Ge Liu

Polysaccharides from Enteromorpha prolifera enhance the immunity of normal mice

The effects of polysaccharides from Enteromorpha prolifera (PEP) on cell-mediated immunity, humoral immunity and mononuclear phagocytic system function were evaluated to assess the immunomodulatory potential of these macromolecules. Relevant immunological mechanisms were verified by biochemical assays and western blot analysis. Results showed that PEP could induce splenocyte proliferation. In vivo experiments on Kunming mice confirmed that PEP could improve cell-mediated immunity, humoral immunity and mononuclear phagocytic system function. To illustrate the mechanism, we determined several immune-related enzymes in the thymus and spleen. The results indicated that PEP could enhance the activities of alkaline phosphatase, superoxide dismutase and lactate dehydrogenase. PEP could also increase the level of NF-κB. These results suggested that PEP exhibited potent immunomodulatory properties and could be used as a novel potential immunostimulant in food and pharmaceutical industries.

A novel polysaccharide from Sargassum integerrimum induces apoptosis in A549 cells and prevents angiogensis in vitro and in vivo.

Many polysaccharides isolated from plants have exhibited promising antitumor activities. The aim of this study is to investigate the antitumor activity of the novel polysaccharide named SPS from Sargassum integerrimum, elucidate the underlying anticancer mechanism in a human lung cancer cell line A549, and evaluate its anti-angiogenic activity both in vitro and in vivo. The results show that SPS significantly reduces A549 cells viability in a dose- and time-dependent manner via MTT method. Flow cytometry analysis indicates that SPS could induce cell apoptosis, the loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and G2/M phase cell cycle arrest of A549 cells. Up-regulation of the expressions of P53 and Bax, down-regulation of the expression of Bcl-2, and activation of cleaved caspase-3, caspase-9 and PARP are also detected by western blotting after the treatment of SPS. In addition, SPS inhibits the proliferation, migration and cord formation of human umbilical vein endothelial cells (HUVECs) in vitro, and prevents the vascular development of zebrafish embryos in vivo. Altogether, our data prove the anticancer and anti-angiogenesis properties of SPS, and provide further insights into the potential pharmacological application of SPS as antitumor and anti-angiogenic agent against lung cancer.

Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling.

Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129‐1

An extracellular alkaline protease produced by marine bacteria strain Pseudoalteromonas sp. 129‐1 was purified by ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The purity of the protease was confirmed by SDS–PAGE and molecular mass was estimated to be 35 kDa. The protease maintained considerable activity and stability at a wide temperature range of 10–60 °C and pH range of 6–11, and optimum activity was detected at temperature of 50 °C and pH of 8. Metallo‐protease inhibitor, EDTA, had no inhibitory effect on protease activity even at concentration up to 15 mM, whereas 15 mM PMSF, a common serine protease inhibitor, greatly inactivated the protease. The high stability of the protease in the presence of surfactants (SDS, Tween 80, and Triton X‐100), oxidizing agent H2O2, and commercial detergents was observed. Moreover, the protease was tolerant to most of the tested organic solvents, and saline tolerant up to 30%. Interestingly, biofilm of Pseudomonas aeruginosa PAO1 was greatly reduced by 0.01 mg ml−1 of the protease, and nearly completely abolished with the concentration of 1 mg ml−1. Collectively, the protease showed valuable feathers as an additive in laundry detergent and non‐toxic anti‐biofilm agent.

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities.

Antagonistic Activity and Mode of Action of Phenazine-1-Carboxylic Acid, Produced by Marine Bacterium Pseudomonas aeruginosa PA31x, Against Vibrio anguillarum In vitro and in a Zebrafish In vivo Model

Phenazine and its derivatives are very important secondary metabolites produced from Pseudomonas spp. and have exhibited broad-spectrum antifungal and antibacterial activities. However, till date, there are few reports about marine derived Pseudomonas and its production of phenazine metabolites. In this study, we isolated a marine Pseudomonas aeruginosa strain PA31x which produced natural product inhibiting the growth of Vibrio anguillarum C312, one of the most serious bacterial pathogens in marine aquaculture. Combining high-resolution electro-spray-ionization mass spectroscopy and nuclear magnetic resonance spectroscopy analyses, the functional compound against V. anguillarum was demonstrated to be phenazine-1-carboxylic acid (PCA), an important phenazine derivative. Molecular studies indicated that the production of PCA by P. aeruginosa PA31x was determined by gene clusters phz1 and phz2 in its genome. Electron microscopic results showed that treatment of V. anguillarum with PCA developed complete lysis of bacterial cells with fragmented cytoplasm being released to the surrounding environment. Additional evidence indicated that reactive oxygen species generation preceded PCA-induced microbe and cancer cell death. Notably, treatment with PCA gave highly significant protective activities against the development of V. anguillarum C312 on zebrafish. Additionally, the marine derived PCA was further found to effectively inhibit the growth of agricultural pathogens, Acidovorax citrulli NP1 and Phytophthora nicotianae JM1. Taken together, this study reveals that marine Pseudomonas derived PCA carries antagonistic activities against both aquacultural and agricultural pathogens, which broadens the application fields of PCA.

