RESUMO
Multidrug resistance (MDR) is a challenge for the treatment of cancer and the underlying molecular mechanisms remain elusive. The current study exposed MG63 osteosarcoma cells to increasing concentrations of vincristine (VCR) to establish four VCRresistant MG63/VCR cell sublines (MG63/VCR1, 2, 3 and 4). The drug resistance indices (RI) of these sublines was detected with the CCK8 assay and determined to be163, 476, 1,247, and 2,707fold higher than that of parental cells, respectively. These sublines also exhibited crossresistance to doxorubicin, paclitaxel and pirarubicin. With increased RI, the proliferative capacity of these sublines was gradually reduced and cell morphology was also altered, characterized by increased formation of pseudopodia and long cytoplasmic processes at opposite poles. However, the migration capacity and expression of certain drug resistanceassociated genes were not in accordance with the increased RI; multidrug resistance protein 1 (MDR1) expression was significantly increased in these sublines compared with parental cells. However, in the highly resistant MG63/VCR3 and MG63/VCR4 cells, MDRassociated protein 1, topoisomerase II and LIM domain kinase 1 levels were significantly reduced compared with the moderately resistant MG63/VCR2 cells. Expression of glutathione Stransferaseπ mRNA was determined using reverse transcriptionquantitative polymerase chain reaction and determined that it was not changed between MG63 and MG63/VCR cells. The data of the present study demonstrated that the molecular alterations of drug resistance may change with the degree of drug resistance. Taking cell morphology into consideration, the intratumor clonal and phenotypic heterogeneity may be responsible for drug resistance. These MG63/VCR sublines may be a valuable tool to assess drug resistance and the underlying mechanisms, and to identify novel drug resistanceassociated genes or strategies to overcome MDR in human osteosarcoma.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Vincristina/farmacologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Osteossarcoma/genética , Osteossarcoma/patologiaRESUMO
Cofilin promotes actin filament turnover by severing and depolymerizing actin filaments. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase1 (LIMK1) and is activated when protein phosphatase Slingshot-1L (SSH1L) dephosphorylates this residue. The authors have shown that Ca-induced cofilin dephosphorylation is mediated by calcineurin (Cn)-dependent activation of SSH1L. In this study, Ca/calmodulin-dependent protein kinase II (CaMKII) is shown to negatively regulate SSH1L activity and bind to SSH1L in a complex with 14-3-3. Phosphorylation of LIMK1 by CaMKII and its subsequent activation regulates the subcellular localization of SSH1L. Based on these findings, the authors suggest that CaMKII and Cn provide a switch-like mechanism that controls Ca-dependent LIMK1, SSH1L and cofilin activation, and subsequently actin cytoskeletal reorganization.
Assuntos
Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cofilina 1/metabolismo , Proteínas 14-3-3/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Calcimicina/farmacologia , Cálcio/metabolismo , Ionóforos de Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Quinases Lim/antagonistas & inibidores , Quinases Lim/genética , Quinases Lim/metabolismo , Células MCF-7 , Modelos Biológicos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Transdução de SinaisRESUMO
To investigate the effect of heroin on purine nucleotides catabolism, a rat model of heroin administration and withdrawal was established. Concentrations of uric acid, creatinine, and urea nitrogen in plasma and ADA in plasma, brain, liver, and small intestine were tested. When two heroin administration groups were compared with the control group, the concentrations of plasma uric acid and ADA in plasma, brain, liver, and small intestine increased, whereas the plasma urea nitrogen concentrations in two heroin administration groups and the plasma creatinine concentration in the 3-day heroin administration group did not increase. It seemed that heroin exposure for a short time did not affect renal clearance rate notably. When two withdrawal groups were compared with two heroin administration groups, the concentrations of plasma uric acid and ADA in liver and small intestine decreased, but there was no significant reduction in ADA concentrations of the brain, while the plasma ADA concentrations in the two withdrawal groups were significantly higher than those of two heroin administration groups. When the two withdrawal groups were compared with the control group, there was no significant difference in the concentrations of plasma uric acid and ADA in liver and small intestine, while the concentrations of ADA in plasma and brain were still higher than those of the control group. The results imply that heroin administration may enhance the catabolism of purine nucleotides in the brain and other tissues by increased concentration of ADA and the effect may last for a long time in the brain.
