Mechanisms of antimelanoma effect of oat β-glucan supported by electroporation

Anna Choromanska a,⁎, Sandra Lubinska b, Anna Szewczyk c, Jolanta Saczko a,d, Julita Kulbacka a,d,⁎
aDepartment of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368 Wroclaw, Poland
bDepartment of Chemistry, Wroclaw University of Science and Technology, Norwida 4, 50-373 Wroclaw, Poland
cDepartment of General Zoology, Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland
dDepartment of Molecular and Cellular Biology, Wroclaw Medical University, Borowska 211A, 50-556 Wroclaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history:
Received 22 November 2017
Received in revised form 19 May 2018 Accepted 5 June 2018
Available online 06 June 2018
There are still not specified mechanisms how beta-glucan molecules are transported into cells. Supposing, beta- glucan toxicity against tumor cells may be related to the overexpression of the transporter responsible for the transport of glucose molecules in the cells. In this case, glucans – polymers composed of glucose units are much more up-taken by tumor than normal cells. Increased GLUT1 (Glucose Transporter Type 1) expression has been demonstrated earlier in malignant melanomas. GLUT1 expression promotes glucose uptake and cell

Oat beta-glucan GLUT1
WZB117 Melanoma
growth in that cells. Also, in human melanoma tissues a significant correlation between GLUT1 expression and mitotic activity was found.
The aim of the study was to verify if oat β-glucan (OβG) is delivered into cells by GLUT-1 membrane protein. To check it out we blocked GLUT1 transporters by an inhibitor WZB117 and then we investigated cells viability with and without reversible electroporation (EP).
The obtained results bring us to elucidate the mechanism of transport of the OβG into the cells is GLUT-1 depen- dent and moreover can be supported by EP method.
© 2018 Elsevier B.V. All rights reserved.


In recent years the interest of the usage of plants for the production of pharmacological compounds has increased. Medicines derived from plants nowadays are becoming even more popular than standard che- motherapeutics. To compete with the growing synthetic drugs market, there is an urgency to scientifically validate more its usefulness in the medication [1]. The study of the metabolic drugs pathways is an essen- tial and important part of the drug development process. During the drug evaluation the research of drug metabolism is of high importance especially when metabolites are pharmacologically active, toxic or when a drug is extensively metabolized [2].
Numerous studies have revealed that β-glucan has many therapeu- tic properties, including anticancer [3, 4]. It is non-cellulosic polymer of β-glucose, which is a glycoside in position β (1-3), (1-4) or β (1-6) [5, 6]. β-Glucans are carbohydrates found in the cell walls of yeast, fungi, algae, lichens, and plants such as oats or barley [6]. There are various sources from which β-glucan can be obtained. This enables to get a large number of formulations of similar or different properties [3].

Immunomodulatory and anti-cancer properties of β-glucans result mainly from their structure and degree of branching, but the mecha- nisms of anticancer activities of β-glucan seem to be multicomplex and still unclear [7–9]. There are still not specifi ed mechanisms how β-glucan molecules are transported into cells. According to our hypoth- eses, β-glucan cytotoxicity against tumor cells may be related to the overexpression of the transporter responsible for the transport of glu- cose molecules to the cells. In this case, glucans – polymers composed of glucose units are much more up-taken by tumor than normal cells [4, 9].
Up to 90% of cancers demonstrate a phenotype of an increased glu- cose uptake and increased dependence on glucose as a source of energy and biosynthesis precursor for cell growth, while normal cells metabo- lize lipids, amino acids and glucose in a more balanced way [10, 11]. The increased glucose uptake by cancer cells is achieved primarily by upreg- ulation of glucose transporters (GLUTs) [12]. The increased GLUT1 (Glu- cose Transporter Type 1) expression was demonstrated in malignant melanomas [13]. GLUT1 expression promotes glucose uptake and cell growth in that cells [14]. Also, in human melanoma tissues a significant correlation between GLUT1 expression and mitotic activity was found

⁎ Corresponding authors at: Department of Medical Biochemistry, Wroclaw Medical University, Chałubińskiego 10, 50-368 Wroclaw, Poland.
E-mail addresses: [email protected], (A. Choromanska), [email protected] (J. Kulbacka).

https://doi.org/10.1016/j.bioelechem.2018.06.005 1567-5394/© 2018 Elsevier B.V. All rights reserved.
[13]. The overexpression of GLUT1 protein is related in poor prognosis in a wide range of solid tumors [15].
We investigated the possible involvement of GLUT1 in the transport of β-glucan to melanoma cells. In our experiments WZB117 was used.

