Fucoidan, a sulfated polysaccharide present in brown seaweed, has demonstrated anticancer activity in lung, breast, liver and colon cells. The insulin-like growth factor (IGF) signaling pathway regulates growth in HT-29 cells through the insulin receptor substrate-1 (IRS-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Ras/Raf/extracellular signal-regulated kinase (ERK) pathways. The aim of the present study was to investigate whether fucoidan downregulates the IGF-IR signaling pathway in HT-29 human colon cancer cells. Fucoidan treatment (0–1,000 µg/ml) was administered for 24 h in HT-29 cells. First, we investigated IRS-1/PI3K/AKT pathway-related protein expression levels following treatment with fucoidan (0–500 µg/ml) using western blot analysis. Fucoidan significantly inhibited the expression of IGF-IR, PTEN, PI3K and AKT as well as their phosphorylated forms (p-IRS-1, p-PI3K and p-AKT). Next, we investigated the effects of fucoidan on Ras/Raf/ERK pathway-related protein expression levels in HT-29 cells. Fucoidan significantly inhibited the expression of IGF-IR, Shc, Ras, SOS, Raf and MEK. HT-29 cells were then incubated in the presence of fucoidan (0 or 250 µg/ml), and IGF-I (10 nM) was added for 0 to 60 min. Immunoprecipitation (IP) experiments showed that fucoidan inhibited IGF-I-induced phosphorylation of IGF-IR, PI3K, Shc (IP, IGF-IR), and phosphorylated IRS-1 and PI3K (IP, IRS-1) compared to the control group. Western blot analysis showed that fucoidan inhibited the expression of IGF-I-induced p-IGF-IR/IGF-IR and p-AKT/AKT, but not p-ERK/ERK. In conclusion, the inhibition of cell viability by fucoidan in HT-29 cells may be due to the downregulation of IGF-IR signaling through the main IRS-1/PI3K/AKT pathway. Fucoidan also partially impacted Ras/Raf signaling in the Ras/Raf/ERK pathway. Therefore, we suggest that fucoidan may be a suitable candidate chemopreventive agent in HT-29 colon cancer cells.
Colon cancer is the most commonly diagnosed cancer and one of the leading causes of deaths in the United States and worldwide (
Fucoidan is a fucose-rich sulfated polysaccharide found in various species of brown seaweed (
The insulin-like growth factor (IGF) signaling system, consisting of ligands (IGF-I and IGF-II), growth factor receptors (IGF-IR and IGF-IIR), and IGF binding proteins (IGFBPs-1-6), regulates cell growth, proliferation, transformation, differentiation, migration and apoptosis (
In previous studies, fucoidan inhibited HT-29 cell proliferation by inducing apoptosis (
Fucoidan purified from
HT-29 human colon adenocarcinoma cells (cat. no. 30038) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; GenDEPOT, Inc.) containing 50 µg/ml penicillin, 25 µg/ml amphotericin B, and 50 µg/ml streptomycin, in an incubator with 5% CO2 at 37°C.
Cell proliferation was estimated using a Cyto X cell viability assay kit (LPS Solution, Daejeon, Korea). Cells were seeded in 96-well plates at a density of 4×104 cells/well and allowed to attach for 24 h. Attached cells were treated with 62.5, 125, 250, 500 or 1,000 µg/ml of fucoidan in serum-free medium for 24 h. The cell proliferation assay solution was added and incubated for 1 h, and the absorbance of each well was measured at a wavelength of 450 nm using a FilterMax F5 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA).
HT-29 cells were cultured with 0, 62.5, 125, 250, or 500 µg/ml of fucoidan for 24 h. Subsequently, cells were washed with phosphate-buffered saline (PBS) and lysed with extraction buffer (1% Nonidet P-40, 1 mM EDTA, 50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM sodium orthovanadate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM NaF, and 1 mM PMSF). The extracts were centrifuged at 9,750 × g for 10 min, and the supernatant was used for western blot analysis.
