Levan is a high-valued polysaccharide of fructose produced by several microbial species. These polysaccharides have been described as effective therapeutic agents in some human disease conditions, such as cancer, heart diseases and diabetes.
Carbohydrate Polymers 273 (2021) 118613 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Production of levan from Bacillus subtilis var natto and apoptotic effect on SH-SY5Y neuroblastoma cells Amanda Mota Vieira a, Farrah Zahed b, Alessandre Carmo Crispim c, Edson de Souza Bento c, Rafael de Freitas Oliveira Franỗa d, Irapuan Oliveira Pinheiro a, Luis A Pardo b, Bruno Melo Carvalho a, * a Biological Sciences Institute, University of Pernambuco (ICB/UPE), Recife, Brazil Max Planck Institute for Experimental Medicine, Gă ottingen, Germany Institute of Chemistry and Biotechnology, Federal University of Alagoas, Maceio, Brazil d Department of Virology, Aggeu Magalh˜ aes Institute (IAM/FIOCRUZ Pernambuco), Recife, Brazil b c A R T I C L E I N F O A B S T R A C T Keywords: Exopolysaccharide Neoplastic cells Live-cell analysis Levan is a high-valued polysaccharide of fructose produced by several microbial species These polysaccharides have been described as effective therapeutic agents in some human disease conditions, such as cancer, heart diseases and diabetes The objective of this study was to examine the effect of levan (β-(2 → 6)-fructan) produced through sucrose fermentation by B subtilis var natto on the proliferation rate, cytotoxicity, and apoptosis of human neuroblastoma SH-SY5Y cells It was obtained 41.44 g/L of levan in 18 h by biotechnological fermen tation and SH-SY5Y cells were exposed to 1000 μg/mL of levan The treatment with 1000 μg/mL of levan induced apoptosis in SH-SY5Y cancer cells by the significant increase in Annexin V/7-AAD and caspase 3/7 activation, but did not decrease proliferation or triggered a cytotoxic effect 1000 μg/mL levan treatment is a promising therapeutic strategy for SH-SY5Y neuroblastoma cells Introduction Cancer is a disease characterized by the abnormal growth of cells, which can invade adjoining parts of the body and/or spread to other organs, affecting almost any part of the body According to the World Health Organization, cancer is one of the leading causes of death glob ally, and it was responsible for 10 million deaths in 2020 (World Health Organization, 2020) This disease rarely occurs before the age of 20 years, but childhood cancers exist, raising a range of medical, psy chological, ethical, and societal concerns (Steliarova-foucher et al., 2017) Neuroblastoma (NB) is the most common neoplasm during infancy, usually diagnosed in the first year of life Prevention and screening are not possible, due to the formation of this tumor during sympathetic nervous system development (Cheung & Dyer, 2013) The tumorigenesis of neuroblastoma involves both embryonic and oncogenic factors, as this is a highly heterogeneous and complex disease (Kågedal, 2009) Many factors, such as age and stage of the disease at diagnosis, and molecular, cellular, and genetic characteristics of the tumor, determine whether it will spontaneously regress or metastasize and become highly malignant (Cheung & Dyer, 2013) The standard treatment for NB is based on the combination of chemotherapeutic drugs such as doxorubicin, vincristine, cyclophos phamide, and cisplatin; however, chemoresistance occurs and the tumor becomes highly aggressive and metastatic (Tibullo et al., 2018) In search of new therapeutic approaches, several compounds from pro cesses have been developed and tested in the SH-SH5Y neuroblastoma cell line (Biedler, Roffler-tarlov, Schachner, & Freedman, 1978) Levan are fructose polysaccharides that are produced by plants and many microorganisms Consisting almost solely of fructosyl residues linked via the β-2, carbons, these fructan molecules are packed into nano-sized, spherical forms, providing them with a remarkably low intrinsic viscosity (Arvidson, Rinehart, & Gadala-Maria, 2006) Micro organisms, such as Bacillus subtilis (Veerapandian, Ramiah, & Varadhan, 2020), Bacillus aryabhattai (Nasir et al., 2020), Brachybacterium pheno liresistens (Moussa, Al-qaysi, Thabit, & Kadhem, 2017), Gluconobacter ăvels, Kosciow, Kniewel, Jakob, & Deppenmeier, 2020) can strains (Ho produce levan from sucrose, syrups, or molasses in submerged cultures, * Corresponding author at Av Gov Agamenon Magalh˜ aes - Santo Amaro, Recife, PE 50100-010, Brazil E-mail address: bruno.carvalho@upe.br (B.M Carvalho) https://doi.org/10.1016/j.carbpol.2021.118613 Received 15 June 2021; Received in revised form 20 August 2021; Accepted 23 August 2021 Available online 27 August 2021 0144-8617/© 2021 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/) A.M Vieira et al Carbohydrate Polymers 273 (2021) 118613 and possible levan structural variability may be induced by different production conditions and molecular weight (Hundschell, Jakob, & Wagemans, 2020) Levansucrase EC 2.4.10 are fructosyltransferases enzyme (E.C.2.4.1.9) that catalyzes β-(2,6)-levan synthesis through su crose hydrolysis to glucose and fructose, and also catalyzes formation of fructooligosaccharides (FOS) It has been considered one of the most important enzymes in levan's biotechnological field, with high levels of efficiency (using 100 g/L of sucrose as substrate in production medium, it was obtained 47 g/L of levan yield) (Ragab et al., 2019) The microbial source of levansucrase determines the molecular weight, degree of branching (Runyon, Nilsson, Ulmius, & Castro, 2014), diameter, intrinsic viscosity, stability, and functionalities such as immunogenic activity and adhesive strength Since the discovery of the molecular versatility of levan, researchers have been attracted by the potential health benefits of these natural product Regarding anticancer activity, levan molecular weight execute an important role (Calazans, Lima, de Franỗa, & Lopes, 2000), although researchers have not yet reached a consensus on the ideal molecular weight for the treatment The antineoplastic activity of levan has been widely investigated for the potential in treating hepatocellular and gastric carcinomas (Yoon, Yoo, Cha, & Lee, 2004) (Abdel-fattah, Gamaleldeen, Helmy, & Esawy, 2012a; Cabral de Melo, Borsato, Macedo, & Celligoi, 2015; Dahech, Belghith, Belghith, & Mejdoub, 2012; Esawy et al., 2013; Sarilmiser & Oner, 2014); however, information regarding the effects of levan in other cancer cell lines is lacking Thus, the aim of this study was to produce levan by microorganism B subtilis var natto and evaluate the effects of levan on cellular proliferation, cytotoxicity, and apoptosis processes in an aggressive neuroblastoma cell line (SHSY5Y) Columbus, Ohio, EUA) and a dissolved oxygen probe (Type In PRO®; Mettler Toledo) Pre-cultured medium (600 mL) were inoculated into 2400 mL of levan production medium and cultured for 18 h Agitation was provided by a three-blade impeller operated at 950 rpm Aeration was provided by a sprinkler, and the aeration rate was maintained at vvm pH was maintained at 7.0 during cultivation and was monitored according to previous pH optimization studies (Chidambaram et al., 2019; Gojgic-Cvijovic et al., 2019; Ragab et al., 2019) To maintain the pH, N NaOH and N HCl were automatically added to the culture broth The fermentation temperature was maintained using a recirculating water bath at 37 ◦ C During fermentation, samples were extracted hourly to evaluate bacterial growth by turbidimetry at λ = 660 nm Levan production were performed in duplicate 2.4 Sucrose concentration Sucrose concentration was determined using the High Performance Liquid Chromatography Serie 1200 (Agilent Technologies, Waldbronn, Germany) using a 300 × 7.8 mm Aminex HPX-87H column, with μm particle size (Bio-Rad, Hercules, CA, USA) The mobile phase used was mM sulfuric acid at a flow rate of 0.6 mL/min and a temperature of 20 ◦ C Sucrose was used for the standard curve (1 g/L–5 g/L), and integration of the peak area was performed using ChemStation Rev B.04.02.98 software (Agilent Technologies) 2.5 Levan molecular weight Levan was isolated from the cell-free culture fluid by centrifugation at 18,500 ×g at ◦ C, followed by 0.22 μm filtration, and precipitated using 70% v/v of cold ethanol The precipitate was redispersed in L of ˜o Paulo, distilled water and dried in a Spray dryer MSDi 1.0 (LabMaq, Sa Brazil), at a flow rate of 0.8 L/h, with a 140 ◦ C inlet temperature and a 113.3 ◦ C outlet temperature At the end of the operation, the powder was weighed, and the levan concentration was estimated in g/L The average molecular weight (Mw), number average molecular weight (Mn), and molecular weight of the highest peak (Mp) polydispersity index (PDI = Mw/Mn) of levan was determined using a highperformance size-exclusion chromatography system (GPC), models BOM007, INJ 003, and CRL008 (Waters, Santa Clara, CA, USA) coupled with four serially connected Shodex columns (SB 806 M HQ, SB 804 HQ, SB 803 HQ, and SB 802 HQ) The polysaccharide sample was dissolved in ultrapure water and filtered through a 0.22 μm Millipore filter (Merck, Newark, NJ, USA) before injection The mobile phase was 0.1 M sodium nitrate at a flow rate of 0.4 mL/min at room temperature, and the Waters 2414 refractive index was used for detection Pullulan (Shodex, Tokyo, Japan) was used as a standard for the correlation curve (MP 5800, MP 12,200, MP 23,700, MP 48,000, MP 44,200, MP 100,000, MP 186,000, and MP 1,660,000 g/mol) Finally, data acquisition and processing were performed using Waters Empower software (Walters, Santa Clara, CA, USA) Material and methods 2.1 Microorganisms and reagents for culture medium B subtilis var natto was purchased from GEM Cultures (Ft Bragg, CA, USA) B subtilis var natto was kept on Nutrient Agar (NA) medium, containing: agar (15 g/L), beef extract (3 g/L), sodium chloride (5 g/L), peptone (5 g/L) And also nutrient broth (NB) was composed of beef extract (3 g/L), peptone (1.5 g/L), and NaCl (5 g/L) were purchased from Becton Dickinson (BD) (Franklin Lakes, New Jersey, USA) MgSO4⋅7H2O, NaH2.PO4.2 H2O, and NaH2⋅PO4⋅12H2O were obtained from Sigma Chemicals (San Luis, Missouri, USA) All reagents used had a high level of purity (≥98% of purity grade) 2.2 Inoculum preparation B subtilis var natto was maintained at ◦ C on NA medium and subcultured every 15 days For activation, bacteria were cultured on NA at 37 ◦ C, pH 7.4, overnight, then colonies were transferred into mL of nutrient broth (pH 7.4) and incubated at 37 ◦ C for 24 h, with agitation at 150 rpm Cell growth was determined by turbidimetry at λ = 660 nm using a spectrophotometer (Bel Photonics, S˜ ao Paulo, Brazil) and the inoculum was 1% (v/v) After incubation, cells were transferred to a 60 mL Erlenmeyer flask for 24 h, shaking at 150 rpm Following, 540 mL of levan production medium, a modification of that previously described by da Costa et al (da Costa, 2005), composed of sucrose (250 g/L), urea (2 g/L), yeast extract (5 g/L), KH2PO4 (1 g/L), K2HPO4 (8 g/L), MgSO4⋅7H2O (1 g/L), FeSO4.7H2O (0.1 g/L), CuSO4⋅5H2O (0.0088 g/L), MnSO4⋅H2O (0.0076 g/L), and ZnSO4⋅7H2O (0.01 g/L), was added under the same conditions 2.6 H and 13 C NMR spectroscopy Chemical structure of levan was confirmed by NMR All NMR spectra were measured on a AvanceTM 600 spectrometer (Bruker, Madison, WI), 600.09 MHz for 1H and 150.89 MHz for 13C nuclei at 25 ◦ C, using a mm PABBO probe Spectra were obtained at 298 K in D2O (10 mg/mL), and the residual H1 chemical shift signal of D2O was used as reference for chemical shift in the hydrogen spectrum Homo- and heteronuclear two-dimensional (2D) spectra H–H COSY (correlation spectroscopy), H–C HSQC (Heteronuclear Single-Quantum Coherence) and H–C HMBC (Heteronuclear Multiple Bond Correlation) applying standard Brucker's pulse sequences were used for full assignment of