The primary aim of this investigation was to determine whether a combination of the fucoidan from Saccharina cichorioides and resveratrol would be a better outcome for colon cancer prevention. As a first step toward accomplishing this aim, we examined the cytotoxicity of each compound alone against human colon carcinoma cell line HCT 116 cells. This cell line is known to be invasive and highly motile in vitro studies.

To define the inhibitory concentration (IC₅₀) of fucoidan ScF2 and resveratrol, HCT 116 cells were treated with ScF2 (100, 200, 400, 800 μg/mL) and resveratrol (5, 10, 20, 40, 80 μM) for 24 h as described in the Experimental section.

Fucoidan ScF2 (800 μg/mL) was less cytotoxic against HCT 116 cells (the cell number was reduced 15%) (Figure 2A), while resveratrol (IC₅₀ value of 55 μM) possessed cytotoxic activity against the tested cell line, as indicated by the dose-response curve (Figure 2B).

These results are in agreement with data from previous studies. The sulfated polysaccharides from other species of brown algae were found to be nontoxic against JB6 Cl41 (epidermal mouse cells), Vero (African green monkey kidney), MCF-10A (human epithelial cells), MCF-7 (human breast cancer cells) and other cells [26,27]. It has been reported that resveratrol was cytotoxic against different types of cancer; for example, the IC₅₀ value of resveratrol against HCT 116 cells ranged from 35 to 100 μM [28,29].

In the present study, we observed that fucoidan ScF2 is nontoxic against HCT 116 cells in doses up to 800 μg/mL, which indicated that the anti-tumor activity of this polysaccharide may be produced by activation of specific receptors and proteins, suppression of signal transduction and/or induction of apoptosis in cancer cells. These findings make the fucoidan an ideal candidate for further studies as an anti-tumor agent, which could enhance the anti-tumor activity of resveratrol at nontoxic doses.

Based on the cytotoxic assay data, a fucoidan concentration of 300 μg/mL (the cell number was reduced less than 10%) and a resveratrol concentration of 40 μM (the cell number was reduced less than 50%) were chosen for the antiproliferative assay.

The effects of each compound alone on the growth of HCT 116 cells were studied. The cells were treated with fucoidan ScF2 (300 μg/mL) and resveratrol (40 μM) for 24, 48 and 72 h (Figure 3A).

The incubation of HCT 116 cells with fucoidan ScF2 affected cell growth slightly (12% growth inhibition after 72 h of treatment). Resveratrol inhibited the proliferation of HCT 116 cells by 34%, 60% and 71% after 24, 48 and 72 h, respectively (Figure 3A).

Based on these results, the optimal treatment time and concentrations of fucoidan ScF2 and resveratrol were chosen to determine whether the fucoidan from Saccharina cichorioides might enhance the antiproliferative effect of resveratrol. Cells were pre-incubated with resveratrol (40 μM) for 24 h and then treated with fucoidan ScF2 (100, 200 and 300 μg/mL) for an additional 24 h. As shown in Figure 3B, the growth inhibitory effect of the combination of fucoidan ScF2 and resveratrol was greater than the effect of either agent alone. The antiproliferative activity of resveratrol increased by 13%, 18% and 22% after the addition of 100, 200 and 300 μg/mL fucoidan ScF2, respectively (Figure 3B). So, it became evident that the inhibition of cell proliferation was stronger as the concentration of fucoidan ScF2 increased.

The growth inhibitory activities of resveratrol and fucoidans from different species of brown algae have been reported in recent years. These natural compounds have been shown to suppress viability in a wide variety of cancer cells, including lymphoid and myeloid, breast, colon, pancreas, stomach, prostate, lung and cervical cancers [8,30].

However, this study is the first to demonstrate the ability of the fucoidan from the brown alga Saccharina cichorioides to enhance the antiproliferative activity of resveratrol at nontoxic doses.

The next experiment was performed to determine whether the comparatively greater growth inhibition of colon cancer cells in response to the combinatorial treatment with fucoidan ScF2 and resveratrol could be the result of increased apoptosis.

