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FUCOIDAN IN THE PREVENTION AND TREATMENT OF CHRONIC DISEASE 1

Fucoidan in the Prevention and Treatment of Chronic Disease

Rebecca Behr

Advanced Food Science

Prof. Eileen Fitzpatrick

Russell Sage College


Introduction

Marine algae have long been a part of food and diet. Algae have also been

documented as being used in oriental medicine for more than 1,000 years (Smit, 2004;

Wijesekara, 2010). All classes of algae contain polysaccharides in their cell walls, which

can be commonly found in food and cosmetics, as compounds such as carrageenans,

alginates and agar. Marine algae are classified according to the type of pigment they

contain. These include Rhodophyta (red algae), Phaeophyta (brown algae), and

Chlorophyta (green algae) (Barros Gomes Camara, et al., 2011). The class Phaeophyta, in

particular, contains sulfated polysaccharides, termed fucan or fucoidan (heterofucans),

which are becoming increasingly studied in biomedical research. Each fucoidan is

structurally unique, so the relationship between structure and biological activity has not

yet been found (Barros Gomes Camara, et al., 2011).

According to Tutor Ale, Mikkelsen and Meyer, Fucans were first isolated from

algae in 1913, and since then, approximately 1,800 studies have been published on the

topic, with a significant increase in the last five to ten years (2011). This increase in

fucoidan interest can be credited to the polysaccharide’s potential health benefits, such as

antioxidant, anti-inflammatory, anticoagulant, antithrombotic, antitumor, and antiviral

(Tutor Ale, et al., 2011). Recently, there has been a rise in related chronic conditions,

such as cardiovascular disease (CVD), diabetes and cancer, in part due to lifestyle factors,

aging and urbanization (Barros Gomes Camara, et al., 2011). The current treatments for

these diseases are often painful and have adverse side effects, so the need for alternative

therapies can also be attributed to the increase in fucoidan research.

Despite this plethora of research and medical interest, the mechanism behind

fucoidan’s beneficial activities has yet to be fully understood and applications in


treatment of diseases have not yet been approved for fucoidan. Extraction and

purification methods vary across studies, and the chemical structures of the extracts vary,

as well (Tutor Ale, et al., 2011). For this reason, it is important to note the species of

algae and extraction methods used in fucoidan research.

Anticoagulant and Antioxidant Activities

According to the Centers for Disease Control and Prevention (CDC), CVD is the

leading cause of death in the United States, with about 600,000 adults dying from the

disease, each year (2013). Related conditions, cancinogenesis and diabetes, also make the

CDC’s list of leading causes of death, with 574,743 yearly deaths from cancer and 69,071

from diabetes (2013). Although the causes these diseases cannot be narrowed down to

one single factor, Freinbichler, et al. claim that oxidative stress and the resulting

inflammation has been linked to diabetes, atherosclerosis and cancinogenesis (as cited in

Barros Gomes Camara, et al., 2011). Oxidative stress can be prevented through the use of

antioxidants. Several synthetic antioxidants have been developed and are commonly used

in food products. These include butylated hydroxyanisole (BHA), butylated

hydroxytoluene (BHT), and tertiary butyl hydroquinone (TBHQ). Such antioxidants have

shown to be effective in preventing lipid peroxidation, however, according to Hu, et al.,

the synthetic compounds have possible cancinogenic and toxic health effects (as cited in

Barros Gomes Camara, et al., 2011). Furthermore, fucan has showed promise as an

antioxidant in several applications and has no adverse health effects (Barros Gomes

Camara, et al., 2011).

In atherosclerosis and CVD, anticoagulants are used to prevent thromboembolic

disorders. Some of the most commonly used anticoagulants are unfractionated heparins

and their low weight derivatives (Barros Gomes Camara, et al., 2011). Unfractionated


heparins are sulfured polysaccharides, which inactivate the clotting factor, thrombin

(Hirsh, et al., 2001). According to Barros Gomes Camara, et al., the drug has several

negative side effects including “frequent activated partial thromboplastin time

monitoring, variable anticoagulant effects, the inability to inhibit clot-bound thrombin

and the occurrence of thrombocytopenia, all of which have led to the search for

alternative sources of anticoagulant agents” (2011).

In 2011, Barros Gomes Camara, et al. published a study in Brazil, which sought to

isolate sulfated polysaccharides from Canistrocarpus cervicornis, and evaluate their

anticoagulant and antioxidant activities, in vitro. The C. cervicornis was collected from

Búzios Beach, Nísia Floresta, Brazil. The samples were then dried in an oven, ground in

a blender, and incubated with acetone to remove lipids and pigments. Next the samples

underwent a pH adjustment, proteolytic enzyme digestion and further incubation. It was

then filtered through a cheese cloth and fractionated, so the resulting precipitate could be

collected by centrifugation, vacuum dried, re-suspended in distilled water, and stained

with toluidine blue, to reveal that the samples consisted solely of sulfated polysaccharides

(Barros Gomes Camara, et al., 2011).

