High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung Cancer

High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial... Pathol. Oncol. Res. (2018) 24:583–592 DOI 10.1007/s12253-017-0278-3 ORIGINAL ARTICLE High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung Cancer 1 1 2 2 Anna Choromanska & Julita Kulbacka & Joanna Harasym & Remigiusz Oledzki & 3 1 Anna Szewczyk & Jolanta Saczko Received: 23 February 2016 /Accepted: 12 July 2017 /Published online: 29 July 2017 The Author(s) 2017. This article is an open access publication Abstract Beta-glucans are widely used in treatment, cos- Introduction metics, and the food industry. Glucans play a significant role in activation of the immune and antioxidant system Despite of the advances methods in medicine and the dynam- and inhibiting tumor proliferation. In the current study the ics of biochemical and biotechnological techniques, it is more antitumor activities of new high and low molecular weight often reached up to the sources of natural medicine. Herbal beta-glucan derived from oats were investigated in two medicine, principles of rational nutrition concerns of growing human lung cancer cell line (A549, H69AR) and normal as an alternative way of treatment and support for pharmaco- keratinocytes (HaCaT). The effect of high and low molec- logical therapy. In recent years interest in the use of plants for ular weight beta-glucan from oat was evaluated by cellular the production of compounds of pharmacological is increased. viability assessment, lipid peroxidation and manganese Additionally various studies have shown that components of superoxide dismutase evaluation and cytoskeleton visual- plant can prevent disease, especially cancer. Using analytical isation. Additionally the level of red blood cells hemolysis methods for pharmacodynamic and which are used to assess was performed. Our results indicate strong anti-tumor the therapeutic properties and their actions, we get more and properties of new beta-glucan from oat and at the same more data on the therapeutic efficiency of herbal supplements time no toxicity for normal cells. [1–3]. Recently, one of the active ingredients responsible for the anticancer therapy of many of these herbs was found to be a form of complex polysaccharides known as Bbeta-D- . . . Keywords Oat beta-glucan Viability Lung cancer glucan^. It is non cellulosic polymers, elevating of the cellular . . Keratinocytes Oxidative stress Cytoskeleton walls, of beta-glucose, which is glycoside in position beta (1– 3), (1–4) or beta (1–6). It occurs in the form of long-chain, three-dimensional molecule polysaccharide side chains of glu- cose molecules built [4]. Beta-glucans are isolated mainly Electronic supplementary material The online version of this article from different fungi. However there are obtained from other (doi:10.1007/s12253-017-0278-3) contains supplementary material, which is available to authorized users. sources, such as cereals, barley, bacteria or seaweeds. Beta- glucans, which are isolated from different sources character- * Anna Choromanska ized similar biological properties but different structures and anna.choromanska@umed.wroc.pl solubility in water or other solvents [5]. The variety of mate- rials from which beta-glucan is obtained, makes it possible to get a large number formulations of similar or different param- Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368 Wroclaw, Poland eters. Immunomodulatory and anti-cancer properties of beta- glucans result from their structure, molecular weight, degree Department of Food Biotechnology, Wroclaw University of Economics, Komandorska 118-120, 53-345 Wroclaw, Poland of branching, conformation and its behavior in gastrointestinal tract [6]. Moreover, these properties depend on an isolation Department of General Zoology, Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland procedure of beta-glucan. Beta-glucans belong to well-known 584 Choromanska A. et al. biologic modifiers, which play immunostimulatory function piling of cells. Even when cells are healthy and