RESUMO
The endothelin-1 (ET-1)/ET A receptor (ETAR) signalling pathway is a well-established driver of epithelial ovarian cancer (EOC) progression. One key process promoted by ET-1 is tumor cell invasion, which requires the scaffolding functions of ß-arrestin-1 (ß-arr1) downstream of the receptor; however, the potential role of ET-1 in inducing invadopodia, which are crucial for cellular invasion and tumor metastasis, is completely unknown. We describe here that ET-1/ETAR, through ß-arr1, activates RhoA and RhoC GTPase and downstream ROCK (Rho-associated coiled coil-forming kinase) kinase activity, promoting actin-based dynamic remodelling and enhanced cell invasion. This is accomplished by the direct interaction of ß-arr1 with PDZ-RhoGEF (postsynaptic density protein 95/disc-large/zonula occludens-RhoGEF). Interestingly, ETAR-mediated invasive properties are related to the regulation of invadopodia, as evaluated by colocalization of actin with cortactin, as well as with TKS5 and MT1-MMP (membrane type 1-matrix metalloproteinase) with areas of matrix degradation, and activation of cofilin pathway, which is crucial for regulating invadopodia activity. Depletion of PDZ-RhoGEF, or ß-arr1, or RhoC, as well as the treatment with the dual ET-1 receptor antagonist macitentan, significantly impairs invadopodia function, MMP activity and invasion, demonstrating that ß-arr1/PDZ-RhoGEF interaction mediates ETAR-driven ROCK-LIMK-cofilin pathway through the control of RhoC activity. In vivo, macitentan is able to inhibit metastatic dissemination and cofilin phosphorylation. Collectively, our data unveil a noncanonical activation of the RhoC/ROCK pathway through the ß-arr1/PDZ-RhoGEF complex as a regulator of ETAR-induced motility and metastasis, establishing ET-1 axis as a novel regulator of invadopodia protrusions through the RhoC/ROCK/LIMK/cofilin pathway during the initial steps of EOC invasion.
Assuntos
Movimento Celular/fisiologia , Neoplasias Ovarianas/metabolismo , Podossomos/fisiologia , Receptor de Endotelina A/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , beta-Arrestinas/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Cortactina/metabolismo , Feminino , Humanos , Immunoblotting , Quinases Lim/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Camundongos Nus , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Podossomos/genética , Podossomos/metabolismo , Interferência de RNA , Receptor de Endotelina A/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais/genética , Transplante Heterólogo , beta-Arrestinas/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína de Ligação a GTP rhoCRESUMO
Cellular mechanical properties constitute good markers to characterize tumor cells, to study cell population heterogeneity and to highlight the effect of drug treatments. In this work, we describe the fabrication and validation of an integrated optofluidic chip capable of analyzing cellular deformability on the basis of the pressure gradient needed to push a cell through a narrow constriction. We demonstrate the ability of the chip to discriminate between tumorigenic and metastatic breast cancer cells (MCF7 and MDA-MB231) and between human melanoma cells with different metastatic potential (A375P and A375MC2). Moreover, we show that this chip allows highlighting the effect of drugs interfering with microtubule organization (paclitaxel, combretastatin A-4 and nocodazole) on cancer cells, which leads to changes in the pressure-gradient required to push cells through the constriction. Our single-cell microfluidic device for mechanical evaluation is compact and easy to use, allowing for an extensive use in different laboratory environments.
Assuntos
Antineoplásicos/administração & dosagem , Bioensaio/instrumentação , Citometria de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/secundário , Apoptose/efeitos dos fármacos , Movimento Celular , Separação Celular/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Análise de Injeção de Fluxo/instrumentação , Neoplasias Experimentais/patologia , Dispositivos ÓpticosRESUMO
We present a novel optofluidic device for real-time sorting on the basis of cell mechanical properties, measured by optical stretching. The whole mechanism, based on optical forces, does not hamper the viability of the tested cells, which can be used for further analysis. The device effectiveness is demonstrated by extracting a sample population enriched with highly metastatic cells from a heterogeneous cell mixture.
Assuntos
Separação Celular/instrumentação , Técnicas Analíticas Microfluídicas , Linhagem Celular Tumoral , Forma Celular , Tamanho Celular , Humanos , Melanoma/patologia , Metástase Neoplásica , Neoplasias Cutâneas/patologiaRESUMO
OBJECTIVES: Curative properties of medicinal plants such as Psidium guajava L. (Myrtaceae) have often been indicated by epidemiological studies on populations in which these fruits are consumed daily. However, complete characterization of the active principles responsible for this ability has never been performed. Here, we have characterized P. guajava's anti-cancer potential and identified the parts of the fruit involved in its anti-neoplastic action. MATERIALS AND METHODS: We studied morphology of our cells, cell cycle characteristics and apoptosis and performed immunostaining, differentiation and western blot analyses. RESULTS: We report that the P. guajava extract exerted anti-cancer control on both haematological and solid neoplasias. P. guajava extract's anti-tumour properties were found to be tightly bound to induction of apoptosis and differentiation. Use of ex vivo myeloid leukaemia blasts corroborated that P. guajava was able to induce cell death but did not exhibit anti-cancer effects on all malignant cells investigated, indicating selective activity against certain types of tumour. Analyses of P. guajava pulp, peel and seeds identified the pulp as being the most relevant component for causing cell cycle arrest and apoptosis, whereas peel was responsible for causing cell differentiation. P. guajava itself and its pulp-derived extract were found to induce apoptosis accompanied by caspase activation and p16, p21, Fas ligand (FASL TNF super-family, member 6), Bcl-2-associated agonist of cell death (BAD) and tumour necrosis factor receptor super-family, member 10b (DR5), overexpression. CONCLUSIONS: Our findings showed that P. guajava L. extract was able to exert anti-cancer activity on cultures in vitro and ex vivo, supporting the hypothesis of its anti malignant pro-apoptotic modulation.