Apoptosis is a representative form of programmed cell death, which is thought to be critical for cancer prevention [31]. Current studies indicate that there are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. These two apoptotic pathways converge on caspase-3 and other subsequent proteases and nucleases that drive the terminal events of apoptosis. Caspases perform critically important roles in the induction of apoptosis. They are classified based on their mode of activation as either initiator (caspase-8 and caspase-9) or effector caspases (caspase-3 and caspase-7). Activated initiator caspases can cleave and activate effector caspases, which in turn cleave a variety of cellular substrates, most notably poly(ADP-ribose) polymerase (PARP). One of the most important functions of PARP is to help repair single-strand DNA nicks; thus, cleaved PARP is a useful marker for apoptosis [32].

Based on these previous results, we attempted to determine whether fucoidan ScF2, resveratrol or a combination of these two compounds activated initiator and effector caspases as well as PARP cleavage via Western blotting and an immunoprecipitation assay.

The results indicated that resveratrol (40 μM) could induce slight activation of caspase-9, -7 and -3 in HCT 116 cells, and fucoidan ScF2 (300 μg/mL) had no effect on the activation of these caspases (Figure 4). However, the combination of fucoidan ScF2 and resveratrol induced a dose-dependent increase in the activation of caspase-9, -7 and -3. Moreover, the combination of fucoidan ScF2 and resveratrol induced a dose-dependent increase in the protein levels of cleaved caspases-9, -7 and -3 and cleaved PARP in HCT 116 cells (Figure 4). These results suggested that the fucoidan from the brown alga Saccharina cichorioides could enhance resveratrol-induced apoptosis in human colon cancer cells by activating initiator and effector caspases and cleaving PARP, which ultimately cause the morphological and biochemical changes observed in apoptotic cells.

To further test this idea, we investigated the effect of fucoidan ScF2 on resveratrol-induced apoptosis by flow cytometry analysis. As illustrated in Figure 5, the treatment with resveratrol alone yielded 28% apoptotic cells. On the other hand, the treatment of resveratrol-treated cells with fucoidan ScF2 at 100, 200 and 300 μg/mL increased the percentage of apoptotic cells to 30%, 43% and 52%, respectively (Figure 5).

These results strongly indicated that fucoidan ScF2 facilitated resveratrol-induced apoptosis in the HCT 116 cell line and that this strategy might be a novel one for improving the treatment of colon cancer.

Because apoptosis induction by nontoxic natural agents was found to be arguably the most potent defense against cancer, many studies have focused on elucidating the proapopotic activity of many biologically active compounds, such as the fucoidans from brown algae and resveratrol. The molecular mechanism of resveratrol-induced apoptosis was studied in detail. The growth-inhibitory effects of resveratrol were thought to be mediated by cell-cycle arrest, the upregulation of p21Cip1/WAF1 and proteins p53 and Bax, the downregulation of survivin, cyclin D1, cyclin E, Bcl-2, Bcl-xL and cIAPs, and the activation of caspases [33].

Previous studies have shown that the fucoidans induced apoptosis in different types of cancer cells via a variety of mechanisms. It was reported that commercially available (Sigma, USA) fucoidan (100 μg/mL) from the brown alga Fucus vesiculosus Linnaeus induced apoptosis via the activation of caspase-3 and downregulation of the ERK pathway in human lymphoma HS-Sultan cells [34] and via the caspase-8-dependent pathway in MCF-7 breast cancer cells [35]. Commercially available fucoidan (820 μg/mL), sold under the product name “Power the fucoidan” from Cladosiphon novae-caledoniae Kylin (Daiichi Sangyo Corporation, Japan) activated a caspase-independent apoptotic pathway in MCF-7 breast cancer cells via the activation of ROS-mediated MAP kinases and the regulation of the Bcl-2 family protein-mediated mitochondrial pathway [17]. Galactofucan (200 μg/mL) from Undaria pinnatifida was found to induce apoptosis in A549 human lung carcinoma cells through the downregulation of Bcl-2 and the activation of the caspase pathway [36]. Several experiments were performed to investigate the anti-tumor effect of the fucoidan from F. vesiculosus on colon cancer. Hyun et al. [37] reported that the fucoidan (100 μg/mL) induced apoptosis in HCT-15 human colon carcinoma cells via the activation of caspase-9 and -3 accompanied by changes in Bcl-2 and Bax; additionally, there were changes in the phosphorylation of ERK, p38 kinase and Akt. Another study demonstrated that this fucoidan (20 μg/mL) induced apoptosis in HT-29 human colon carcinoma cells and HCT 116 cells via both the death receptor-mediated apoptotic pathway and mitochondria-mediated apoptotic pathway [16].