well-attached, against infections and cancer [7, 8]. This compound activated there are many viable floating cells. the response of adaptive immune cells such as CD4+ or/and All cell lines were grown in polystyrene flasks with 25 cm CD8+ T cells and in addition B cells. It is one of the possible cell culture surface (Eppendorf, Germany) as a monolayer in mechanisms of protective and anticancer action. However, the Dulbecco modified Eagle medium (DMEM, Sigma-Aldrich, mechanisms of anticancer activities of beta-glucan seem to be USA) for HaCaT and A549 cell line or RPMI-1640 Medium, multicomplex and still understood. Beta-glucan extracted (Gibco, USA) for H69AR cell line. Mediums were enhanced from cell wall of fungi is characterized by low purity and 10% (DMEM) or 20% (RPMI-1640) fetal bovine serum additionally causes unfavorable effect. Moreover, there are (FBS, Sigma-Aldrich, USA) and 50 μg/ml streptomycin water insoluble fraction [9]. (Sigma-Aldrich, USA). Cells were incubated at 37 °C in 5% Recently more remark is concentrated on beta-glucan from CO . Before the every experiments the cells were removed by cereals mainly from oat. This soluble fibre fraction is the 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, USA). greatest find of recent years [10]. Numerous researches con- firmed the significant anticancer activity of beta-glucan HMW and LMW Beta-Glucans Recovery Procedure [11–13]. In the treatment of breast cancer, and also in areas undergoing irradiation after amputation, more rapid healing In this examination beta-glucan from oats was derivedin form was obtained of inflammation. The use of beta-glucan in these of white powder. HMW and LMW beta-glucan was obtained appeared unaffected healthy tissue [14]. Still is well docu- at the University of Economics in Wroclaw. HMW oat beta- mented only the immune activation role of oat beta-glucan glucan was obtained due to procedure described elsewhere in killing cancer cells [15, 16]. We postulated that anticancer [17] with beta-glucanase inactivation during lipid removal activity of oat beta-glucan is more complicated and complex. step, alkaline extraction, protein removal in isoelectric point, Many of the compounds used in the methods of standard solution neutralization to pH = 7,0 and beta-glucan precipita- anticancer therapy activated oxidative stress in cancer cells, tion with ethanol. Low molecular oat 1–3, 1–4–D-beta-glucan which leads to cell death through lipid and thiol groups of was manufactured due to procedure described elsewhere [10] protein peroxidation. Moreover, beta-glucan can influence with multistep freeze-milling of raw materials (20% beta- on cells cytoskeleton and antioxidant preventing system. glucan fiber, Microstructure, Poland), fat removal with etha- The aim of the study was to evaluate the cytotoxicity and nol extraction, alkaline extraction (pH 8–10) of beta-glucan antitumor activities of high-molecular weight (HMW) and and oat proteins, protein precipitation and separation in iso- low-molecular weight of beta-glucan (LMW) derived from electric point, and finally beta-glucan precipitation with etha- oats, based on the two cell lines, normal and cancerous. nol in equilibrium. Beta-glucan preparations was then dried Additionally, the effect of the plant polysaccharides, such for 24 h. Purity was determined with according to AOAC the beta-glucan, on human red blood cell hemolysis as an 995.16 method with test kit (Megazyme, Ireland) and was indicator of membrane stability, was investigated. 