Cytotoxicity of Fucoidan from Three Tropical Brown Algae Against Breast and Colon Cancer Cell Lines

Introduction: Fucoidan is a sulfated polysaccharide that has a wide range of bioactivities including anti-cancer. This polysaccharide commonly is extracted from marine brown seaweed. There is lack of information on the fucoidan extracted from tropical brown algae and its anti-cancer activity. Objectives: The objectives of this study were to purify fucoidan from Sargassum sp., Turbinaria sp. and Padina sp., and to evaluate their cytotoxicity against breast cancer (MCF-7) and colon cancer cells (WiDr). Materials and Methods: Fucoidan extraction was conducted by using acid extraction method. Purified fucoidans were obtained by DEAE cellulose column chromatography and confirmed by HPLC and FT-IR spectrometry. The cytotoxicity was evaluated by using the MTT (3-[4,5-dimethylthiazol-2-yl] -2,5diphenyltetrazolium bromide) assay. Results: Fucoidan from Sargassum sp. and Turbinaria sp. showed low cytotoxicity with IC50 ranging between 461-663 μg/mL. Higher cytotoxicity against MCF-7 and WiDr was showed by fucoidan from Padina sp. with IC50 of 144 and 118 μg/mL, respectively. While its IC50 against Vero cells was 501 μg/mL.Standard fucoidan from Fucus vesiculosus exhibited IC50 of 60, 63 and 211 μg/mL against MCF-7, WiDr and Vero Cells, respectively. Although the IC50 was higher than that of standard fucoidan, Padina sp. fucoidan showed cytotoxicity comparable with standard fucoidan at concentrations below 100 μg/mL. Conclusion: These results indicated that Padina sp.fucoidan showed potential selective cytotoxicity, and promising for the development of an anti-cancer compound.


INTRODUCTION
Fucoidan is a polysaccharide of brown seaweed and marine invertebrates (such as sea urchin and sea cucumber) with a main component of L-fucose and sulfate ester group. 1 Chemical structure of fucoidan from brown seaweed is very complex and varies among seaweeds species. 2 The main difference is in the backbone structure of fucoidan. 3,4Monosaccharides component of fucoidan affect their physiological and biological activities. 5Fucoidan has a wide range of bioactivity among others as immunostimulan, [6][7][8][9] and antitumor/anticancer. 4,10,11 Cancer is a disease that attacks the modern society globally, and as a major disease today and in the future.WHO have reported that there are 14 million new cases and 8.2 million cancer related death in 2012, and annual cancer cases will rise to 22 million within the next two decades. 12Two cancers that cause the major deaths in men are lung cancer and colorectal cancer, whereas in women are breast cancer and lung cancer. 13irish et al 14 have found the potential activity of Pavonia odorata Willd extract against human breast and lung cancers.Breast cancer is also effectively inhibited by β-mangostin isolated from Cratoxylum arborescens either in vitro or in vivo. 15How ever, research on tropical brown seaweeds as sources of fucoidan for anti-cancer agent is still limited.Cumashi et al 5 have found the potent anti-angiogenesis activity of fucoidan from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, F. vesiculosus, Ascophyllum nodosum and Cladosiphon okamuranus.Fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocks MDAMB-231 breast carcinoma cell adhesion to platelets.A commercial fucoidan from F. vesiculosus also active against HCT-15 colon carcinoma cells through apoptosis-inducing activity. 16Senthilkumar et al 17 have reviewed the fucoidan from F. vesiculosus, C. novae-caledoniae, C. okamuranus, S. japonica, U. pinnatifida with cytotoxicity against cancer cells.Fucoidan from Sargassum sp. and F. vesiculosus also reduces viability of lung carcinoma and melanoma cells. 18Fucoidan from Eclonia cava, Sargassum hornery, and Costaria costata from South Korea inhibits colony formation in human melanoma and colon cancer cells. 19urphy et al 20 have reviewed anticancer substance including fucoidan from brown seaweeds.Marudhupandi et al 21 reported the cytotoxicity of fucoidan from a tropical brown seaweed, Turbinaria sp.collected from Tamilnadu India against the human lung cancer A549 cell line.In the present study we purified fucoidan from Sargassum sp., Turbinaria sp. and Padina sp., and evaluated its anticancer activity against colon and breast cancer cell lines.To the best of our knowledge, the anticancer activities of fucoidan from these three brown seaweeds species against these two cancer cell lines have not been reported before.

