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International Journal of Biological Macromolecules 72 (2015) 757–763

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

ro-apoptotic properties of (1,3)(1,4)-�-d-glucan from Avena sativan human melanoma HTB-140 cells in vitro

ndrzej Parzonkoa,∗, Magdalena Makarewicz-Wujecb, Edyta Jaszewskab,oanna Harasymc, Małgorzata Kozłowska-Wojciechowskab

Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw,olandDepartment of Clinical Pharmacy and Pharmaceutical Care, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, PolandDepartment of Food Biotechnology, Wroclaw University of Economics, Komandorska 118/120, 53-345 Wroclaw, Poland

r t i c l e i n f o

rticle history:eceived 12 February 2014eceived in revised form2 September 2014ccepted 22 September 2014vailable online 5 October 2014

eywords:

a b s t r a c t

In this study, the growth-inhibitory effect of polysaccharide (1,3)(1,4)-�-d-glucan from oat, Avena sativaL. grains was explored on the human skin melanoma HTB-140 cells in vitro. The oat �-d-glucan (OBG)exerted cytotoxic action on HTB-140 cells. After 24 h of incubation, LD50 (concentration at which 50% of thecells were found dead) was obtained of 194.6 ± 9.8 �g/mL. The oat �-d-glucan caused a concentration-dependent increase of caspase-3/-7 activation and appearance of phosphatidylserine on the externalsurface of cellular membranes where it was bound to annexin V-FITC, demonstrating the induction ofapoptosis. Intracellular ATP level decreased along with the mitochondrial potential, which suggested a

1,3)(1,4)-�-d-Glucanvena sativapoptosis

mitochondrial pathway of apoptosis. A cell cycle analysis showed increase in the number of apoptoticcells, increase in the number of cells in G1 phase and decrease in the number of cells in G2/M. Althoughthe detailed mechanism for the anti-tumor activity of the oat �-d-glucan still needs further investigation,this study provides preliminary insights into this direction along with perspectives of developing it as ananti-tumor agent.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

�-d-Glucan is a specific fiber-type complex polysaccharidehich can be derived from the cell wall of baker’s yeast, and manyedicinal mushrooms or bacteria, as well as widely consumed

ereals, such as oats and barley [1]. Immunomodulation by �-d-lucan was confirmed both in vitro and in vivo in numerous animalnd human studies involving a wide range of tumors, includingreast, lung and gastrointestinal cancers [2]. The immunomodu-

ating and cancerostatic properties make �-d-glucan one of theubstances with great potential in cancer treatment [3]. Reportsave shown that �-d-glucans are effective in restraining and killingany of cancer cells such as gastric [4], prostate [5], and breast

ancer cells [6]. There is some evidence that the anti-tumor effectf �-d-glucans may be due to a combination of immunomodula-

ory and cytotoxic activities [7]. However, the mechanisms of theirctions are not fully known and appear complex due to differencesn source, chemical structure, insufficiently defined preparation,

∗ Corresponding author. Tel.: +48 22 572 09 85; fax: +48 22 572 09 85.E-mail address: aparzonko@wum.edu.pl (A. Parzonko).

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.09.033141-8130/© 2014 Elsevier B.V. All rights reserved.

and molecular weight, hence the inconsistent and often contradic-tory results obtained [8]. Most studies concerning on the effects of�-d-glucans on cancer cells applies to �-d-glucans from microor-ganisms and mushrooms composed mainly of a linear centralbackbone of d-glucose linked in the � (1 → 3)-position with glucoseside branches (linkage �1 → 6), such as schizophyllan and lentinan[9,10]. Since a prevailing view that �-(1–3) linkages in the mainchain of the glucan and additional �-(1–6) branch points are neededfor activating their anti-tumor properties [11], there are only singlestudies on the cytotoxic effects of (1,3)(1,4)-�-d-glucans on tumorcell lines so far, and they concentrate only on the synergistic effectwith monoclonal antibodies [12–15].

Oat, Avena sativa L. grains contain relatively high levels of �-d-glucan, approx. 5% [16], even up to 7.5% [17]. Oat �-d-glucan (OBG)is a natural polymer comprised of individual glucose moleculesthat are linked together by a series of �-(1 → 3) and �-(1 → 4)linkages, which has outstanding functional and nutritional prop-erties exhibiting high viscosities at relatively low concentrations

[18]. OBG triggers immune functions both in vitro and in vivo withthe effectiveness comparable to the yeast �-d-glucan composed of(1–3)(1–6)-linkages [19]. Studies on the tumor metastasis modelin mice have suggested that consumption of OBG can decrease the

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etastatic spread of injected B16 melanoma cells to the lungs [20],ut the direct effects of oat �-d-glucan on human melanoma cellsave not been studied yet.