For immunoprecipitation (IP), cells were incubated for 24 h with 0 or 250 µg/ml of fucoidan, and 10 nM of IGF-I (recombinant human IGF-I; Invitrogen Life Technologies, Frederick, MD, USA) was added. At 0, 5, 30 or 60 min after the addition of IGF-I, the cell lysates were centrifuged at 9,750 × g for 10 min. Supernatant (0.90 mg protein) were incubated with 3 µl of anti-IGF-IRβ or IRS-1 antibody overnight at 4°C. Protein A-agarose beads (GenDEPOT, Inc.) were added to the lysate-antibody mix, which was then incubated for 4 h at 4°C. The beads were washed 3 times with extraction buffer. The immunoprecipitates and total protein (40 µg) were electrophoresed using 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 1% bovine serum albumin (BSA; GenDEPOT, Inc.) in Tris-buffered saline-Tween-20 (TBS-T; 5 mM Tris-HCl, 20 mM sodium chloride pH 7.4, and 0.1% Tween-20) incubated with primary antibodies (1:1,000) in 1% BSA in TBS-T with gentle shaking overnight at 4°C. Membranes were washed twice for 15 min in TBS-T, and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) for 2 h at room temperature and washed again. Immunoreactive bands were detected using an enhanced chemiluminescence substrate (Advansta, Inc., Menlo Park, CA, USA) and visualized using the GeneSys imaging system (SynGene Synoptics, Ltd., London, UK). The following primary antibodies from Santa Cruz Biotechnology, Inc., and Invitrogen were used: anti-p-IGF-IR (sc-101703, anti-rabbit), anti-IGF-IR (sc-390130, anti-mouse), anti-phospho-tyrosine (PY99; sc-7020, anti-mouse), anti-p-IRS-1 (sc-17200, anti-goat), anti-IRS-1 (sc-185, anti-mouse), anti-p-AKT (sc-7985, anti-rabbit), anti-AKT (sc-8312, anti-rabbit), anti-p-PI3K (PA5-17387, anti-mouse), anti-PI3K (sc-374534, anti-mouse), anti-Ras (sc-520, anti-rabbit), anti-Raf (sc-227, anti-rabbit), anti-p-MEK (sc-81503, anti-mouse), anti-MEK (sc-81504, anti-mouse), anti-p-ERK (sc-7383, anti-mouse), anti-ERK (sc-292838, anti-rabbit), anti-SOS (sc-259, anti-rabbit), anti-Grb2 (sc-255, anti-rabbit), anti-Shc (sc-967, anti-mouse), anti-PTEN (sc-7974, anti-mouse), and anti-β-actin (sc-47778, anti-mouse). The secondary antibodies used were HRP-conjugated anti-mouse IgG (32430), anti-rabbit IgG (31460), and anti-goat IgG (31400) (all from Invitrogen Life Technologies).
The results are presented as means ± standard deviation of three independent experiments. The difference in protein expression levels was determined by quantifying the density of the bands using ImageJ (NIH). Significant differences among multiple mean values were assessed using one-way or two-way analysis of variance followed by Bonferroni's multiple comparison test using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
To investigate the effects of fucoidan on cell viability, HT-29 cells were incubated with various concentrations (0–1,000 µg/ml) of fucoidan for 24 h. Fucoidan treatment significantly decreased the viability of HT-29 cells in a concentration-dependent manner (
IGFs signaling is known to affect cell survival, and IGF-I mRNA is increased in colon cancer cells (
Next, we investigated Ras/Raf/ERK pathway-related protein expression levels, the other main IGF-IR downstream signaling pathway. Fucoidan treatment significantly decreased the expression of IGF-IR, phospho-tyrosine, Shc, Ras, SOS, Grb2, and Raf in HT-29 cells in a concentration-dependent manner (
We examined whether fucoidan downregulates IGF-I-induced tyrosine phosphorylation of IGF-IR. HT-29 cells were treated for 24 h with 0 or 250 µg/ml fucoidan, and IGF-IR was stimulated with 10 nM IGF-I for 0, 5, 30, or 60 min. Total cell lysates were prepared and immunoprecipitated using an IGF-IRβ antibody. The immune complexes were used in western blot analysis with an anti-phospho-tyrosine antibody (PY99). IGF-I induced tyrosine phosphorylation of IGF-IR at 5 min, and tyrosine phosphorylation levels persisted at 60 min in control cells. Cells treated with fucoidan exhibited significantly inhibited IGF-IRβ phosphorylation up to 30 min after IGF-I stimulation (
To investigate the association of the Shc and p85 subunits of PI3K with IGF-IR, we performed IP of cell lysates with an IGF-IRβ antibody and subsequent western blot analysis with p85 and Shc antibodies. IGF-I stimulated the association of the p85 regulatory subunit of PI3K with IGF-IR. Expression of the p85 regulatory subunit of PI3K in the control group was induced within 5 min, but fucoidan treatment delayed its expression by up to 30 min. IGF-I also stimulated the association of the Shc subunit with IGF-IR. The expression of Shc in the control cells was induced within 1 min, but in cells treated with fucoidan, expression was induced at 5 min.