Sampling and Identification of Brown Seaweeds
Sampling was carried out in the intertidal zone during low tide.Sargassum sp., Turbinaria sp. and Padina sp. were collected and then put in a cool box and transported to the laboratory.Identification was carried out based on Dhargalkar, 22 Trono. 23

Extraction and Purification
The seaweed samples were washed with fresh water, then air dried in the laboratory to obtain the dry biomass, and stored in plastic bags.Extraction was carried out by the method of Kim et al 13 Purification was carried out by DEAE cellulose column chromatography (Sigma-Aldrich, St. Louis, MO, USA) eluted with distilled water and gradient 0.5 to 3 M NaCl 24 The total carbohydrate of the purified fucoidan in the fractions was determined by the phenol-sulfuric acid method according to Masuko et al 25 using L-fucose as a reference.The fucoidan fractions were evaporated, dialysed (MW cut-off 12,300 Da) for 48 hours, then freeze-dried and stored at 4°C until use.The purified fucoidan was analyzed by HPLC using Agilent Hi-Plex columns Ligand-Exchange (Agilent Hi-Plex H for Carbohydrate) 7.7 × 100 mm, with a refractive index detector (RID-10A, Shimadzu, Japan) with a flow rate of 0.7 mL/min at 60°C.HPLC using the autosampler (SIL-10ADVP, Shimadzu, Japan) with injection volume of 20 µL.Fucoidan sample concentration was 5 µg/mL.The measurement of sulphate in the fucoidan was based on the barium sulphate (BaSO 4 ) determination using barium chloride (BaCl 2 ), 26 using Na 2 SO 4 as a standard.

FTIR spectra
FTIR spectra were recorded by a FTIR spectrometer in KBr with a wave number range of 4000-450 cm -1 .The ratio of fucoidan and KBr was approximately 1: 100 (2 mg sample + 200 mg KBr).The IR spectra of fucoidan from the seaweeds were compared with the IR spectrum of standard fucoidan (Sigma-Aldrich, St. Louis, MO, USA).

Cytotoxicity Assay
Cell lines used in this assay were a colon cancer cells (human collon adenocarcinoma cells) (WiDr), breast cancer cells (human breast adenocarcinoma cells) (MCF-7) and a normal cells, an African green monkey kidney cells (vero cells).Each of these cells were cultured in RPMI or M199 medium (Sigma-Aldrich, St. Louis, MO, USA) with the addition of 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), antibiotics Panstrep (Gibco, Grand Island, NY, USA) and Fungison (Gibco).The cells were cultured at 37°C in a CO 2 incubator until confluent.After incubation, the medium was replaced with a new medium, then the cells were harvested and re-cultured in 96-well microplate at a density of 10 4 cells/100 µl/well using a medium containing 2% FBS.Then fucoidan with a serial dilution was added to wells.As a negative control cells were treated with sterile dimethyl sulfoxide (DMSO), whereas the positive control was treated with a standard fucoidan from Fucus vesiculosus (Sigma-Aldrich, St. Louis, MO, USA).Cells were then incubated in the same conditions as above.Cells proliferation was observed by MTT assay (3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide [Sigma-Aldrich, St. Louis, MO, USA]). 27Growth was detected with a microplate reader at a wavelength of 570 nm.The growth rate of cells exposed to fucoidan compared to the positive and negative controls, and then the cells mortality expressed in percent (%) was calculated.

