RESUMO
Trafficking of inflammatory T cells into the brain is associated with interactions of certain chemokines with their receptors, which plays an important role in the pathogenesis of multiple sclerosis (MS). We examined whether interferon-beta (IFN-beta) had the ability to regulate the production of chemokines and the expression of their receptors in T cells derived from patients with MS. It was demonstrated for the first time that in vitro exposure of T cells to IFN-beta-1a selectively inhibited mRNA expression for RANTES and MIP-1alpha and their receptor CCR5. T cell surface expression of CCR5 was significantly reduced in MS patients treated with IFN-beta, correlating with decreased T cell transmigration toward RANTES and MIP-1alpha. The study provides new evidence suggesting that IFN-beta treatment impairs chemokine-induced T cell trafficking by reducing the production of RANTES and MIP-1alpha and the expression of their receptors CCR5.
Assuntos
Quimiocina CCL5/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon beta/farmacologia , Proteínas Inflamatórias de Macrófagos/biossíntese , Receptores CCR5/biossíntese , Adulto , Movimento Celular , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/genética , Feminino , Humanos , Proteínas Inflamatórias de Macrófagos/genética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , RNA Mensageiro/análise , Receptores CCR5/genética , Linfócitos T/fisiologiaRESUMO
Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/fisiologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana Transportadoras/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Animais , Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Feminino , Corantes Fluorescentes/farmacologia , Inativação Gênica , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.
Assuntos
Permeabilidade Capilar/genética , Implantação do Embrião , Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/genética , Isoformas de Proteínas/genética , Útero/metabolismo , Animais , Sequência de Bases , Primers do DNA , Feminino , Hibridização In Situ , Camundongos , Neuropilina-1 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Trafficking of inflammatory T cells into the central nervous system (CNS) plays an important role in the pathogenesis of multiple sclerosis. The directional migratory ability of peripheral T cells is associated with interactions of chemokines with their receptors expressed on T cells. In this study, transmigration of peripheral T cells toward a panel of chemokines was examined in patients with multiple sclerosis and healthy individuals using Boyden chemotactic transwells. A significantly increased migratory rate preferentially toward RANTES and MIP-1alpha, but not other chemokines, was found in T cells obtained from multiple sclerosis patients as opposed to healthy individuals (P: < 0.001). The migratory T-cell populations represented predominantly Th1/Th0 cells while non-migratory T cells were enriched for Th2-like cells. The study demonstrated further that aberrant migration of multiple sclerosis-derived T cells toward RANTES and MIP-1 alpha resulted from overexpression of their receptors (CCR5) and could be blocked by anti-CCR5 antibodies. These findings have important implications for our understanding of the mechanism underlying aberrant T cell trafficking in multiple sclerosis.