RESUMEN
PURPOSE: To investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation. METHODS AND MATERIALS: Human pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation. RESULTS: Carbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2 major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion--irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion--irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA--mediated XIAP knockdown, indicating that XIAP is involved in C-ion--induced inhibition of cell motility. CONCLUSION: In contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells.
Asunto(s)
Movimiento Celular/efectos de la radiación , Radioterapia de Iones Pesados/métodos , Invasividad Neoplásica , Neoplasias Pancreáticas/radioterapia , Proteína Inhibidora de la Apoptosis Ligada a X/efectos de la radiación , Proteína de Unión al GTP rac1/efectos de la radiación , Proteína de Unión al GTP rhoA/efectos de la radiación , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Neoplasias PancreáticasRESUMEN
We report an optogenetic method based on Arabidopsis thaliana cryptochrome 2 for rapid and reversible protein oligomerization in response to blue light. We demonstrated its utility by photoactivating the ß-catenin pathway, achieving a transcriptional response higher than that obtained with the natural ligand Wnt3a. We also demonstrated the modularity of this approach by photoactivating RhoA with high spatiotemporal resolution, thereby suggesting a previously unknown mode of activation for this Rho GTPase.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Multimerización de Proteína , Vía de Señalización Wnt , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efectos de la radiación , Western Blotting , Técnicas de Cultivo de Célula , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Criptocromos/genética , Criptocromos/efectos de la radiación , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Ensayo de Inmunoadsorción Enzimática , Recuperación de Fluorescencia tras Fotoblanqueo , Células HEK293 , Humanos , Luz , Fototransducción , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/efectos de la radiación , Ratones , Células 3T3 NIH , Multimerización de Proteína/efectos de la radiación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/efectos de la radiación , Transcripción Genética , Transfección , Vía de Señalización Wnt/efectos de la radiación , Proteína Wnt3A/genética , Proteína Wnt3A/efectos de la radiación , beta Catenina/genética , beta Catenina/efectos de la radiación , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/efectos de la radiación , Proteína Fluorescente RojaRESUMEN
INTRODUCTION: Radioresistant brain tumor vasculature is thought to hamper the efficiency of adjuvant cancer therapies. However, little is known regarding the signalling pathways involved in the angiogenic response to brain tumor-derived growth factors in irradiated human brain microvascular endothelial cells (HBMEC). The goal of this study is to assess the effect of ionizing radiation (IR) on HBMEC survival, migration and tubulogenesis. METHODS: HBMEC were cultured and irradiated at sublethal single doses. Cell survival was assessed by nuclear cell counting and flow cytometry. HBMEC migration in response to brain tumor-derived growth factors (U-87 GF) and tubulogenesis were assayed using modified Boyden chambers and Matrigel, respectively. RESULTS: We observed that single administration of 3-10 Gy IR doses only reduced cell survival by 30%. Radioresistant HBMEC overexpressed RhoA, a small GTPase protein regulating cellular adhesion and migration, and Rho-kinase (ROK), a serine-threonine protein kinase and one of RhoA's major targets. HBMEC migration was induced by vascular endothelial growth factor (VEGF), but even more so in response to sphingo-sine-1-phosphate (S1P) and to U-87 GF. Following IR exposure, HBMEC basal migration increased more than two-fold, whereas the response to S1P and to U-87 GF was significantly diminished. Similarly, the inhibitor of ROK Y-27632 decreased HBMEC migration in response to S1P and U-87 GF. Overexpression of RhoA decreased tubulogenesis, an effect also observed in irradiated HBMEC. CONCLUSION: Our results suggest that radioresistant HBMEC migration response to tumor-secreted growth factors and tubulogenesis are altered following IR. The RhoA/ROK signalling pathway is involved in the IR-altered angiogenic functions and may represent a potential molecular target for enhancing the impact of radiotherapy on tumor-associated endothelial cells.