. Materials and methods

.1. Materials

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumromide), ribonuclease A and propidum iodide were purchasedrom Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Triton-100 solution and Cytotoxicity Detection Kit (LDH) were pur-hased from Roche Diagnostics (Mannheim, Germany). Annexin-FITC Apoptosis Detection Kit I and BD MitoScreen (JC-1) wasurchased from BD Biosciences (San Diego, USA). Fluo Cell Doubletaining Kit was purchased from MoBiTec (Goettingen, Germany).iaLight Plus Cell Proliferation and Cytotoxicity Bio Assay Kit wereurchased from Lonza (Verwiers, Belgium). Caspase-Glo-3/7 assayas purchased from Promega (Madison, USA).

Phosphate buffered saline (PBS), penicillin/streptomycin andccutase were purchased from PAA Laboratories (Pasching,ustria). DMEM medium (Dulbecco’s modified essential medium)nd HEPES buffer were purchased from Lonza (Verwiers, Belgium).BS (fetal bovine serum) was purchased from Thermo ScientificLogan, USA).

.2. (1,3)(1,4)-ˇ-d-Glucan extraction

(1,3)(1,4)-�-d-Glucan (Fig. 1) was extracted from oat bran con-entrate (20% of �-glucan) from grains of A. sativa L. (Poaceae)ccording to Harasym et al. [21] with some modification. Deoilingnd enzyme inactivation was conducted in 80 ◦C for 1 h, solid:liquidatio was 1:40 and pH of extraction was 9.5. After precipitation-d-glucan was removed with vibrating sieves, dewaterized byashing twice with 90% EtOH and dried in tray dryer. After drying-d-glucan was milled and passed through 500 �m sieve.

.3. Determination of (1,3)(1,4)-ˇ-d-glucan

(1,3)(1,4)-�-d-Glucan determination was performed accord-ng to AOAC 995.16 method with test kit (Megazyme, Ireland)nd procedure modification due to high �-d-glucan concentra-ion. Molecular weight of �-d-glucan was determined with aigh-performance size-exclusion chromatography system HPSEC-ALLS. To calibrate the HPSEC system, a oat �-d-glucan standards

Megazyme, Ireland) was used. The liquid chromatograph consistedf an gradient pump (Beckman System Gold 125 Solvent Mod-le), a sample injection valve (Rheodyne, model 7725i) with

50 �l loop, column oven (Waters, model TCM), two PL-GFC

Fig. 1. Chemical structure of o

gical Macromolecules 72 (2015) 757–763

columns in series (l000 ´A, 8 �m, 7.5 mm × 300 mm and 4000 ´A,8 �m, 7.5 mm × 300 mm) with a guard column (7.5 mm × 50 mm)and a differential refractive index detector (Waters, model 410).Temperature of operating for columns was 80 ◦C whereas the tem-perature of the refractometer cell was 40 ◦C. The mobile phase wasdegassed Mini-Q water at a flow rate of 1 ml/min. A multi-anglelaser-light scattering photometer (Wyatt, model Dawn F) equippedwith an argon-ion laser-light source (A = 488 nm), connected tothe liquid chromatograph through a flow cell (Wyatt, model F-2),was used for the on-line measurement of �-d-glucan molecularweights. The temperature of the photometer cell was 25 ◦C. A com-patible personal computer using LC Software (Lipopharm Ltd.) wasapplied to collect and analyze data.

The differential refractive index increment (dn/dc) of oat �-d-glucan was determined with an interferometric refractometer(Wyatt, model Optilab 903) calibrated with aqueous NaCl andoperating at the laser photometer wavelength. At each elutionvolume, Vi, the signal from the differential refractive index detec-tor is proportional to �-d-glucan concentration, ci. Also, at eachelution volume, Vi, we have a set of signals from the light scatter-ing detector, one for each scattering angle, �. These signals giveRi(�), the excess Rayleigh ratio at angle � for elution volume Vi(where Ri(�) = Ri(�) solution − Ri(�) solvent). The extrapolation to0 angle is performed in the Debye type plot, namely, Ri(�)Kci versussin2 �/2, from which Mw,i is obtained as intercept, K being the opti-cal constant, K = (4�2no

2/�o4NA)·(dn/dc)2. The initial slope of such

extrapolation also yields the mean square radius of gyration, 〈s2〉it of the macromolecule having Mw,i. The molecular weight aver-age and macromolecular radius corresponding to the samples canbe calculated according to the appropriate equations. Aqueous �-d-glucan solutions used were in the range 0.1 × 10−3 to 1 × 10−3 g/ml,and the dn/dc value, calculated by linear regression analysis data,was 0.153 ml/g.

