Figure 6

Caspase inhibitors suppress fucoidan-induced apoptosis in HT-29 cells. Cells were incubated in the absence or presence of 20 μmol/L of caspase-8 inhibitor (Z-IETD-FMK) (A) or caspase-9 inhibitor (Z-LEHD-FMK) (B) 4 h prior to 48 h of treatment with 20 μg/mL of fucoidan. Cells were stained with 7-amino-actinomycin D and annexin V, then analyzed via flow cytometry. The numbers of living cells and early apoptotic cells are expressed as a percentage of the total cell number. Each bar represents the mean ± SEM (n = 6). (C) Cells were treated with the caspase-8 inhibitor (Z-IETD-FMK) and then 20 μg/mL of fucoidan, as described in (A). Total cell lysates were analyzed via Western blotting with the indicated antibodies. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified, and the control levels were set to 1. The adjusted means ± SEM (n = 3) of each band are shown above each blot. Means without a common letter differ, P < 0.05.