Figure 3
2-DG up-regulates TRAIL death receptors in melanoma cells. A, Mel-RM and MM200 cells treated with 2-DG (10 μM) for indicated periods were subjected to measurement of the cell surface expression of TRAIL-R2 (upper panel) and -R1 (lower panel) using flow cytometry. The data in y axes represent mean fluorescence intensity (MFI). B, Representative flow cytometry histograms showing up-regulation of TRAIL-R2 by 2-DG in melanoma cells. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. C, 2-DG up-regulates TRAIL-R2 and -R1 in a panel of melanoma cell lines, melanocytes, and fibroblasts. Cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 (upper panel), and -R1 (lower panel) was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells treated with 2-DG (10 μM) for indicated periods were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells were treated with the 2-DG (10 μM) for indicated periods. Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. The increases in TRAIL-R2 mRNA at 16, 24, and 36 hours after treatment in both cell lines were statistically significant (p < 0.05); Right panel: Mel-RM and MM200 cells were treated with actinomycin D (3 μg/ml) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Total RNA was isolated and subjected to Real-time PCR analysis for TRAIL-R2 mRNA expression. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Up-regulation of TRAIL-R2 mRNA by 2-DG was significantly inhibited by actinomycin D in both cell lines (p < 0.05). The data shown are either the mean ± SE (A, C, & E), or representative (B & D), of three individual experiments.