Characterization Of Multidrug Resistant Osteosarcoma Sublines And Molecular Events

Osteosarcoma(OS) is the first most common malignant bone tumor, and always shown resistant to many chemo-drugs. But the definite mechanism of multidrug resistance(MDR) is not clear, especially the study in OS is rare. So it is urgent to establish a multidrug resistant osteosarcoma cell line by simulation of clinical therapy, and investigate their biological characteristics and the mechanism of multidrug resistance.We established four different multidrug resistant MG63/VCR sublines: MG63/VCR1-4, by exposing the human osteosarcoma cell line MG63 to gradient increased vincristine. The drug resistant index was detected by CCK-8 assay, the derivative-resistant sublines were 163, 476, 1247, 2707-fold resistant to vincristine in comparison with the parental MG63 cells respectively, and also exhibited cross-resistance to DOX, THP and PTX.Some biological characteristics of the cell lines showed a gradual change, with the increase of drug resistance index. First, we detect the cellular morphological difference by invert microscope, resistant sublines showed spindle-shaped morphology, the long cytoplasmic processes at the opposite poles of the cell were markedly increased. Then the proliferation ability is measured, they are negative correlate with the resistance index, this is reflected on the CCK-8 proliferation assay, the volume of the colons on the plate and the cell cycle analysis.But the migration ability and the standard of some drug resistant related genes are not in line with the increase in resistance index. The RT-PCR and real-time quantitative PCR was performed to determine the m RNA expression levels of drug resistance-related genes, MDR1, MRP1, Topo-II, LIMK1, GST-π, in the five sublines. The MDR1 and MRP1 expression was significantly increased in the derivative-resistant sublines when compared with that in MG63 cells. But in the highly resistant MG63/VCR3, MG63/VCR4 cells, the level of MRP1 was significantly reduced than the moderate resistant MG63/VCR2. This trend is also shared by TOPO-II and LIM kinase 1. The latter was thought to be related to cell motility and cyto-dynamic. And there were no significant difference in GST-π m RNA expression between the MG63 and MG63/VCR cells.The results demonstrate that, in the different degree of resistant cells, the mechanism may be changed. Take the morphology into consideration, we hold that the intra-tumor clonal and phenotypic heterogeneity is the fundamental reason. In conclusion, MG63/VCR cells are invaluable tools with which to assess the biochemical and genetic mechanisms of drug resistance in human osteosarcoma, identify novel drug resistance-related genes and the methods to overcome MDR in tumors.