Targeting epithelial-to-mesenchymal transition of cancer cells by sulfated glycans from marine algae

The sulfated glycans in the tunic of Styela plicata differ from the glycosaminoglycans of animal tissues and also from the sulfated polysaccharides isolated from marine algae. The ascidian glycans occur primarily as three fractions that differ markedly in molecular weight and chemical composition. The high molecular weight fraction encompasses a broad range of molecular weights but is chemically homogeneous and contains an unusual amount of galactose. The 20,000 molecular weight polysaccharide is rich in galactose and glucose while the 8,000 molecular weight fraction is rich in amino sugars and contains the neutral hexoses galactose, glucose, and mannose. All fractions contain large amounts of sulfate esters. The ascidians polysaccharides can be extracted from the tissue by proteolytic enzyme or by guanidine hydrochloride solutions. The high molecular weight fraction is preferentially extracted by papain while guanidine hydrochloride removes mainly the low molecular weight polysaccharides. We speculate that these sulfated glycans are essential for maintaining the structural integrity of the tunic, in analogy with the glycosaminoglycans of vertebrate connective tissues.

In recent decades, there has been an increased interest in the utilization of polysaccharides showing biological activity for various novel applications owing to their biocompatibility, biodegradability, non-toxicity, and some specific therapeutic activities. Increasing studies have started in the past few years to develop algal polysaccharides-based biomaterials for various applications. Saccharide mapping or enzymatic profiling plays a role in quality control of polysaccharides. Whereby, in vitro and in vivo tests as well as toxicity level discriminating polysaccharides biological activities. Extraction and purification methods are performed in obtaining algal derived polysaccharides followed by chromatographic profiles of their active compounds, structural features, physicochemical properties, and reported biological activities. Marine algae are capable of synthesizing Glycosaminoglycans (GAGs) and non-GAGs or GAG mimetics such as sulfated glycans. The cell walls of algae are rich in sulfated polysaccharides, including alginate, carrageenan, ulvan and fucoidan. These biopolymers are widely used algal-derived polysaccharides for biological and biomedical applications due to their biocompatibility and availability. They constitute biochemical compounds that have multi-functionalization, therapeutic potential and immunomodulatory abilities, making them promising bioactive products and biomaterials with a wide range of biomedical applications. Algal-derived polysaccharides with clearly elucidated compositions/structures, identified cellular activities, as well as desirable physical properties have shown the potential that may create new opportunities. They could be maximally exploited to serve as therapeutic tools such as immunoregulatory agents or drug delivery vehicles. Hence, novel strategies could be applied to tailor multi-functionalization of the polysaccharides from algal species with vast biomedical application potentials.

Fucoidan ameliorates acute and sub-chronic in vivo toxicity of the fungicide cholorothalonil in Oreochromis niloticus (Nile tilapia)

Objectives To review red algae bioactive compounds and their pharmaceutical applications. Content Seaweed sources are becoming attractive to be used in health and therapeutics. Among these red algae is the largest group containing bioactive compounds utilized in cosmetic, pharmaceutical, food industry, manure and various supplements in food formula. Various significant bioactive compounds such as polysaccharides (aginate, agar, and carrageenan), lipids and polyphenols, steroids, glycosides, flavanoids, tannins, saponins, alkaloids, triterpenoids, antheraquinones and cardiac glycosides have been reported in red algae. The red algae have rich nutritional components Different polysaccharides of red algae possess the antiviral potential namely agarans, carrageenan, alginate, fucan, laminaran and naviculan. Sulfated polysaccharides and carraginans of red algae are rich source of soluble fibers which can account for antitumor activities depending upon chemistry of various secondary metabolites and metabolism of cell line. Flavons-3-ols containing catechins from many red algae block the telomerase activity in colon cancer cells. Contraceptive agents were tested from red algae as a source for post-coital. Lectin of red algae showed pro-healing properties and anti-ulcerogenic activities. Carragenates from red algae also conferred a positive influence on diabetes. Red algae depicted a reducing effect on plasma lipids and obesity. Porphyran from red alga can act as anti-hyperlipidemic agent also reduces the apolipoprotein B100 via suppression of lipid synthesis in human liver. Summary The polyphenolic extracts of Laurencia undulate, Melanothamnus afaqhusainii and Solieria robusta extract show anti-inflammatory effects against multiple genera of devastating fungi. Antioxidants such as phlorotannins, ascorbic acids, tocopherols, carotenoids from red algae showed toxicity on some cancer cells without side effects. Red algae Laurencia nipponica was found insecticidal against mosquito larvae. Red algae fibers are very important in laxative and purgative activities. Gracilaria tenuistipitat resisted in agricultural lands polluted with cadmium and copper. Outlook In the recent decades biotechnological applications of red algae has been increased. Polysaccharides derived from red algae are important tool for formulation of drugs delivery system via nanotechnology.

