The control cells showed a normal pattern of DNA content that reflected G0/G1, S and G2/M phases of the cell cycle (Figure 5A). Open in a separate window Figure 5 Effect of dihydromethysticin on the cell cycle phase distribution in human osteosarcoma cells. observed mitochondrial transmembrane depolarization along with decreased phosphorylation levels for Nav1.7-IN-2 PI3K, AKT (Ser 473), AKT (Thr 308), GSK-3, and BAD. These reductions were associated with down regulation of AKT and upregulation of both GSK-3 and BAD. < 0.05 was considered to be statistically significant. Results and discussion Effect of dihydromethysticinon on the viability of human osteosarcoma (MG-63) cells MG-63 cells were treated with different concentrations (0, 2.5, 5, 25, 75 and 100 M) of dihydromethysticin for 12, 24, and 48 hours and cell viability was evaluated using an MTT assay. Figure 1 shows the dose-dependent as well as time dependent growth inhibitory effects of dihydromethysticin on the cell viability of MG-63 osteosarcoma cells. The percentages of growth inhibition at various concentrations in osteosarcoma cells were determined as the percentage of viable treated cells in comparison with viable cells of untreated controls. At lower doses of dihydromethysticin, time periods of 12, 24 and 48 h had a less effect on tumor cell growth inhibition. However, at higher doses, exposure of tumor cells to greater duration of time resulted in higher growth inhibition and 48 h exposure at 100 M dose led to over 90% growth inhibition. Open in a separate window Figure 1 Growth inhibitory effect of dihydromethysticin against human osteosarcoma (MG-63) cells. *P < 0.05 vs. control group. **P < 0.01 vs. control group. Apoptosis detection by nuclear staining with Hoechst 33258 for cellular morphological study In order to evaluate whether the dihydromethysticin induced apoptotic effects in human osteosarcoma cells (MG-63), we examined the effect of dihydromethysticin on nuclear morphology using Hoechst 33258 staining involving a fluorescence microscope. The MG-63 cells were treated with different doses of dihydromethysticin (Figure 2A-D). Figure 2A, represents untreated cells which showed normal nuclear morphology without any signs of chromatin condensation, Figure 2B-D represent 25, 75 and 100 M doses of dihydromethysticin respectively. Dihydromethysticin treated cells Rabbit polyclonal to VDP showed dose dependent chromatin condensation which increases from A-D. The nuclei of untreated control MG-63 cells were stained in less bright blue and homogeneous color. By contrast, after treatment with different doses of dihydromethysticin for 48 h, most cells exhibited very intense staining of condensed and fragmented chromatin. The white arrows pointed at the condensed chromatin with Nav1.7-IN-2 typical apoptotic bodies. Open in a separate window Figure 2 Effect of dihydromethysticin on nuclear morphology (chromatin condensation) in human osteosarcoma cells (MG-63). MG-63 cells were treated with 0 M (A, untreated control), 25 M (B), 75 M (C) and 100 M (D) respectively for 48 h and stained with Hoechst 33258. The nuclear morphology was observed by fluorescent microscope (magnification 500 ). White arrows represent nuclear chromatin condensation and apoptotic body formation. Quantitative videomicroscopy analysis by inverted phase microscope Quantitative videomicroscopy or computer-assisted phase contrast microscopy allows us to differentiate between different kinds of growth inhibitory effects (cytostatic or cytotoxic effects) of dihydromethysticin against human osteosarcoma cells. Figure 3A-D depicts the results of videomicroscopy assay in MG-63 cells. Cells that perished appeared rounded and refringent under quantitative videomicroscopy analysis. The proportion of this cell type in MG-63 cells was higher following treatment with dihydromethysticin at higher doses (B, C and D represent 25, 75 and 100 M concentration of dihydromethysticin) indicating higher cell death at higher concentration of dihydromethysticin. This assay showed that dihydromethysticin induced cell death through both cytotoxic and cytostatic effects inducing designated vacuolization processes which finally led to cell Nav1.7-IN-2 death. Open in a separate window Number 3 Quantitative videomicroscopy analysis human being osteosarcoma cell collection (MG-63) treated with numerous concentrations of dihydromethysticin. The arrows show rounded cells which represent the deceased (through cytostatic and cytotoxic effects) cells. A, represents untreated Nav1.7-IN-2 cells; B, represents 25 M, C represents 75 M and D represents 100 M concentration of dihydromethysticin. Data are indicated as the means SEM. The morphological analyses were carried out at a 100 magnification. Quantification of cell apoptosis by annexin V-FITC/PI assay This assay gives a quantitative measure of cell apoptosis as well as differentiates between numerous forms of cell death like necrosis and apoptosis. Annexin V staining can determine phosphatidyl serine and as such can be used for its analysis. After cells are stained with annexin V in tandem with propidium iodide (PI), this reagent enters the cell only when the plasma cell membrane is definitely damaged. The results of this assay are demonstrated in Number 4A-D and reveal the apoptotic effects of dihydromethysticin on MG-63 cells are dose-dependent. The percentage of apoptotic cells (early apoptosis + late apoptosis)raises from.