Figure 1.
Fucoidan inhibits cell growth and promotes cell apoptosis.
(A) Cell growth. Cells were treated with fucoidan at the indicated concentration and the viable cells were assessed using the CellTiter96 AQueous One Solution Cell Proliferation Assay. (B) Caspase 3 activation. Cells were treated with fucoidan for 3 days and the expression of cleaved caspase 3 was examined by Western blot. (C) TUNEL. For TUNEL analysis, cells were treated with fucoidan at 100 µg/ml for 3 days and the cell apoptosis was analysed using TUNEL assay. Apoptotic cells (green) were counted in five randomly selected fields (300 cells per field) and presented as a mean ± SD of percentage of apoptotic cells over the total cells (DAPI, blue). Data are expressed as a mean ± SD in triplicate experiments. *p<0.05; **p<0.01.
Figure 2.
Effect of fucoidan on the expression GRP78 and ERp29.
Cells at 70–80% confluence were treated with fucoidan at the indicated concentrations for 3 days and both the attached and suspended cells were harvested for protein expression analysis. (A) The expression of GRP78, but not ERp29, was inhibited by fucoidan in MDA-MB-231 cells; (B) The expression of ERp29 was significantly attenuated by fucoidan in HCT116 cells, while GRP78 was only slightly decreased (∼1.5-fold) in cells treated with fucoidan at >50 µg/ml. As a positive control, cells were treated with ER stress inducer TG (2.0 µM) for 24 h. Data represent mean ± SD in triplicate experiments. *p<0.05; **p<0.01.
Figure 3.
Effect of fucoidan on the relative phosphorylation of CaMKII (p-CaMKII/CaMKII) and Bax expression in MDA-MB-231 cells (A) and HCT116 cells (B).
(A) Relative phosphorylation of CaMKII and Bax expression were progressively increased by fucoidan in MDA-MB-231 cells; (B) Relative phosphorylation of CaMKII was moderately stimulated (∼1.5-fold) at 10 µg/ml, followed by inhibition (∼1.4-fold) of fucoidan at 100 µg/ml. Bax expression was not affected by fucoidan in HCT116 cells. (C) Caspase 12 expression and cleavage. Fucoidan efficiently induces caspase 12 expression in MDA-MB-231 cells, rather than in HCT116 cells. No cleavage of caspase 12 was found in both types of cells. Data represent mean ± SD from triplicate experiments. *p<0.05; **p<0.01.
Figure 4.
Fucoidan suppresses p-IRE-1\XBP-1 splicing in MDA-MB-231 cells (A) and HCT116 cells (B).
Cells were treated with fucoidan as described in the “Materials and Methods” and in Figure 2. The phosphorylation of IRE-1 (p-IRE-1) and XBP-1 splicing were remarkably reduced by fucoidan as assessed by immunoblot. Its downstream target, p58IPK, was not subsequently decreased. Data are expressed as mean ± SD in triplicate experiments. Note that TG-treated cells (2.0 µM, 24 h) showed an increased p-IRE-1 and XBP-1s in both cells. *p<0.05; **p<0.01.
Figure 5.
Fucoidan activates p-eIF2α\CHOP in MDA-MB-231 cells (A) and HCT116 cells (B).
Cells were treated with fucoidan as described in the “Materials and Methods” and in Figure 2. The relative phosphorylation of eIF2α (p-eIF2α/eIF2α) and CHOP expression were remarkably increased by fucoidan as assessed by immunoblot. TG treatment (2.0 µM, 24 h) induced activation of p-eIF2α and CHOP expression. Data are expressed as mean ± SD in triplicate experiments. *p<0.05; **p<0.01.
Figure 6.
Combinatory treatment of fucoidan and salubrinal enhances eIF2α phosphorylation and CHOP expression in MDA-MB-231 cells and HCT116 cells.
Cells were treated with salubrinal (50 µM) alone or in combination with fucoidan (100 µg/ml) for 48 h. Cell lysates (30 µg) were applied for the analysis of the expression of p-eIF2α and CHOP by immunoblot. **p<0.01; ***p<0.001.
Figure 7.
Effect of silencing CHOP on fucoidan-induced DNA damage and cytotoxicity.
(A) CHOP knockdown by siRNA inhibits fucoidan-induced expression of cleaved PARP. Cells at 70–80% confluence were treated with CHOP siRNA or scramble siRNA for 48 h, followed by fucoidan treatment (0 or 100 µg/ml) for 2 days. The levels of CHOP and cleaved PARP were assessed by immunoblot. (B) Silencing CHOP antagonizes fucoidan-induced cell death. Cells pre-treated with CHOP siRNA or control siRNA were treated with fucoidan (0 or 100 µg/ml) for 2 days and the cell viability was examined using the trypan blue exclusion assay. Cell viability was expressed as a percentage of the siRNA control cells without fucoidan treatment (column 3 in MDA-MB-231 cells; column 7 in HCT116 cells). Note that CHOP knockdown alone does not affect cell viability in both types of cells (column 1 vs. 3; column 5 vs. 7). Instead, CHOP knockdown remarkably prevents cell apoptosis induced by fucoidan at 100 µg/ml (column 2 vs. 4 in MDA-MB-231 cells; column 6 vs. 8 in HCT116 cells). Results are interpreted as mean (±SD) of triplicate experiments. *p<0.05; **p<0.01.