Figure 3 | Oncogene

Figure 3

From: Redistribution of CD95, DR4 and DR5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells

Figure 3

Pretreatment with resveratrol sensitizes colon cancer cells to caspase activation by death receptor agonists. (a) HT29 cells were left untreated (Co) or treated with 30 μ M resveratrol for 48 h (R30). When indicated, CH11 Ab or TRAIL were added to the culture medium for the last 24 h in the absence or presence of 50 μ M z-VAD-fmk (zVAD), before measuring the ability of cell lysates to hydrolyse DEVD-AMC, LEHD-AFC and IETD-AFC caspase substrates, respectively (mean±s.d. of three independent experiments). Enzyme activities: ‘fold increase’ related to the control value. (b) Fluorescence microscopy analysis of active caspase-3 (C3a), caspase-9 (C9a) and caspase-8 (C8a) in HT29 cells treated as in (a). Active caspases were assigned the color green and nuclei, labeled with Hoechst 33342, were assigned the color blue. Representative cells of three independent experiments are shown. (c) HT29 cells were left untreated (Co) or treated with 30 μ M resveratrol (R30) for 48 h or CH11 for 24 h or the combination of both. In this latter case, cells were treated with resveratrol for 48 h and CH11 was added for the last 24 h, in the absence (Co) or presence of indicated permeant caspase inhibitors (50 μ M). Right panel: flow cytometry analysis of active caspase-3 (Gray areas: Isotype-matched control Ab; white areas: antiactive caspase-3 Ab). Left panel: The percentage of apoptotic cells (Hoechst 33342 staining) was measured at the end of cell treatment (mean±s.d. of three independent experiments; 300 cells/point). Similar observations were made when TRAIL was added to resveratrol-pretreated cells (not shown)

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