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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 VCR-resistant MG63/VCR cell sublines (MG63/VCR1, 2, 3 and 4). The drug resistance indices (RI) of these sublines was detected with the CCK-8 assay and determined to be163, 476, 1,247, and 2,707-fold higher than that of parental cells, respectively. These sublines also exhibited cross-resistance 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 resistance-associated 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, MDR-associated protein 1, topoisomerase II and LIM domain kinase 1 levels were significantly reduced compared with the moderately resistant MG63/VCR2 cells. Expression of glutathione S-transferase-⨅ mRNA was determined using reverse transcription-quantitative 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 resistance-associated genes or strategies to overcome MDR in human osteosarcoma. [ABSTRACT FROM AUTHOR]

Osteosarcoma is a type of malignant bone tumor with high metastasis and poor prognosis. Previous studies have demonstrated the involvement of LIM kinase 1 (LIMK1) in the proliferation of osteosarcoma cells. LIMK1 is over expressed in human osteosarcoma tissues and cell lines. To further study LIMK1-associated mechanisms, we used shRNA targeted to the LIMK1 gene to block its expression in the osteosarcoma cell lines MG63 and U2OS. Insulin promoted the proliferation of MG63 cells in a time- and dose-dependent manner, however, this insulin induced proliferation was significantly inhibited by transfection of shRNA targeted to the LIMK1 gene, as well as by the PI3K inhibitor LY294002, but not by the mitogen-activated protein kinase (MAPK) inhibitor PD98059. The level of cofilin phosphorylation was increased significantly following stimulation of insulin for 24 h, indicating the activation of LIMK1. MG63 cell proliferation was also significantly inhibited by 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) in a time-dependent manner. Furthermore, 1,25(OH)2D3 negated the inhibitory effect of LIMK1 shRNA, indicating that LIMK1 is important in the inhibitory pathway of 1,25(OH)2D3. The present study confirms that LIMK1 is important in regulating osteosarcoma cell proliferation via the insulin/PI3K/LIMK1 signaling pathway, thus the development of gene therapy for osteosarcoma targeting LIMK1 is warranted. [ABSTRACT FROM AUTHOR]