Six families of heterofucans were obtained from C. cervicornis, denoted CC-0.3,

CC-0.5, CC-0.7, CC-1.0, CC-1.2, and CC-2.0 (see Appendix A, Figure A1) (Barros

Gomes Camara, et al., 2011). Anticoagulant activity was measured using prothrombin

time (PT) and activated partial thromboplastin time (aPPT) coagulation assays. In the PT

test, no clotting activity was found for any of the families. In contrast, each family

extended aPPT, in a dose-dependent manner. “Heterofucans CC-0.3, CC-0.5, CC-0.7, and

CC-1.0 doubled aPPT with only 0.1 mg/mL of plasma, a result similar to that of

Clexane®, a commercial low molecular weight heparin that had the same effect with 0.08


mg/mL of plasma” (Barros Gomes Camara, et al., 2011). Researchers also concluded that

heterofucans act via the intrinsic pathway for coagulation and effects are specific to the

sulfation site and/or glycosidic linkage within fucoidan, not sulfate charge density or

amount (Barros Gomes Camara, et al., 2011).

Antioxidant activity was evaluated using total antioxidant capacity, superoxide

radical scavenging, hydroxyl radical scavenging, and metal-chelating property. Although

none of the heterofucans showed significant antioxidant effects on hydroxyl radicals, it is

important to note that CC-1.2 showed excellent superoxide radical scavenging activity

(see Figure A2) (Barros Gomes Camara, et al., 2011). The superoxide radical is a

precursor to the hydroxyl radical; therefore, heterofucans may be able to inhibit this

radical by preventing its formation. For the metal-chelating property test, antioxidant

activity was varied among the heterofrucans, with CC-0.5, CC-0.7, and CC-1.0 showing

the best capacity (see Figure A3) (Barros Gomes Camara, et al., 2011). The researchers

concluded that heterofucans, specifically CC-0.7 and CC-1.2, have strong anticoagulant

and antioxidant activities, and therefore are “potential multipotent drugs” (Barros Gomes


Antitumor and Antimetastatic Activities

Not only does fucoidan show promise as an antioxidant, which can help prevent

cancer, but it also has shown the potential to prevent the spread of existing cancer cells.

Metastasis occurs when cancer cells break off the primary tumor and move through the

blood and lymphatic vessels to invade other organs. This phenomenom causes up to 90

percent of cancer-related deaths (Lee et al., 2012). Matrix metalloproteinases (MMPs)

play a key role in the mechanism behind tumor metastasis, by degrading extracellular

matrix proteins such as collagen, proteoglycan, elastin, laminin, and fibronectin. Cancers


with the highest levels of MMPs, such as lung cancer, are particularly aggressive and

metastatic (Lee et al., 2012).

Due to the harsh nature of current antitumor treatments, such as chemotherapy,

more research is being conducted to identify potential therapies from natural sources (Lee

et al., 2012). A South Korean study conducted by Lee, J. Kim, and E. Kim (2012) sought

to assess and understand the mechanism of fucoidan anti-metastatic effects, using a

highly metastatic form of human lung cancer, denoted A549. For this study, fucoidan

extract was obtained from Fucus vesiculosus, of Sigma, St. Louis, Missouri. The powder

was dissolved in phosphate buffer solution and sterilized, using a filter. First, Twelve-

well plates were inoculated with A549 cells, and allowed to grow for 24 hours. After

incubation, non-invading cells were removed, and the remaining invasive cells were

inoculated with various concentrations of fucoidan and incubated for 48 hours (Lee et al.,

2012).

At concentrations of 400-1,000 µg/ml, “fucoidan inhibited cell proliferation and

decreased cell viability in a concentration-dependent manner” (see Figure B1) (Lee et al.,

2012). At a concentration of 200 µg/ml, fucoidan reduced MMP-2 activity by 72 percent

and inhibited MMP-2 protein expression by 60 percent (see Figure B2) (Lee et al., 2012).

The effects of fucoidan on A549 cell invasion were evaluated using an invasion chamber

kit. At 200 µg/ml, cell invasion was inhibited by 86 percent (see Figure B3) (Lee et al.,

2012). Finally researchers found that fucoidan inhibited several pathways by which

cancer cells migrate and invade in humans, such as the ERK1/2 and PI3K pathways.

From these results the researchers concluded that because fucoidan is indeed an anti-

metastatic reagent and shows low side effect in normal cells, further in vivo studies and

clinical investigations are needed to apply fucoidan as a new cancer therapy.


Antiviral Activities

Fucoidan’s inhibitory abilities extend beyond just tumor inhibitions. A study by

Baba, Snoeck, Pauwels, and de Clercq (1988) found that fucoidan has the potential to

inhibit viral replication including herpes simplex virus and human immunodeficiency

virus (as cited in Mori, Nakasone, Tomimori, & Ishikawa, 2012). Hepatitis C virus

(HCV) currently has infected about 170 million people globally, with an estimated 3.5

million people newly diagnosed, each year (Mori, et al., 2012). This virus is extremely

virulent, devastating to the liver, and chronic infection can cause liver cirrhosis (LC),

which can lead to liver cancer (Chen, et al., 2013). HCV strains vary greatly, so no

vaccine currently exists. Current antiviral treatments can be sometimes results in serious

side effects and a significant portion of those treated are non-responders (Mori, et al.,

2012).

In Japan, Mori, et al. conducted a study, which assessed the antiviral activity of

fucoidan on HCV, both in vivo and in vitro (2012). The fucoidan used was extracted

from Cladosiphon okamuranus Tokida, cultivated from Okinawa, Japan. The alga was

cooked in a water-acid solution, neutralized and cooled, centrifuged, filtered, then dried.

Study participants consisted of 15 chronic liver disease patients, all infected with HCV.

Six of the patients were non-responders to standard treatments and six suffered from LC

(Mori, et al., 2012).

