Effect of PI3K-mediated autophagy in human osteosarcoma MG63 cells on sensitivity to chemotherapy with cisplatin

Objective: To investigate the influence of autophagy on sensitivity to the chemotherapy drug cisplatin (DDP) and the role of PI3K in autophagy. Methods: MTT methods and flow cytometer, with rapamycin up-regulating the autophagy and 3-MA down-regulating the autophagy, were employed to measure the proliferation inhibition rate on DDP-treated osteosarcoma cells and the change in cell cycle. The expression of intracellular protein was detected by Western blot. The autophagy of MG63 cell was observed using fluorescence microscope and transmission electron microscope. Results: Western blot showed that basic autophagy level of MG63 cell was significantly lower than that of hFOB cell. MTT test revealed that the cell proliferation inhibition rate in the group treated with rapamycin and DDP, group treated with 3-MA and DDP, and group only treated with DDP was significantly different. It was demonstrated by the flow cytometry that in group treated with DDP, inhibition on autophagy can increase the cell numbers in G

Effect of PI3K-mediated autophagy in human osteosarcoma MG63 cells on sensitivity to chemotherapy with cisplatin

OBJECTIVE@#To investigate the influence of autophagy on sensitivity to the chemotherapy drug cisplatin (DDP) and the role of PI3K in autophagy.@*METHODS@#MTT methods and flow cytometer, with rapamycin up-regulating the autophagy and 3-MA down-regulating the autophagy, were employed to measure the proliferation inhibition rate on DDP-treated osteosarcoma cells and the change in cell cycle. The expression of intracellular protein was detected by Western blot. The autophagy of MG63 cell was observed using fluorescence microscope and transmission electron microscope.@*RESULTS@#Western blot showed that basic autophagy level of MG63 cell was significantly lower than that of hFOB cell. MTT test revealed that the cell proliferation inhibition rate in the group treated with rapamycin and DDP, group treated with 3-MA and DDP, and group only treated with DDP was significantly different. It was demonstrated by the flow cytometry that in group treated with DDP, inhibition on autophagy can increase the cell numbers in G1 phase and reduce the cell numbers in S phase of cell cycle. Increase of autophagosome in MG63 cytoplasm was observed under fluorescence microscope.@*CONCLUSIONS@#Up-regulating the autophagy significantly reduced the sensitivity of MG63 cell to chemotherapy with DDP. DDP induced autophagy of MG63 cell and blocked the cell cycle at G1 phase.

Cytotoxicity and apoptosis of human osteosarcoma U2OS cells induced by recombinant soluble AZURIN / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of recombinant AZURIN protein of P. aeruginosa on growth and apoptosis of U2OS cells.</p><p><b>METHODS</b>The AZURIN gene was amplified from the genome of P.aeruginosa by PCR, and cloned into prokaryotic expression vector pQE30. The soluble AZURIN protein was expressed in E. coli cells M15, then purified and refolded. After treatment of AZURIN, the cell cycle, proliferation and apoptosis were determined by morphological observation, MTT assay, flow cytometry(FCM) and DNA fragmentation assay. Mitochondrial membrane potential(DeltaPsim) was measured by FCM.</p><p><b>RESULTS</b>The purity of recombinant protein AZURIN reached to 99.1%. Proliferation of U2OS cells were significantly inhibited 12 h after AZURIN (100-200 mg/L) treatment. Apoptosis peak and DNA ladder were observed. Mitochondrial membrane potential decreased gradually from 12 h to 72 h after AZURIN treatment.</p><p><b>CONCLUSION</b>The recombinant AZURIN inhibit the growth of the human osteosarcoma U2OS cells and inducs apoptosis in vitroìwhich may be associated with the decrease of mitochondrial membrane potential.</p>

Synergistic induction of apoptosis by the combination of TRAIL and low dose adriamycin in human osteosarcoma cell line U2OS / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate whether TRAIL can synergize with adriamycin (ADM) to kill osteosarcoma cells (U2OS) in vitro, and its possible molecular mechanism.</p><p><b>METHODS</b>MTT was used to evaluate the cytotoxic effect of TRAIL and ADM either used alone or in combination at 24 hours after treatment to U20S cells. Cell apoptosis and its proportion were detected by flow cytometry assay. Acridine orange fluorescence microscopy and transmission electron microscopy were used to examine cellular and ultrastructural changes of apoptosis. The changes of cFLIP in mRNA and protein level were semi-quantified by RT-PCR and Western blot analysis.</p><p><b>RESULTS</b>(1) U2OS cells were not sensitive to TRAIL (IC(50) > 1 mg/L); the cells were relatively more responsive to ADM in an apparent dose-effect fashion. (2) The combination of TRAIL and ADM presented a synergistic effect on U2OS cells. Subtoxic concentration of TRAIL (0.1 mg/L) combined with subtoxic concentration of ADM (1.0 micromol/l) killed (49.54 +/- 2.79)% of U2OS cells. (3) The cytotoxicity was mainly attributed to cell apoptosis as demonstrated by flow cytometry assay, fluorescence microscopy and electron microscopy.</p><p><b>CONCLUSION</b>Subtoxic dose of TRAIL can effectively kill osteosarcoma cells (U2OS) in combination with subtoxic dose of ADM, but not effective when used alone. Apoptosis is the main mechanism of this killing effect induced by combination of TRAIL and ADM. Down regulation of cFLIP at mRNA and protein level is involved in this apoptosis pathway.</p>

Effect of graded composite zirconia-hydroxyapatite on viability of rat osteoblast cells cultured in vitro / 中华创伤杂志

Objective To evaluate the effect of a novel orthopedic biomaterial,graded composite zireonia(ZrO2)hydroxyapatite(HAP)on activity of rat osteoblast ceils(OB)cultured in vitro. Methods The pure zirconia material was used as control to measure surface roughness of the composite material that was examined by using scanning electron microscope(SEM)and X-ray diffraetometer (XRD).The rat osteoblast cells were cultured on the two materials.Alkali phosphatase(ALP)of the two groups was measured and ELISA was used to detect IL-6 and TGF-?eoncentration of the supematant of OB cells.Tumor growth factor-?(TGF-?)mRNA was detected by RT-PCR.SEM was used to observe OB cells on the two materials.The extract of the composite material was used for a eytotoxicity test to cal- culate the relative proliferation rate(RGR)and classify the toxicity.Results The surface roughness of the gradual composite materials was significantly higher than that of the control materials(P＜0.01). The ALP of the gradual material group was markedly higher than that of the control group at different in- tervals.There was significant difference of the IL-6 and TGF-?concentrations 2-4 days after culture be- tween two groups(P＜0.05,P＜0.01).The mRNA level of TGF-?of the two OB groups also showed marked statistical difference(P＜0.01).The ossification of the OB cells on the composed material was marked after 14 days.The MTT color experiments showed no statistic significance between materials group and negative group,with the toxicity at levelⅠand 0(P＜0.05).Conclusion Graded composite ZrO2 HAP can significantly promote proliferation and differentiation of OB cells cultured in vitro and has good biocompatibility.