2.4. Cell culture

Human melanoma cells, line Hs 294T (HTB-140TM), were pur-chased from American Type Culture Collection (Rockville, USA).Cells (from the third to sixth passage) were seeded on 12-wellplates (3.5 × 104 cells per well) or 96-well plates (5 × 103 cells perwell) and incubated for 3–4 days in the DMEM medium with 10%FBS, penicillin and streptomycin, in 5% CO2 and at 37 ◦C. Then themedium was changed to serum-free DMEM. Thus, fresh workingsolutions of (1,3)(1,4)-�-d-glucan in DMEM were added to the cul-ture medium to obtain final concentration of 50–200 �g/mL.

2.5. (1,3)(1,4)-ˇ-d-Glucan preparation

(1,3)(1,4)-�-d-Glucan was dissolved in propylene glycol on hotwater bath after dispersion on ultrasonic bath. Then PBS buffer at

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between means was established by ANOVA with Tukey post hoctest. p values below 0.05 were considered statistically significant.All analyses were performed using Statistica 10.

A. Parzonko et al. / International Journal o

H 7.4 and DMEM medium was added to obtain the proper con-entration of compound in sample. The same amount of propylenelycol was also added to the control and did not influence the per-ormed assays in concentration used (<1%).

.6. Mitochondrial function assessment

The function of mitochondria of cells was assessed with these of MTT, which is converted in viable cells under the effectf mitochondrial dehydrogenase into insoluble formazan. In brief,TB-140 cells cultivated on 24-well plates were incubated withat �-d-glucan for 24 h and then MTT solution was added for 2 h.he converted dye was then solubilized with the use of acidic iso-ropanol (0.04 M HCl in absolute isopropanol), and absorbance waseasured at 570 nm with background subtraction at 650 nm, using

ynergy 4 microplate reader (Biotek, USA). Cell viability was calcu-ated as a percent versus the control (cells incubated in serum-freeMEM without glucan).

.7. Cellular membrane integrity assessment

To assess cellular membrane integrity at 24 h of incubation ofhe cells on 12-well plates with the OBG, the activity of lactateehydrogenase (LDH) released from cytosol of damaged cells tohe supernatant was measured using the Cytotoxicity Detectionit test according to the manufacturer protocol. Absorbance valueseasured at 490 nm, using microplate reader, were translated into

ytotoxicity rate, where negative controls were cells in serum-freeMEM without OBG, and positive controls were cells incubated in

erum-free DMEM with 10% Triton X-100 (100% LDH release).

.8. ATP level measurement

Measurement of the intracellular level of ATP was performedsing the ViaLight Plus Assay Kit test according to the manufacturerrotocol. Briefly, cells were incubated on 96-well white-walledlates in serum-free DMEM with various concentrations of OBG50–200 �g/mL) for 24 h, and then the cells were lysed on plate,TP level monitoring reagent was added to lysate, and lumines-ence was measured using the microplate reader. The result wasresented as the percentage of ATP level in comparison with theontrol (cells incubated in serum-free DMEM without glucan).

.9. Mitochondrial membrane potential analysis

Mitochondrial membrane potential was measured using Mito-creen Flow Cytometry Mitochondrial Membrane Potential Detec-ion Kit according to manufacturer protocol. Briefly, cells werencubated on 12-well plates in serum-free DMEM with the OBGt various concentrations (50–200 �g/mL) for 24 h. Then cells werearvested, washed with PBS, centrifuged and 5,5′,6,6′-tetrachloro-,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) solu-ion was added. After 30 min of incubations, cells were centrifugednd analyzed using FACSCalibur flow cytometer and the CellQuestoftware (BD Biosciences, USA).

.10. Annexin V-FITC/PI staining

To detect apoptosis/necrosis, HTB-140 cells were incubated on2-well plates in serum-free DMEM with the OBG at various con-

entrations (50–200 �g/mL) for 24 h. The cells were rinsed with PBSnd detached using accutase. Then the protocol for the Annexin-FITC apoptosis detection kit test described by the manufactureras followed. The samples were analyzed by flow cytometry, using

gical Macromolecules 72 (2015) 757–763 759

FACSCalibur flow cytometer and the CellQuest software (BD Bio-sciences, USA). The results are presented as rates of apoptotic cells.