Researchers found from the in vitro testing that fucoidan inhibited HCV

replication, as indicated by luciferase (LUC) activity, but did not exhibit cytotoxic

effects, as cell viability was not significantly affected (see Figure C1) (Mori, et al., 2012).

These results were similar to those found in vivo. There was no significant decrease in

HCV RNA levels or clinical symptoms in the participants, however, none of the patients


showed progression of LC during the 12-month treatment (see figure C2). It is important

to note, however, that the patient pool was small and contained non-responders to

standard treatments (Mori, et al., 2012).

In Taiwan, a similar study was conducted by Chen, et al., which assessed the

effects of Gracilaria tenuistipitata (AEGT) extract on HCV replication, in vitro, and

indentified the mechanism behind these effects (2013). The extracts were cultivated from

Kouhu beach, Taiwan, soaked in distilled water and dried. They were then pulverized and

filtered. The researchers found that AEGT inhibited HCV protein synthesis and RNA

synthesis in a dose-dependent manner however; these effects were not cytotoxic (see

Figure C3). The researchers then combined AEGT with the standard HCV treatment,

pegylated-interferon-α (IFN-α), which resulted in synergistic inhibition. Researchers

concluded that AEGT was useful in treatment and prevention of chronic HCV infection,

however, since a crude extract was used, further purification would be necessary for

future studies (Chen, et al., 2013).

Extraction Methods and Molecular Structure

Although fucoidan has shown immense potential for the treatment of

aforementioned chronic diseases, the relationship between the beneficial activities of

fucoidan and the structure of the molecules has not yet been completely understood.

According to a review by Tutor Ale, et al., this understanding has been delayed by the

lack of extraction and purification standards and protocol in fucoidan research (2011).

The authors also state that the methods of extraction affect the final structure of the

fucoidan molecules, which has an effect on their biological properties. It has been noted

across various studies, that the degree of sulfation and size of the fucoidan molecules has

a direct impact on their antitumor and anticoagulant properties (Tutor Ale, et al., 2011).


Tutor Ale, et al., suggest that standard extraction procedures should be developed, which

include “hydrolysis treatment, purification and fractionation methodology, preferably

with specific steps adapted to the particular botanical order of the seaweed” (2011).

Conclusion

Current research has shown that fucoidan, extracted from brown algae, has

anticoagulant, antioxidant, antitumor, and antiviral properties. Fucoidan is a potential

effective treatment for chronic disease, such as CVD and cancer, and is a more healthful

alternative to current treatments. Despite ample research on the topic, standard protocol

for fucoidan extraction and purification must be developed, so that fucoidan can be better

understood and eventually approved for use in disease prevention and treatment (Tutor

Ale, et al., 2011).


References

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potent and selective inhibitors of various enveloped viruses, including herpes

simplex virus, cyto- megalovirus, vesicular stomatitis virus, and human immu-

nodeficiency virus. Antimicrobial Agents and Chemotherapy 32, 1742-1745.

Barros Gomes Camara, R.; Silva Costa, L.; Pereira Fidelis, G.; Duarte Barreto Nobre,

L.T.; Dantas-Santos, N.; Lima Cordeiro, S.; Santana Santos Pereira Costa, M.;

Guimaraes Alves, L.; Oliveira Rocha, H.A. (2011). Heterofucans from the Brown

Seaweed Canistrocarpus cervicornis with Anticoagulant and Antioxidant

Activities. Marine Drugs, 9, 124-138. doi:10.3390/md9010124

Centers for Disease Control and Prevention. (2013). Heart Disease Fact Sheet. Retrieved

from http://www.cdc.gov/dhdsp/data_statistics/fact_sheets/fs_heart_disease.htm

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Death. Retrieved from http://www.cdc.gov/nchs/fastats/lcod.htm

Chen, K.J., Tseng, C.K., Chang, F.R., Yang, J.I., Yeh, C.C., Chen, W.C., Wu, S.F.,

Chang, H.W., Lee, J.C. (2013). Aqueous extract of the edible Gracilaria

tenuistipitata inhibits hepatitis C viral replication via cyclooxygenase-2

suppression and reduces virus-induced inflammation. PLoS One, 8(2). doi:

10.1371/journal.pone.0057704

Freinbichler, W., Bianchi, L., Colivicchi, M.A., Ballini, C., Tipton, K.F., Linert, W.,

Corte, L.D. (2008). The detection of hydroxyl radicals in vivo. Journal of

Inorganic Biochemistry 102, 1329-1333.


Hirsh, J., Anand, S.S., Halperin, J.L., & Fuster, V. (2001). Mechanism of Action and

Pharmacology of Unfractionated Heparin. Arteriosclerosis, Thrombosis, and

Vascular Biology 21: 1094-1096. doi: 10.1161/hq0701.093686

Hu, T., Liu, L., Chen, Y., Wu, J., Wang, S. (2010). Antioxidant activity of sulfated

polysaccharide fractions extracted from Undaria pinnatifida in vitro. International

Journal of Biological Macromolecules 46, 193-198.

Lee H., Kim J.S., Kim E. (2012) Fucoidan from Seaweed Fucus vesiculosus Inhibits

Migration and Invasion of Human Lung Cancer Cell via PI3K-Akt-mTOR

Pathways. PLoS ONE 7(11). doi:10.1371/journal.pone.0050624

Mori N., Nakasone K., Tomimori K., Ishikawa C. (2012). Beneficial effects of fucoidan

in patients with chronic hepatitis C virus infection. World Journal of

Gastroenterology, 18(18): 2225-30. doi: 10.3748/wjg.v18.i18.2225.