the signals TOPSPIN 3.2 (Bruker) was used for data acquisition and processing 2.3 Levan production by batch fermentation Fermentation was carried out in a fully instrumented and computercontrolled 5-L SL-135/E stirred tank bioreactor (Solab, S˜ ao Paulo, Brazil), equipped with a pH probe (Type In PRO® 3255; Mettler Toledo, A.M Vieira et al Carbohydrate Polymers 273 (2021) 118613 2.7 Cell culture mean ± SEM Cell fluorescence was represented by the green object fluorescence (GOF) unit The Student's t-test and one-way analysis of variance (ANOVA) were used to compare differences between the groups Values of p < 0.05 were considered statistically significant SH-SY5Y neuroblastoma cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in culture with Dulbecco's Modified Eagle Medium (1×) + GlutaMAX (GIBCO-Invitrogen, Carlsbad, CA, USA) supplemented with 15% FCS and antibiotic/antimycotic (1% penicillin/streptomycin), in a humidified atmosphere at 37 ◦ C and 5% CO2 The medium was changed as needed, and cells were sub-cultured upon reaching ~85% confluence Result and discussion 3.1 Evaluation of the levan produced by B subtilis var natto The growth curve of B subtilis var natto and sucrose concentration in media changed during fermentation, as shown in Fig The microor ganisms remained in the lag phase for the first h, grew linearly for h, and declined from the tenth hour until the end of levan production (Fig 1) In medium with an initial 250 g/L of sucrose, the substrate was completely consumed after 14 h of fermentation The final levan con centration after drying was estimated at 41.44 g/L Similar findings were previously reported using fermentation with the same microor ganism and initial sucrose levels, reaching 61 g/L at 24 h (Wu, Chou, & Shih, 2013) In addition, using Bacillus licheniformis NS03, which is of the same genus, approximately 53 g/L of levan was produced after 48 h of fermentation (Gojgic-Cvijovic et al., 2019) Taking into account the total concentration of levan and the duration of fermentation time, B subtilis var natto effectively produced levan in bioreactor 2.8 Cell proliferation assay Cellular proliferation rate was measured using a live-cell imaging system (IncuCyte ZOOM, Essen Bioscience, Birmingham, UK) and the corresponding software application Cells (1.0 × 105) were seeded into 96-well plates, and levan treatment was initiated after 24 h, in triplicate Levan concentration ranged from 200 μg/mL to 1000 μg/mL, and im ages were recorded hourly over 1–5 days Proliferation rate (%) was determined as cell confluence over time every hour 2.9 Caspase 3/7 activity assay Apoptotic processes dependent on caspase 3/7 activation were evaluated by live-cell imaging (IncuCyte ZOOM®; Essen Bioscience) using the CellPlayer96-Well Kinetic Caspase-3/7 reagent containing the caspase substrate DEVD coupled to the DNA intercalating dye NucView 488 (Incucyte Caspase 3/7 Green reagent; Essen Bioscience) at a con centration of μM, which was added along with cell culture medium to the different levan test concentrations (200, 600, 800, and 1000 μg/mL) in 96-well plates The wells were analyzed until they reached 80%–90% cell confluence The number of apoptotic cells per well was determined in triplicate, as the number of green positive signals (in the green channel; 488 nm)/mm2 over time 3.2 Levan molecular weight description Levan generated in this study showed a bimodal distribution of two distinct Mw; the larger proportion had a lower Mw (8.8 kDa), and the smaller portion had a higher Mw (~2201 kDa), and both had a relatively low polydispersity index (around 1.3) (Supplementary data, Fig S1; Supplementary data, Table S1) Levan's physicochemical characteristics and biological potential are ă determined by the microorganism and production conditions (Oner, ´ndez, & Combie, 2016; Tanaka, Oi, & Yamamoto, 1980) A narrow Herna polydispersity index is important for the suitability to various applica tions, and values