We examined whether fucoidan downregulates IGF-I-induced tyrosine phosphorylation of IRS-1. HT-29 cells were treated for 24 h with 0 or 250 µg/ml fucoidan, and IGF-IR was stimulated with 10 nM IGF-I for 0, 5, 30, or 60 min. Total cell lysates were prepared and immunoprecipitated using an IRS-1 antibody. The immune complexes were used in western blot analysis with anti-phospho-tyrosine antibody. IGF-I induced tyrosine phosphorylation of IRS-1 at 5 min, and tyrosine phosphorylation levels persisted for 30 min in control cells. Treatment with fucoidan significantly inhibited the phosphorylation of IRS-1 for up to 60 min after IGF-I stimulation (
To investigate the association of the p85 subunit of PI3K with IRS-1, we performed IP of cell lysates with an IRS-1 antibody and subsequent western blot analysis with a p85 antibody. IGF-I stimulated the association of the p85 regulatory subunit of PI3K and IRS-1 with IRS-1. Expression of the p85 regulatory subunit of PI3K and IRS-1 in control cells were stimulated within 5 min, but fucoidan treatment delayed expression for up to 60 min.
AKT and ERK1/2 are known to play vital roles in cell survival and are activated by IGF-I (
Colon cancer is one of the most common cancers in both men and women and is prevalent worldwide (
Studies related to colon cancer have found that laminarin inhibits cancer cells via Fas and IGF-IR signaling through the intrinsic apoptotic and ErbB pathways, and induces Fas-mediated apoptosis by regulating Fas and Fas-associated protein with death domain (FADD) protein levels (
In previous studies, fucoidan induced apoptosis through the apoptotic pathway and cytotoxicity, and inhibited migration and proliferation in HT-29 colon cancer cells (
The present study provided the first evidence that fucoidan reduces IGF-IR protein expression and IGF-IR-mediated signaling through the IRS-1/PI3K/AKT pathway. We examined cell proliferation using a cell viability assay kit with various concentrations of fucoidan (0–1,000 µg/ml). The results indicated that fucoidan inhibited cell proliferation in a dose-dependent manner in HT-29 cells (
IGF signaling plays a vital role in promoting normal cell proliferation, tumorigenesis, and cancer cell proliferation. IGF-I is also known to inhibit cell death and promote growth in various cancer cells, including colon cancer cells. IGF-IR expression is increased in colon cancer compared to normal mucosal tissues (
The present study suggests that fucoidan inhibits phosphorylation of the β-subunit of IGF-IR by downregulating the α-subunit, directly interfering with the binding of IGF-I to IGF-IR. In immunoprecipitation assays and western blot analysis, fucoidan reduced IGF-I-induced tyrosine phosphorylation of IGF-IR and IRS-1, leading to reduced interaction between PI3K p85 and IGF-IRβ, and the subsequent activation of PI3K/AKT but not ERK1/2 (
Recent data indicate that fucoidan triggers G1 phase arrest and apoptosis in HCT116 colon cancer cells through a p53-independent pathway. In particular, it has been suggested that fucoidan is able to enhance p21 expression at transcriptional level in a p53-independent manner (
Based on these findings, we conclude that fucoidan inhibits cell proliferation and induces apoptosis by inhibiting the IGF-I-induced IGF-IR signaling pathway, including the IRS-1/P13K/AKT pathway, in HT-29 cells. These results suggest that downregulation of IGF-IR/IRS-1/PI3K/AKT signaling may be one of the mechanisms by which fucoidan impacts HT-29 cells. These results suggest that the anti-proliferative effect of fucoidan may be mediated through downregulation of the IGF-I/IGF-IR/IRS-1/PI3K/AKT signaling pathway. Therefore, fucoidan may be useful as a chemopreventive agent in colon cancer cells.
