RESUMEN
Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.
Asunto(s)
Antibacterianos/metabolismo , Química Farmacéutica , Túbulos Renales/metabolismo , Microscopía Fluorescente/métodos , Polimixinas/metabolismo , Análisis de la Célula Individual/métodos , Sincrotrones , Animales , Antibacterianos/análisis , Células Cultivadas , Colorantes Fluorescentes , Humanos , Radioisótopos de Yodo , Túbulos Renales/citología , Modelos Moleculares , Estrés Oxidativo , Polimixinas/análisis , Ratas , Rayos XRESUMEN
Drug resistance is a major obstacle for the successful treatment of many malignancies, including neuroblastoma, the most common extracranial solid tumor in childhood. Therefore, current attempts to improve the survival of neuroblastoma patients, as well as those with other cancers, largely depend on strategies to counter cancer cell drug resistance; hence, it is critical to understand the molecular mechanisms that mediate resistance to chemotherapeutics. The levels of LIM-kinase 2 (LIMK2) are increased in neuroblastoma cells selected for their resistance to microtubule-targeted drugs, suggesting that LIMK2 might be a possible target to overcome drug resistance. Here, we report that depletion of LIMK2 sensitizes SHEP neuroblastoma cells to several microtubule-targeted drugs, and that this increased sensitivity correlates with enhanced cell cycle arrest and apoptosis. Furthermore, we show that LIMK2 modulates microtubule acetylation and the levels of tubulin Polymerization Promoting Protein 1 (TPPP1), suggesting that LIMK2 may participate in the mitotic block induced by microtubule-targeted drugs through regulation of the microtubule network. Moreover, LIMK2-depleted cells also show an increased sensitivity to certain DNA-damage agents, suggesting that LIMK2 might act as a general pro-survival factor. Our results highlight the exciting possibility of combining specific LIMK2 inhibitors with anticancer drugs in the treatment of multi-drug resistant cancers.
Asunto(s)
Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Quinasas Lim/fisiología , Neuroblastoma/patología , Acetilación , Línea Celular Tumoral , Daño del ADN , Humanos , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The Rho-associated coiled-coil kinase (ROCK) family of proteins, including ROCK1 and ROCK2, are key regulators of actin and intermediate filament morphology. The newly discovered ROCK substrate Tubulin polymerization promoting protein 1 (TPPP1) promotes microtubule polymerization and inhibits the activity of Histone deacetylase 6 (HDAC6). The effect of TPPP1 on HDAC6 activity is inhibited by ROCK signaling. Moreover, it was recently demonstrated that ROCK activity increases the cellular expression of the oncogene ß-catenin, which is a HDAC6 substrate. In this study, we investigated the interplay between ROCK-TPPP1-HDAC6 signaling and ß-catenin expression. We demonstrate that ß-catenin expression is increased with ROCK signaling activation and is reduced with increased TPPP1 expression in U2OS cells. Further investigation revealed that ROCK-mediated TPPP1 phosphorylation, which prevents its binding to HDAC6, negates TPPP1-mediated reduction in ß-catenin expression. We also show that increased HDAC6 activity resulting from ROCK signaling activation reduced ß-catenin acetylation at Lys-49, which was also accompanied by its decreased phosphorylation by Caesin kinase 1 (CK1) and Glycogen synthase kinase 3ß (GSK3ß), thus preventing its proteasomal degradation. Overall, our results suggest that ROCK regulates ß-catenin stability in cells via preventing TPPP1-mediated inhibition of HDAC6 activity, to reduce its acetylation and degradation via phosphorylation by CK1 and GSK3ß.
Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Osteosarcoma/metabolismo , beta Catenina/metabolismo , Acetilación , Línea Celular Tumoral , Histona Desacetilasa 6 , Humanos , Osteosarcoma/enzimología , Osteosarcoma/patología , Fosforilación , Transducción de Señal , Quinasas Asociadas a rho/metabolismoRESUMEN
The small Rho GTPase family of proteins, encompassing the three major G-protein classes Rho, Rac and cell division control protein 42, are key mitogenic signaling molecules that regulate multiple cancer-associated cellular phenotypes including cell proliferation and motility. These proteins are known for their role in the regulation of actin cytoskeletal dynamics, which is achieved through modulating the activity of their downstream effector molecules. The Rho-associated coiled-coil kinase 1 and 2 (ROCK1 and ROCK2) proteins were the first discovered Rho effectors that were primarily established as players in RhoA-mediated stress fiber formation and focal adhesion assembly. It has since been discovered that the ROCK kinases actively phosphorylate a large cohort of actin-binding proteins and intermediate filament proteins to modulate their functions. It is well established that global cellular morphology, as modulated by the three cytoskeletal networks: actin filaments, intermediate filaments and microtubules, is regulated by a variety of accessory proteins whose activities are dependent on their phosphorylation by the Rho-kinases. As a consequence, they regulate many key cellular functions associated with malignancy, including cell proliferation, motility and viability. In this current review, we focus on the role of the ROCK-signaling pathways in disease including cancer.
Asunto(s)
Quinasas Asociadas a rho/metabolismo , Animales , Citoesqueleto/metabolismo , Humanos , Neoplasias/enzimología , Fosforilación/genética , Fosforilación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Quinasas Asociadas a rho/genéticaRESUMEN
Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Quinasas Asociadas a rho/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Fase G1 , Regulación de la Expresión Génica , Humanos , Microscopía Fluorescente/métodos , Mitosis , Modelos Biológicos , Fenantrenos , Fosforilación , Propidio/farmacología , Unión Proteica , Fase SRESUMEN
The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.