7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits...
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New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY O3-4 Polysaccharides production by autotrophic cultures of microalgae Giuseppe Olivieri 1 , Renato S. Coellho 2 , Telma T. Franco 2 , Antonino Pollio 3 , Antonio Marzocchella 1,∗ 1 DICMaPI – Università degli Studi di Napoli Federico II, Italy 2 Faculdade de Engenharia Química – University of Campinas, Brazil 3 Biological Science Department – Università degli Studi di Napoli “Federico II”, Italy Light energy and CO 2 may be considered as the most abun- dant and cheap feedstock for bioprocesses aimed to produce energy vectors as well as green chemical building blocks. Microalgae can be cultivated on waste streams of gas (CO 2 polluted) and liquids (industrial effluents and salt supplements) for producing biofuels and carbohydrates, a building block for bioplastics production. The aim of this contribute is to report about recent joint progress of a research activity carried out in Campinas (BR) and in Napoli (IT). The study regards autotrophic cultures of microalgae selected to produce a significant amount of carbohydrates coupled, if possible, with lipids as biofuel resource. Selected strains were autochthon in Brazil and from the collection available at the University of Napoli ACUF (http://www.biologiavegetale.unina.it/acuf.html). Microalgae were grown in flasks and then inoculated in the photobioreactors. Both the broth and the cells have been characterized for cultures carried out under a wide interval of operating conditions. Main measured data were cell concentration, total organic carbon (TOC) in the liquid phase, polysaccharide concentration and protein concentration. The attention was paid on both the extracellular and intracellular polysaccharides. http://dx.doi.org/10.1016/j.nbt.2014.05.1651 O3-5 Synthesis of potential prebiotics using Pseudomonas syringae DC3000 levansucrase Lsc3 Triinu Visnapuu 1,∗ , Anneli Aasamets 1 , Karin Mardo 1 , Eerik Jõgi 1 , Heiki Vija 2 , Tiina Alamäe 1 1 University of Tartu, Estonia 2 National Institute of Chemical Physics and Biophysics, Estonia Plant-derived inulin-type oligofructans are considered to be effective prebiotics that function as specific growth substrates for beneficial gut bacteria. The effects of levan-type FOS (fruc- tooligosaccharides) containing -2,6 linkages are poorly studied as they are not commercially available. Some studies prove their enhanced prebiotic effect [1]. Polymeric levan has potential appli- cations as anti-cancer, anti-inflammatory and immune stimulating agent. Levansucrases (EC 2.4.1.10) are bacterial enzymes belonging to GH68 family of glycoside hydrolases. Pseudomonas syringae DC3000 encodes three levansucrases (Lsc1, Lsc2, Lsc3). Recom- binant Lsc3 splits sucrose and synthesizes -2,6-linked FOS, polymeric levan and also heterooligofructans when transfructosy- lating alternative acceptors [2]. Lsc3 has very high catalytic activity (k cat ∼500 1/s), stability and therefore a high biotechnological potential. In this study, we optimized levan and FOS production by Lsc3 protein. High-performance liquid chromatography system cou- pled with ELS detector was used to detect and quantify saccharides, levan was quantified spectrophotometrically. We showed that Lsc3 produced up to 15.4g of FOS per mg of protein under optimized conditions. Product yield and spectrum depended on reaction conditions. Also, permeabilized bacterial cells expressing levansucrase were shown to serve as effective cat- alyst for FOS production. We developed a method for enzymatic production of levan-type FOS from sucrose and a simple yeast-based method for the removal of monosaccharides that form as by-products of the reaction. Acknowledgements: This work was supported by ERF grant 3.2.0701.12-0041 (SLOMR12215T) managed by Archimedes Foun- dation and an ETF grant GLOMR9072. References [1].Marx SP, et al. FEMS Microbiol Lett 2000;182:163–9. [2].Visnapuu T, et al. J Biotechnol 2011;155:338–49. http://dx.doi.org/10.1016/j.nbt.2014.05.1652 O3-6 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits metasta- sis in vitro and in vivo Hui Min Wang Kaohsiung Medical University, Taiwan Melanoma is the deadliest cancer. We identified 7- hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bioactive agent against human melanoma A375.S2 cells. 7- hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and anti- migratory activity of melanoma in vitro and in vivo. Cell proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysis were applied to observe cell morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein iso- thiocyanate (FITC)/propidium iodide (PI) double stained. Western blot was applied to examine protein expressions whereas wound healing assay was to examine cell activity. Strong anticancer effects of 7-HDNF exhibited in a dose-dependent manner, and displayed minor cytotoxicities on normal human skin cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF increased the cellular arrest in cell cycle at G2/M phase. Cellular membrane asymme- try lost was confirmed. Protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. 7-HDNFpresented the high-quality anti-migratory activity. 7-HDNF inhibited melanoma tumor growth in mice xenograft model, accompanied with a decrease www.elsevier.com/locate/nbt S17
Transcript of 7-Hydroxydehydronuciferine induces human melanoma A375.S2 autophagy and apoptosis and inhibits...
New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY
O3-4
Polysaccharides production by autotrophic cultures ofmicroalgae
Giuseppe Olivieri 1 , Renato S. Coellho2, Telma T. Franco2, AntoninoPollio3, Antonio Marzocchella1,∗
1 DICMaPI – Università degli Studi di Napoli Federico II, Italy2 Faculdade de Engenharia Química – University of Campinas, Brazil3 Biological Science Department – Università degli Studi di Napoli “Federico II”,Italy
Light energy and CO2 may be considered as the most abun-dant and cheap feedstock for bioprocesses aimed to produce energyvectors as well as green chemical building blocks. Microalgae canbe cultivated on waste streams of gas (CO2 polluted) and liquids(industrial effluents and salt supplements) for producing biofuelsand carbohydrates, a building block for bioplastics production.