2.11. Morphology

Changes in morphology of cells were assessed with the use ofthe Nikon Eclipse TS100F microscope fitted with the Nikon Digi-tal Sight DS-U2 camera, using the NIS Elements BR 2.30 software(Nikon, USA), in visible light and in fluorescence after their priordyeing with calcein-AM and propidium iodide according to themanufacturer protocol.

2.12. Cell cycle analysis

For cell cycle analysis, cells cultured on 12-well plates wereincubated in the presence of OBG at various concentrations(50–200 �g/mL) in serum-free DMEM. After 24 h of incubation,cells were collected, washed with PBS, fixed with cold 70% ethanoland conserved at −20 ◦C overnight. After the fixation, the ethanolwas removed and the cells were washed with PBS. 100 �L of RNaseA (1 mg/mL) were then added, and after 30 min of incubation at37 ◦C, cells were stained with a solution containing 10 �g/mL ofpropidium iodide (PI). The samples were analyzed using a FACSCal-ibur flow cytometer and the CellQuest software (BD Biosciences,USA).

2.13. Caspase-3/-7 activity assay

In order to assess mechanism of apoptosis induction in cellscaspase-3 and -7 activation was measured using Caspase-Glo-3/-7assay according to manufacturer protocol. Cells were cultured on96-well white-walled plates and then OBG was added at variousconcentrations (50–200 �g/mL) in serum-free DMEM. After 24 h ofincubation, 100 �l of Caspase-Glo-3/-7 reagent to each well wasadded, and luminescence was measured using microplate reader.

2.14. Statistical analysis

The results were expressed as a mean ± SEM of the indi-cated number of experiments. Statistical significance of differences

Fig. 2. MTT assay results from HTB-140 cells treated with the indicated concentra-tion of glucan for 24 h. Results were expressed as mean ± SEM; performed in fiveindependent experiments, assayed in triplicate. Statistically significant differences:*p < 0.05, **p < 0.01 versus control (untreated cells).

760 A. Parzonko et al. / International Journal of Biological Macromolecules 72 (2015) 757–763

Fig. 3. Lactate dehydrogenase (LDH) release after 24 h of incubation with the indi-cated concentration of glucan. Results were expressed as mean ± SEM; performedif

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Fig. 4. Changes in ATP levels of HTB-140 cells after 24 h treatment with indi-

about 50% of the control level at 200 �g/ml of the glucan (Fig. 2).

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n five independent experiments, assayed in triplicate. Statistically significant dif-erences: *p < 0.05, **p < 0.01 versus control (untreated cells).

. Results

.1. Determination of (1,3)(1,4)-ˇ-d-glucan

(1,3)(1,4)-�-d-Glucan concentration in used glucan prepara-

ion was 93%. The calculated overall molar-mass averages Mw

13.38 × 105 g/mol, Mn – 4.0 × 103 g/mol and the dispersityD = Mw/Mn) was 334.

ig. 5. The effects of oat �-d-glucan on mitochondrial transmembrane potential in HTB-14aused by the accumulation of JC-1 in mitochondrial and green fluorescence caused by treated with 50, 100 and 200 �g/mL of �-d-glucan, respectively. Representative cytogram

cated concentrations of oat �-d-glucan. Data are presented as the means ± SEM;performed in three independent experiments assayed in triplicate. Statistically sig-nificant differences: *p < 0.05; **p < 0.01 refer to the control (untreated cells).

3.2. Effects of (1,3)(1,4)-ˇ-d-glucan on cell viability and cellmembrane integrity

In this study, the effect of (1,3)(1,4)-�-d-glucan on prolifer-ation and viability of the human melanoma HTB140 cells wereinvestigated using the MTT method. At 24 h of treatment, (1,3)(1,4)-�-d-glucan reduced the proliferation and viability of the cancercells concentration-dependently, so that they were decreased by

Tested OBG caused also concentration-dependent increase in therelease of lactate dehydrogenase (LDH) into the culture medium.After 24 h of incubation, the LD50 (concentration at which 50% of

0 cells. Mitochondrial potential detection was based on the ratio of red fluorescencehe accumulation of JC-1 in cytoplasm. (A) control (untreated cells) and (B–D) cellss are presented.

