Fucoidan present in brown algae induces apoptosis of human colon cancer cells

Abstract

Background: Fucoidan is a sulfated polysaccharide found in brown algae; it has been shown to exhibit a number of biological effects, including anti-tumor effects. In this study, we evaluated the effects of fucoidan on apoptosis in HT-29 and HCT116 human colon cancer cells.

Methods: HT-29 and HCT116 cells were cultured with various concentrations of fucoidan (0 - 20 microg/mL). Apoptosis was assayed via Hoechst staining and Annexin V staining followed by flow cytometric analysis. Western blot analyses and JC-1 staining were conducted to determine the levels of apoptosis-regulating proteins and mitochondrial membrane permeability, respectively.

Results: Fucoidan induced substantial reductions in viable cell numbers and apoptosis of HT-29 and HCT116 cells in a dose-dependent manner. In HT-29 cells, fucoidan also increased the levels of cleaved caspases-8, -9, -7, and -3, and cleaved poly (ADP-ribose) polymerase (PARP) levels. The levels of the X-linked inhibitor of apoptosis protein and survivin were attenuated in the fucoidan-treated cells. Fucoidan was also shown to enhance mitochondrial membrane permeability, as well as the cytochrome c and Smac/Diablo release from the mitochondria. Fucoidan increased the levels of the Bak and truncated Bid proteins, but reduced the levels of Mcl-1. Additionally, fucoidan increased the levels of the tumor necrosis factor-related apoptosis-inducing ligand, Fas and death receptor 5 proteins. The caspase-8 and -9 inhibitors Z-IETD-FMK and Z-LEHD-FMK induced a reduction in fucoidan-mediated apoptosis. Caspase-8 inhibitor inhibited the fucoidan-induced cleavage of Bid, caspases-9 and -3, and PARP.

Conclusion: The findings of this study indicate that fucoidan induces apoptosis in HT-29 and HCT116 human colon cancer cells, and that this phenomenon is mediated via both the death receptor-mediated and mitochondria-mediated apoptotic pathways. These results suggest that fucoidan may prove useful in the development of a colon cancer-preventive protocol.

Figure 1

Fucoidan reduces the viability of HT-29 and HCT116 cells. HT-29 (A), HCT116 (B) and FHC (C) cells were plated in 24-well plates at a density of 50,000 cells/well with DMEM/F12 supplemented with 10% FBS. One day later, the monolayers were serum-deprived with DMEM/F12 supplemented with 1% charcoal-stripped FBS (serum-deprivation medium) for 24 h. Following serum deprivation, the cells were incubated in serum-deprivation medium containing 0 - 20 μg/mL of fucoidan. Viable cell numbers were estimated via MTT assays. Each bar represents the mean ± SEM (n = 6). Means at a time without a common letter differ, P < 0.05.

Figure 2

Fucoidan induces apoptosis of HT-29 and HCT116 cells. (A) HT-29 cells were plated on cell culture coverslips at 50,000 cells/cover and treated for 72 h with fucoidan, as shown in Figure 1. The cells were stained with Hoechst 33258 and the microphotograph images are representative of three independent experiments. Magnification, × 200. (B) HT-29 cells were treated with or without 20 μg/mL of fucoidan for the indicated periods as described in Figure 1. HT-29 (C) and HCT116 (D) cells were treated with various concentrations of fucoidan for 72 h. (B, C, D) The cells were trypsinzed, stained with 7-amino-actinomycin D and annexin V, and then analyzed via flow cytometry. The numbers of living cells and early apoptotic cells are expressed as percentages of the total cell number. Results are expressed as the means ± SEM (n = 6). HT-29 (E), HCT116 (F), and FHC (G) cells were plated in 100 mm dishes and treated with various concentrations of fucoidan for 60 h, as described in Figure 1. The cell lysates were analyzed via Western blotting with the indicated antibodies. A photograph of chemiluminescent detection of the blots, which were representative of three independent experiments, are provided. The relative abundance of each band to its own β-actin was quantified, and the control levels were set to 1. The adjusted mean ± SEM (n = 3) of each band is shown above each blot. (B) *Significantly different from 0 μg/mL of fucoidan, P < 0.05. (C, D, E, F) Means without a common letter differ, P < 0.05.

Figure 3

Fucoidan increases the levels of cleaved caspases and reduces the levels of the inhibitor of apoptosis proteins in HT-29 cells. Cells were treated with various concentrations of fucoidan for 60 h, as shown in Figure 1. 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 (0 μg/mL fucoidan) were set to 1 except for caspase-3, for which 1 is set to 5 μg/mL of fucoidan. The adjusted means ± SEM (n = 3) are shown above each blot. Means without a common letter differ, P < 0.05.

Figure 4

Fucoidan induces mitochondrial membrane depolarization, increases the release of cytochrome c and Smac/Diablo from the mitochondria, and alters the levels of the Bcl-2 family proteins in HT-29 cells. (A) Cells were treated with various concentrations of fucoidan for 48 h as described in Figure 1, then subjected to subcellular fractionation. The resultant cytosolic fractions 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 α-tubulin was quantified, and the control levels were set to 1. The adjusted mean ± SEM (n = 3) of each band is shown above each blot. (B) Cells were treated with fucoidan for 48 h. Cells were loaded with JC-1 and then analyzed via flow cytometry. The numbers of cells with normal polarized mitochondrial membranes (red) and with depolarized mitochondrial membranes (green) are expressed as a percentage of the total cell number. Each bar represents the mean ± SEM (n = 6). (C) Cells were treated for 36 h with various concentrations of fucoidan as described in Figure 1. 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 provided. 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. (A, B, C) Means without a common letter differ, P < 0.05.

Figure 5

Fucoidan increases the protein levels of cell death receptors and their ligands in HT-29 cells. Cells were treated for 36 h with various concentrations of fucoidan, as described in Figure 1. 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.

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.