| ObjectivesTo investigate the mechanism of the apoptosis of osteosarcoma U2-OS cells and MG-63cells induced by tetrandrine and its clinical value.Materials and methods1. MTT assay was used to test the viability of osteosarcoma cells treated with TET.2. Transmission electronic microscopy was used to observe the apoptosis of osteosarcoma cells treated with TET.3. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to quantify the apoptosis of osteosarcoma cells treated with TET.4. Caspase activity assay, was used to to investigate whether the apoptosis induced by TET was correlated with the activation of caspase enzymes.5. Western blot analysis was used to investigate the apoptotic-related proteins in apoptosisResults:1. TET inhibited cell proliferation in a dose-and time-dependent manner. Following incubation with TET for24h, the IC50values for TET were13.36μM for the MG-63cells and9.98μM for the U-2OS cells.2. Transmission electronic microscopy showed typical apoptotic morphological features:Cell shrinkage, nuclear condensation, nuclear fragmentation, chromatin condensation, a crescent-shaped nucleus and cytoplasmic vacuoles. Flow cytometry experiments similarly showed that TET is capable of inducing apoptosis in human osteosarcoma cells. A caspase enzyme activity assay revealed that TET caused time-dependent activation of caspase-3and caspase-8activity3. Western blot analysis showed that treatment with TET for24h significantly increased the levels of the pro-apoptotic proteins Bid and Bax, but decreased the levels of the anti-apoptotic proteins Bcl-2and Bcl-xL, in a dose-dependent manner. Furthermore, TET increased the protein levels of cytochrome-c and Apaf-1.Conclusions:1. TET inhibited cell proliferation in a dose-and time-dependent manner.2. Tetrandrine induces apoptosis and triggers a caspase cascade in osteosarcoma cells through the intrinsic and extrinsic pathways. |
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