This is a small molecule, that effectively inhibits GLUT1 and cancer cell growth in vitro and in vivo [16, 17]. Previous studies showed that WZB117 impeded glucose transport in human red blood cells, in which GLUT1 is the only glucose transporter expressed [18]. This con- clusively shows that WZB117 inhibits GLUT1. Different studies have shown that after the exposure to WZB117 cancer cells experienced an immediate reduction in glucose transport and it is the consequence of the decrease in the GLUT1 protein level after the WZB117 treatment [19].
For the verification how β-glucan is transported to cells, different variants of viability test were performed: after incubation with glucan, after blocking GLUT1, after incubation with glucan and simultaneous blocking of GLUT1 and finally after blocking GLUT1 and delivering glu- can to cells by reversible electroporation.


2.1.Cell culture

Two human malignant melanoma cell lines were used – Me45 and MeWo cell line. Me45 cell line (derived from a lymph node metastasis of skin melanoma in a 35-year-old male) was established in 1997 at the Radiobiology Department of the Center of Oncology in Gliwice, Poland, MeWo cell line was purchased from ATCC® (LGC Standards, Poland). The cells were grown as a monolayer in Dulbecco modifi ed Eagle medium (DMEM, Sigma-Aldrich, USA) which was supplemented by 2 mM L-glutamine, 10% fetal bovine serum (FBS, Sigma-Aldrich, USA) and 50 μg/ml streptomycin (Sigma-Aldrich, USA) at 37 °C in 5% CO2. Before every experiment cells were removed by 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, USA).


Oat β-glucan was courtesy of Mrs. J. Harasym from the University of Economics in Wroclaw and it was obtained due to the procedure de- scribed previously [20] with beta-glucanase inactivation during lipid re- moval step, alkaline extraction, protein removal in isoelectric point, solution neutralization to pH = 7.0 and beta-glucan precipitation with ethanol. The different concentrations of OβG were used to the studies (50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml, 500 μg/ml).

The inhibitor WZB117 was purchased from Sigma-Aldrich (Poland). The following concentrations were used for experimental protocols: 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM.

2.3.Cellular viability – MTT assay

The viability of cells was determined by MTT assay (Sigma-Aldrich, USA) after experiments with different concentrations of beta-glucan (50–500 μg/ml), different concentrations of WZB117 (2.5 μM–25 μM) and after experiments where oat β-glucan, WZB117 or its combination was supported by electroporation. The MTT assay was used to the esti- mation of mitochondrial metabolic function through the measurement of mitochondrial dehydrogenase after 24 h incubation after experi- ments. For the experiment the cells were seeded into 96-well microcul- ture plates at 1 × 104 cells/well. After incubation with selected concentrations of beta-glucan the experiments were realized according to the manufacture’s protocol. The absorbance was determined using a multiwell scanning spectrophotometer at 570 nm (Enspire Perkin Elmer Multiplate reader, USA). Mitochondrial metabolic function was expressed as a percentage of viable treated cells in relation to untreated control cells.

2.4.Electroporation protocol

The electroporation was carried out using Gene Pulser Xcell total electroporation system (Bio-Rad, cat. number: 165-2660, purchased from Bio-Rad Poland). The electroporation protocol was selected ac- cording to the previous studies [19]. Cells in suspension were centri- fuged for 5 min at 800 rpm and resuspended in the electroporation buffer with low electrical conductivity (10 mM phosphate, 1 mM MgCl2, 250 mM sucrose, pH 7.4) with selected drug. After pulsation (eight 100 μs pulses, 400–1200 V/cm) cells were left for 10 min with an addition of DMEM at 37 °C, then centrifuged and seeded into culture 96-well plates for the MTT assay described in Section 2.3.