84%. Molecular weight of oat beta-glucan was measured with HPLC-SEC with guard column (OHpak SB-G, Shodex), a GPC column (SB-806 M HQ, Shodex) and was 69,650 g/ mol. Firstly the stock solution of beta-glucan was prepared. Material and Methods Two mg of beta-glucan was dissolved in 1 ml of sterile dis- tilled water and one drop of 10% NaOH was added. Then the Cell Culture stock solution was incubated at 37 °C for 24 h. The different concentrations of this compound were used to the studies Three human cell lines, (purchased from the American Type (5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, 50 μg/ml, 75 μg/ Culture Collection Cell-ATCC-American Type Culture ml, 100 μg/ml, 150 μg/ml and 200 μg/ml). Collection) were used in this investigations: HaCaT - normal, immortal cell line of the transformed phenotype in vitro, the Cellular Viability - MTT Assay first stable line of adult human keratinocytes presenting nor- mal differentiation. That is typical adherent cell line, growing The viability of cells was determined by MTT assay (Sigma- in monolayer. A549 - human epithelial lung cancer cell line, Aldrich, USA) after experiments with different concentrations derived from the 58-year-old Caucasian man. That is typical of beta-glucan. The MTT assay was used to estimation of mi- adherent cell line, growing in monolayer. H69AR - human tochondrial metabolic function through the measurement of multidrug resistant small cell lung cancer cell line, derived mitochondrial dehydrogenase. For the experiment the cells from the 55-year-old Caucasian man. H69AR cells begin to were seeded into 96-well micoculture plates at 1 × 10 cells/ grow as aggregates which attach as domed patches. Most cells well and grown overnight. After incubation with selected con- flatten into an epithelial monolayer, however some areas have centrations of LMW and HMW beta-glucan the experiments High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 585 were realized according to the manufacture’sprotocol. Those conventional bright-field microscopy (peroxidase-ABC label- cells were incubated 24 h. The absorbance was determined ling), the samples were incubated with a diaminobenzidine- using a multiwell scanning spectrophotometer at 570 nm H O mixture to visualize the peroxidase label and counter- 2 2 (Enspire Perkin Elmer Multiplatereader, USA). Mitochondrial stained with haematoxylin for 30 s. The samples were ana- metabolic function was expressed as a percentage of viable lyzed with the upright microscope (Olympus BX51, Japan). treated cells in relation to untreated control cells. Stained cells were determined by counting 100 cells in ran- domly selected fields. The result was judged positive if stain- Lipid Peroxidation ing was observed in more than 5% of cells. The intensity of immunohistochemical staining was evaluated as: (−) negative, The measurement of lipid peroxidation assay is based on the (+) weak, (++) moderate and (+++) strong. reaction of malondialdehyde with thiobarbituric acid (TBA). The cells were treated with selected concentrations of LMW Red Blood Cells Hemolysis and HMW beta-glucan. After 24 h incubation the cells were removed by trypsinization and suspended in phosphate buff- The level of hemolysis was determined by spectrophotometry. ered saline (PBS, Sigma Aldrich, USA). The final product of Red blood cell hemolysis was induced using sodium chloride lipid peroxidation, malondialdehyde (MDA), reacts with TBA solutions of various degrees of hypotonicity. The effect of to form a colored complex. The level of MDA was measured each type of beta-glucan (LMW and HMW beta-glucan frac- by the absorbance at a wavelength of 535 nm. The concentra- tion) on erythrocytes hemolysis was examined at two concen- tion of MDA was quantified spectrophotometrically based on trations (300 μg/ml and 400 μg/ml) and incubation periods of a set of MDA standards of known concentration [18]. 24 h. Immunofluorescencent Assessment of Cytoskeleton – Preparation of red Blood Cells Suspensions CLSM Study Blood samples from healthy volunteers were obtained in hep- The confocal laser scanning microscopy (CLMS) was used to arinized tubes. The red blood cells were separated from leu- assess the morphology of treated cells. A549, H69AR and kocytes by centrifugation (3000 rpm for 10 min.) and subse- HaCaT cells were prepared for immunofluorescence reaction. quently, washed three times with 0.9% sodium chloride solu- The cells were grown on coverslips, than fixed 4% parafor- tion (saline). Then prepared mixture of erythrocytes was fill- maldehyde (PFA, Sigma-Aldrich, USA) in PBS, perme- ing by saline until the correct hematocrit for an individual abilized with 0.5% Triton X-100 (Sigma-Aldrich, USA) in subjects (approximately 40% in healthy females and 43% in PBS (v/v) for 5 min. And blocked with 1% FBS in PBS (for healthy men) [19]. 