RESUMEN
BACKGROUND: Recent prospective clinical trials of low molecular weight heparins (LMWHs) have demonstrated that these agents may provide significant advantages in terms of progression-free and overall survival in certain subgroups of cancer patients. The mechanisms of improved survival associated with LMWHs are not known, and may involve direct and/or indirect effects on tumor growth. The purpose of this study was to investigate the effects of LMWH and a sulfated non-anticoagulant LMWH (S-NACH) on tumor chemotherapeutic uptake and chemoresponse. MATERIALS AND METHODS: LMWH and S-NACH were tested for their ability to reduce tumor growth and tumor-associated angiogenesis using three different in vivo models. Biodistribution studies were undertaken to determine the effect of these agents on uptake of paclitaxel (PACL) and doxorubicin (Dox) by breast cancer tumor xenografts. RESULTS: LMWH and S-NACH (10 mg/kg s.c. daily) effectively limited tumor growth of human A549 lung adenocarcinoma xenografts in the nude mouse. In an MDA453/LCC6 breast tumor xenograft model, PACL plus S-NACH showed significant (p < 0.01) tumor growth suppression and improved survival when compared to PACL alone. LMWH increased [(124-)I]-PACL uptake into MDA453/LCC6 tumors, with tumor:muscle ratios several fold greater than that of [(124-)I]-PACL alone 24 h post-injection. Similarly, LMWH and S-NACH significantly (p < 0.01) increased the uptake of Dox by 1.5-2 fold in MCF7 Dox-resistant tumor xenografts. CONCLUSION: Protocols utilizing adjuvant or neo-adjuvant therapy with LMWH or S-NACH could lead to increased tumor chemo responsiveness, potentially overcoming tumor chemoresistance.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Doxorrubicina/farmacocinética , Heparina de Bajo-Peso-Molecular/farmacología , Paclitaxel/farmacocinética , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Animales , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Anticoagulantes/farmacología , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Embrión de Pollo , Doxorrubicina/farmacología , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Femenino , Humanos , Radioisótopos de Yodo , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Paclitaxel/farmacología , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We used a two-compartment coculture model comprising human endothelial cells (EC) and non-small cell lung carcinoma (CA) cells to study capillary formation. Elevated NO concentrations, contributed in part by CA cells, lead to inhibited capillary formation (Phillips PG, Birnby LM, Narendran A, and Milonovich WL. Am J Physiol Lung Cell Mol Physiol 281: L278-L290, 2001). Here we demonstrate using gelatin substrate zymography that high NO concentrations, whether produced endogenously or by NO donor spermine-NONOate or peroxynitrite-generating compound SIN-1, significantly inhibit MMP-9 expression and activation. Furthermore, high NO concentrations decrease Cav-1 abundance and alter its cellular distribution in EC. Cav-1 is essential for capillary formation in this model because Cav-1 antisense treatments targeted to EC significantly inhibit capillary formation. Laser confocal microscopy demonstrated extensive colocalization of MMP-9 with Cav-1 in sprouting EC, primarily at the basolateral surfaces of EC in focal structures associated with directed migration. This codistribution was NO concentration dependent, and elevated NO concentrations lead to marked dissociation of these two proteins. We propose that compartmentalization of MMP-9 within caveolar structures does occur, and that this could facilitate directed proteolysis essential for early migratory and invasive processes. Our data suggest elevated NO concentrations could impact on capillary formation via a combination of direct effects on MMP activation and by altering the distribution or abundance of Cav-1. Consequences of Cav-1 alterations may include impaired activation of proteolytic enzymes that utilize caveolar structure for stabilization and/or compartmentalization of MMP-9 as well as other putative members of an ECM proteolytic cascade.
Asunto(s)
Caveolinas/genética , Endotelio Vascular/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/genética , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/farmacología , Capilares/efectos de los fármacos , Capilares/enzimología , Capilares/fisiopatología , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Caveolina 1 , Caveolinas/metabolismo , Línea Celular Tumoral , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Activación Enzimática , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Metaloproteinasa 9 de la Matriz/metabolismo , Venas UmbilicalesRESUMEN
The lack of commercially available primary murine endothelial cells prompted us to isolate and cultivate this cell type. We report here the effect of sex steroids on the in vitro growth of murine aortic endothelial cells. Murine aortic endothelial cells were isolated by a combination of explant outgrowth from aortic rings and enzymatic digestion. The endothelial nature of the cells was verified by uptake of acylated low-density lipoprotein and positive staining for CD-31. Murine aortic endothelial cell growth is stimulated by physiological concentrations of estrogen. Progesterone, when given simultaneously with estrogen, inhibited the stimulatory growth effect of estrogen. Murine aortic endothelial cells grown in vitro continue to express messenger ribonucleic acid for proteins related to endothelial growth. These include vascular endothelial growth factor, its receptors Flt-1 and Flk-1, and the angiogenesis-associated transcription factor, Ets-1.
Asunto(s)
Endotelio Vascular/citología , Estradiol/farmacología , Animales , Aorta , Técnicas de Cultivo de Célula/métodos , División Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hormonas Esteroides Gonadales/farmacología , Cinética , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We employed an in vitro angiogenesis model that simulates the in vivo milieu for tumor capillary formation to study the direct effects of estrogen. 17beta-estradiol (E2) treatment significantly stimulated capillary sprouting within 8 h in co-cultures of rat aortic endothelial cells (RAECs) and mouse mammary tumor cells. Co-cultures treated with either progesterone (P4) or E2+P4 showed minimal endothelial cell (EC) sprouting when compared to E2 treated cultures. Treatment with the E2 agonist ICI 182,780 dramatically inhibited capillary formation, demonstrating E2-specificity. Within hours, of E2 treatment ECs isolated from tumor cell/EC co-cultures demonstrated a statistically significant increase in both mRNA and protein levels of the transcription factor Ets-1. We observed increased matrix metalloproteinase (MMP) and decreased tissue inhibitor of metalloproteinase (TIMP) mRNA levels in these ECs following E2 treatment. Ets-1 upregulates expression of the vascular endothelial growth factor (VEGF) receptor, Flt-1 and we detected increased Flt-1 mRNA levels in ECs co-cultured with tumor cells following E2 treatment. Expression of Ets-1 contributes to destabilization of a quiescent EC phenotype in favor of an invasive angiogenic one, in part, by increasing expression of MMPs and integrin molecules that favor migration and invasion. Transfection of ECs with Ets-1 antisense prior to co-culture with E2 resulted in a 95% inhibition in capillary formation. We demonstrate here, for the first time that nanomolar concentrations of E2 directly and rapidly induced new capillary formation in a mammary tumor/EC co-culture system and suggest that this response may be mediated, in part, by an E2-induced increase in Ets-1 expression.