Identification of differentially expressed genes in MG63 osteosarcoma cells with drug­resistance by microarray analysis.

Osteosarcoma is the most common type of primary malignant bone tumor, with extremely poor prognosis in patients with metastatic disease and resistance to therapy, such as multidrug regimens. The mechanisms of drug resistance are quite complex and have not been fully elucidated; thus, novel therapeutic targets should be identified to alleviate drug resistance in osteosarcoma. In the present study, the transcriptomes of the human osteosarcoma cell line MG63 and vincristine (VCR)­resistant MG63 cells were compared by microarray analysis. A total of 1,300 genes (602 upregulated and 698 downregulated) were reported to be differentially expressed in MG63/VCR compared with MG63 cells. Bioinformatics analysis predicted that the differentially expressed genes were mainly enriched in the B cell receptor, UVA­induced mitogen­activated protein kinases and receptor tyrosine kinase 2/3 signaling pathways. In the present study, 10 of the dysregulated genes, including roundabout homolog 1, death­associated protein kinase 1 and A­kinase anchor protein 12 were further evaluated by reverse transcription­quantitative polymerase chain reaction. These results may aid the validation of candidate biomarkers for the treatment and prognosis of osteosarcoma, and provide novel insight into the molecular mechanisms underlying the drug resistance of osteosarcoma cells.

LIM kinase 1 serves an important role in the multidrug resistance of osteosarcoma cells.

Multidrug resistance (MDR) is a major challenge for the management of the majority of cancers. The precise molecular mechanisms of MDR remain elusive. In a previous study, a multidrug resistant osteosarcoma model [MG63/vincristine (VCR)] was established by intermittent exposure of MG63 cells to gradually increasing concentrations of VCR. These cells exhibited cross-resistance to multiple structurally and mechanistically unrelated chemotherapeutic agents. The development of MDR was associated with increased expression of LIM kinase 1 (LIMK1). Compared with that in normal human fetal osteoblasts (hFOB) 1.19, the messenger RNA and protein expression of LIMK1 was significantly elevated both in MG63 and U2OS osteosarcoma cells. To observe the expression pattern of LIMK1 in osteosarcoma, immunohistochemical analyses were performed on specimens derived from 6 patients. The results indicated that LIMK1 was expressed to a greater extent in the tumor parenchyma than in the mesenchyme. The role of LIMK1 in MDR was confirmed by transfecting plasmids coding LIMK1-small interfering RNA (siRNA), wild-type-LIMK1 or empty vector into MG63/VCR cells, and measuring the expression of LIMK1 and multidrug resistance protein 1 (MDR1), also known as P-gycoprotein (P-gp). The results demonstrated that the level of MDR1/P-gp was positively correlated with the level of LIMK1. This correlation was also shown with the doxorubicin efflux assay and by measuring apoptosis. Specifically, after 6 h of incubation with VCR, 25.6% of the cells transfected with the LIMK1-siRNA plasmid were apoptotic compared with 6.2% in the empty vector group and 1.3% in the group of cells transfected with the wild-type-LIMK1 plasmid. Thus, it was concluded that LIMK1 serves a key role in the MDR of osteosarcoma and functions through MDR1.

Characterization of multidrug­resistant osteosarcoma sublines and the molecular mechanisms of resistance.

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.