RESUMO
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.
Assuntos
Movimento Celular/efeitos da radiação , Radioterapia com Íons Pesados/métodos , Invasividade Neoplásica , Neoplasias Pancreáticas/radioterapia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/efeitos da radiação , Proteínas rac1 de Ligação ao GTP/efeitos da radiação , Proteína rhoA de Ligação ao GTP/efeitos da radiação , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Neoplasias PancreáticasRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Multimerização Proteica , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Western Blotting , Técnicas de Cultura de Células , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Criptocromos/genética , Criptocromos/efeitos da radiação , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Ensaio de Imunoadsorção Enzimática , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Luz , Transdução de Sinal Luminoso , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/efeitos da radiação , Camundongos , Células NIH 3T3 , Multimerização Proteica/efeitos da radiação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Transcrição Gênica , Transfecção , Via de Sinalização Wnt/efeitos da radiação , Proteína Wnt3A/genética , Proteína Wnt3A/efeitos da radiação , beta Catenina/genética , beta Catenina/efeitos da radiação , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/efeitos da radiação , Proteína Vermelha FluorescenteRESUMO
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.