos , Efecto Espectador/genética , Efecto Espectador/fisiología , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular Tumoral , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/fisiología , Terapia Genética , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/virología , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-bcl-2 , Simplexvirus/enzimología , Simplexvirus/genética , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/enzimología , Esferoides Celulares/patología , Timidina Quinasa/metabolismo , Transfección , Proteínas Virales/metabolismo , Proteína X Asociada a bcl-2 , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
BACKGROUND: The normal duct and lobular system of the mammary gland is lined with luminal and myoepithelial cell types. Although evidence suggests that myoepithelial cells might suppress tumor growth, invasion and angiogenesis, their role remains a major enigma in breast cancer biology and few models are currently available for exploring their influence. Several years ago a spontaneous transplantable mammary adenocarcinoma (M38) arose in our BALB/c colony; it contains a malignant myoepithelial cell component and is able to metastasize to draining lymph nodes and lung. METHODS: To characterize this tumor further, primary M38 cultures were established. The low-passage LM38-LP subline contained two main cell components up to the 30th subculture, whereas the higher passage LM38-HP subline was mainly composed of small spindle-shaped cells. In addition, a large spindle cell clone (LM38-D2) was established by dilutional cloning of the low-passage MM38-LP cells. These cell lines were studied by immunocytochemistry, electron microscopy and ploidy, and syngeneic mice were inoculated subcutaneously and intravenously with the different cell lines, either singly or combined to establish their tumorigenic and metastatic capacity. RESULTS: The two subpopulations of LM38-LP cultures were characterized as luminal and myoepithelium-like cells, whereas LM38-HP was mainly composed of small, spindle-shaped epithelial cells and LM38-D2 contained only large myoepithelial cells. All of them were tumorigenic when inoculated into syngeneic mice, but only LM38-LP cultures containing both conserved luminal and myoepithelial malignant cells developed aggressive papillary adenocarcinomas that spread to lung and regional lymph nodes. CONCLUSION: The differentiated histopathology and metastatic ability of the spontaneous transplantable M38 murine mammary tumor is associated with the presence and/or interaction of both luminal and myoepithelial tumor cell types.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Mamarias Animales/genética , Mioepitelioma/genética , Adenocarcinoma/enzimología , Adenocarcinoma/secundario , Adenocarcinoma Papilar/enzimología , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patología , Adenocarcinoma Papilar/ultraestructura , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , ADN de Neoplasias/genética , Modelos Animales de Enfermedad , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/patología , Metástasis Linfática/genética , Metástasis Linfática/patología , Neoplasias Mamarias Animales/enzimología , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión/métodos , Trasplante de Neoplasias/métodos , Péptido Hidrolasas/biosíntesis , Ploidias , Esferoides Celulares/química , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestructura , Células Tumorales CultivadasRESUMEN
Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.