2.5.β-Glucan-WZB117-EP – combination therapy

Melanoma cells were trypsinized and suspended in electroporation buffer containing WZB117 in 25 μM concentration and β-glucan in con- centrations: 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml, 500 μg/ml. Than cells were exposed to electroporation according protocol described in

Fig. 1. viability of Me45 and MeWo cell line after 24 h and 72 h incubation following increasing concentrations of oat β-glucan (panel A), after incubation with the increasing concentrations of WZB117 (panel B), following increasing concentrations of oat β-glucan in combination with 25 μM WZB117 (panel C). Viability is expressed as the percentage of the control cells (cells without oat β-glucan). Error bars shown are means ±SD for n ≥ 3. *Statistically significant for p ≤ 0.05.

Section 2.4. Then viability assay after 24 and 72 h was performed as de- scribed in Section 2.3.

2.6.Immunofl uorescent assessment of GLUT1 – confocal laser scanning microscopy (CLSM) study

For the immunofluorescent reaction the cells were seeded on micro- scopic cover slides. Then after 24 h, an inhibitor WZB117 was added in the following concentrations 2.5–25 μM for 24 h incubation. After this time cells were fixed 4% paraformaldehyde (PFA, Sigma-Aldrich, USA) in PBS, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, USA) in

PBS (v/v) for 5 min. and blocked with 1% FBS in PBS (for 30 min. at RT). The cells were washed in PBS on every step of procedure. The fol- lowing antibodies were used: primary monoclonal antibody anti- GLUT1 produced in mouse (overnight incubation at 4 °C; 1:100; ThermoFisher Scientifi c, Germany); secondary antibody goat anti- mouse IgG FITC conjugated (for 60 min. at RT; 1:50; Sigma-Aldrich, USA). DNA was stained with DAPI (4,6-diamidino-2-phenylindole) contained in mounting medium (Fluoroshield™, Sigma-Aldrich, USA). For imaging, Olympus FluoView FV1000 confocal laser scanning micro- scope (Olympus, Japan) was used. The images were recorded by employing a Plan-Apochromat 60× oil-immersion objective.

Fig. 2. Viability of Me45 and MeWo cell line after 24 h and 72 h incubation following different parameters of electroporation (panel A), increasing concentrations of oat β-glucan in combination with electroporation (panel B), after incubation with 25 μM WZB117 and increasing concentrations of oat β-glucan in supported by electroporation (panel C) and after increasing concentrations of WZB117 in combination with electroporation (panel D). Viability is expressed as the percentage of the control cells (cells without oat β-glucan). Error bars shown are means ±SD for n ≥ 3. *Statistically significant for p ≤ 0.05.

2.7.Glucose uptake assay

The verification if GLUT1 transporters were effi ciently blocked by WZB117, was performed by the glucose uptake assay (Promega, USA). It is a non-radioactive, plate-based, homogeneous bioluminescent method for measuring glucose uptake in mammalian cells based on the detection of 2-deoxyglucose-6-phosphate (2DG6P). Test reagents include: glucose-6-phosphate dehydrogenase (G6PDH), NADP+, Re- ductase, Ultra-Glo™ Recombinant Luciferase and proluciferin substrate. G6PDH oxidizes 2DG6P to 6-phosphodeoxygluconate and simulta- neously reduces NADP+ to NADPH. The reductase uses NADPH to con- vert the proluciferin to luciferin, which is then used by Ultra-Glo™ Recombinant Luciferase to produce a luminescent signal that is propor- tional to the concentration of 2DG6P.

2.8.GLUT1 inhibition protocol

Based on MTT assay, immunofluorescent assessment of GLUT1 and glucose uptake assay for GLUT1 inhibition in melanoma cells 25 μM con- centration of WZB117 was selected.

