Abstract
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 melanoma HTB-140 cells in vitro. The oat β-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 μg/mL. The oat β-d-glucan caused a concentration-dependent 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 β-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.
Graphical abstract
Introduction
β-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 β-d-glucan 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 β-d-glucan one of the substances with great potential in cancer treatment [3]. Reports have shown that β-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 β-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, and molecular weight, hence the inconsistent and often contradictory results obtained [8]. Most studies concerning on the effects of β-d-glucans on cancer cells applies to β-d-glucans from microorganisms and mushrooms composed mainly of a linear central backbone of d-glucose linked in the β (1 → 3)-position with glucose side branches (linkage β1 → 6), such as schizophyllan and lentinan [9], [10]. Since a prevailing view that β-(1–3) linkages in the main chain of the glucan and additional β-(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)-β-d-glucans on tumor cell lines so far, and they concentrate only on the synergistic effect with monoclonal antibodies [12], [13], [14], [15].
Oat, Avena sativa L. grains contain relatively high levels of β-d-glucan, approx. 5% [16], even up to 7.5% [17]. Oat β-d-glucan (OBG) is a natural polymer comprised of individual glucose molecules that are linked together by a series of β-(1 → 3) and β-(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 β-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 metastatic spread of injected B16 melanoma cells to the lungs [20], but the direct effects of oat β-d-glucan on human melanoma cells have not been studied yet.
Section snippets
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
Determination of (1,3)(1,4)-β-d-glucan
(1,3)(1,4)-β-d-Glucan concentration in used glucan preparation was 93%. The calculated overall molar-mass averages Mw – 13.38 × 105 g/mol, Mn – 4.0 × 103 g/mol and the dispersity (D = Mw/Mn) was 334.
Effects of (1,3)(1,4)-β-d-glucan on cell viability and cell membrane integrity
In this study, the effect of (1,3)(1,4)-β-d-glucan on proliferation and viability of the human melanoma HTB140 cells were investigated using the MTT method. At 24 h of treatment, (1,3)(1,4)-β-d-glucan reduced the proliferation and viability of the cancer cells concentration-dependently, so that they were
Discussion
Although anti-cancer and immune-enhancing activities of (1,3)(1,6)-β-glucans from mushrooms and bacteria are quite well described in the literature, there is little knowledge about β-d-glucans occurring in widely consumed cereals. In this study, we showed for the first time the cytotoxicity, cell cycle arrest and pro-apoptotic properties of oat (1,3)(1,4)-β-d-glucan (OBG) on human melanoma HTB-140 cells in vitro. OBG exerted a potent anti-proliferative effect on HTB-140 cells, with IC50 of 196.7
References