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Fucoidan from brown alga Fucus evanescens and its product of enzymatic hydrolysis have precisely established structure and possess significant biological activities. The aim of present study was to determine radiosensitizing activity of fucoidan from brown alga F. evanescens and its derivative in human melanoma, breast adenocarcinoma, and colorectal carcinoma cell lines and elucidate mechanism of their action. The fucoidan from F. evanescens and its derivative had a comparable radiosensitizing activity and increased the inhibiting effect of X-ray radiation on proliferation and colony formation of human cancer cells, with significant inhibition of melanoma cells. The molecular mechanism of this action was associated with the induction of apoptosis by activating the initiator and effector caspases, suppressing the expression of the anti-apoptotic protein, and enhancing the fragmentation of DNA. The obtained data confirm the prospects of using fucoidan's derivative in combination with radiation therapy for the improvement of the schemes of cancer therapy.

Laminarans are currently the focus of attention in regard to the selection of prospective agents for the prevention and treatment of cancer. Laminaran from Saccharina cichorioides was aminated to heighten anticancer and radiosensitizing activities and elucidate its molecular mode of action. Aminated laminaran, ScLNH2, was identified as 1,3-β- d -glucan with −CH2−CH(OH)−CH2-NH2 group at the C6 of branches. ScLNH2 selectively inhibited the viability and colony formation in the MDA-MB-231 cell line of triple negative breast cancer cells. ScLNH2 possessed synergism with radiation, resulting in a decreased number of colonies of MDA-MB-231 cells. The mechanism underling the radiosensitizing effect of ScLNH2 was associated with apoptosis induction via regulation of caspases 9 and 3 and PARP enzyme, preventing the repair of DNA damage in irradiated cells. These findings confirmed that combination therapy by aminated laminaran and radiation might play a role in the optimization of therapy for an aggressive form of human breast cancer.

Structure of the fucoidan from Sargassum horneri and products of its enzymatic transformation with molecular weight over 20 kDa were investigated. Fucoidan was hydrolyzed by recombinant fucoidanase FFA1 and its fraction of higher molecular weight was fractionated using anion-exchange chromatography, resulting in three sulphated polysaccharides of various molecular weight (63-138 kDa). Their structures were analyzed using NMR spectroscopy, showing the fucoidan (ShF) to be a branched polysaccharide with the backbone consisting of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment and side chains including the α-l-Fucp-1→2-α-l-Fucp-1→ or α-l-Fucp-1→3-α-l-Fucp(4SO3-)-1→ fragments attached to the main chain at C4. The fragment F3 differing by molecular weight and side chain from other fucoidans fragments possessed the most significant anticancer and radiosensitizing activities.

In this report, polysaccharides - alginate, fucoidan, laminaran - were isolated from marine algae Saccharina cichorioides and Fucus evanescens and their activity as a reducing and stabilizing agents in the biogenic synthesis of silver nanoparticles was evaluated. The cytotoxic and antibacterial properties of obtained nanoparticles were also assessed. It was found that all tested polysaccharides could be used as a reducing agent; however, their catalytic activities varied significantly in the following range alginate < fucoidan < laminaran. Nanoparticles demonstrated cytotoxicity against rat C6 glioma cells. It was considerably higher for alginate- and laminaran-obtained nanosilver samples compared to fucoidan. Additionally, silver nanoparticles possessed considerable antibacterial properties more pronounced in fucoidan-obtained samples. Our data demonstrate that different algal polysaccharides can be used for the synthesis of silver nanoparticles with varying bioactivities.

Enzymatic depolymerization of fucoidans attracts many researchers due to the opportunity of obtaining standardized fucoidan fragments. Fucoidanase catalyzes the cleavage of fucoidan from Fucus evanescens (FeF) to form low molecular weight products (LMP) and a polymeric fraction (HMP) with 50.8 kDa molecular weight and more than 50% yield. NMR spectroscopy shows that the HMP fraction has regular structure and consists of a repeating fragment [→3)-α- l -Fucp2,4OSO3−-(1 → 4)-α- l -Fucp2,4OSO3−-(1 → 4)-α- l -Fucp2OSO3−-(1→]n. The anticancer effects of FeF fucoidan and its derivative (HMP) were studied in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The anticancer activity of the HMP fraction was found to be slightly lower than that of the FeF fucoidan. Research and practical applications of the enzyme include modification of native fucoidans for purposes of regular and easier characterized derivatives acquisition.