2.9.Statistical analysis

Statistical signifi cance was determined by 2-way ANOVA test vs. control untreated cells where p b 0.05 values were assumed as statisti- cally significant. The results were analyzed statistically with GraphPad Prism 7.03. All samples were analyzed in triplicate.

3.Results and discussion

The results obtained from viability test showed a decrease in survival of melanoma cells with increasing β-glucan concentration (Fig. 1A). In a variant where the transport of glucan was assisted by electroporation the cytotoxicity was signifi cantly higher (25% decrease). Me45 cell line was more sensitive to β-glucan. After 72 h of incubation with the highest concentration of β-glucan the cell viability reached 43% and after EP-OβG it indicated 33%. In the same conditions the viability of MeWo cells revealed 57% and 44% respectively (Fig. 2B). This part of study presented similar outcomes as the previous reported [9, 21, 22]. WZB117 efficiently inhibited the expression of GLUT1 protein, as was demonstrated by immunofluorescence expression of GLUT1 (Fig. 3A) and by bioluminescent method for measuring glucose uptake (Fig. 3B). Based on viability test WZB117 in concentration 25 μM was chosen for study (Fig. 1B). Cell viability in samples incubated with glu- can and inhibitor was signifi cantly higher than in samples without WZB117 (in both tested lines the viability was higher N10% after 24 h and about 30% higher after 72 h incubation) (Fig. 1C). Distinctly the highest cytotoxic effect in both cell lines was observed after the syner- gistic effect of WZB117 and reversible electroporation with β-glucan (Fig. 2C).

The previous investigation demonstrated that β-glucans have low cytotoxicity in normal cells and they are well tolerated by oncological patients [9, 23]. Despite this there is still not enough information about its transport to cells. The results of our study showed that OβG significantly reduced melanoma cells viability and EP-OβG significantly increased the cytotoxic effect. WZB117 successfully reduced GLUT1 ex- pression. The viability assessment after blocking GLUT1 and OβG incu- bation confirmed our assumption, that β-glucan can be transported to the cells via GLUT1 transporters. Cell viability was significantly higher compared to cells where GLUT1 was not blocked. Furthermore, in ex- periments where GLUT1 was blocked and OβG was delivered into cells by electroporation there was observed the highest cytotoxic effect. In this case influx of energy substrate (glucose molecules) into the cells was limited, but at the same time glucan was efficiently brought up to the cells and caused the cytotoxic effect. These results indicate that glu- can is transported into cells via GLUT1 transporters. We also came to the interesting conclusion that the synergistic effect of WZB117 and revers- ible electroporation with OβG causes much better results than using glucan alone or after EP-OβG. Enhanced GLUT1 expression and acceler- ated glycolysis have been found to promote an aggressive growth in a range of tumor entities [15]. Blocking the main path for transporting the substrate for glycolysis and at the same time delivering the cytotoxic OβG into cells resulted in a marked reduction in melanoma cells viabil- ity. Importantly, as was shown previously, OβG is non-toxic for human normal cells [24], whereas GLUT1 protein is a key rate-limiting factor in the transport and metabolism of glucose in cancer cells [10]. GLUT1 ex- pression is primarily undetectable in normal epithelial tissues [25, 26]. Thus, the use of GLUT1 inhibitor does not signifi cantly affect normal cells. Koch et al. showed significantly higher levels of GLUT1 in 78 mel- anomas compared to 128 melanocytic nevi. Most importantly they found that GLUT1 expression in primary melanomas was an indicator for progression free- and overall survival [15]. Liu et al. presented that WZB117 treatment resulted in significantly more cell growth inhibition in lung cancer A549 cells than in nontumorigenic lung NL20 cells. Com- parable results were also observed in breast cancer MCF7 cells and their nontumorigenic MCF12A cells [12]. Another study reported various as- sociations between GLUT1 expression and tumor aggressiveness in other malignancies including ovarian, colorectal, pancreatic, pulmo- nary, hepatocellular and squamous cell carcinoma [15]. However, it should be mentioned that the usage of the GLUT1 inhibitor doesn’t completely block the possibility of glucose penetration into melanoma cells because other glucose transporters are present in the melanoma cell membrane – for example GLUT9 protein [26]. This may result in a slight decrease in the survival of melanoma cells after inhibition of GLUT1 protein in our study.