30 min. at room temperature). The cells were washed in PBS on the every steps of procedure. The following antibodies Preparation of Beta-Glucan Solutions were used: primary antibody monoclonal anti-F-actin anti- body produced in mouse (overnight incubation at 4 °C; The solutions of beta-glucan (LMW and HMW beta-glucan 1:100; Sigma-Aldrich, USA); secondary antibody goat anti- fraction) were prepared by dissolving the requisite amount of mouse IgG FITC conjugated (for 60 min. at room tempera- solute in mixture of erythrocytes to obtain the desired concen- ture; 1:50; Sigma-Aldrich, USA). DNA was stained with trations (300 μg/ml and 400 μg/ml). DAPI (4,6-diamidino-2-phenylindole; 0.2 μg/ml in mounting Each of the prepared test mixtures of red blood cells with medium with Mowiol-0.1% and DABCO). For imaging, beta-glucan (0.02 mL) was introduced into a tube with respec- Olympus FluoView FV1000 confocal laser scanning micro- tive concentration of hypotonic sodium chloride solution. scope (Olympus, Japan) was used. The images were recorded Then the tubes were incubated for 0.5 h at 23 °C (at room by employing a Plan-Apochromat 60× oil-immersion temperature) and then centrifuged (2000 rpm for 5 min.). The objective. absorbance of the supernatant solution was measured spectro- photometrically at 540 nm using a microplate reader (EnSpire, Immunocytochemical MnSOD Evaluation PerkinElmer, USA) [20]. Immunocytochemistry was performed using the ABC meth- Statistical Analysis od. The cultures were fixed and dehydrated using 4% PFA during 10 min. The samples were then permeabilized and Data are reported as mean ± standard deviation (SD). Analysis blocked by incubation with 0.1% Triton X-100 in PBS. The utilized the one-way repeated measures analysis of variance enzymes expression were visualized with polyclonal antibody (ANOVA). As a post-hoc test when results of the above test (1:100, anti-MnSOD; SOD 2; Santa Cruz, USA). For were significant Fisher’s Least Significant Difference (LSD), 586 Choromanska A. et al. Tukey’s Honestly Significant Difference (HSD) and Scheffe’stest wereapplied, where Scheffe’s is the most con- servative of the three. A level of P < 0.05 was considered to be statistically significant. The results from analysis are pre- sented in supplementary material (SM). Results Cellular Viability The MTTassay showed that LMW beta glucan derived from oats hasn’t demonstrated cytotoxic effect against normal HaCaT cells, while in A549 and H69AR cells it caused slightly decreased in cells viability (to about 80%) (Fig. 1a). In turn HMW beta-glucan hasn’t demonstrated cytotoxic effect against normal cells (Fig. 1b), but for can- cerous it cytotoxic. The cytotoxicity was increased with ris- ing concentration of beta-glucan. The greatest decrease of Fig. 2 The effect of and LMW (panel A) and HMW (panel B) beta- cells viability was observed for 200 μg/ml and reached glucan on the MDA level in cells. Error bars shown are means ±SD for about 60% for A549 cells and 70% for H69AR. That cells n ≥ 3. *P ≤ 0.05 were more sensitive to HMW beta-glucan from oats (Fig. 1b). cell line this increase was almost the same in all HMW beta- glucan concentrations. In H69AR cell line MDA level was Lipid Peroxidation increasing with increase of HMW beta-glucan concentrations, while for HaCaT cells MDA level was increasing also, but only LMW beta glucan didn’t cause significant changes in MDA to 50 μg/ml concentration of HMW beta-glucan. Above that level in tested cells (Fig. 2a), where HMW beta glucan clearly concentration the level of MDA was decreasing (Fig. 2b). caused increase of MDA level in all tested cell lines. In A549 Assessment of Changes in the Cell Cytoskeleton A low (50 μg/ml) as well as high (200 and 400 μg/ml) con- centrations of LMW beta-glucan cause dramatic changes in cell morphology manifested by nucleus perturbations such as nuclear blebbing and