A. Parzonko et al. / International Journal of Biological Macromolecules 72 (2015) 757–763 761

Fig. 6. Number of HTB-140 cells in apoptosis evaluated using Annexin V-FITC/PIstaining followed by flow cytometric analysis. Cells were treated with oat beta-glucan for 24 h. Data are presented as the means ± SEM; performed in threei*

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Fig. 7. Activation of caspase-3/-7 in HTB-140 cells. Cells were treated with indicatedconcentrations of oat �-d-glucan for 24 h. Data are presented as the means ± SEM;

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ndependent experiments assayed in triplicate. Statistically significant differences:p < 0.05; **p < 0.001 versus control (untreated cells).

ells were dead) value of 194.65 ± 9.8 �g/mL was obtained at theested concentrations (50–200 �g/mL) of the (1,3)(1,4)-�-d-glucanFig. 3). On the other hand, in the parallel experiment conducted onormal human dermal fibroblasts cell line, no significant cytotoxicffect was observed (data no shown).

.3. Effects of (1,3)(1,4)-ˇ-d-glucan on mitochondrial functions

At 24 h of incubation of HTB-140 cells with the OBG, the intra-ellular ATP level was significantly lowered with concentrations of0–200 �g/mL, attaining the lowest value of 50.8 ± 7.8% (versus the

ontrol) at a concentration of 200 �g/mL. The ATP reduction wastrictly concentration-dependent (Fig. 4). Incubation of melanomaells with glucan caused decrease of mitochondrial membraneotential. As shown on Fig. 5, increase in the percentage of cells

ig. 8. Cell cycle analysis of HTB-140 cells treated with oat �-d-glucan. (A) control (untreatepresentative histograms are presented.

performed in three independent experiments assayed in triplicate. Statistically sig-nificant differences: *n.s.; **p < 0.001 verus control (untreated cells).

with lowered red fluorescence was observed after incubation withglucan (by 56.0 ± 3.3%, 78.3 ± 4.6% and 96.8 ± 2.2%, at the concen-tration of 50, 100 and 200 �g/mL respectively, in comparison withcontrol).

3.4. Effects of (1,3)(1,4)-ˇ-d-glucan on caspase-3/-7 activationand apoptosis

To check the effect of the OBG on the quantity of HTB-140 cells inapoptosis at 24 h of incubation, cell staining with annexinV labeledwith FITC and propidium iodide was performed (Fig. 6). The totalquantity of cells stained with annexin was 12.6%, 21.7%, and 37.7%at 50, 100, 200 �g/mL of glucan, respectively. Appearance of phos-

phatidylserine on the external surface of cellular membranes whereit was bound to annexin V, giving an increased number of annexin-positive (An+) cells, is a sign that the cells entered apoptosis. Toassess the mechanism of further changes, caspase-3/-7 activity was

ed cells) and (B–D) cells treated with 50, 100 and 200 �g/mL of glucan, respectively.

762 A. Parzonko et al. / International Journal of Biological Macromolecules 72 (2015) 757–763

Fig. 9. Morphologic changes of HTB-140 cells. (A) Untreated cells observed in the light microscope (100×); (B) in fluorescence (200×) after calcein-AM staining; (C) influorescence (200×) after propidium iodide staining; (D) cells treated with oat �-d-glucan (200 �g/mL) for 24 h observed in the light microscope; (E, F) in fluorescence aftercalcein-AM or propidium iodide staining, respectively. The calcein in viable cells emits green fluorescence, while PI emits red fluorescence only in dead cells.

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ested. After the 24 h period of incubation of HTB-140 cells with theBG, near 2.5-fold and 3-fold increase of caspase-3 and -7 activityas observed at 100 and 200 �g/mL of glucan, respectively, in com-arison with control (cells incubated in serum-free DMEM) (Fig. 7).

.5. Effect of (1,3)(1,4)-ˇ-d-glucan on cell cycle

Cell cycle comprises a series of cellular events that an eukary-tic cell has to proceed orderly before it can divide into daughterells; the rate of cell proliferation is therefore determined by theate that the cell proceeds through the different cell-cycle phases

and checkpoints. Results from the DNA-flow cytometry showedthat 200 �g/ml of glucan caused significant decrease of percentageof cells in G2/M phase of the melanoma cells at 24 h of incu-bation (Fig. 8). Apoptosis is a mode of programmed cell death,which plays an important role in cancer chemotherapy. DNA frag-mentation is one of the characteristic features of apoptotic cells,so that they are shown as subG1 cells in the DNA histogram of

flow cytometry. Oat �-d-glucan was found to induce apoptosisconcentration-dependently in the melanoma cells. The number ofsubG1 cells elevated by 2-fold of the control level at 100 �g/mL andby 3-fold at 200 �g/mL after 24 h of incubation.