Smit, AJ. (2004). Medicinal and pharmaceutical uses of seaweed natural products: a

review. Journal of Applied Phycology 16, 245–262.

Tutor Ale, M., Mikkelsen, J.D., Meyer, A.S. (2011). Important Determinants for

Fucoidan Bioactivity: A Critical Review of Structure-Function Relations and

Extraction Methods for Fucose-Containing Sulfated Polysaccharides from Brown

Seaweeds. Marine Drugs 9(10), 2106-2130. doi:10.3390/md9102106

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Polymers 84, 14–21.


Appendix A

Fucoidan Anticoagulant and Antioxidant Activities

Figure A1

“Anticoagulant activity by aPTT test. Results were expressed as aPTT ratio, obtained by

dividing clotting time achieved with the anticoagulant by that obtained with the control.

Data are expressed as means ± standard deviation of four determinations; a,b,c,d

Different letters indicate a significant difference between each concentration of the same

sulfated polysaccharide using one-way Anova followed by the Student-Newman-Keuls

test (p < 0.05). Cx: Clexane®” (Barros Gomes Camara, et al., 2011)

Figure A2

“Total antioxidant capacity of sulfated polysaccharides extracted from the marine brown

seaweed C. cervicornis. The results are expressed as ascorbic acid equivalents. Each

value is the mean ± SD of three determinations: a,b,c Different letters indicate a

significant difference (p < 0.05) between sulfated polysaccharides” (Barros Gomes


Mar. Drugs 2011, 9

129

F igure 1. Anticoagulant activity by aPTT test. Results were expressed as aPTT ratio, obtained by dividing clotting time achieved with the anticoagulant by that obtained with the control. Data are expressed as means ± standard deviation of four determinations; a,b,c,d Different letters indicate a significant difference between each concentration of the same sulfated polysaccharide using one-way Anova followed by the Student-Newman-Keuls test (p < 0.05). Cx: Clexane®.

2.3. Antioxidant Activity

Marine algae inhabit primarily intertidal areas, a harsh environment where they are subjected to repeated immersion and emersion due to tidal fluctuations. As a result, they are exposed twice daily to a wide range of environmental stress, including exposure to ultraviolet radiation, rapid temperature fluctuations, osmotic stress, and desiccation [29,30]. Some of these factors contribute to free radical generation. However, these organisms are not seriously damaged by the presence of these reactive species, indicating that they possess protective mechanisms mediated by enzymes or antioxidant compounds [1,30]. Corroborating these data, several sulfated polysaccharides from marine algae have been recently described as potential antioxidant compounds [2,13 15].

In the present study, in vitro antioxidant activity of sulfated polysaccharides from C . cervicornis was evaluated using the following assays: total antioxidant capacity, superoxide radical scavenging, hydroxyl radical scavenging and metal-chelating property.

2.3.1. Total Antioxidant Capacity

As total antioxidant capacity is indicative of oxidative stress or increased susceptibility to oxidative damage, this assay was used to determine the antioxidant potential of polysaccharides from C . cervicornis (Figure 2).

Total antioxidant capacity of sulfated polysaccharides ranged from 20.9 (CC-1.0) to 39.4 mg/g (CC-0.3) of ascorbic acid equivalents. Acetone fractionation increased total antioxidant capacity of the heterofucans, since sulfated polysaccharide-rich extract from C . cervicornis activity was not higher than 20 mg/g of ascorbic acid equivalent [2]. In a previous study using extracts from seaweeds


Figure A3

“Ferrous chelating activity of sulfated polysaccharides from the brown seaweed C.

cervicornis. Each value is the mean ± standard deviation of three determinations: a,b,c

Different letters indicate a significant difference (p < 0.05) between each concentration of

the same sulfated polysaccharide” (Barros Gomes Camara, et al., 2011).

Mar. Drugs 2011, 9

130

Padina tetrastomatica and Turbinaria conoides, Chandini and co-workers found 9.79 and 9.65 mg/g of ascorbic acid equivalent, respectively, which was considered an elevated total antioxidant capacity [31]. Thus, the values detected here for sulfated polysaccharides from C . cervicornis are extremely interesting, leading us to further testing of different antioxidant assays to determine their possible antioxidant mechanisms.

F igure 2. Total antioxidant capacity of sulfated polysaccharides extracted from the marine brown seaweed C . cervicornis. The results are expressed as ascorbic acid equivalents. Each value is the mean ± SD of three determinations: a,b,c Different letters indicate a significant difference (p < 0.05) between sulfated polysaccharides.

2.3.2. Hydroxyl and Superoxide Radical Scavenging

Hydroxyl radicals and superoxide anions are reactive oxygen species (ROS) implicated in cell damage. The occurrence of oxidative stress is correlated with numerous pathologies, including neurodegenerative diseases, ischemic or traumatic brain injuries, cancer, diabetes, liver injury, and AIDS [11].

The results of hydroxyl and superoxide scavenging activity are exhibited in Table 3. All sulfated polysaccharides extracted from C . cervicornis showed low hydroxyl radical scavenging activity, not exceeding 3% inhibition.

The absence or presence of low activities in the hydroxyl radical scavenging assay is common in sulfated polysaccharides extracted from brown algae [2,5]. This shows that hydroxyl radical scavenging is probably not the major antioxidant mechanism of these heterofucans.

