Activation of Wnt/β-Catenin Signaling Increases Apoptosis in Melanoma Cells Treated with Trail
Figure 1
WNT3A sensitizes melanoma cell lines to TRAIL in a β-catenin-dependent manner.
A) A375 melanoma cells were treated with indicated doses of rhTRAIL for 24 hours in the presence of WNT3A or control L-cell (L) CM (10%). Apoptotic cells were detected by Annexin V binding assay using FACS. Representative FACS histograms with Annexin V-positive gates are shown. Percent apoptotic cells are indicated. B) A375 cells were treated with indicated doses of rhTRAIL in the presence of WNT3A or L CM (10%). Data represents mean % apoptotic cells ± SEM as determined by Annexin V positivity at 24 hours. An (*) indicates that the difference between L and WNT3A CM treated cells at the indicated TRAIL dose is significant with a p-value of <0.01, calculated using Student’s t-test. C) A375 cells were treated with rhTRAIL and WNT3A in the absence and presence of the pan-caspase inhibitor zVAD-FMK (100 µM), and then analyzed for cleaved PARP at 24 hours. C) A375 cells were pre-treated with siRNA specific for β-catenin (CTNNB1) or non-targeting control siRNA for 48 hours. Cells were then treated with rhTRAIL (20 ng/mL) in the presence of WNT3A CM or L CM. Data represents mean percentages of Annexin V-positive cells (+/- SEM) at 24 hours post-treatment as detected by FACS. P-values were calculated using one way ANOVA and Tukey’s post-test analysis. A parallel immunoblot (lower panel) confirms knockdown of β-catenin. The experiments in A and B are representative of at least three independent experiments with similar results.