The Effect And Mechanism Of Pien Tze Huang On Adriamycin Resistant Human Osteosarcoma Cells

ObjectiveUsing human osteosarcoma adriamycin resistant U20S/ADM cells and the corresponding animal model for the study, to evaluate the effects of Pien Tze Huang on U2OS/ADM2cells and the associated mechanisms, to provide experimental basis for Pien Tze Huang to reverse osteosarcoma drug-resistance in clinical.Methods1. Establishment of an adriamycin resistant human osteosarcoma U2OS/ADM cells and explore its drug resistance mechanism.Two resistant cell lines U20S/ADM1and U2OS/ADM2were established by impacting with high dose of adriamycin for short time and gradually increasing concentration of adriamycin from the parent cell line U2OS in vitro, observed their morphological features by optical microscope and transmission electron microscope, checked their resistance index (RI) and drug sensitivity by MTT assay, gathered their growth curve and calculated their doubling time. The accumulation of Rh123was evaluated by flow cytometry. Expressions of MDR1/P-gp、MRP1and Survivin were measured quantitatively by RT-PCR and Western blot.2. Study of reversal effects of Pien Tze Huang (PZH) on U2OS/ADM2cellsPZH alone or combined with adriamycin were used to treated U2OS/ADM2cells, the growth inhibitions were detected by MTT assays, the combination effect of PZH with ADM on U2OS/ADM2cells was calculated by index Q and reversal index; the apoptosis was detected by Hoechst33258staining assay and Annexin V-FITC/PI double staining analysis by flow cytometry.3. Study of the reversal mechanisms of PZH on U2OS/ADM2cellsPZH alone or combined with adriamycin were used to treated U2OS/ADM2cells, the accumulation of Rh123or ADM was evaluated by flow cytometry. Expressions of Bcl-2、 Bax、Survivin and MDRl/P-gp were studied by RT-PCR and Western blot.4. Study on the reversal effects of PZH on U2OS/ADM xenograft in nude miceThe nude mice modes were built by injecting U2OS/ADM2cells in nude mice subcutaneous. PZH or ADM or PZH combined with adriamycin were used to treated the nude mice. Tumors volume and tumors weight were measured during the drug therapy, the growth curve was gathered and tumor volume and weight inhibition rate were calculated. The apoptosis was detected by In situ end labeling (TUNEL) and the expressions of Bcl-2、Bax、 MDR1mRNA were measured by RT-PCR.Result1. The RI of U2OS/ADM1and U2OS/ADM2cells were79.63and91.06to adriamycin. They also demonstrated cross resistance to paclitaxel and cisplatin. Both resistant cell lines grew slowly, their doubling time were prolonged (P<0.01,vs U2OS cells) and exhibited changes in morphology. The accumulation of Rh123was lower in two resistant cell lines than in the parent cells (P<0.01, vs U2OS cells). The expressions of MDR1, MRP1and Survivin were enhanced in both resistant cell lines (P<0.05, vs U2OS cells). The expression of MRP1was higher in U2OS/ADM2cells than in U2OS/ADM1cells (P<0.05).2. The IC50value of PZH has no significant difference on the U2OS cells and U2OS/ADM2cells, indicating that drug resistant U2OS/ADM2cells have no cross-resistance to PZH. The proliferation of U2OS/ADM2cells was inhibited by PZH in a dose-time-dependent manner. The IC10of PZH to U2OS/ADM2cells is0.4mg/ml. The U2OS/ADM cells growth inhibition rate was significantly increased compared the combined effect of0.4mg/ml PZH and ADM with PZH and ADM separately. The IC50value of ADM in U2OS/ADM cells was (14.57±0.15)μg/ml, it decreased to (6.75±0.29)μg/ml after combined with PZH, the reverse index is2.16, the combined index Q was2.29>1.15. The percentage of U2OS/ADM2cells apoptosis were significantly increased by PZH in a dose-dependent manner. Cooperated the apoptosis rate of PZH and ADM with PZH and ADM separately, the combination of PZH and ADM significantly increased (P<0.05)3. PZH attenuated the expressions of Bcl-2, Survivin and MDR1/P-gp, activated the expression of Bax at mRNA and protein levels, enhanced the accumulation of Rh123in U2OS/ADM2cells in a does-dependent manner (P<0.05, vs control). The fluorescence intensity of ADM in U2OS/ADM2cells treated by0.4mg/ml PZH combined with ADM was increased1.27times compared by AMD. The mRNA and protein expressions of Bcl-2、 Survivin and MDRl/P-gp were downregulated and the mRNA and protein expression of Bax was upregulated in0.4mg/ml PZH combined with ADM group (P<0.05, vs. control) 4. The tumor models of human osteosarcoma U2OS/ADM2cells xenograft were successfully constructed. Compared the combined effect of PZH and ADM group with PZH and ADM group separately, volume and weight of U2OS/ADM2cells xenograft were significantly decreased (P<0.05), the apoptotic index was increased detected by TUNEL assay (P<0.01). RT-PCR results showed that the mRNA expression level of Bcl-2and MDR1were downregulated and the mRNA expression level of Bax was upregulated in the PZH and ADM group, compared to the PZH group and ADM group (P<0.05)Conclusion1. Using high dose of adriamycin for short time and gradually increasing concentration of adriamycin from the parent cell line U2OS in vitro can establishment the adriamycin resistant human osteosarcoma cell lines U2OS/ADM1and U2OS/ADM2, the drug-resistance mechanisms maybe relate to overexpressions of MDR1/P-gp, MRP1and Survivin. The resistant ability of U2OS/ADM2cells was higher than U2OS/ADM1cells.2. PZH can inhibit the growth of U2OS/ADM2cells and promote apoptosis in time and dose-dependent manner, and PZH has a synergistic effect with ADM, increase the sensitivity of U2OS/ADM2cells to ADM. The mechanism is related to inhibit the expression of P-gp, change the proportion of Bcl-2and Bax, and down regulate the expression of Survivin.3. PZH can enhance the anti-tumor effect of ADM on U2OS/ADM2cells xenograft, the mechanism is related to downregulate the expressions of Bcl-2and MDR1gene and upregulate the expression of Bax gene.