However, it should be noted that most of these studies have used a commercially available fucoidan from Fucus vesiculosus, which contains more than 16 different fucans with varying proportions of the individual monosaccharide [38]. The anti-tumor activity of the fucoidans, including apoptosis induction, is known to depend on their structural characteristics. These characteristics could vary according to the algae species, season of harvest, age of the plants and even parts of a single algal thallus [39]. Each newly isolated fucoidan is therefore a new compound with unique structural characteristics, thus representing a potential novel drug. To the best of our knowledge, this work is the first to show that purified fucoidan from the brown alga Saccharina cichorioides significantly facilitated resveratrol-induced apoptosis in human colon cancer cells via the caspase-3 activation-dependent pathway.

The search for compounds that sensitize cancer cells to chemotherapeutic agents is an important task in the modern strategy of anticancer therapy. Recently, it has been reported that sulfated polysaccharides from brown algae inhibited colony formation of human breast cancer and melanoma cells [40,41]. Resveratrol is able to suppress cell transformation induced by different stimulants [42,43]. Based on these studies, we suggest that a combination therapy that includes the fucoidan from the brown alga Saccharina cichorioides and resveratrol will be highly effective in inhibiting colony formation of colon cancer cells.

To evaluate the inhibitory effects of fucoidan ScF2, resveratrol, and fucoidan ScF2 and resveratrol together on colony formation, we performed soft agar clonogenic assays using HCT 116 cells (Figure 6). In the preliminary study, we found that fucoidan ScF2 concentration effective against colony formation was 200 μg/mL; this concentration was therefore used in additional experiments. Resveratrol was used at a concentration of 40 μM as in the antiproliferative assay.

The treatment of HCT 116 cells with resveratrol (40 μM) and fucoidan ScF2 (200 μg/mL) alone reduced the colony number by 34% and 27%, respectively, whereas the combination of these two agents reduced the colony number by 60% (Figure 6).

In this study, we also attempted to investigate whether the fucoidan could sensitize colon cancer cells to resveratrol. It was found that pre-incubation of HCT 116 cells with fucoidan ScF2 (200 μg/mL) for 24 h significantly sensitized colon cancer cells to resveratrol. This pre-incubation reduced the colony number by more than 80% and reduced colony size when compared to cells treated with either resveratrol or fucoidan ScF2 alone and even treatment with a combination of the fucoidan and resveratrol at the same time (Figure 6). The present findings demonstrate for the first time that human colon cancer cells treated with the fucoidan and resveratrol underwent a significant reduction in their ability to express their transformed phenotype.

Information about the ability of the fucoidans to sensitize cancer cells to chemotherapeutic drugs and/or enhance their anti-tumor activity is very limited. Philchenkov et al. [44] used human malignant MT-4 lymphoid cells to study the ability of the fucoidan from Fucus evanescens C.Agardh to modulate the apoptosis induced by etoposide, which is a potent chemotherapeutic agent with clinical activity against a broad range of human malignancies. It was shown that the incubation of MT-4 lymphoid cells with the fucoidan prior to etoposide treatment increased the apoptosis percentage and activated caspase-3, which was not observed with etoposide treatment only. However, the fucoidan’s ability was assessed in cells pretreated for 14 days with a high concentration of the fucoidan (500 μg/mL). Therefore, our findings that a low concentration of the highly sulfated α-L-fucan from the brown alga Saccharina cichorioides significantly sensitized the HCT 116 cells to resveratrol may lead to it as a novel therapy for colon cancers in the future.

Isolation of polysaccharides from the brown alga Saccharina cichorioides was carried out using the modified method [45]. Briefly, the alga was ground to a particle size 1 mm and treated with ethanol, acetone and chloroform sequentially. Samples of defatted, dried alga (100 g) were extracted with 0.1 N HCl (2 L) at room temperature. The extracts were concentrated to 1/5 of the volume by ultrafiltration using a 10 kDa Millipore membrane, and the polysaccharides were then precipitated with four volumes of 96% ethanol. The precipitates were washed with 96% ethanol and air-dried.

Polysaccharide solutions in 0.04 N HCl (1 g/20 mL) were applied onto a DEAE-cellulose column (Cl⁻ form, 3 × 14 cm) equilibrated with 0.04 N HCl. Sulfated polysaccharides were successively eluted with liner gradient of 250 mL H₂O/250 mL NaCl (2 M). The fractions were dialyzed and lyophilized to obtain the polysaccharide fractions ScF1 and ScF2.

