Assuntos
Plaquetas/fisiologia , Proteínas Ativadoras de GTPase/genética , Megacariócitos/citologia , Regulação para Cima , Animais , Plaquetas/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Proteínas Ativadoras de GTPase/metabolismo , Inativação Gênica , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Selectina-P/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Cultura Primária de Células , Trombina/farmacologiaRESUMO
Multiple myeloma (MM) is characterised by intense protein folding and, consequently endoplasmic reticulum (ER) stress. The prostaglandin 15d-PGJ2 is able to raise oxidative stress levels within the cell and potentially trigger cell death. The aim of this study was to evaluate the antineoplastic effect of 15d-PGJ2 on MM in vitro and in vivo via ER and oxidative stress pathways. MM.1R and MM.1S cell lines were treated with 15d-PGJ2 at 1-10µM and evaluated with regard to proliferation, mRNA expression of PRDX1, PRDX4, GRP78, GRP94, CHOP, BCL-2 and BAX. Stress data was validated via oxidized glutathione assays. MM.1R cells were inoculated into NOD/SCID mice, which were subsequently treated daily with 15d-PGJ2 at 4mg/kg or vehicle (control), with tumour volume being monitored for 14days. 15d-PGJ2 reduced cell proliferation, induced cell death and apoptosis at 5µM and 10µM and Stress-related genes were upregulated at the same doses. Oxidized glutathione levels were also increased. 15d-PGJ2 at 4mg/kg in vivo halted tumour growth. In conclusion, 15d-PGJ2 induced myeloma cell death via ER stress in vitro. 15d-PGJ2 in vivo also inhibited tumour growth.
Assuntos
Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Prostaglandina D2/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Prostaglandina D2/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Vasodilator-stimulated phosphoprotein (VASP) and Zyxin are interacting proteins involved in cellular adhesion and motility. PKA phosphorylates VASP at serine 157, regulating VASP cellular functions. VASP interacts with ABL and is a substrate of the BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. However, the function of VASP and Zyxin in BCR-ABL pathway and the role of VASP in CML cells remain unknown. In vitro experiments using K562 cells showed the involvement of VASP in BCR-ABL signaling. VASP and Zyxin inhibition decreased the expression of anti-apoptotic proteins, BCL2 and BCL-XL. Imatinib induced an increase in phosphorylation at Ser157 of VASP and decreased VASP and BCR-ABL interaction. VASP did not interact with Zyxin in K562 cells; however, after Imatinib treatment, this interaction was restored. Corroborating our data, we demonstrated the absence of phosphorylation at Ser157 in VASP in the bone marrow of CML patients, in contrast to healthy donors. Phosphorylation of VASP on Ser157 was restored in Imatinib responsive patients though not in the resistant patients. Therefore, we herein identified a possible role of VASP in CML pathogenesis, through the regulation of BCR-ABL effector proteins or the absence of phosphorylation at Ser157 in VASP.
Assuntos
Benzamidas/farmacologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Zixina/metabolismo , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Células Clonais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.