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

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Pro-apoptotic properties of (1,3)(1,4)-␤-d-glucan from Avena sativa on human melanoma HTB-140 cells in vitro Andrzej Parzonko a,∗ , Magdalena Makarewicz-Wujec b , Edyta Jaszewska b , Joanna Harasym c , Małgorzata Kozłowska-Wojciechowska b a Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland b Department of Clinical Pharmacy and Pharmaceutical Care, Medical University of Warsaw, Banacha 1, 02-097 Warsaw, Poland c Department of Food Biotechnology, Wroclaw University of Economics, Komandorska 118/120, 53-345 Wroclaw, Poland

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Article history: Received 12 February 2014 Received in revised form 22 September 2014 Accepted 22 September 2014 Available online 5 October 2014 Keywords: (1,3)(1,4)-␤-d-Glucan Avena sativa Apoptosis

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 sativa L. 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 the cells were found dead) was obtained of 194.6 ± 9.8 ␮g/mL. The oat ␤-d-glucan caused a concentrationdependent increase of caspase-3/-7 activation and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC, demonstrating the induction of apoptosis. Intracellular ATP level decreased along with the mitochondrial potential, which suggested a mitochondrial pathway of apoptosis. A cell cycle analysis showed increase in the number of apoptotic cells, increase in the number of cells in G1 phase and decrease in the number of cells in G2/M. Although the 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 an anti-tumor agent. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ␤-d-Glucan is a specific fiber-type complex polysaccharide which can be derived from the cell wall of baker’s yeast, and many medicinal mushrooms or bacteria, as well as widely consumed cereals, such as oats and barley [1]. Immunomodulation by ␤-dglucan was confirmed both in vitro and in vivo in numerous animal and human studies involving a wide range of tumors, including breast, lung and gastrointestinal cancers [2]. The immunomodulating and cancerostatic properties make ␤-d-glucan one of the substances with great potential in cancer treatment [3]. Reports have shown that ␤-d-glucans are effective in restraining and killing many of cancer cells such as gastric [4], prostate [5], and breast cancer cells [6]. There is some evidence that the anti-tumor effect of ␤-d-glucans may be due to a combination of immunomodulatory and cytotoxic activities [7]. However, the mechanisms of their actions are not fully known and appear complex due to differences in source, chemical structure, insufficiently defined preparation,

∗ Corresponding author. Tel.: +48 22 572 09 85; fax: +48 22 572 09 85. E-mail address: [email protected] (A. Parzonko). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.033 0141-8130/© 2014 Elsevier B.V. All rights reserved.

and molecular weight, hence the inconsistent and often contradictory results obtained [8]. Most studies concerning on the effects of ␤-d-glucans on cancer cells applies to ␤-d-glucans from microorganisms and mushrooms composed mainly of a linear central backbone of d-glucose linked in the ␤ (1 → 3)-position with glucose side branches (linkage ␤1 → 6), such as schizophyllan and lentinan [9,10]. Since a prevailing view that ␤-(1–3) linkages in the main chain of the glucan and additional ␤-(1–6) branch points are needed for activating their anti-tumor properties [11], there are only single studies on the cytotoxic effects of (1,3)(1,4)-␤-d-glucans on tumor cell lines so far, and they concentrate only on the synergistic effect with monoclonal antibodies [12–15]. Oat, Avena sativa L. grains contain relatively high levels of ␤-dglucan, approx. 5% [16], even up to 7.5% [17]. Oat ␤-d-glucan (OBG) is a natural polymer comprised of individual glucose molecules that are linked together by a series of ␤-(1 → 3) and ␤-(1 → 4) linkages, which has outstanding functional and nutritional properties exhibiting high viscosities at relatively low concentrations [18]. OBG triggers immune functions both in vitro and in vivo with the effectiveness comparable to the yeast ␤-d-glucan composed of (1–3)(1–6)-linkages [19]. Studies on the tumor metastasis model in mice have suggested that consumption of OBG can decrease the