The carbohydrate content was determined colorimetrically by the phenol-sulfuric acid method [46].

The sulfate group content was determined by the turbidimetric method after hydrolysis of the corresponding fractions with HCl (1 N) [47].

Total acid hydrolysis of fucoidan ScF2 (5 mg) was carried out using trifluoroacetic acid (2 N, 0.5 mL, 6 h, 100 °C), and the acid was evaporated using a 2.5% ammonia solution on a rotary evaporator.

The monosaccharide composition of the polysaccharides after acidic hydrolysis was determined on a Biotronik IC-5000 carbohydrate analyzer (Germany) using a Shim-pack ISA-07/S2504 (0.4 × 25 cm) column. Elution was performed with a potassium borate buffer at an elution rate of 0.6 mL/min. Detection was carried out by the bicinchoninate method and integration on a Shimadzu C-R2 AX system. Monosaccharides (Rha, Man, Fuc, Gal, Xyl, and Glc) were used as HPLC standards [48].

Fucoidan ScF2 (10 mg/mL) was applied onto a cation-exchange column (Timberlite CG-120, 200–400 mesh, Serva, Germany), and pyridine (0.5 mL) was added after elution of the samples. The samples were lyophilized, and 18 mL of DMSO and 2 mL of methanol were added to the fucoidan. This mixture was heated at 100 °C for 3 h. To remove DMSO at the end of the reaction, the mixture was dialyzed then lyophilized.

The molecular weight of fucoidan ScF2 was determined by HPLC on a Shimadzu LC-20A instrument (Japan) with a RID-10A refractometric detector. Polysaccharides were separated over Shodex Asahipak GS-520 HQ and Shodex Asahipak GS-620 columns (7.5 mm × 300 mm) (Showa, Denko, Japan) at 50 °C and eluted with water (0.8 mL/min). The columns were calibrated using a set of P-82 Shodex Standards Pullulan (Japan) and Blue Dextran (Amersham, Sweden).

The IR spectrum of the fucoidan was registered in KBr pellets on a Carl Zeiss IR-75 spectrometer.

NMR spectra for solutions of the fucoidan in D₂O were obtained on a Bruker Avance DPX-500 NMR spectrometer with a working frequency of 75.5 MHz at 60 °C.

HCT 116 human colon cancer cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin solution. The cultures were maintained at 37 °C in a 5% CO₂ incubator (MCO-18AIC, “Sanyo”, Japan).

Resveratrol was dissolved in DMSO and subsequently diluted with McCoy’s 5A medium (final DMSO concentration in experiments was 0.05%), and fucoidan ScF2 was dissolved in McCoy’s 5A medium.

The MTS assay was used as an indicator of cell viability as determined by the mitochondrial-dependent reduction of formazan [49]. Briefly, HCT 116 cells (1 × 10⁴ cells/200 μL) were seeded in 96-well plates and cultured for 24 h at 37 °C in a 5% CO₂ incubator. McCoy’s 5A medium was replaced with 200 μL of fresh medium (control), medium containing fucoidan ScF2 (100, 200, 400, 800 μg/mL) or medium containing resveratrol (5, 10, 20, 40, 80 μM). After 24 h, the MTS reagent (20 μL) was added to each well, and cells were incubated for 3 h at 37 °C in a 5% CO₂ incubator. The intensity of the developed color, which is a reflection of number of live cells, was measured at a wavelength of 490/630 nm in a microplate reader (Power Wave XS, USA). The 50% inhibitory concentration (IC₅₀) was defined as the concentration of the fucoidan or resveratrol that caused a 50% reduction in cell viability. All tested samples were assayed in triplicate.

HCT 116 human colon cancer cells were seeded at a density of 8.0 × 10³ cells/200 μL of complete McCoy’s 5A medium in 96-well plates. After 24 h, incubation was continued for 24, 48 or 72 h in the absence (control) or presence of fucoidan ScF2 (300 μg/mL) or resveratrol (40 μM) alone. To estimate whether fucoidan ScF2 might enhance the antiproliferative effect of resveratrol, HCT 116 cells (8.0 × 10³ cells/200 μL) were pre-treated with resveratrol (40 μM) for 24 h. Culture medium was then substituted with medium containing fucoidan ScF2 (100, 200 and 300 μg/mL), and the cells were incubated for an additional 24 h. At the end of the incubation, the reaction was terminated by adding MTS reagent (20 μL) to each well. Absorbance was measured at 490/630 nm. All assays were performed in triplicate.