The laminaran DdL and fucoidan DdF were obtained from the brown alga Dictyota dichotoma. DdF was a sulfated (28.9%) and acetylated heteropolysaccharide containing fucose, galactose, mannose and glucose (57.9, 20.4, 12.4 and 9.2mol%, respectively). DdL was a 1,3;1,6-β-d-glucan with the main chain built from 1,3-linked glucose residues and single glucose residue in branches at C6 (one branch on three glucose residues of the main chain). Sulfated (43.7%) laminaran DdLs was obtained from DdL by sulfation. It was determined that sulfates occur at C2, C4 and C6 of glucose residues. The anticancer effect of DdF, DdL, and DdLs (200μg/mL) was studied in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The effect of polysaccharides (40μg/mL) on colony formation of DLD-1 cancer cells after irradiation (4Gy) was investigated first. All polysaccharides showed a synergistic effect with X-ray irradiation against cancer cells, decreasing the amount and size of cancer cells colonies.

Fucoidans are a class of sulfated fucose-containing bioactive polysaccharides produced by brown algae. The biological effects exhibited by fucoidans are thought to be related to their sulfation. However, the lack of methods for sulfation control does not allow for a reliable conclusion about the influence of the position of certain sulfate groups on the observed biological effects. We identified the gene encoding the endo-acting fucoidan sulfatase swf5 in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. This is the first report on the sequence of fucoidan endo-sulfatase. Sulfatase SWF5 belongs to the subfamily S1_22 of the family S1. SWF5 was shown to remove 4O-sulfation in fucoidans composed from the alternating α-(1→3)- and α-(1→4)-linked residues of sulfated L-fucose but not from fucoidans with the α-(1→3)-linked backbone. The endo-sulfatase was used to selectively prepare 4O-desulfated fucoidan derivatives. It was shown that the 4O-desulfated fucoidans inhibit colony formation of DLD-1 and MCF-7 cells less effectively than unmodified fucoidans. Presumably, 4O-sulfation makes a significant contribution to the anticancer activity of fucoidans.

The laminaran SdL and fucoidan SdF were isolated from brown algae Sargassum duplicatum. SdL was 1,3;1,6-β-d-glucan (1,3:1,6=6:1) with a main chain, represented by 1,3-linked glucose residues, due to NMR spectroscopy data. Single glucose residues could form branches at C6. Unusual structure of fucoidan SdF was studied by chemical and enzymatic methods, NMR spectroscopy of desulfated and deacetylated polysaccharide and mass spectrometry of fucoidan fragments labeled with 18O. Fucoidan was sulfated (31.7%) and acetylated galactofucan (Fuc:Gal∼1:1) with a main chain of 1,4-linked alternating α-l-fucose and β-d-galactose residues. Side chains were represented by extensive (DP≥5) 1,3-linked 2,4-disulfated α-l-fucose residues with branching points at C2. Fucose residues in the main chain were sulfated at C2 and less at C3, while galactose residues were sulfated at C2, C3, and less at C4, C6. The fucoidan SdF was effective against colony formation of colon cancer cells in vitro.

A fucoidan ScF from brown alga Saccharina cichorioides was extracted, purified and partially depolymerized by autohydrolysis at 37°C for 24, 48 and 72h. Supernatant (SN) and pellet (PL) fractions were obtained by ethanol precipitation of each sample. Unlike spectral data of ScF, NMR of PL derivatives clearly suggested the structure: 1,3-linked α-l-Fucp-4-OSO3- repeating unit. Molecular weights (MWs) of PL fractions were 30, 26 and 18kDa for 24, 48 and 72h of autohydrolyis, respectively. MALDI-TOFMS, size-exclusion HPLC and carbohydrate polyacrylamide-gel electrophoresis (C-PAGE) indicated a similarity of SN mixtures. They consisted mainly of a polysaccharide part (MW 6kDa, C-PAGE data) with a structure similar to PL components (NMR data) and monosaccharides α-l-Fucp-4-OSO3-, α-l-Fucp-2,4-di-OSO3-. PL fractions exhibited almost identical antiproliferative activity in vitro as native fucoidan, while an SN sample for 72h of autohydrolysis was slightly more active against colony formation of colorectal carcinoma cells HT-29.