In the superoxide scavenging assay, fucan CC-1.0 showed the lowest antioxidant activity up to the maximum concentration evaluated. CC-0.3, CC-0.5, CC-0.7, and CC-2.0 had only moderate activities, ranging from 13.0% (CC-0.5) to 18.4% (CC-0.7) at 0.1 mg/mL. Heterofucan CC-1.2 deserves special attention, since it had excellent superoxide radical scavenging activity (43.1%) at 0.1 mg/mL, higher than gallic acid, a positive control, at the same concentration (41.8%) and higher than the sulfated polysaccharide-rich extract from C . cervicornis [2].

The literature has systematically reported several sulfated polysaccharides extracted from algae with superoxide radical scavenging activity [2,14,32]. The reactivity of the superoxide anion is too low and

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2.3.3. Chelating Effect on Ferrous Ions

Antioxidants inhibit interaction between metals and lipids through the formation of insoluble metal complexes with ferrous ion or generation of steric resistance. Furthermore, transition metal ions can react with H2O2, generating hydroxyl radicals, the most reactive free radicals [33].

The chelating effect on ferrous ions exhibited by sulfated polysaccharides extracted from C . cervicornis is shown in Figure 3. The results revealed that heterofucan CC-0.3 did not display significant ferrous chelating capacity, while CC-1.2 and CC-2.0 showed moderate activity at the maximum concentration tested, chelating 33.3% and 39.7% of ferrous ions, respectively. CC-0.5, CC-0.7, and CC-1.0 showed similar dose-dependent activity, reaching maximum activity (about 47.0%) at a concentration of 2.0 mg/mL. This was only 1.8 times lower than EDTA activity at the same concentration under the same experimental conditions (data not shown). The purification process did not increase the chelating effect of the heterofucans compared to the chelating effect of sulfated polysaccharide-rich extract from C . cervicornis [2].

F igure 3. Ferrous chelating activity of sulfated polysaccharides from the brown seaweed C . cervicornis. Each value is the mean ± standard deviation of three determinations: a,b,c Different letters indicate a significant difference (p < 0.05) between each concentration of the same sulfated polysaccharide.

The results obtained by heterofucans, with the exception of CC-0.3, were higher than those shown previously by Costa and co-workers where a sulfated polysaccharide-rich extract from C . cervicornis had a chelating capacity of about 30.0% at a concentration of 2.0 mg/mL [2]. These heterofucans still had superior activity to that of sulfated polysaccharides extracted from Laminaria japonica [34], low molecular and high sulfated derivatives from polysaccharides extracted from Ulva pertusa [32], and sulfated polysaccharide-rich extracts from the algae Dictyopteris delicatula, Dictyota menstrualis, Sargassum filipendula, Spatoglossum schroederi, Gracilaria caudata, Caulerpa cupressoides and Codium isthmocladum [2].


Appendix B

Fucoidan Antimetastatic and Antitumor Activities

“The data shown are the means 6 SD of three experiments. Significant difference from

control group, *p,0.05 and **p,0.01” (Lee et al., 2012).

Figure B1

“MMP-2 activity by analysis of zymography was quantified by measuring the band

intensities using Image J software” (Lee et al., 2012).

Figure 1. Effect of fucoidan on MMP-2 activity of A549 cells. (A) Subconfluent A549 cells were incubated for 48 hr in the absence or presenceof fucoidan (0, 12.5, 25, 50, 100, and 200 mg/ml) in serum-free RPMI media. The conditioned media were collected, and their MMP-2 activity wasestimated using gelatin zymography. (B) MMP-2 activity by analysis of zymography was quantified by measuring the band intensities using Image Jsoftware. (C) Total cell lysates were subjected to Western blot analysis probed with anti-MMP-2 antibody. (D) Expression of MMP-2 by analysis ofwestern blot was quantified by measuring the band intensities using Image J software. The data shown are the means 6 SD of six experiments.Significant difference from control group, *p,0.05 and **p,0.01.doi:10.1371/journal.pone.0050624.g001

Fucoidan Inhibits Cancer Cell Metastasis

PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e50624


Figure B2

“Expression of MMP-2 by analysis of western blot was quantified by measuring the band

intensities using Image J software” (Lee et al., 2012).

Figure B3

“The amount of invading cells was quantified using Image J software” (Lee et al., 2012).

Figure 1. Effect of fucoidan on MMP-2 activity of A549 cells. (A) Subconfluent A549 cells were incubated for 48 hr in the absence or presenceof fucoidan (0, 12.5, 25, 50, 100, and 200 mg/ml) in serum-free RPMI media. The conditioned media were collected, and their MMP-2 activity wasestimated using gelatin zymography. (B) MMP-2 activity by analysis of zymography was quantified by measuring the band intensities using Image Jsoftware. (C) Total cell lysates were subjected to Western blot analysis probed with anti-MMP-2 antibody. (D) Expression of MMP-2 by analysis ofwestern blot was quantified by measuring the band intensities using Image J software. The data shown are the means 6 SD of six experiments.Significant difference from control group, *p,0.05 and **p,0.01.doi:10.1371/journal.pone.0050624.g001



Western BlottingThe protein components (50 mg) were separated on 12% SDS-

polyacrylamide gel, transferred to polyvinylidene difluoridemembranes (Bio-Rad, C.A. USA), and subsequently subjected toimmunoblot analysis using specific primary antibodies for over-night at 4uC. After washing, the membranes were incubated withhorseradish peroxidase-conjugated secondary antibody (Cell Sig-naling Technology, Beverly, MA) for 1 hr at room temperature.The blots were then visualized by using the enhanced chemilu-minescence method (ECL, Amersham Biosciences, Buckingham-shire, UK) on blue light-sensitive film (Fujifilm Corporation,Tokyo, Japan). If necessary, the blotted membranes were strippedand reprobed using other primary antibodies. Densitometryanalysis was performed with a Hewlett-Packard scanner andNIH Image software (Image J).