Our studies indicate an inhibition effect between WZB117 and EP- OβG. The EP process signifi cantly overcame this consequence by

Fig. 3. The level of GLUT1 expression in Me45 and MeWo cell line after 24 h incubation following increasing concentrations of WZB117 or negative control (A). Results of the Glucose Uptake-Glo™ Assay after 24 h incubation following increasing concentrations of WZB117 or negative control (B).

inducing expected increased anticancer activity. GLUT1 transporters ex- pression induces progression and metastasis of the primary melanoma [12, 15], because of increased glucose molecules uptake. So, we suggest that the application of a GLUT1 inhibitor in combination with a cyto- toxic agent, can be an effective way of eliminating melanoma cells. The other therapeutic solution point to natural substances as OβG used in this study, which are “glucose-like” drugs. However, the best an- ticancer effect can be expected when glucose inhibitors, anticancer drugs and electroporation are utilized.


This work was supported by the project obtained from NUTRICIA Foundation (FNU.A.040.16.001). The authors thank Mrs. Joanna Harasym from Wroclaw University of Economics for oat β-glucan and professor Katarzyna Bogunia-Kubik from Institute of Immunology and Experimental Therapy in Wroclaw for the possibility of performing electroporation on GenePulser in our research.

[1]L.R. Atmakuri, S. Dathi, Current trends in herbal medicines, J. Pharm. Res. 3 (2010) 109–113.
[2]R. Roškar, T. Trdan Lušin, Analytical methods for quantification of drug metabolites in biological samples, in: L. de Azevedo Calderon (Ed.), Chromatography – The Most Versatile Method of Chemical Analysis, In Tech 2012, pp. 79–80.
[3]A. Choromanska, J. Kulbacka, J. Harasym, R. Oledzki, A. Szewczyk, J. Saczko, High- and low- molecular weight oat beta-glucan reveals antitumor activity in human ep- ithelial lung cancer, Pathol. Oncol. Res. (2017)https://doi.org/10.1007/s12253-017- 0278-3 (Epub ahead of print).
[4]S. Rahar, G. Swami, M.A. Nagpal, G.S. Singh, Preparation, characterization and biolog- ical proporties of β-glucans, J. Adv. Pharm. Technol. Res. 2 (2011) 94–103.
[5]A. Piotrowska, B. Waszkiewicz-Robak, F. Świderski, Possibility of beta-glucan from spent brewer’s yeast addition to yoghurts, Pol. J. Food Nutr. Sci. 59 (2009) 299–302.
[6]N. Fatima, T. Upadhyay, D. Sharma, R. Sharma, Particulate beta-glucan induces early and late phagosomal maturation in murine macrophages, Front Biosci. (Elite Ed) 9 (2017) 129–140.
[7]K. Tarun, N.F. Upadhyay, S. Deepak, V. Saravanakumar, S. Rolee, Preparation and characterization of beta-glucan particles containing a payload of nanoembedded rifabutin for enhanced targeted delivery to macrophages, EXCLI J. 16 (2017) 210–228.
[8]G.D. Brown, S. Gordon, Fungal β-glucans and mammalian immunity, Immunity 19 (2003) 311–315.
[9]A. Choromanska, J. Kulbacka, N. Rembialkowska, J. Pilat, R. Oledzki, J. Harasym, J. Saczko, Anticancer properties of low molecular weight oat beta-glucan – an in vitro study, Int. J. Biol. Macromol. 80 (2015) 23–28.