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

metastatic spread of injected B16 melanoma cells to the lungs [20], but the direct effects of oat ␤-d-glucan on human melanoma cells have not been studied yet. 2. Materials and methods 2.1. Materials MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), ribonuclease A and propidum iodide were purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Triton X-100 solution and Cytotoxicity Detection Kit (LDH) were purchased from Roche Diagnostics (Mannheim, Germany). Annexin V-FITC Apoptosis Detection Kit I and BD MitoScreen (JC-1) was purchased from BD Biosciences (San Diego, USA). Fluo Cell Double Staining Kit was purchased from MoBiTec (Goettingen, Germany). ViaLight Plus Cell Proliferation and Cytotoxicity Bio Assay Kit were purchased from Lonza (Verwiers, Belgium). Caspase-Glo-3/7 assay was purchased from Promega (Madison, USA). Phosphate buffered saline (PBS), penicillin/streptomycin and accutase were purchased from PAA Laboratories (Pasching, Austria). DMEM medium (Dulbecco’s modified essential medium) and HEPES buffer were purchased from Lonza (Verwiers, Belgium). FBS (fetal bovine serum) was purchased from Thermo Scientific (Logan, USA). 2.2. (1,3)(1,4)-ˇ-d-Glucan extraction (1,3)(1,4)-␤-d-Glucan (Fig. 1) was extracted from oat bran concentrate (20% of ␤-glucan) from grains of A. sativa L. (Poaceae) according to Harasym et al. [21] with some modification. Deoiling and enzyme inactivation was conducted in 80 ◦ C for 1 h, solid:liquid ratio was 1:40 and pH of extraction was 9.5. After precipitation ␤-d-glucan was removed with vibrating sieves, dewaterized by washing twice with 90% EtOH and dried in tray dryer. After drying ␤-d-glucan was milled and passed through 500 ␮m sieve. 2.3. Determination of (1,3)(1,4)-ˇ-d-glucan (1,3)(1,4)-␤-d-Glucan determination was performed according to AOAC 995.16 method with test kit (Megazyme, Ireland) and procedure modification due to high ␤-d-glucan concentration. Molecular weight of ␤-d-glucan was determined with a high-performance size-exclusion chromatography system HPSECMALLS. To calibrate the HPSEC system, a oat ␤-d-glucan standards (Megazyme, Ireland) was used. The liquid chromatograph consisted of an gradient pump (Beckman System Gold 125 Solvent Module), a sample injection valve (Rheodyne, model 7725i) with a 50 ␮l loop, column oven (Waters, model TCM), two PL-GFC

´˚ 8 ␮m, 7.5 mm × 300 mm and 4000 A, ´˚ columns in series (l000 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 temperature of the refractometer cell was 40 ◦ C. The mobile phase was degassed Mini-Q water at a flow rate of 1 ml/min. A multi-angle laser-light scattering photometer (Wyatt, model Dawn F) equipped with an argon-ion laser-light source (A = 488 nm), connected to the liquid chromatograph through a flow cell (Wyatt, model F-2), was used for the on-line measurement of ␤-d-glucan molecular weights. The temperature of the photometer cell was 25 ◦ C. A compatible personal computer using LC Software (Lipopharm Ltd.) was applied 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 and operating at the laser photometer wavelength. At each elution volume, Vi , the signal from the differential refractive index detector is proportional to ␤-d-glucan concentration, ci . Also, at each elution volume, Vi , we have a set of signals from the light scattering detector, one for each scattering angle, . These signals give Ri (), the excess Rayleigh ratio at angle  for elution volume Vi (where Ri () = Ri () solution − Ri () solvent). The extrapolation to 0 angle is performed in the Debye type plot, namely, Ri ()Kci versus sin2 /2, from which Mw,i is obtained as intercept, K being the optical constant, K = (4␲2 no 2 /o 4 NA )·(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 average and macromolecular radius corresponding to the samples can be calculated according to the appropriate equations. Aqueous ␤-dglucan 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 purchased from American Type Culture Collection (Rockville, USA). Cells (from the third to sixth passage) were seeded on 12-well plates (3.5 × 104 cells per well) or 96-well plates (5 × 103 cells per well) and incubated for 3–4 days in the DMEM medium with 10% FBS, penicillin and streptomycin, in 5% CO2 and at 37 ◦ C. Then the medium was changed to serum-free DMEM. Thus, fresh working solutions of (1,3)(1,4)-␤-d-glucan in DMEM were added to the culture 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 hot water bath after dispersion on ultrasonic bath. Then PBS buffer at

Fig. 1. Chemical structure of oat (1,3)(1,4)-␤-d-glucan.

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

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pH 7.4 and DMEM medium was added to obtain the proper concentration of compound in sample. The same amount of propylene glycol was also added to the control and did not influence the performed assays in concentration used (

Pro-apoptotic properties of (1,3)(1,4)-β-D-glucan from Avena sativa on human melanoma HTB-140 cells in vitro.

In this study, the growth-inhibitory effect of polysaccharide (1,3)(1,4)-β-D-glucan from oat, Avena sativa L. grains was explored on the human skin me...
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