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A. Parzonko et al. / International Journal o

.6. Effect of (1,3)(1,4)-ˇ-d-glucan on cell morphology

In the light microscope, significant changes in morphologyf HTB-140 cells were observed already under the influencef 50 �g/mL �-d-glucan, while higher concentration caused aoncentration-dependent change in morphology of the cells (Fig. 9).fter 24 h of incubation, cells were contracted, and concentrationf chromatin was observed. In fluorescence, after prior staining ofTB-140 cells with calcein-AM and propidium iodide, a progres-

ive and strictly concentration-dependent increase in the numberf PI+ cells was observed.

. Discussion

Although anti-cancer and immune-enhancing activities of1,3)(1,6)-�-glucans from mushrooms and bacteria are quite wellescribed in the literature, there is little knowledge about �-d-lucans occurring in widely consumed cereals. In this study, wehowed for the first time the cytotoxicity, cell cycle arrest andro-apoptotic properties of oat (1,3)(1,4)-�-d-glucan (OBG) onuman melanoma HTB-140 cells in vitro. OBG exerted a potent anti-roliferative effect on HTB-140 cells, with IC50 of 196.7 ± 5.9 �g/mLogether with the increase in cell mortality, with LD50 amountingo 194.6 ± 9.8 �g/mL already at 24 h of incubation. In the same con-itions significant changes in cell morphology and increase in theumber of dead cells further confirmed its cytotoxicty.

Multiple observations support the opinion that cancer cellsave higher mitochondrial membrane potential and they are

ess susceptible to mitochondrial membrane permeabilization andctivation of the mitochondrial pathway of apoptosis. Thereforeuccessful elimination of a cancer may be based on the abil-ty of anti-cancer treatment to destabilize the mitochondria bynduction of the respiratory chain malfunction, reduction in mito-hondrially produced ATP and disruption of the mitochondrialembrane potential [22]. In this study, we revealed that incu-

ation of HTB-140 cells with OBG results in significant decreasef intracellular ATP level and collapse of mitochondrial mem-rane potential (approx. by 97% at 200 �g/mL). What is in lineith the results found by Hirahara et al. who observed loss ofitochondrial transmembrane potential after treatment of PSK,-d-glucan consisting of �-1,4 linkages in the main chain, con-luding that decreased membrane potential is possibly the resultf apoptotic cell death [23]. Our study showed a concentration-ependent effect of OBG on the initiation of apoptosis in HTB-140ells with a loss of membrane phospholipid asymmetry andhosphatidylserine exposure on the external surface of the cells.oreover, we also detected a significant (threefold at 200 �g/mL)

ctivation of effector caspase-3 and -7, the main enzymes involvedn the apoptotic process. Similar findings on caspase-3 activa-ion were reported by Jimenez-Medina et al. who incubated B16

urine melanoma and Ando-2 human melanoma cells with PSK7].

Consistently with mentioned findings by Jimez-Medina et al.

nd reports by Zhang et al. who described the activity of �-d-lucan from Poria cocos, comprising mainly (1–3)(1–4)-linkages,ur analysis of the cell cycle revealed a significant accumulation ofpoptotic cells containing fragmented DNA in the G1 phase, with

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gical Macromolecules 72 (2015) 757–763 763

simultaneous decrease in the proportion of cells in the G2/M phase,which indicates a simultaneous cell division arrest.

Recent reports concerning (1,3)(1,4)-�-d-glucan derived frombarley suggested its efficacy in potentiation the treatment of cancerdisease such as Lewis lung carcinoma [24], lymphoma [14] and neu-roblastoma [12]. Our study provides preliminary insights into themechanism of OBG activity with perspectives of expanding knowl-edge in this direction, developing OBG as an adjuvant to knownanti-tumor agents; however, further studies are needed especiallywith coadministration of cytostatics.

In conclusion, this study for the first time demonstrates that�-d-glucan isolated from widely consumed oats exerts direct cyto-toxic action on human melanoma cells leading to impaired ATPsynthesis and loss of mitochondrial membrane potential. Oat �-d-glucan induces cell cycle G1 arrest and the apoptosis of humanmelanoma cells through the mitochondrial pathway. Our studyprovides preliminary insights into perspectives of developing it asan adjuvant in the treatment of cancer disease.

Acknowledgment

This work was supported in part by non-restricted grant fromFuturum s.c., producer of oat �-d-glucan.

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