Statistical AnalysisThe results are expressed as the mean 6 standard deviation

(S.D.). One-way analysis of variance (ANOVA) was used toevaluate the significance of difference between the two meanvalues. *P,0.05 and **P,0.01 were considered to be statisticallysignificant.

Results

Fucoidan Inhibits the Activity of MMP-2 in A549 HumanLung Cancer CellsIn order to determine non-cytotoxic concentration of fucoidan,

A549 cells were incubated with various concentrations of fucoidan(0–1000 mg/ml) for 24 hr or 48 hr, and then cell viability wasdetermined using MTT assay (data not shown). Fucoidan did notshow any significant toxic effect on A549 cells within theconcentration range of 0–200 mg/ml. At higher concentrations(400–1000 mg/ml), however, fucoidan inhibited cell proliferationand decreased cell viability in a concentration-dependent manner.Therefore, the present study applied the treatment of fucoidan atconcentrations equal to or lower than 200 mg/ml, at which nosignificant cytotoxic effect of A549 cells was observed.The degradation of extracellular matrix (ECM) is crucial for

cellular invasion, indicating the inevitable involvement of matrix-degrading proteinases for the process. Hence, it was assessed theeffect of fucoidan on MMP-2 activity, as a key molecule of ECMdegradation, by gelatin zymography. As shown in Figure 1A,fucoidan suppressed MMP-2 activity in a concentration-depen-dent manner. On the other hand, the impact of fucoidan on theMMP-9 activity was inconclusive, since there is only an extremelylow level of intrinsic MMP-9 activity detectable in A549 cells (datanot shown). MMP-2 activity was reduced by 36%, 53%, and 72%at 50, 100, and 200 mg/ml, respectively, after treatment with the

Figure 2. Effects of fucoidan on migration of A549 cells. (A)A549 cells were plated on 12-well and grown to 90% confluence inRPMI containing 10% FBS. Cells were scratched with a pipette tip andthen treated with fucoidan (0, 50, 100, and 200 mg/ml). (B) Cellmigration distance was estimated by measuring the width of thewound. The data shown are the means 6 SD of six experiments.Significant difference from control group, *p,0.05 and **p,0.01.doi:10.1371/journal.pone.0050624.g002

Figure 3. Effect of fucoidan on invasion of A549 cells. (A) Effectsof fucoidan on cancer cells invasion were examined using Cell InvasionAssay kit (BD Bioscience, San Jose, CA, USA). For this, A549 cells wereseeded onto the upper chamber of Matrigel-coated filter and incubatedin the absence or the presence of fucoidan (0, 50, 100, and 200 mg/ml)for 48 hr. The invading cells on the lower surface of the membranewere visualized by crystal-violet staining and observed with lightmicroscope. (B) The amount of invading cells was quantified usingImage J software. The data shown are the means 6 SD of threeexperiments. Significant difference from control group, *p,0.05 and**p,0.01.doi:10.1371/journal.pone.0050624.g003




Appendix C

Fucoidan Antiviral Activity

Figure C1

“Anti-hepatitis C virus effects of fucoidan in hepatitis C virus replicon cells. Luciferase

(LUC) activity (a marker of replication level) and cell viability of FLR3-1 cells, which

constitutively express hepatitis C virus replicon, were measured in the presence of

various concentrations of fucoidan. LUC and WST-8 assays were performed in triplicate.

Data are mean ± SD” (Mori, et al., 2012).

Figure C2

“Effects of treatment with fucoidan on hepatitis C virus RNA and alanine

aminotransferase levels in patients with liver diseases. Serum alanine aminotransferase

(ALT) levels, indicative of HCV RNA activity. Values are mean ± SD. bP < 0.01 vs

pretreatment value” (Mori, et al., 2012). Eight patients were not eligible for IFN treatment be-cause of LC, complication (depression), or advanced age. Seven patients had received IFN therapy in the past. Six patients were non-responders to IFN and 1 discontinued therapy because of side effect (depression). All patients assessed the tolerability as excellent.

During fucoidan treatment, 11 patients received a -

hide Bio Co., Okinawa, Japan) was given orally as cap-sules containing 166 mg of dry extract from C. okamura-nus Tokida12 mo. Informed consent was obtained from all patients enrolled in the study, after a thorough explanation of the

Test parametersThe outcome parameters included the course of alanine aminotransferase (ALT), aspartate aminotransferase (AST), quantitative HCV RNA levels, subjective symp-toms associated with CHC, LC, and HCC (such as fa-tigue, abdominal discomfort, depression, and dyspepsia), safety, and compliance. Data on all clinical parameters were documented at each visit. HCV RNA levels were determined using the AMPLICOR GT HCV Monitor

a lower limit of quantitation of 0.5 kIU/mL at a linear range up to 850 kIU/mL.