The sulfated α-l-fucans ScF and LlF were obtained from brown algae of the Laminariaceae family (Saccharina cichorioides and Laminaria longipes). According to spectroscopy NMR, the LlF fucan predominantly contained the →3)-α-l-Fucp-(2SO3-)-(1→4)-α-l-Fucp-(1→2)-α-l-Fucp-(4SO3-)-(1→ repeating units, with small amounts of disaccharide 1,4-linked fragments and 3-sulfated fucose residues. Mass spectrometric analysis revealed the presence of the following fragments in the fucan structure: α-l-Fucp-(2SO3-)-(1→4)-α-l-Fucp-(2SO3-)-(1→3)-α-l-Fucp-(4SO3-); α-l-Fucp-(2,4SO3-)-(1→3)-α-l-Fucp-(1→3)-α-l-Fucp-(4SO3-); α-l-Fucp-(2SO3-)-(1→2)-α-l-Fucp; α-l-Fucp-(2SO3-)-(1→2)-α-l-Fucp-(4SO3-); α-l-Fucp-(2SO3-)-(1→3)-α-l-Fucp; α-l-Fucp-(2,4SO3-)-(1→3)-α-l-Fucp; α-l-Fucp-(4SO3-)-(1→4)-α-l-Fucp; and α-l-Fucp-(4SO3-)-(1→4)-α-l-Fucp-(2SO3-). Both ScF and LlF fucoidans inhibited colony formation and growth of melanoma and colon cancer cells and sensitize-tested cancer cells to X-ray radiation to a comparable degree.

Laminaran and three fucoidan fractions were obtained from the brown alga Alaria marginata . Alaria angusta, studied earlier by us, has the same polysaccharide composition. Galactofucan AmF3 from A. marginata has a main chain of → 3)-α- l -Fuc p -(2,4- S O 3 − )-(1 → residues, similar to galactofucan from A. angusta . However, the structure of the branches in fucoidan AmF3 can differ from those in the fucoidan from A. angusta. The following fragments were identified in AmF3: HexA-(1 → 2)-Fuc, HexA-(1 → 2)-Gal, Gal-(1 → 4)-HexA, Fuc-(1 → 2)-Gal-6-SO 3 − , Fuc-4-SO 3 − -(1 → 6)-Gal, Gal-(1 → 2)-Gal-2-SO 3 − , Gal-4-SO 3 − -(1 → 6)-Gal, Gal-4-SO 3 − -(1 → 3)-Fuc-(1 → 3)-Fuc, Fuc-4-SO 3 − -(1 → 6)-Gal-(1 → 4)-Gal, Gal-(1 → 4)-Gal-(1 → 3)-Fuc, Gal-2-SO 3 − -(1 → 4)-Gal-(1 → 4)-Gal, Gal-(1 → 4)-Gal-6-SO 3 − -(1 → 2)-Gal. Chains of galactose residues (DP up to 9) were found in AmF3 fucoidan. The laminarans, galactofucans and their derivatives from both algae exhibited no cytotoxicity in vitro . Polysaccharides from A. angusta were more effective against colony formation of HT-29 cells, while those from A. marginata had a greater effect on T-47D cells. Sulfated and desulfated fucoidans possessed weak antitumor activity using SK-MEL-28 cells.

Laminaran, fucoidan, and alginate were isolated from the brown alga Coccophora langsdorfii collected in the Japan Sea. The structural characteristics of polysaccharides were investigated by NMR spectroscopy. The laminaran was determined as β-d-glucan, which consisted of 80% of 1,3- and 20% of 1,6-linked residues and was terminated with mannitol. The alginate was a guluronic acid-rich polysaccharide (M/G=0.85). Fucoidan, sulfated α-l-fucan, contained a linear backbone of alternating (1→3)- and (1→4)- linked α-l-fucopyranose residues with sulfate at C2 and C4 of (1→3)-α-l-fucopyranose residues. Anticancer activity of this fucoidan was investigated in comparison with activity of fucoidan having similar linear backbone from the brown alga Fucus evanescens. The fucoidan from C. langsdorfii significantly inhibited colony formation of SK-MEL-5 and SK-MEL-28 melanoma cells (the percentage of inhibition was 28 and 76, respectively) and weakly inhibited colony formation of breast adenocarcinoma cells MDA-MB-231 (the percentage of inhibition was about 5). Similar results were obtained for fucoidan from F. evanescens; the percentage of inhibition of colony formation of SK-MEL-5 and SK-MEL-28 melanoma cells was 54 and 56, respectively. The inhibition of colony formation of breast adenocarcinoma cells MDA-MB-231 was weak. We suppose that other sulfated and partially acetylated fucoidans consisting of (1→3)- and (1→4)-linked α-l-fucopyranose residues may suppress progression of melanoma cell colony formation similar to fucoidans of C. langsdorfii and F. evanescens.