Mansouramycin C kills cancer cells through reactive oxygen species production mediated by opening of mitochondrial permeability transition pore

Cancer is one of the deadliest diseases in the world and the search for novel anticancer agents is urgently required. Marine-derived isoquinolinequinones have exhibited promising anticancer activities. However, the exact mechanisms of cytotoxic activities of these isoquinolinequinones are poorly characterized. In this study, we investigated the anticancer effects and molecular mechanisms of mansouramycin C (Mm C), a cytotoxic isoquinolinequinone isolated from a marine streptomycete. We demonstrated that Mm C preferentially killed cancer cells and the cytotoxic effects were mediated by reactive oxygen species (ROS) generation. Mass spectrometry based proteomic analysis of Mm C-treated A549 cells revealed that many ROS-related proteins were differentially expressed. Proteomic-profiling after Mm C treatment identified oxidative phosphorylation as the most significant changes in pathways. Analysis also revealed extensive defects in mitochondrial structure and function. Furthermore, we disclosed that Mm C-induced ROS generation was caused by opening of mitochondrial permeability transition pore. Notably, Mm C synergized with sorafenib to induce cell death in A549 cells. Hence, we propose that the marine-derived natural compound Mm C is a potent inducer of the mitochondrial permeability transition and a promising anticancer drug candidate. Moreover, molecular mechanisms of Mm C shed new light on the understanding of the cytotoxic mechanisms of marine-derived isoquinolinequiones.

The structure-activity relationship between polysaccharides from Sargassum thunbergii and anti-tumor activity

Polysaccharides derived from Sargassum thunbergii were prepared to investigate the structure-activity relationship between polysaccharides and anti-tumor activity in vitro. Many factors were examined. Overall, STW (polysaccharide extracted by hot water) had the best activity, followed by STJ (polysaccharide extracted by dilute alkali), and then STA (polysaccharide extracted by dilute acid). Location of algae had no effect at 500μg/mL and 1000μg/mL, while STW-QD (algae collected from Qingdao, China) had the best activity, followed by STW-WZ (algae collected from Wenzhou, China) and STW-LJ (algae collected from Lianjiang, China) and then STW-DL (algae collected from Dalian, China) and STW-RC (algae collected from Rongcheng, China) at 250μg/mL. Moreover, molecular weight had no effect at 1000μg/mL, while higher molecular weights were associated with better activities at 250μg/mL and 500μg/mL. Sulfate content had no effect at 1000μg/mL, while anti-tumor activities decreased accompanying with the changes of sulfate content. Uronic acid content was an important factor influencing activity. The fractions of STW showed little anti-tumor activity; however, the mixture of the fractions of STW showed approximately 60% inhibition. Overall, these findings suggested that the anti-tumor activity of polysaccharides required multilateral cooperation and that some of the effective components were lost.

Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities

Monthly variations of polysaccharides from Sargassum thunbergii and their anti-complement and anti-tumour activities were investigated. It was observed that an increase in fucose and total sugar contents occurred during the growth period (from early April to mid-June), accompanied by a decrease in molar ratios of other monosaccharides to fucose. The highest yields were obtained from early July to early September, which was in accordance with the significant increase in molar ratio of glucose to fucose and decrease in molar ratio of other monosaccharides to fucose. And the above results suggested that S. Thunbergii synthesized large amount of laminaran, the storage substance of brown algae, during the senescence period. However, sulfate contents were relatively stable in the life cycle of S. thunbergii. These results suggested that S. thunbergii synthesized complex sulfated heteropolysacchairdes during inactive period, while during other periods, it synthesized more sulfated galactofucan. All polysaccharides showed anti-complement activity, suggesting that the harvesting time did not influence the anti-complement activities. In the anti-tumour assay in vitro, the polysaccharides taken during the senescence period had much lower anti-tumour activity, suggesting that fucoidan, but not laminaran, determined the anti-tumour activities. Therefore, polysaccharides from S. thunbergii might have great potential in anti-complement and anti-tumour application.

Marine Bacterial Polysaccharide EPS11 Inhibits Cancer Cell Growth via Blocking Cell Adhesion and Stimulating Anoikis

Tumor cells that acquire metastatic potential have developed resistance to anoikis, a cell death process, after detachment from their primary site to the second organ. In this study, we investigated the molecular mechanisms of a novel marine bacterial polysaccharide EPS11 which exerts its cytotoxic effects through affecting cancer cell adhesion and anoikis. Firstly, we found that EPS11 could significantly affect cell proliferation and block cell adhesion in A549 cells. We further demonstrated that the expression of several cell adhesion associated proteins is downregulated and the filiform structures of cancer cells are destroyed after EPS11 treatment. Interestingly, the destruction of filiform structures in A549 cells by EPS11 is in a dose-dependent manner, and the inhibitory tendency is very consistent with that observed in the cell adhesion assay, which confirms that filiform structures play important roles in modulating cell adhesion. Moreover, we showed that EPS11 induces apoptosis of A549 cells through stimulating βIII-tubulin associated anoikis: (i) EPS11 inhibits the expression of βIII-tubulin in both transcription and translation levels; and (ii) EPS11 treatment dramatically decreases the phosphorylation of protein kinase B (PKB or AKT), a critical downstream effector of βIII-tubulin. Importantly, EPS11 evidently inhibits the growth of A549-derived tumor xenografts in vivo. Thus, our results suggest that EPS11 may be a potential candidate for human non-small cell lung carcinoma treatment via blocking filiform structure mediated adhesion and stimulating βIII-tubulin associated anoikis.