This study was supported by Pukyong National University (grant no. C-D-2016-0267), Busan, Republic of Korea.
Fucoidan treatment induced death in HT-29 cells. HT-29 cells were seeded in 96-well plates at a density of 4×104 cells/well and subsequently treated with 0–1,000 µg/ml fucoidan for 24 h. Data are presented as means ± standard deviation (SD) of three independent experiments. Data were analyzed using one-way analysis of variance. *P<0.05 vs. control.
Fucoidan treatment reduces the levels of proteins in the IRS-1/PI3K/AKT pathway in HT-29 cells. HT-29 cells were incubated with various concentrations of fucoidan (0–500 µg/ml) for 24 h. The protein expression levels of IGF-IR, IRS-1, PTEN, PI3K, and AKT were examined by western blot analysis. Data were analyzed using one-way analysis of variance. *P<0.05 vs. control. IRS-1, insulin receptor substrate-1; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; IGF, insulin-like growth factor. (Values at the left of the western blot indicate the molecular weights of the proteins in kDa).
Fucoidan reduces the levels of proteins in the Ras/Raf/ERK pathway in HT-29 cells. HT-29 cells were incubated with various concentrations of fucoidan (0–500 µg/ml) for 24 h. The protein expression levels of IGF-IR, Shc, Ras, SOS, Grb2, Raf, MEK, and ERK were examined by western blot analysis. Data were analyzed using one-way analysis of variance. *P<0.05 vs. control. ERK, extracellular signal-regulated kinase. (Values at the left of the western blot indicate the molecular weights of the proteins in kDa).
Fucoidan treatment reduces IGF-I-induced tyrosine phosphorylation of IGF-IR in HT-29 cells. HT-29 cells were incubated for 24 h with 0 or 250 µg/ml of fucoidan and lysed with or without stimulation with IGF-I (10 nM) for 0, 5, 30 or 60 min. Total cell lysates (0.90 mg total protein) were incubated with anti-IGF-IRβ antibody and the immune complexes were precipitated with protein A-agarose beads. The immunoprecipitated proteins were analyzed with western blot analysis with antibodies against phospho-tyrosine (PY99), IGF-IR, PI3K p85 or Shc. IGF, insulin-like growth factor; PI3K, phosphatidylinositol 3-kinase.
Fucoidan treatment reduces IGF-I-induced tyrosine phosphorylation of IRS-1 in HT-29 cells. HT-29 cells were incubated for 24 h with 0 or 250 µg/ml fucoidan and lysed with or without stimulation with IGF-I (10 nM) for 0, 5, 30 or 60 min. Total cell lysates (0.90 mg total protein) were incubated with anti-IRS-1 antibody and the immune complexes were precipitated with protein A-agarose beads. The immunoprecipitated proteins were analyzed with western blot analysis with antibodies against p-Tyr, IRS-1 or PI3K p85. IGF, insulin-like growth factor; IRS-1, insulin receptor substrate-1; PI3K, phosphatidylinositol 3-kinase.
Fucoidan reduces the levels of phosphorylated IGF-IR, AKT, and ERK in IGF-I-stimulated HT-29 cells. HT-29 cells were incubated for 24 h with 0 or 250 µg/ml fucoidan and lysed with or without stimulation with IGF-I (10 nM) for 0, 5, 30 or 60 min. Total cell proteins were analyzed with antibodies against IGF-IR, AKT, and ERK using western blot analsysis. Data were analyzed using two-way analysis of variance. *P<0.05 vs. fucoidan (0 µg/ml) (0 min); #P<0.05 vs. fucoidan (250 µg/ml) (0 min); **P<0.05 vs. fucoidan (0 µg/ml) (same time). (Values at the left of the western blot indicate the molecular weights of the proteins in kDa).