Changes in the structures and quantities of sulfated glycans play important roles in inflammatory and neurological diseases and cancer. Therefore, sulfated glycans are expected to become diagnostic markers for a variety of diseases such as Alzheimer's disease and cancer. On the other hand, structural abnormalities in the phosphorylated glycans on lysosomal enzymes cause a number of lysosomal diseases, while novel phosphorylated glycans have been found in other proteins. As with sulfated glycans, structural and quantitative changes in these phosphorylated glycans and their associations with disease are also of interest. In this article, we introduce a new method for the simultaneous analysis of sulfated and phosphorylated glycans. We first employ a serotonin-immobilized column to enrich these glycans. Glycans obtained in this manner were sequentially subjected to other analytical techniques without desalting. We employed hydrophilic interaction chromatography to distinguish the sulfate and phosphate groups of the glycans and were able to analyze sulfated and phosphorylated N-glycans expressed on thyroglobulin, ovalbumin, and myozyme. We showed that our method not only analyzes sulfated and phosphorylated glycans, but also glycans containing the GlcNAc-HPO3 residue. We comparatively analyzed sulfated glycans, phosphorylated glycans, and GlcNAc-HPO3-residue-containing glycans expressed on MKN7 cells (well-differentiated gastric cancer cells) and MKN45 cells (poorly differentiated gastric cancer cells). To the best of our knowledge, this is the first report of a method for the simultaneous and quantitative analysis of sulfated and phosphorylated glycans.

Glycosaminoglycans (GAGs) are sulfated glycans capable of regulating various biological and medical functions. Heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate and hyaluronan are the principal classes of GAGs found in animals. Although GAGs are all composed of disaccharide repeating building blocks, the sulfation patterns and the composing alternating monosaccharides vary among classes. Interestingly, GAGs from marine organisms can present structures clearly distinct from terrestrial animals even considering the same class of GAG. The holothurian fucosylated chondroitin sulfate, the dermatan sulfates with distinct sulfation patterns extracted from ascidian species, the sulfated glucuronic acid-containing heparan sulfate isolated from the gastropode Nodipecten nodosum, and the hybrid heparin/heparan sulfate molecule obtained from the shrimp Litopenaeus vannamei are some typical examples. Besides being a rich source of structurally unique GAGs, the sea is also a wealthy environment of GAG-resembling sulfated glycans. Examples of these mimetics are the sulfated fucans and sulfated galactans found in brown, red and green algae, sea urchins and sea cucumbers. For adequate visualization, representations of all discussed molecules are given in both Haworth projections and 3D models.

In the present study, overcome on liver cancer and other types of cancer by foods that have bioactive chemical components is the most important trend. The marine organisms can give us this opportunity. In this study, we collected some water samples to assess the environmental impacts on the biota existed in red sea cost. The measurements demonstrated that, the habitat was healthy and not effected by any biotic or abiotic stressors. Also, we screened some methanolic extracts of some marine macro-algae on liver cancer cell line. The results showed a good impression about Jania Rubens – red algae – has LC 50 8.61 µg/mL followed by Hydroclathrus Clathratus – brown algae – 17 µg/mL, then Sargassum Cinereum – brown algae – 18.2 µg/mL. Finally, Scinaia Fascularis , – red algae – at value 43 µg/mL. We recommend that, the isolating of the bioactive compounds extracted from these algae will be needed in traditional medicinal field.

Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)β1-3Galβ1-4GlcNAc(6-SO3-)β1-3Galβ1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.

Lectins or carbohydrate-binding proteins, are widely distributed in nature, including in marine algae. It may have been considered that binding specificity of lectins to some carbohydrates provokes to produce many unique biological activities, including cell agglutination, mitogenic activity, and antitumor activity. The aim of this study was to determine the cytotoxicity of crude lectins from red macroalgae collected from the southern coast of Java Island, Gunung Kidul Regency, Yogyakarta against MCF-7 and HeLa cancer cells. In vitro MTT assay was used in this study. The results showed that less than 50% of MCF-7 and HeLa cancer cells growth were inhibited by the crude lectins from five species of red macro algae used in this study. The highest inhibition ability shown in the red alga A. nana was able to kill 47.68% of HeLa cervical cancer cells.

Purpose: To evaluate the cytotoxicity of n-hexane extract and its metabolites obtained from the red alga, Laurencia majuscula, against three cancer cell lines HCT-116 (colon cancer), PC-3 (prostate cancer) and HepG2 (liver cancer) cells; and to identify the phytochemical compound(s) involved.
 Methods: Solvent extraction, thin layer chromatography, aluminum oxide column chromatography, and preparative thin layer chromatography (PTLC) were employed for isolating pure compounds from nhexane extract of Laurencia majuscula. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) measurements were used for structural elucidation of the compounds. The cytotoxicity of the nonpolar extract and isolated compounds were evaluated against HCT, PC-3, and HepG2 cells using MTT assay, relative to the standard cytotoxic drug (cisplatin).
 Results: Three sesquiterpenes (1, 2 and 8), and five acetogenins (3-7) were isolated from the n-hexane extract. The n-hexane extract showed higher potent cytotoxic effect than sesquiterpenes and the acetogenins (3-7).
 Conclusion: These results indicate that the n-hexane extract of Laurencia majuscula exerts significant cytotoxicity against HCT-116, PC-3 and HepG2 cell lines, thus suggesting that the plant extract may be effective chemotherapeutic agents for the management of colon, postrate and liver cancer.
 Keywords: Red Sea alga, Rhodomelaceae, Polyketides, Terpenes, Anticancer

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.

With the advent of exploration in finding new sources for treating different diseases, one possible natural source is from marine algae. Having an array of potential benefits, researchers are interested in the components which comprise one of these activities. This can lead to the isolation of active compounds with biological activities, such as antioxidation of free radicals, anti-inflammation, antiproliferation of cancer cells, and anticoagulant to name a few. One of the compounds that are isolated from marine algae are sulfated polysaccharides (SPs). SPs are complex heterogenous natural polymers with an abundance found in different species of marine algae. Marine algae are known to be one of the most important sources of SPs, and depending on the species, its chemical structure varies. This variety has important physical and chemical components and functions which has gained the attention of researchers as this contributes to the many facets of its pharmacologic activity. In this review, recent pharmacologic application potentials and updates on the use of SPs from marine algae are discussed.

Here, we review potential determinants of the anticancer efficacy of innate immune peptides (ACPs) for cancer cells. These determinants include membrane-based factors, such as receptors, phosphatidylserine, sialic acid residues, and sulfated glycans, and peptide-based factors, such as residue composition, sequence length, net charge, hydrophobic arc size, hydrophobicity, and amphiphilicity. Each of these factors may contribute to the anticancer action of ACPs, but no single factor(s) makes an overriding contribution to their overall selectivity and toxicity. Differences between the anticancer actions of ACPs seem to relate to different levels of interplay between these peptide and membrane-based factors.

Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.

Chapter 13 - Brominated Molecules From Marine Algae and Their Pharmacological Importance