abnormal chromatin condensation in A549 and HaCaT cell lines (Fig. 3). In H69AR cells LMW beta-glucan did not cause significant changes in the cytoskel- eton (Fig. 3). In case of a HMW beta-glucan abnormalities in actin fibers arrangement were observed at each tested concen- trations in H69AR and HaCaT cell line (Fig. 4). MnSOD Expression LMW and HMW beta glucan increased the expression of MnSOD compared to control cells in all tested cell lines, but the highest expression was visible in A549 cells after incuba- tion with LMW beta-glucan for concentration 50 μg/ml, 400 μg/ml (90 and 100% respectively) and HMW beta- glucan for 400 μg/ml (100%). HaCaT and H69AR cells dem- Fig. 1 The effect of and LMW (panel A) and HMW (panel B) beta- onstrated lower expression than A549 cells for experimental glucan on the cells viability. Error bars shown are means ±SD for n ≥ 3.*P ≤ 0.05 conditions (Fig. 5 ant Table 1). High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 587 Fig. 3 The cytoskeleton visualization in A549, H69AR and HaCaT cell line after treatment with LMW beta-glucan. Red fluorescence: F-actin fibers, blue fluorescence: cell nucleus Red Blood Cells Hemolysis mechanisms by which beta-glucan can destroy cancer cells are very complex and still not fully understand. Some results Our results showed that the inhibition of hemolysis in the suggest that immunomodulatory and anti-cancer features of b- presence of beta-glucan and the inhibition of hemolysis was glucans consequence from their structure, molecular weight, concentration dependent (Figs. 6 and 7). The highest level of degree of branching and conformation. At present most inves- inhibition of hemolysis was also observed at the higher of the tigation has been focus on cereals’ extract due to its good two tested concentrations of HMW beta-glucan (Figs. 6 and water-solubility. It is commonly known that beta-glucan is 7). The red blood cells hemolysis in water free of sodium useful in adjuvant or supplementation therapy but not as a chloride (in distilled water) was inhibited by 36,95%, standard recurrent treatment. Management of the standard 49,5%, 38,18% and 51,64% at 300 μg/mL concentrations of cancer treatment protocol is still required [14, 21]. LMW beta-glucan, 300 μg/ml concentrations of HMW beta- Even though there is no complete evidence that beta-glucan glucan, 400 μg/ml concentrations of LMW beta-glucan, and can be effectively used as anti-cancer factors, there are many 400 μg/ml concentrations of HMW beta-glucan, respectively. interesting investigations confirming its usefulness to affect Results were considered statistically significant at p >0.05. cancer cells in vitro and in vivo [22–24]. Our data showed The concentrations of chloride solution needed to cause he- that the high molecular weight beta-glucan similar to low molysis of red blood cell in the presence of beta-glucan are didn’t affect the normal human keratinocytes in concentration shown in Table 2. 50 to 200 μg/ml. In both tested lung cancer cell line we observed the propor- tional decrease of cell viability after incubation with HMW Discussion beta-glucan. The LMW beta-glucan causes only a slight cyto- toxicity in these cells. The research of Hong et al. [9]iscon- Beta-glucan is frequently a natural products used in medicine, centrated on the antitumor effect of glucan from microorgan- cosmetics, and the food industry. Glucans can activate the isms (Paenibacillus polymyxa JB115) on four different cancer lines (A549, Hela, Hep3B, Sarcoma 180) [9]. Significant immune system and demonstrate antitumor effects. The 588 Choromanska A. et al. Fig. 4 The cytoskeleton visualization in A549, H69AR and HaCaT cell line after treatment with HMW beta-glucan. F-actin fibers, blue fluorescence: cell nucleus cytotoxicity was observed in Hela and Sarcoma 180 cells. The cancer cells is dependent on the applied dose of beta-glucan. cytotoxicity of beta-glucan was confirmed by Kim et al. [21]. They tested with MTT assay usage indicated that 200 μg/ml They studied colon cancer cells and postulated that viability of dose