Polysaccharide fractions of alginate, laminarans and fucoidans were obtained from the brown alga Tauya basicrassa. Yields of alginate and laminarans were large (19.7 % and 5.62 %, respectively), whereas the content of fucoidans (0.52 %) was not significant. Alginate and laminarans had typical structures for those substances. Fucoidans were low- and medium-sulfated heterogeneous polysaccharides. The fucoidan fraction 1TbF1 was sulfated fucogalactan containing a backbone from 1,6-linked residues of β- d -galactopyranose with branches at C3 and C4, terminal fucose and galactose residues and fragments from 1,3-; 1,4-; and 1,2-fucose residues. Sulfate groups were found at positions 2 and 4 of fucose, and positions 2, 3 and 4 of galactose residues. Laminaran 2TbL was subjected to a sulfation to obtain the derivative 2TbLS with partial sulfation (46 %) at C2, C4 and C6. It was shown that 2TbL and 2TbLS inhibited colony formation of sensitize-tested colon cancer cells HT-29 and HCT-116 to X-ray radiation.

The laminarans are neutral water-soluble β-D-glucans of brown algae possessing potent immunomodulating, radioprotective, and anticancer activities. The aim of the present study was to investigate in vitro anticancer, radioprotective, and radiosensitizing activities of laminaran from brown alga Dictyota dichotoma and its sulfated derivative. The native and sulfated laminarans by themselves at non-toxic doses possessed significant anticancer activity against melanoma cells. Both polysaccharides protected normal epidermal cells, while only sulfated laminaran was able to sensitize melanoma cells to X-rays irradiation resulting in significant inhibition of cell proliferation, colony formation, and migration of cancer cells. The molecular mechanism of this action was related to the inhibition of MMP-2 and MMP-9 proteinases activity as well as down-regulation of kinases' phosphorylation of ERK1/2 signaling cascade. Taken together, the combination of sulfated derivative of laminaran from D. dichotoma with X-ray may serve as a potential treatment strategy for human melanoma.

Laminaran and three fractions of fucoidan were isolated from brown alga Alaria angusta. The laminaran AaL was characterized as a typical 1,3;1,6-β-D-glucan (ratio of bonds 1,3:1,6 = 10:1). Fucoidans AaF1 and AaF2 are sulfated heteropolysaccharides, containing fucose, galactose, mannose and xylose. The fraction AaF3 is sulfated and acetylated galactofucan with the main chain represented by a repeating unit → 3)-α-L-Fucp-(2,4-SO3(-))-(1 →. According the data of methylation analysis, AaF3 contains mainly 1,3-linked fucose, less 1,4-linked and 1,4,6-linked galactose residues. The autohydrolysis (37 °C) of fucoidan AaF3 allowed to obtain selectively 2-desulfaled polysaccharide fraction, built up of fucose only, and low molecular weight (LMW) fraction. The negative-ion tandem mass spectrometry of LMW fraction, further hydrolyzed by acid hydrolysis identified the following fragments: Gal-2-SO3(-)-(1 → 4)-Gal, Gal-4-SO3(-)-(1 → 4)-Gal, Gal-(1 → 2)-Gal-4-SO3(-), Fuc-2-SO3(-)-(1 → 4)-Gal, Gal-2-SO3(-)-(1 → 3)-Fuc-(1 → 3)-Fuc, Fuc-2-SO3(-)-(1 → 3)-Fuc-(1 → 4)-Gal. The laminaran AaL and the fucoidan AaF3 exhibited no cytotoxicity in vitro for HT 29, T-47D, and SK-MEL-28 cell lines. The AaF3 fraction suppressed colony formation of HT 29 and T-47D cells, AaL-only HT 29 cells.

