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Abstract
Purpose
We investigated the effects of phosphatase and tensin homologue deleted on chromosome 10 gene phosphatase and tensin homologue deleted on chromosome 10 gene (PTEN) expression on the cell proliferation and on the responsiveness of troglitazone in osteosarcoma cells.
Materials and Methods
Western blotting alnalysis was performed to detect the expression of PTEN in U-2OS cells treated with troglitazone. WST (water-soluble tetrazolium) assay was used to evaluate cell proliferation. Flow cytometry was used to determine cell apoptosis. Further, transfection of wild-type PTEN plasmid DNA was used to upregulate PTEN expression.
Results
Troglitazone treatment induced growth inhibition of U2-OS cells in a dose- and time-dependent manner. Troglitazone increased the expression of PTEN in a dose-dependent manner. PTEN upregulation induced by troglitazone treatment resulted in cell growth inhibition and apoptosis in U-2OS cells. PTEN over-expression by plasmid transfection enhanced these effects of troglitazone. Moreover, no changes were observed in the mutant type-PTEN group.
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Figure 1.
Growth inhibition effects of troglitazone in the human osteosarcoma U-2OS cell line in a dose (A) and time (B) -dependent manner. Cells (1×105) were treated with various concentrations of troglitazone. Cell viability was determined by the WST assay and was presented as a calculated percentage of viable cells between troglitazone-treated and -untreated control cells. Each points represents the mean SEM of three determinations. †p<0.01 vs. untreated control.
Figure 2.
Effect of troglitazone on the cell survival of osteosarcoma cells by flow cytometry. Histogram patterns of U-2OS cells treated with various concentrations (0, 10, 20, 30, 40, and 50 μM) of troglitazone for 48 hrs by FACS. Cell cycle distribution was analyzed by flow cytometry after coupled staining with Annexin V conjugated to fluorescein isothiocyanate (FITC) and Propidium iodide (PI) as described in Materials and Methods.
Figure 3.
Dose-dependent effect of troglitazone on the PTEN expression. U2-OS cells (5×106) were treated with 5, 10, 20, and 30 μM of troglitazone for the indicated concentrations. A representative result was presented from among at least three separate experiments yielding similar results. Each points represents the mean SEM of three determinations. ∗p<0.05 vs. untreated control. †p<0.01 vs. untreated control.
Figure 4.
Establishment of PTEN over-expression osteosarcoma cells using transfection. The PTEN expression in the U-2OS cells declined gradually after being treated with troglitazone for a longer time period (eg 72 hrs), since the activity of plasmid trasfection was decreased with time.
Figure 5.
Transfection of active PTEN in U2-OS cells enhanced the inhibitory effect of troglitazone on cell growth. The effect of troglitazone-decreased cell viability was enhanced in the wild type-PTEN group, whereas no changes were observed in the mutant type-PTEN group. Each points represents the mean SEM of three determinations. ∗p<0.05 vs. untreated control. †p<0.01 vs. untreated control.
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