| Objective:To establish human osteosarcoma multidrug resistant cell line 143B/ADM, and analyze its biological characteristics.Methods:In our study, increasing concentrations and intermittent of adriamycin (ADM) were applied to establish 143B/ADM resistant strain. The half of inhibiting concentrations (IC50) and resistance indexs (RI) of different antitumor drugs were detected by WST-8 in human osteosarcoma wild-type or resistance cells. The stability of resistant strain was measured by cryopreservation/thawing and withdrawal test. Moreover, the cell adherence rate, doubling time, growth curve and double layer soft-agar colony formation rates were observed in 143B/WT and 143B/ADM cells. And the cell viability was also observed by trypan blue dye and crystal violet staining in the two cell lines. The quantitative analysis of Cell cycle, apoptosis and rhodamine efflux were detected by flow cytometry. The mRNA and protein expression of MDR-1, MRP-1 and LRP were detected by realtime PCR or western blotting respectively.Results:The RI (resistance index) of human osteosarcoma 143B/ADM resistance cells to ADM was 15.7, with different degrees of resistance to other antitumor drugs. Meanwhile, cryopreservation/thawing and withdrawal of drug didn't affect the RI of 143B/ADM. Compared with 143B/WT cell line, there were slow growth curve, prolonged cell doubling time, significant increase in cell cloning efficiency, less cells in G2/M phase and more cells in G1 and S phase in 143B/ADM cell line. Realtime PCR and western-blot analysis showed that MDR-1 expression was significantly increased in 143B/ADM cells without MRP-1 and LRP changed. Compared with 143B/WT cells, the Intracellular rho123 was markedly decreased in the 143B/ADM cells. Flow cytometry and confocal laser microscopy analysis showed that 72 h after treatment with ADM (10μg/ml), the number of cell apoptosis in 143B/ADM cells was less than that in 143B/WT cells.Conclusion:We successfully built human osteosarcoma resistance cell line 143B/ADM. The drug resistance may be related to increased MDR-1 expression, which induce increased efflux of antitumor drugs. Objective:To study the role of heme oxygenase-1 (HO-1) and protein kinase C (PKC) in multidrug resistance in 143B/ADM cells, and explore the relationship between HO-1, PKC and MDR-1, revealing the molecular mechanism of multidrug resistance in human osteosarcoma cells.Methods:1. The expression of HO-1, PKC and MDR-1 in the 143B/WT or 143B/ADM cells were determined by realtime PCR and/or western blotting respectively, with HO-1 activity measured by spectrophotometer.2. After 143B/WT cells were transfected with HO-1 plasmid, or 143B/ADM cells were transfected with HO-1 siRNA, the expression of HO-1, PKC and MDR-1 were detected by realtime PCR and/or western-blot. Meanwhile, HO-1 activity, Rho123 efflux, IC50 and RI, intracellular ADM and cell apoptosis were also measured by the methods also used in Part I.3. 72 h after 143B/ADM cells or 143B/WT cells transfected with HO-1 were treated with PKC inhibitor staurosporine, the expression of HO-1, PKC and MDR-1 were detected by realtime PCR and/or western-blot. Meanwhile, HO-1 activity, Rho123 efflux, IC50 and RI, intracellular ADM and cell apoptosis were also measured by the methods also used in Part I.Results:1. Compared with 143B/WT cells, the expression of HO-1 and PKC were increased significantly in 143B/ADM cells, which was consistent with increased MDR-1.2. Compared with 143B/WT cells transfected with empty vector, the expression of HO-1, PKC and MDR-1 were increased significantly in 143B/WT cells transfected with HO-1 plasmid. Furthmore, HO-1 activity, IC50 and RI against ADM, and Rho123 efflux were also increased significantly in 143B/WT+HO-1 cells, with decreased intracellular ADM and cell apoptosis. Compared with 143B/ADM cells, the expression of HO-1, PKC and MDR-1 were decreased significantly in 143B/ADM cells transfected with HO-1 siRNA. Meanwhile, HO-1 activity, IC50 and RI against ADM, and Rho123 efflux were also reduced significantly in 143B/WT+HO-1 siRNA cells, with increased intracellular ADM and cell apoptosis.3. 72 h after 143B/ADM cells or 143B/WT cells transfected with HO-1 were treated with PKC inhibitor staurosporine, the expression of PKC and MDR-1 were reduced significantly, without HO-1 expression and activity changed. Meanwhile, IC50 and RI against ADM, and Rho123 efflux were also decreased, with increased intracellular ADM and cell apoptosis. Conclusion:1. In human osteosarcoma 143B cells, HO-1 overexpression may induce drug resistance by modulating MDR-1.2. In human osteosarcoma 143B cells, PKC overexpression may induce drug resistance through activated MDR-1.3. In human osteosarcoma 143B cells, HO-1 can activate PKC which can upregulate MDR-1 to induce drug resistance. Objective:To investigate the effects of HO-1 on drug resistance in nude mice model of human osteosarcoma, and explore its mechanism.Methods:Nude mice model of human osteosarcoma was established by injecting human osteosarcoma 143B cells into nude mice subcutaneously. All the mice were divided into four groups: 143B/WT wild-type group, 143B/WT +HO-1 overexpression group, 143B/ADM group, 143B/ADM+HO-1 siRNA group. 10 days after transplantation, 4 mice of each group were treated with ADM. During this study, body weight and tumor size were observed. At the end of the study, tumor weight, pathological changes, and caspase-3 and -8 activity were measured. Meanwhile, HO-1, PKC and MDR-1 expression in tumor tissues were detected by realtime PCR, and/or western blot, with HO-1 activity measured by spectrophotometer.Results:1. Compared with 143B/WT group, tumor regression and pathological changes in 143B/WT+HO-1 group and 143B/ADM group were not significant with lower caspase-3 and -8 activities after receiving ADM treatment. Meanwhile, HO-1, PKC and MDR-1 expression and HO-1 activity were significantly higher than 143B/WT group.2. Compared with 143B/ADM group, tumor regression and pathological changes in 143B/ADM+HO-1 siRNA group were more significant with higher caspase-3 and -8 activities after receiving ADM treatment. Meanwhile, HO-1, PKC and MDR-1 expression and HO-1 activity were significantly decreased than 143B/WT group.Conclusion:HO-1 plays a great role in drug resistance in nude mice model of human osteosarcoma and may be a potential target to reverse drug resistance. |
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