The aim of this contribute is to report about recent jointprogress of a research activity carried out in Campinas (BR) and inNapoli (IT). The study regards autotrophic cultures of microalgaeselected to produce a significant amount of carbohydrates coupled,if possible, with lipids as biofuel resource.
Selected strains were autochthon in Brazil and fromthe collection available at the University of Napoli ACUF(http://www.biologiavegetale.unina.it/acuf.html). Microalgaewere grown in flasks and then inoculated in the photobioreactors.Both the broth and the cells have been characterized for culturescarried out under a wide interval of operating conditions. Mainmeasured data were cell concentration, total organic carbon (TOC)in the liquid phase, polysaccharide concentration and proteinconcentration. The attention was paid on both the extracellularand intracellular polysaccharides.
http://dx.doi.org/10.1016/j.nbt.2014.05.1651
O3-5
Synthesis of potential prebiotics using Pseudomonassyringae DC3000 levansucrase Lsc3
Triinu Visnapuu1,∗ , Anneli Aasamets1, Karin Mardo1, Eerik Jõgi1,Heiki Vija2, Tiina Alamäe1
1 University of Tartu, Estonia2 National Institute of Chemical Physics and Biophysics, Estonia
Plant-derived inulin-type oligofructans are considered to beeffective prebiotics that function as specific growth substratesfor beneficial gut bacteria. The effects of levan-type FOS (fruc-tooligosaccharides) containing �-2,6 linkages are poorly studiedas they are not commercially available. Some studies prove theirenhanced prebiotic effect [1]. Polymeric levan has potential appli-cations as anti-cancer, anti-inflammatory and immune stimulatingagent.
Levansucrases (EC 2.4.1.10) are bacterial enzymes belongingto GH68 family of glycoside hydrolases. Pseudomonas syringaeDC3000 encodes three levansucrases (Lsc1, Lsc2, Lsc3). Recom-binant Lsc3 splits sucrose and synthesizes �-2,6-linked FOS,polymeric levan and also heterooligofructans when transfructosy-
lating alternative acceptors [2]. Lsc3 has very high catalytic activity(kcat ∼500 1/s), stability and therefore a high biotechnologicalpotential.
In this study, we optimized levan and FOS production by Lsc3protein. High-performance liquid chromatography system cou-pled with ELS detector was used to detect and quantify saccharides,levan was quantified spectrophotometrically.
We showed that Lsc3 produced up to 15.4 g of FOS per mg ofprotein under optimized conditions. Product yield and spectrumdepended on reaction conditions. Also, permeabilized bacterialcells expressing levansucrase were shown to serve as effective cat-alyst for FOS production.
We developed a method for enzymatic production of levan-typeFOS from sucrose and a simple yeast-based method for the removalof monosaccharides that form as by-products of the reaction.
Acknowledgements: This work was supported by ERF grant3.2.0701.12-0041 (SLOMR12215T) managed by Archimedes Foun-dation and an ETF grant GLOMR9072.
References
[1].Marx SP, et al. FEMS Microbiol Lett 2000;182:163–9.[2].Visnapuu T, et al. J Biotechnol 2011;155:338–49.
http://dx.doi.org/10.1016/j.nbt.2014.05.1652
O3-6
7-Hydroxydehydronuciferine induces human melanomaA375.S2 autophagy and apoptosis and inhibits metasta-sis in vitro and in vivo
Hui MinWang
Kaohsiung Medical University, Taiwan
Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from theleaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be abioactive agent against human melanoma A375.S2 cells. 7-hydroxydehydronuciferine (7-HDNF) was known to induceautophagy and apoptosis response mechanisms, and anti-migratory activity of melanoma in vitro and in vivo. Cellproliferation assay was used to test cell viability. Acridine orange(AO) staining and flow analysis were applied to observe cellmorphology. The apoptotic cell death ratio was measured viatwo-dimensional flow cytometry by annexin V-fluorescein iso-thiocyanate (FITC)/propidium iodide (PI) double stained. Westernblot was applied to examine protein expressions whereas woundhealing assay was to examine cell activity. Strong anticancer effectsof 7-HDNF exhibited in a dose-dependent manner, and displayedminor cytotoxicities on normal human skin cells.7-HDNF inducedthe formation of intracellular vacuoles and the augmentation ofacidic vesicular organelles (AVO). 7-HDNF increased the cellulararrest in cell cycle at G2/M phase. Cellular membrane asymme-try lost was confirmed. Protein expressions were discovered toverify autophagy and apoptosis response mechanisms sharingthe associated pathways. 7-HDNFpresented the high-qualityanti-migratory activity. 7-HDNF inhibited melanoma tumorgrowth in mice xenograft model, accompanied with a decrease
www.elsevier.com/locate/nbt S17
SYMPOSIUM 3: GLYCOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014
of phosphorylation of AKT. We demonstrated the mechanismof this compound starting with the formation and accumula-tion of AVO leading to autophagy. 7-HDNF caused the cellularmembrane asymmetry lost, triggering the G2/M cell cycle arrestin caspase-dependent apoptosis. 7-HDNF presented high-quality
anti-migratory bio-functions and inhibited melanoma tumorgrowth in mice xenograft model.
http://dx.doi.org/10.1016/j.nbt.2014.05.1653
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