Statistical analysis Data are expressed as mean ± SD. The results of bio-chemical tests and HCV RNA levels were compared by the Student’s t test. A P

!"#$%Fucoidan suppresses HCV replication To assess the effects of fucoidan on intracellular replica-

tion of the HCV genome, HCV subgenomic replicon cells FLR3-1 were cultured in the presence of various concentrations of fucoidan in the medium. The luciferase activities of the FLR3-1 cells showed a concentration-dependent suppression of replication of HCV replicon by fucoidan. The WST-8 assay showed that fucoidan had negligible effect on cell viability (Figure 1). These results suggest that fucoidan inhibits HCV replication but does not have cytotoxic effects.

Effect of fucoidan therapy on HCV RNA and alanine aminotransferase levels Changes in HCV RNA and serum ALT levels in patients treated with fucoidan are shown in Figure 2. The mean HCV RNA for the 15 patients was 736 ± 118 kIU/mL (range, 100-850 kIU/mL) before fucoidan therapy. As shown in Figure 2A, fucoidan tended to reduce the mean HCV RNA level with time relative to the baseline,

However, HCV RNA increased after 11 and 12 mo. Bio-chemical tests showed that the mean serum ALT level, but not AST, correlated with the mean HCV RNA level, although the decrease in ALT level was not significant relative to the baseline (Figure 2B). Whereas the above changes were not associated with improvement in clinical symptoms in every patient, none of the patients showed progression of LC or adverse events.

'(#)$##(*+It is estimated that 170 million people worldwide are infected with HCV[3], and some 2 million (1%) of these reside in Japan[23]. Of the HCC cases in Japan, around 80% are caused by HCV infection. The increase in the number of HCC patients contributes to the increase in total deaths in Japan from HCC. This trend is expected to continue until 2015[23]. The general strategies followed in the treatment of CHC include eradication of HCV and suppression of hepatitis.

2227 May 14, 2012|Volume 18|Issue 18|WJG|www.wjgnet.com

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Figure C3

“Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCV

replicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular

gapdh mRNA levels following AEGT treatment for 3 days. Cellular toxicity was

evaluated by the MTS assay after 3 days of incubation with AEGT” (Chen, et al., 2013).

Sulfated polysaccharides including fucoidan are report-ed to inhibit the growth of various enveloped viruses[16-18]. Fucoidan is thought to inhibit virus adsorption to the cell surface by binding to the cell surface, with subsequent prevention of cell infection[18]. In addition, fucoidan in-teracts directly with the envelope glycoprotein on dengue virus type Ⅱ[17]. In the present study, we demonstrated a novel mechanism of action for fucoidan. Using the HCV replicon system, we demonstrated here that fucoidan in-hibits intracellular replication of the HCV genome in vitro.

To our knowledge, this is the first clinical study to investigate the effects of fucoidan in patients with liver diseases. The rationale for this study was stimulated by

cell cultures. Patients with chronic HCV infection, who were not eligible for, did not respond to, or were intoler-ant of IFN treatment, were treated for 12 mo with fu-coidan at 830 mg/d to investigate the effect of this treat-ment on HCV RNA level. In Case 6 (baseline HCV RNA 380 kIU/mL), fucoidan treatment successfully eradicated HCV at 9 mo, although HCV RNA was 5 kIU/mL at 10 mo. Thus, fucoidan was effective in lowering HCV RNA level in this study, although its effect was tempo-rary. There was a significant decrease in HCV RNA at month 8, 9 and 10 of fucoidan commencement (P < 0.01). However, the level increased later at month 12 to become equivalent to the baseline.

We also measured serum IFN levels to determine the indirect effect of fucoidan on IFNs, especially wheth-er it increases the antiviral activity of IFNs. However, IFN could not be detected in the serum of patients treated with fucoidan. Furthermore, fucoidan did not en-hance IFNs expression in FLR3-1 replicon cells (data not shown). It has been reported that the protective effect of fucoidan is based on direct inhibition of viral replica-tion and stimulation of both innate and adaptive immune defense functions[24]. We are currently investigating the effect of fucoidan on the host immune system including natural killer cell cytotoxic activity.

Our study has certain limitations. First, the study comprised only a small number of patients, including 6 patients who were known non-responders to IFN thera-py. Second, all patients harbored HCV virus genotype Ⅰb and 6 had cirrhosis. Thus, at least some patients in this cohort could be classified as likely non-responders to IFN therapy[25,26]. Thus, the selection criteria employed in the present study may have favored a poor response to fucoidan.

The abnormally high levels of ALT tended to decrease temporarily during fucoidan treatment, suggesting a cor-relation between viral load and indices of hepatic dysfunc-tion. Thus, fucoidan may be effective in the management of HCV-related chronic liver diseases, although long-term clinical improvement was not observed in the present study. Importantly, no adverse events were observed in all patients, similar to the results reported in a previously study on fucoidan[19], suggesting that daily oral administra-tion of fucoidan for 12 mo is safe and tolerable.

There is no doubt that patients who fail to respond to conventional treatments often seek alternative therapies. In conclusion, our study demonstrated that fucoidan from C. okamuranus Tokida has HCV replication suppressive ef-fects in a replicon cell system. Furthermore, our relatively small uncontrolled pilot study showed that fucoidan has

that fucoidan may be a useful health-food additive with antiviral activity to be used in the treatment of chronic liver diseases. To suppress the viral titer as much and for

dosage. Further studies on the mechanism of fucoidan-induced HCV inhibition may provide alternative strategies for the design of novel anti-HCV drugs.