HCT 116 human colon cancer cells (6.0 × 10⁵/10 mL) were seeded in 10-cm dishes for 24 h. Cultures were then treated with McCoy’s 5A medium (control) or resveratrol (40 μM). After 24 h, the cultures were treated with McCoy’s 5A medium (control) or fucoidan ScF2 (100, 200, 300 μg/mL) for an additional 24 h. The cells were collected and washed with ice-cold phosphate-buffered saline (1× PBS) and lysed with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 5 mM phenylmethanesulfonylfluoride (PMSF), 1 mM dithiothreitol (DTT) and 1% Triton X-100). Insoluble debris was removed by centrifugation at 12,000 rpm for 10 min, and protein content was determined using Bradford reagent Bio-Rad (Hercules, CA, USA) [50].

Equal amounts of protein (30 μg) were separated electrophoretically by 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF). Membranes were subsequently blocked with 5% skim milk in PBST (1× PBS, 0.05% Tween 20) and hybridized with the primary antibodies against caspase-9 (1:1000), caspase-7 (1:1000), caspase-3 (1:1000), cleaved caspase-9 (1:1000), cleaved caspase-7 (1:1000), cleaved caspase-3 (1:1000) and β-actin (1:500) overnight at 4 °C. After thorough washing with PBST, horseradish peroxidase (HRP)-conjugated secondary antibody from rabbit or mouse (1:5000) was applied, and immune complexes were visualized using the enhanced chemiluminescence (ECL) detection system according to the manufacturer’s instructions Amersham (Buckinghamshire, HP7 9NA, UK).

HCT 116 cells were treated with resveratrol and/or fucoidan ScF2 as stated above (Western blotting assay) and then lysed with lysis buffer (1× PBS, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mg/mL aprotinin, 10 mg/mL leupeptin and 1.6 mg/mL protease inhibitor cocktail. Cell lysates were incubated with antibodies against PARP and cleaved PARP overnight at 4 °C and then with protein A/G-Sepharose beads (Santa Cruz Biotechnology) for an additional 1 h. After centrifugation, the beads were washed three times with wash buffer and resuspended in SDS sample buffer. After heating at 95 °C for 5 min, the samples were loaded onto a 10% gel, and the Western blotting assay was performed.

Apoptosis was detected using the annexin V-FITC apoptosis detection kit as recommended by the manufacturer MBL International Corp. (USA). Apoptosis was compared in HCT 116 cells that were not treated (control) or treated with fucoidan ScF2 (300 μg/mL), resveratrol (40 μM) or resveratrol (40 μM) and fucoidan ScF2 (100, 200, 300 μg/mL) for 48 h. Cells were harvested with 0.025% trypsin/5 mM EDTA in PBS, and 2.5% FBS in PBS was added as soon as the cells were released from the dish. The cells were then transferred to a centrifuge tube, washed with PBS and incubated for 5 min at room temperature with annexin V-FITC plus propidium iodide according to the manufacturer’s protocol. Cells were analyzed on a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, USA), placing the FITC signal in FL1 and the propidium iodide signal in FL2. Intact cells were gated in the forward/side scatter plot to exclude small debris.

HCT 116 human colon cancer cells (2.4 × 10⁴/mL) were seeded in 6-well plates and not treated (control) or treated with fucoidan ScF2 (200 μg/mL), resveratrol (40 μM) or resveratrol and fucoidan ScF2 in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 μg/mL gentamicin. The cultures were maintained at 37 °C in a 5% CO₂ incubator for 14 days and the cell colonies were scored using a Motic AE 20 microscope (China) and the Motic Image Plus computer program.

To estimate the ability of the fucoidan to promote resveratrol-inhibited colony formation, HCT 116 сells (6.0 × 10⁵/10 mL) were plated in 10-cm dishes and cultured in McCoy’s 5A medium with 10% FBS at 37 °C. After incubation for 24 h, the cells were pretreated with fucoidan ScF2 (200 μg/mL) for 24 h. The cells were collected by trypsinization and subjected to the soft agar clonogenic assay described above [51].