RESUMEN
Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-ß, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Citocinas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Invasividad Neoplásica/genética , Neoplasias del Cuello Uterino/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Femenino , Humanos , Glicoproteínas de Membrana/genética , Ratones , Invasividad Neoplásica/patología , Transcriptoma , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
The axon guidance cues semaphorins (Semas) and their receptors plexins have been shown to regulate both physiological and pathological angiogenesis. Sema4A plays an important role in the immune system by inducing T cell activation, but to date, the role of Sema4A in regulating the function of macrophages during the angiogenic and inflammatory processes remains unclear. In this study, we show that macrophage activation by TLR ligands LPS and polyinosinic-polycytidylic acid induced a time-dependent increase of Sema4A and its receptors PlexinB2 and PlexinD1. Moreover, in a thioglycollate-induced peritonitis mouse model, Sema4A was detected in circulating Ly6C(high) inflammatory monocytes and peritoneal macrophages. Acting via PlexinD1, exogenous Sema4A strongly increased macrophage migration. Of note, Sema4A-activated PlexinD1 enhanced the expression of vascular endothelial growth factor-A, but not of inflammatory chemokines. Sema4A-stimulated macrophages were able to activate vascular endothelial growth factor receptor-2 and the PI3K/serine/threonine kinase Akt pathway in endothelial cells and to sustain their migration and in vivo angiogenesis. Remarkably, in an in vivo cardiac ischemia/reperfusion mouse model, Sema4A was highly expressed in macrophages recruited at the injured area. We conclude that Sema4A activates a specialized and restricted genetic program in macrophages able to sustain angiogenesis and participates in their recruitment and activation in inflammatory injuries.
Asunto(s)
Macrófagos Peritoneales/inmunología , Neovascularización Fisiológica , Semaforinas/fisiología , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Movimiento Celular , Quimiocinas/biosíntesis , Quimiocinas/inmunología , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Activación de Macrófagos , Macrófagos Peritoneales/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Miocardio/patología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Peritonitis/inmunología , Peritonitis/metabolismo , Peritonitis/patología , Daño por Reperfusión/inmunología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Semaforinas/farmacología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Fase G1 , Inmunidad Innata , Interleucina-12/fisiología , Fase de Descanso del Ciclo Celular , Técnicas de Cocultivo , Humanos , ARN Interferente PequeñoRESUMEN
Antiretrovirals belonging to the human immunodeficiency virus (HIV) protease inhibitor (HIV-PI) class exert inhibitory effects across several cancer types by targeting tumor cells and its microenvironment. Cervical carcinoma represents a leading cause of morbidity and mortality, particularly in women doubly infected with high-risk human papillomaviruses (HR-HPV) and HIV; of note, combined antiretroviral therapy has reduced cervical carcinoma onset and progression in HIV-infected women. We evaluated the effectiveness and mechanism(s) of action of HIV-PI against cervical carcinoma using a transgenic model of HR-HPV-induced estrogen-promoted cervical carcinoma (HPV16/E2) and found that treatment of mice with ritonavir-boosted HIV-PI, including indinavir, saquinavir, and lopinavir, blocked the growth and promoted the regression of murine cervical carcinoma. This was associated with inhibition of tumor angiogenesis, coupled to downregulation of matrix metalloproteinase (MMP)-9, reduction of VEGF/VEGFR2 complex, and concomitant upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3). HIV-PI also promoted deposition of collagen IV at the epithelial and vascular basement membrane and normalization of both vessel architecture and functionality. In agreement with this, HIV-PI reduced tumor hypoxia and enhanced the delivery and antitumor activity of conventional chemotherapy. Remarkably, TIMP-3 expression gradually decreased during progression of human dysplastic lesions into cervical carcinoma. This study identified the MMP-9/VEGF proangiogenic axis and its modulation by TIMP-3 as novel HIV-PI targets for the blockade of cervical intraepithelial neoplasia/cervical carcinoma development and invasiveness and the normalization of tumor vessel functions. These findings may lead to new therapeutic indications of HIV-PI to treat cervical carcinoma and other tumors in either HIV-infected or uninfected patients.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidor Tisular de Metaloproteinasa-3/agonistas , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Papillomavirus Humano 16 , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/etiología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aberrant cholesterol homeostasis and biosynthesis has been observed in different tumour types. This paper investigates the role of the post-squalenic enzyme of cholesterol biosynthesis, oxidosqualene cyclase (OSC), in regulating tumour angiogenesis and metastasis dissemination in mouse models of cancer. We showed that Ro 48-8071, a selective inhibitor of OSC, reduced vascular density and increased pericyte coverage, with a consequent inhibition of tumour growth in a spontaneous mouse model of pancreatic tumour (RIP-Tag2) and two metastatic mouse models of human colon carcinoma (HCT116) and pancreatic adenocarcinoma (HPAF-II). Remarkably, the inhibition of OSC hampered metastasis formation in HCT116 and HPAF-II models. Ro 48-8071 induced tumour vessel normalization and enhanced the anti-tumoral and anti-metastatic effects of 5-fluorouracil (5-FU) in HCT116 mice. Ro 48-8071 exerted a strong anti-angiogenic activity by impairing endothelial cell adhesion and migration, and by blocking vessel formation in angiogenesis assays. OSC inhibition specifically interfered with the PI3K pathway. According to in vitro results, Ro 48-8071 specifically inhibited Akt phosphorylation in both cancer cells and tumour vasculature in all treated models. Thus, our results unveil a crucial role of OSC in the regulation of cancer progression and tumour angiogenesis, and indicate Ro 48-8071 as a potential novel anti-angiogenic and anti-metastatic drug.
Asunto(s)
Colesterol/biosíntesis , Transferasas Intramoleculares/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzofenonas/administración & dosificación , Benzofenonas/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacología , Células HCT116 , Humanos , Transferasas Intramoleculares/antagonistas & inhibidores , Masculino , Ratones , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 +/- 2 nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cytotoxicity occurred at approximately 10-fold higher drug concentrations (maximal toxicity at 1-10 microM), was sensitive to mevalonate, and was significantly enhanced in the presence of NG-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition.
Asunto(s)
GMP Cíclico/fisiología , Células Endoteliales/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Óxido Nítrico/fisiología , Transducción de Señal/efectos de los fármacos , Western Blotting , Borohidruros/farmacología , Caveolina 1/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , L-Lactato Deshidrogenasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Interferente Pequeño , Fracciones Subcelulares/efectos de los fármacos , Transfección , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
Octacosa-10,19-dien-1-ol is a newly synthesized long-chain alcohol, an unsaturated analogue of 1-octacosanol, the major component of policosanol, the purified natural mixture of different higher aliphatic alcohols obtained from sugarcane wax. Our efficient synthetic protocol (five steps with 50% overall yield) is well suited for gram scale preparations and a rapid generation of analogues with different degrees of unsaturation. Beneficial effects of policosanol in the prevention of atherosclerosis and thromboembolic disorders have been reported and related to the inhibition of sterol biosynthesis possibly by the regulation of the activity of HMGCoA reductase mediated by AMP-dependent kinase AMPK. We have compared the effect of octacosadienol and policosanol on the regulation of HMGCoA reductase in HUVEC and HepG2 human hepatoma cells. Octacosadienol was as effective as policosanol in inhibiting the upregulation of HMGCoA reductase, in inducing the phosphorylation of AMPK and in downregulating the HMGCoA reductase mRNA.
Asunto(s)
Alcoholes Grasos/síntesis química , Alcoholes Grasos/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Alcoholes Grasos/química , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/síntesis química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesisRESUMEN
Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.