caused decreasing of viability of cancer cells about 50% CONTROL LMW beta-glucan LMW beta-glucan HMW beta-glucan HMW beta-glucan 50μg/ml 400μg/ml 50μg/ml 400μg/ml Fig. 5 MnSOD expression in A549, H69AR and HaCaT cell line after treatment with LMW and HMW beta-glucan (×400) HaCaT H69AR A549 High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 589 Table 1 The The intensity of % of positively immunocytochemistry analysis of immunocytochemical staining stained cells MnSOD expression in normal HaCaT and lung cancer A549 and A549 control − 0 H69AR cell lines after exposure to the LMW and HMW beta- LMW beta-glucan 50 μg/ml + 90 glucan LMW beta-glucan 400 μg/ml ++ 100 HMW beta-glucan 50 μg/ml ++ 40 HMW beta-glucan 400 μg/ml ++/+++ 100 H69AR control − 0 LMW beta-glucan 50 μg/ml + 30 LMW beta-glucan 400 μg/ml + 30 HMW beta-glucan 50 μg/ml +/++ 50 HMW beta-glucan 400 μg/ml +/++ 50 HaCaT control − 0 LMW beta-glucan 50 μg/ml + 40 LMW beta-glucan 400 μg/ml + 80 HMW beta-glucan 50 μg/ml +/++ 50 HMW beta-glucan 400 μg/ml + 50 Results were presented as the percentage of stained cells. The evaluation of stained reaction: (−) negative, no reaction, (+) weak, (++) moderate and (+++) strong. Result present as a mean of cells number counted from 3 fields with ± SD (×400) [21]. Other studies showed in contrast the inhibition rate of We found some straight cytotoxic effects of beta-glucan in beta-(1–3) glucan isolated from Poria cocos mycelia on A549 and H69AR cell line in opposite to other investigation Sarcoma 180 as less than 10% [25, 26]. Moreover Zhang in which any direct decrease of tumor cells proliferation was et al. [27] used water-soluble beta-glucan including mainly initiated [6, 28]. We examined the oxidative markers such 1 → 3and1 → 4 linkages obtained from the mycelia of lipid peroxidation, expression of mitochondrial superoxide Poria cocos (PCM3-II). The dose effect of PCM3-II on dismutase MnSOD and cytoskeletal changes. In contrast to MCF-7 cell line was studied by incubating these cells with normal human keratinocytes the level of MDA was increased 12.5–400 μg/ml of the glucan for 72 h. In this case the MTT both in human adenocarcinoma lung cell line and in multidrug examination showed that PCM3-II reduced proliferation and resistant small cell lung cancer cell line in every concentration. viability of the MCF-7 cells dose-dependently, so that the Yamamoto et al. [29] described that beta-glucan from mush- cancer-cell growth was reduced by 50% of the control level room activated suppression of angiogenesis and metastasis in at 400 μg/ml of the beta-glucan [27]. orally controlled model. Also it is well documented that beta- Fig. 6 Inhibition of hypotonic sodium chloride solution-induced Fig. 7 Inhibition of hypotonic sodium chloride solution-induced hemolysis of human erythrocytes by beta-glucan (300 μg/ml). On the hemolysis of human erythrocytes by beta-glucan (400 μg/ml). On the graph marked course of hemolysis with LMW beta glucan (dash line) graph marked course of hemolysis with LMW beta glucan (dash line) and HMW beta glucan (small dots line) after 24 h of incubation. Course and HMW beta glucan (small dots line) after 24 h of incubation. Course of hemolysis without beta-glucan (control) marked on the graph solid line of hemolysis without beta-glucan (control) marked on the graph solid line 590 Choromanska A. et al. Table 2 The percent hemolysis of human erythrocytes treated with Bone marrow hemopoietic suppression and decrease of exemplary hypotonic sodium chloride solution. The rate of hemolysis blood cell populations represent major damaging conse- decreased with increasing concentrations of the protective agent – oat quences in anticancer chemotherapy. Therefore, we beta-glucan wanted to evaluate the effect of beta-glucan not only on Form and concentration Concentration of % hemolysis cancer cells but also on normal human red blood cells. Our of beta-glucan [μg/ml] hypotonic sodium of human erythrocytes studies have also showed that the protective effects of chloride solution [g/L] beta-glucan against hemolysis increased in