RESULTS AND DISCUSSION
Crude fucoidan extracts were purified by DEAE cellulose eluted with distilled water followed by 0.5 to 3 M of NaCl.Each fraction was measured for total sugar content with fucose as a standard (Figure 1).Fractionation of Sargassum sp.crude fucoidan showed the separation of the dominant fraction and two minor fractions.The main fraction was used for subsequent study.The fractionation of Turbinaria sp.crude fucoidan, also found a similar pattern with that of crude fucoidan from Sargassum sp., but there was a medium peak eluted before the main peak.The similar pattern was also found in the fractionation of Padina sp.crude fucoidan where a major fraction was obtained when the crude fucoidan was eluted with 0.5 M NaCl.This DEAE cellulose column chromatogtaphy showed that a major peak was appeared whith 0.5 M NaCl elution either for Sargassum sp., Turbinaria sp.or Padina sp. or fucoidan crude extracts.Each major peak was pooled and used for the subsequent experiments.Pure fucoidans obtained from the DEAE cellulose column chromatography were confirmed by using HPLC (Figure 2) and FTIR spectra (Figure 3).HPLC produced a single peak with the same retention time with standard fucoidan from F. vesiculosus.FTIR spectra also confirmed that the obtained compound were fucoidan.The spectra showed the typical bands of polysaccharide.9][30] The band at 2939 cm -1 was attributed the C-H stretching pyranoid ring and C6 of fucose and/or galactose units. 13,30The band at 1614 cm -1 indicated the stretching of asymmetric carboxylate O-C-O vibration. 28,29The band at 1424 cm -1 suggested C-OH deformation vibration with contribution of O-C-O symmetric stretching vibration of carboxylate group. 29The presence of O=S=O stretching vibration of sulphate esters was indicated by a band at 1258 cm-113, 28,29 The band at 1040 cm -1 corresponded to stretching vibration of C-O-C/C-OH. 309][30][31] The band at 580 cm -1 indicated the asymmetric and symmetric O=S=O deformation of sulfate. 31These FT-IR spectra analysis proved that the extraction and purification processes used and purification process used in this study were effective to obtain fucoidan from the three tropical brown algae.To evaluate the potential bioactivity of fucoidan as an anti-cancer, a cytotoxicity test against colon cancer cells (human collon adenocarcinoma cells) (WiDr), breast cancer cells (human breast adenocarcinoma cells) (MCF-7) and a normal cells of green monkey kidney cells (Vero cells) was conducted.The cytotoxicity was compared with a standard fucoidan from F. vesiculosus.Results showed that standard fucoidan exhibited high cytotoxic activity against colon cancer and killed almost all cells at a dose of 1000 µg/mL (Figures 4 and 5).Lower cytotoxicity activity was found for three fucoidans from brown algae examined in this study.Fucoidan from Padina sp.showed cytotoxicity comparable with standard fucoidan at concentrations below 100 µg/mL.However, increase in the concentrations above 100 µg/mL did not significantly increase in the cytotoxicity activity.The same pattern of cytotoxicity was obtained against breast cancer cells (MCF-7) (Figure 4).The results of cytotoxicity test also indicated that the cytotoxicity of fucoidan varied depending on the seaweeds species as their sources, and cell lines used.Fucoidan from Sargassum sp. and Turbinaria sp.exhibited low cytotoxic activity with IC 50 values in the range of 461-663 µg/mL.The cytotoxic activities were lower comparing to the standard fucoidan Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017  from F. vesiculosus.Fucoidan from Padina sp.exhibited higher cytotoxic activity with IC 50 value of 144 and 118 µg/mL against breast and colon cancer cells, respectively (Table 1).Cytotoxicity of Padina sp.fucoidan was higher than that of standard fucoidan against a normal Vero cells with IC 50 511 µg/mL and 211 µg/mL, respectively, suggested that the former one might be more selective cytotoxic than the later one.Ale et al 18 have evaluated anti-cancer activity of fucoidan from Sargassum sp., against Lewis Lung Carcinoma cells (LLC) and melanoma B16 cells (MC) in low concentration of Fetal Bovine Serum (FBS) (2%) as used in this study.Results indicated thatSargassum sp.fucoidan reduces the viability of LLC cells up to 40 ± 7% at 1,000 µg/mL.The cell viability reduction is more signifancantly noted against MC cells, resulting in only 56 ± 5% viable cells upon addition of 100 and 200 µg/mL.Our study results confirmed that the different fucoidan sources and different cell lines used in cytotoxicity test resulted the variation of cytotoxicity activities.Padina sp.fucoidan used in our study has higher cytotoxicity comparing to that of Sargassum sp. and Turbinaria sp.Without considering the cell lines used in cytotoxicity test, fucoidan from Sargassum sp.against WiDr and MCF-7 exhibited the similar cytotoxicity level with Sargassum sp.used by Ale et al 18 against LLC cells.Several studies have evaluated cytotoxicity of fucoidan against collon and breast cancer. 5,10,16,19Cumashi et al 5 found that fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus and F. vesiculosus at 100 μg/mL strongly blocked MDAMB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumormetastasis.Hyun et al 16 reported that fucoidan from F. vesiculosus at 100 μg/mL demonstrates apoptotic activity against HCT-15 cells (human colon carcinoma cells).Fucoidans from S. hornery, Eclonia cava and Costaria costata have been reported non significant cytotoxic activity against human skin melanoma (SK-MEL-28) and colon cancer cells (DLD-1) after treatment during 24 h at concentration from 1 to 200 μg/mL. 19Fukahori et al 32 also have proved by evaluating cytotoxicity of fucoidan against 15 cancer cell lines with the broad range IC 50 between  18.71and 299.20 μg/mL.The above reports indicate that the cytotoxicities of fucoidan are greatly vary depending on the sources of fucoidan and cell lines used in cytotoxicity assay.Results of our study confirmed fucoidans from three tropical brown seaweeds exhibits cytotoxicity variation.Fucoidan from Padina sp.shows selective cytotoxicity with IC 50 118 to 144 μg/mL against cancer cell lines and 501 μg/mL against a normal cell line suggested that this brown seaweed may a prospective source of fucoidan for developing cytotoxic agent in cancer therapy especially against breast and collon cancer.The further studies are needed to investigate the variability of fucoidan related to the season, sampling location and extraction method, and also evaluation of the cytotoxicity against more broad range cancer cell lines.Human breast adenocarcinoma (MCF-7) )