!"#$%&'()*+($,-

replicon cells and technical assistance. We are grateful to

"%++($,-BackgroundHepatitis C virus (HCV) is a major cause of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The standard care

2228 May 14, 2012|Volume 18|Issue 18|WJG|www.wjgnet.com

."%++($,-

Figure 2 Effects of treatment with fucoidan on hepatitis C virus RNA and alanine aminotransferase levels in patients with liver diseases. A: Hepatitis C virus (HCV) RNA levels; B: Serum alanine aminotransferase (ALT) levels. Values are mean ± SD. bP < 0.01 vs pretreatment value.

900

800

700

600

500

400

300

200

100

0

HCV

RN

A (k

IU/m

L)

b

b b

0 1 2 3 4 5 6 7 8 9 10 11 12

Duration of treatment (mo)

A

120

100

80

60

40

20

0

ALT

(IU

/L)

0 1 2 3 4 5 6 7 8 9 10 11 12

Duration of treatment (mo)

B

Mori N et al . Fucoidan therapy for patients with HCV

Transient Transfection and Luciferase Activity AssayAva5 cells were seeded in 24-well plates at a density of

56104 per well and were transfected with 1 mg of plasmid pCOX-2-Luc or pNF-kB-Luc (BD Biosciences Clontech, Palo Alto, CA)using T-ProTM reagent (Ji-Feng Biotechnology Co. Ltd., Taiwan)in accordance with the manufacturer’s instructions. Six hours post-transfection, the cells were treated with various concentrations ofAEGT for 3 days. To further investigate COX-2 regulation byAEGT, Ava5 cells were transfected with serially increasedconcentrations of COX-2 expression vector (pCMV-COX-2-Myc) from 0.5 to 2 mg, and then treated with 600 mg/ml ofAEGT for 3 days. The luciferase activity of cell extracts from eachsample was measured using the Bright-GloTM Luciferase AssaySystem (Promega) according to the manufacturer’s protocol. Todetermine the relationship between AEGT and NS5A-inducedinflammatory genes (TNF-a, IL-1b, iNOS and COX-2), Huh-7cells were transfected with 1 mg of plasmid pCMV-NS5A-Myc inthe presence of AEGT (200 and 800 mg/ml) for 3 days.Subsequently, total RNA was subjected to qRT-PCR with specificprimers.

Intracellular Prostaglandin E2 (PGE2) MeasurementsAva5 cells were treated with AEGT at various concentrations

for 3 days. Following drug treatment, cultured cells werethoroughly washed with cold phosphate-buffered saline (pH 7.4),and cell membranes were ruptured using lysis reagent containingC15H34BrN to release intracellular PGE2. The levels of PGE2

levels were then analyzed by PGE2 enzyme-linked immunosorbentassay system (Biotrak, Amersham Bioscience) according to themanufacturer’s protocol.

Preparation of Nuclear FractionNuclear extracts were prepared using NE-PER Nuclear and

Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc.,

Figure 1. Inhibitory effect of AEGT on HCV protein synthesis, RNA replication and HCV infection. (A) Concentration-dependentreduction of HCV NS5B protein levels in AEGT-treated HCV replicon cells. Ava5 cells were treated with AEGT for 3 days at the indicatedconcentrations. Treatment with 100 U/ml IFN-a served as a positive control. Equal amounts of protein extracts were subjected to Western blottingwith anti-NS5B and anti-GAPDH (a loading control) antibodies. (B) Concentration-dependent reduction of HCV RNA replication in AEGT-treated HCVreplicon cells. HCV RNA levels were quantified by qRT-PCR and normalized to cellular gapdh mRNA levels following AEGT treatment for 3 days.Cellular toxicity was evaluated by the MTS assay after 3 days of incubation with AEGT. (C) Concentration-dependent reduction of infectious HCV JFH-1 replication in AEGT-treated Huh-7 cells. After 6 h of JFH-1 infection, Huh7-infected cells were treated with AEGT for 3 days. The levels of intracellularHCV RNA were determined by qRT-PCR following normalization of cellular gapdh mRNA. Results are represented as the mean 6 SD (error bar) ofthree independent experiments. *P,0.05; ** P,0.01.doi:10.1371/journal.pone.0057704.g001

Table 1. Combined AEGT and IFN-a inhibitory effects on HCVRNA replication.

IFN-a:AEGT CI Values at Influence

ED50 ED75 ED90

1:5 0.86 0.79 0.73 Synergistic

1:10 0.85 0.82 0.78 Synergistic

1:20 0.52 0.67 0.89 Synergistic

Ava5 cells were treated with combinations of various concentrations of AEGTand IFN-a (1:5, 1:10 or 1:20) for 3 days. Anti-HCV activity was reflected by thereduction of HCV RNA level using qRT-PCR analysis. Mutual influence betweenAEGT and IFN-a was calculated using the CalcuSynTM software, which producesthe combination index (CI) values for various effective doses of 50% (ED50), 75%(ED75) and 90% (ED90). CI values indicate the degree of interaction of potentialdrugs; values of ,1, 1 and .1 indicate synergistic, additive, or antagonisticeffect, respectively.doi:10.1371/journal.pone.0057704.t001

Anti-HCV of Gracilaria tenuistipitata Extract

PLOS ONE | www.plosone.org 3 February 2013 | Volume 8 | Issue 2 | e57704

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Fucoidan in the Prevention and Treatment of Chronic ...rebecccabehr.weebly.com/.../7/5/9/27594381/fucoidan... · The effects of fucoidan on A549 cell invasion were evaluated using