The sulfated and acetylated fucoidan fraction, containing fucose, galactose, mannose, glucose and uronic acid residues, was isolated from the brown alga Padina boryana. The structure of galactofucan part was studied after different modifications by NMR spectroscopy and mass spectrometry. It was shown that galactofucan contained the main chain of alternating 1,4-linked α- l -fucopyranose and 1,3-linked β- d -Galactopyranose. Single fucose residues were found as branches at C4 of galactose residues. Also, fucoidan contained 1,3- or 1,4-linked Fuc-Fuc and Gal-Gal fragments. The sulfate groups occupied positions C2, C3 and C4 of both fucose and galactose residues, which was shown by tandem mass spectrometry of fragments, labeled with heavy-oxygen. The anticancer effect of native and modified fucoidan fractions was studied in vitro on the colorectal carcinoma cells DLD-1 and HCT-116. All fucoidans had no cytotoxicity under 400 μg/mL and inhibited colony formation of cancer cells at concentration of 200 μg/mL.

Structure and anticancer activity of fucoidan from Sargassum horneri and from products of its enzymatic transformation were investigated. A gene that encodes fucoidanase ffa1 in the marine bacteria F. algae was identified, cloned and the protein (FFA1) was produced in Escherichia coli. The mass of the gene product FFA1 is 111kDa. Sequence analysis has revealed that fucoidanase FFA1 belongs to the GH107 (CAZy) family. Recombinant fucoidanase FFA1 was used to produce fucooligosaccharides. Structure of 5 sulphated oligosaccharides with polymerization degree 4-10 was established by NMR-spectroscopy. The fucoidan extracted from S. horneri is almost pure fucan. The main chain of the fucoidan is established to consist mostly of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment, with insertions of →3-α-l-Fucp(2,4SO3-)-1→ fragment. Unsulphated side chains with the α-l-Fucp-1→2-α-l-Fucp-1→ structure connect to the main one at the C4 of monosaccharide residue.

The structure of high molecular weight laminaran from brown alga Eisenia bicyclis was investigated by chemical and enzymatic methods, NMR spectroscopy and mass spectrometry. The laminaran from E. bicyclis was characterized as 1,3;1,6-β- d -glucan with the high content of 1,6-linked glucose residues (ratio of bonds 1,3:1,6 = 1.5:1), which are both in the branches and in the main chain of the laminaran. The degree of polymerization of fragments, building from 1,3-linked glucose residues with single glucose branches at C-6 or without it, was no more than four glucose residues. The main part of 1,3-linked glucose blocks was builded from disaccharide fragments. 1,6-Linked glucose residues were localized basically on non-reduced ends of molecules. The degree of polymerization of 1,6-linked blocks was not greater than three glucose residues. Laminaran contained laminarioligosaccharides, gentiobiose, gentiotriose and single glucose residues in the branches at the C-6. Laminaran and its products of enzymatic hydrolysis inhibited a colony formation of human melanoma SK-MEL-28 and colon cancer DLD-1 cells. It was shown that decreasing the molecular weight of native laminaran to a determined limit (degree of polymerization 9–23) and increasing the content of 1,6-linked glucose residues increased the anticancer effect. Therefore, they may be perspective antitumor agents.

Fucoidan was isolated and purified from the brown algae Fucus evanescens and subjected to autohydrolysis to obtain low-molecular-weight fragments. MALDI-TOFMS analysis has shown that monosulfated fucose and more heavily sulfated (up to 5) fucooligosaccharides with polymerization degree (DP) 2, 4 and 6, including galactose-containing sulfated oligosaccharides were the products of autohydrolysis. The structural features of these fragments were elucidated by negative-ion potential lift tandem MALDI-TOF mass-spectrometry using arabinoosazone as a matrix, which allowed reducing the in-source fragmentation. Native fucoidan has been shown to exert anticancer activities in both human malignant melanoma cell lines SK-MEL-28 and SK-MEL-5. Low-molecular-weight fragments exhibited almost no action to cell proliferation in both cell lines and colony formation on SK-MEL-5 cells, but its inhibition activity to the colony formation of SK-MEL-28 cell lines was as high as was demonstrated by native fucoidan (70%). Probably, the inhibiting activity for SK-MEL-28 depended on the presence of sulfates and (1→4)-linked α-l-Fucp residues in the main chain of fucoidan/oligosaccharides.