The Mechanism By Which Pien Tze Huang Reverses Drug Resistance Of ABC Transportprotein Of Human Osteosarcoma Stem Cell

PurposeThis paper choose human osteosarcoma resistant cell line.MG63/ADM, using a method in serum-free suspension cultures, isolated from among the cell spheres having stem cell properties, the use of a series of related experiments detection methods to identify characteristics of tumor stem cells, and finally Pien Tze Huang(PZH) vitro experimental observation of osteosarcoma MG63/ADM cell spheres proliferative activity of, and on the ABC membrane transporter ABCB1, ABCG2 expression regulation, and thus more in-depth analysis of the relationship between PZH osteosarcoma multidrug resistance (MDR) reversal and the stem cells in osteosarcbma,to make this basic experiment can more effectively serve the clinical treatment of osteosarcoma.Method1, the establishment of human osteosarcoma MG63 cells Adriamycin resistant strains and serum-free culture cell sphere stem cell characterization.The use of high dose (2μg/ml) of adriamycin(ADM) for intermittent short-time effects from the human osteosarcoma MG63 cells, establish the doxorubicin resistant human osteosarcoma cell lines MG63/ADM, the use of experiment of MTT assay and its resistance index to ADM, as well as drug sensitivity to cisplatin,and to paclitaxel, etc.; serum-free suspension culture MG63/ADM cells to obtain third-generation cell spheres, CCK.8 detection ADM to the third-generation cell spheres proliferation; cell clone in vitro experiments, the ability to form a single cell into a cell sphere experiment, testing MG63 cells, MG63/ADM cell,the third-generation cell spheres proliferation forming ability; confocal laser scanning and Western blot stem cell markers Oct-4, Nanog, SOX2 in MG63, MG63/ ADM, the third-generation cell spheres expression.2, ABCB1, ABCG2 in the third-generation of cell spheres of expression and the effect of the PZH in the third-generation of cell spheres.Laser confocal ABCB1, ABCG2 in MG63, MG63/ADM, the third-generation cell spheres expression. CCK8 detection PZH to the third-generation cell spheres proliferation and were observed PZH to influence the third-generation of cell spheres; RT-PCR and Western blot detect PZH to the third-generation cell spheres ABCB1, ABCG2 Expression.Result1, induced by high-dose doxorubicin, resistant strains of human osteosarcoma MG63/ ADM has been successfully established.MTT assay MG63/ADM cells to ADM resistance index (RI) was 11.47, significantly higher than the MG63 cells (P<0.01), while. MG63/ ADM cells never touched cisplatin and paclitaxel are also cross-resistance, resistance index were 2.27,5.32, significantly higher than the osteosarcoma MG63 cells (P<0.01).2,serum-free suspension culture in the third-generation cell spheres compared with the first generation of cell morphology rules, uniform size, no dead cells and single cells are present; the third generation cell spheres colony formation most obvious, followed by drug-resistant cells, The parent cell only a few cells to form colonies; MG63 parent single cell into a cell sphere was 18.80%±2.51, MG63/ADM single cell into a cell sphere was 36.15% ± 4.98, the third-generation cell spheres single cell into a cell sphere was 83.55% ± 10.39, described the third-generation cell spheres with a strong single cell into a cell sphere ability. ADM with the increasing concentration of the drug can inhibit the proliferation of the third-generation cell spheres, but required higher concentrations of ADM, the third-generation cell spheres IC50 is (3.35 ± 0.36) μg/ml, compared to the MG63/ADM cells to ADM IC50 (1.98 ± 0.58) μg/ml, statistically there are obvious differences (p<0.05), describes the third generation cell spheres obvious resistance to ADM. Confocal and Western blot analysis show that the third-generation cell spheres in SOX2, Oct-4, Nanog expression was high, indicating that obtain by the third-generation of cell spheres suspension culture have stem cell properties.3, MG63 cells basically no expression of ABCB1, ABCG2; MG63/ADM cells partially express ABCB1, ABCG2, and the third-generation cell spheres abundantly express ABCB1, ABCG2, describ the third-generation cell spheres and ABC membrane resistance high expression of transporter proteins. The growth of osteosarcoma stem cells drug-resistant can be inhibited by PZH, with the increase of the concentration of intervention, the inhibition rate increase; RT-PCR and Western blot results show that with increasing concentration of PZH, the ABCG2, ABCB1 gene expression in the third-generation cell spheres was significantly decreased, indicating that PZH can inhibit the expression of ABC transporter protein ABCB1, ABCG2, reversal of osteosarcoma MDR.Conclusion1, through the use of high dose of ADM for short time successfully establish the osteosarcoma doxorubicin resistant strains MG63/ADM;serum-free suspension culture MG63/ADM resistant osteosarcoma cells can be enriched of stem cells.2, resistant osteosarcoma stem cells with high expression ABCB1, ABCG2 proteins, the proliferation of drug-resistant osteosarcoma stem cells could be inhibited by PZH, PZH also can inhibit the expression of ABCB1, ABCG2 proteins, reverse the drug resistance.