RESUMO
Elderly cancer patients treated with ionizing radiation (IR) or chemotherapy experience more frequent and greater normal tissue toxicity relative to younger patients. The current study demonstrates that exponentially growing fibroblasts from elderly (old) male donor subjects (70, 72, and 78 years) are significantly more sensitive to clonogenic killing mediated by platinum-based chemotherapy and IR (â¼70%-80% killing) relative to young fibroblasts (5 months and 1 year; â¼10%-20% killing) and adult fibroblasts (20 years old; â¼10%-30% killing). Old fibroblasts also displayed significantly increased (2-4-fold) steady-state levels of O2â¢-, O2 consumption, and mitochondrial membrane potential as well as significantly decreased (40%-50%) electron transport chain (ETC) complex I, II, IV, V, and aconitase (70%) activities, decreased ATP levels, and significantly altered mitochondrial structure. Following adenoviral-mediated overexpression of SOD2 activity (5-7-fold), mitochondrial ETC activity and aconitase activity were restored, demonstrating a role for mitochondrial O2â¢- in these effects. Old fibroblasts also demonstrated elevated levels of endogenous DNA damage that were increased following treatment with IR and chemotherapy. Most importantly, treatment with the small-molecule, superoxide dismutase mimetic (GC4419; 0.25 µmol/L) significantly mitigated the increased sensitivity of old fibroblasts to IR and chemotherapy and partially restored mitochondrial function without affecting IR or chemotherapy-induced cancer cell killing. These results support the hypothesis that age-associated increased O2â¢- and resulting DNA damage mediate the increased susceptibility of old fibroblasts to IR and chemotherapy that can be mitigated by GC4419. Cancer Res; 77(18); 5054-67. ©2017 AACR.
Assuntos
Cisplatino/efeitos adversos , Fibroblastos/patologia , Mitocôndrias/patologia , Radiação Ionizante , Pele/patologia , Superóxidos/metabolismo , Adulto , Fatores Etários , Idoso , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Estresse Oxidativo , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Superóxido Dismutase/metabolismo , Adulto JovemRESUMO
Fission and fusion events dynamically control the shape and function of mitochondria. The activity of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is finely tuned by several post-translational modifications. Phosphorylation of Ser-656 by cAMP-dependent protein kinase (PKA) inhibits Drp1, whereas dephosphorylation by a mitochondrial protein phosphatase 2A isoform and the calcium-calmodulin-dependent phosphatase calcineurin (CaN) activates Drp1. Here, we identify a conserved CaN docking site on Drp1, an LXVP motif, which mediates the interaction between the phosphatase and mechanoenzyme. We mutated the LXVP motif in Drp1 to either increase or decrease similarity to the prototypical LXVP motif in the transcription factor NFAT, and assessed stability of the mutant Drp1-CaN complexes by affinity precipitation and isothermal titration calorimetry. Furthermore, we quantified effects of LXVP mutations on Drp1 dephosphorylation kinetics in vitro and in intact cells. With tools for bidirectional control of the CaN-Drp1 signaling axis in hand, we demonstrate that the Drp1 LXVP motif shapes mitochondria in neuronal and non-neuronal cells, and that CaN-mediated Drp1 dephosphorylation promotes neuronal death following oxygen-glucose deprivation. These results point to the CaN-Drp1 complex as a potential target for neuroprotective therapy of ischemic stroke.