[10]T. Bui, C.B. Thompso, Cancer’s sweet tooth, Cancer Cell 9 (2006) 419–420.
[11]K. Garber, Energy deregulation: licensing tumors to grow, Science 312 (2006) 1158–1159.
[12]Y. Liu, W. Zhang, Y. Cao, Y. Liu, S. Bergmeier, X. Chen, Small compound inhibitors of basal glucose transport inhibit cell proliferation and induce apoptosis in cancer cells via glucose-deprivation-like mechanisms, Cancer Lett. 298 (2010) 176–185.
[13]V.C. Angadi, P.V. Angadi, GLUT-1 immunoexpression in oral epithelial dysplasia, oral squamous cell carcinoma, and verrucous carcinoma, J. Oral Sci. 57 (2015) 115–122.
[14]F.R. Ayala, R.M. Rocha, K.C. Carvalho, A.L. Carvalho, I.W. da Cunha, S.V. Lourenço, F.A. Soares, GLUT1 and GLUT3 as potential prognostic markers for Oral Squamous Cell Carcinoma, Molecules 15 (2010) 2374–2387.
[15]A. Koch, S.A. Lang, P.J. Wild, S. Gantner, A. Mahli, G. Spanier, M. Berneburg, M. Müller, A.K. Bosserhoff, C. Hellerbrand, Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells, Oncotarget 32 (2015) 32748–32760.
[16]Y. Qian, X. Wang, X. Chen, Inhibitors of glucose transport and glycolysis as novel an- ticancer therapeutics, World J. Transl. Med. 3 (2014) 37–57.
[17]Y. Liu, Y. Cao, W. Zhang, S. Bergmeier, Y. Qian, H. Akbar, R. Colvin, J. Ding, L. Tong, S. Wu, A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo, Mol. Cancer Ther. 11 (2012) 1672–1682.
[18]J.P. Harasym, J. Brach, J.L. Czarnota, M. Stechman, A. Słabisz, A. Kowalska, M. Chorowski, M. Winkowski, J. Rać, A. Madera, European patent WO/2011/078711 (2011).
[19]A. Choromanska, J. Kulbacka, N. Rembialkowska, J. Pilat, M. Drag-Zalesinska, T. Wysocka, A. Garbiec, M. Kotulska, J. Saczko, Effects of electrophotodynamic therapy in vitro on human melanoma cells—melanotic (MeWo) and amelanotic (C32), Mel- anoma Res. 25 (2015) 210–224.
[20]A. Parzonko, M. Makarewicz-Wujec, E. Jaszewska, J. Harasym, M. Kozłowska- Wojciechowska, Pro-apoptotic properties of (1,3)(1,4)-β-D-glucan from Avena sativa on human melanoma HTB-140 cells in vitro, Int. J. Biol. Macromol. 72 (2015) 757–763.
[21]H. Xu, S. Zou, X. Xu, L. Zhang, Anti-tumor effect of β-glucan from Lentinus edodes and the underlying mechanism, Sci. Rep. 6 (2016), 28802. .
[22]R.E. Airley, A. Mobasheri, Hypoxic regulation of glucose transport, anaerobic metab- olism and angiogenesis in cancer: novel pathways and targets for anticancer thera- peutics, Chemotherapy 53 (2007) 233–256.
[23]T. Amann, C. Hellerbrand, GLUT1 as a therapeutic target in hepatocellular carcinoma, Expert Opin. Ther. Targets 13 (2009) 1411–1427.
[24]Y.J. Jun, S.M. Jang, H.L. Han, K.H. Lee, K.S. Jang, S.S. Paik, Clinicopathologic significance of GLUT1 expression and its correlation with Apaf-1 in colorectal adenocarcinomas, World J. Gastroenterol. 17 (2011) 1866–1873.
[25]M. Kunkel, M. Moergel, M. Stockinger, J.H. Jeong, G. Fritz, H.A. Lehr, T.L. Whiteside, Overexpression of GLUT-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity, Oral Oncol. 43 (2007) 796–803.
[26]A. Godoy, V. Ulloa, F. Rodríguez, K. Reinicke, A.J. Yañez, L. García Mde, R.A. Medina, M. Carrasco, S. Barberis, T. Castro, F. Martínez, X. Koch, J.C. Vera, M.T. Poblete, C.D. Figueroa, B. Peruzzo, F. Pérez, F. Nualart, Differential subcellular distribution of glu- cose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues, J. Cell. Physiol. 207 (2006) 614–627.