a dose- LMW 300 0 63,05 dependent manner. The test performed has allowed to HMW 300 0 50,50 demonstrate that relatively low concentrations of beta-- LMW 400 0 61,82 glucan (300 μg/ml) have been shown to efficiently inhibit the hemolytic action of hypotonic sodium chloride solu- HMW 400 0 48,36 LMW 300 1 61,45 tion and distilled water. It can suggest that hemolysis of the red blood cells can be blocked by virtue of reversible HMW 300 1 42,36 binding of the beta-glucan to the erythrocyte membrane. LMW 400 1 55,64 Hemolysis by hypotonic solution of sodium chloride ap- HMW 400 1 44,55 peared to be inhibited by beta-glucan, presumably by a LMW 300 3 26,73 mechanical enhancement mechanism of erythrocyte HMW 300 3 7,26 phospholipid. LMW 400 3 29,09 The effect of beta-glucan is consistent with our suggestion HMW 400 3 10,00 that this plant polysaccharide prevent hemolysis by binding loosely to the erythrocyte, presumably to the erythrocyte surface. Clarification of this issue would require carrying glucan from mushrooms has reduced pulmonary metastasis out microscopic examination to show whether the beta- and inhibited the growth of metastatic cancer in the lung [29]. glucan is present in the form of one larger conglomerate or Possibly beta-glucan can induces oxidative stress into the more discrete molecules physically associated with erythro- tumor cells. The high expression of mitochondrial superoxide cyte plasma membranes. Possibly in these instances, single dismutase and significant changes in cytoskeleton of A549 beta-glucan molecules appear to encompass the plasma and H69AR lung cancer cell line confirm our suggestion. membrane of erythrocytes in the form of network. The mor- Some research demonstrated that apoptosis is activated in phology analysis of the red blood cells surface would pro- cancer cells by beta-glucan through an increase the expression vide confirmation of this thesis that the beta-glucan can cre- ate insoluble linkages with the red blood cell membrane of caspase-3 enzyme. Additionally beta-glucan can lead to changes morphology and of the expression of proapoptotic constituents. It can be seen that HMW beta-glucan was more gene [21]. The apoptosis death pathways can be activated effective in inhibition of hemolysis. Probably this fraction of multifactorial. One of the ways of inducing apoptosis in tumor beta-glucan has greater avidity for erythrocyte membrane cells is oxidative stress. Some studies show that bioactive phospholipids. beta-glucan polysaccharide of the Maitake mushroom has cy- Presumably the protective effect of the beta-glucan in totoxic outcome probably through oxidative stress on prostatic part was dependent also by virtue of impact against lipid cancer cells, which lead to apoptosis. peroxidation [32, 33]. Beta-glucan is known to have pro- To explore more effective treatment for hormone-refractory tective effects on antioxidant status and peroxidation of prostate cancer, they investigated the potential antitumor ef- phospholipids. Several studies have shown that oral con- fect of beta-glucan, on prostatic cancer cells in vitro. sumption of beta-glucan in human and animals can be Enhancement of cytotoxic effect of glucan by vitamin C and effective in preventing oxidative stress which damages carmustine can also have clinical application [30]. Previous other relevant blood components such as platelets [34]. results show that beta-glucan can induced apoptosis by inter- Obtained results allow to suggest that plant polysac- nal pathway, due to modulation of Bcl-2 family and activation charides as beta-glucan may afford beneficial effects in of caspase 3 expression [21]. Soluble beta-glucan from preventing oxidative damage to membranes of erythro- Candida albicans induced significantly apoptosis and oxida- cytes. These observations may be useful in preventing tive stress, enhanced the formation of 8-OHdG and HO-1in or treating various disease conditions relating to erythro- the lung isolated from mice, which is associated with lung cytes in which lipid peroxidation and mechanical damage injury [31]. Kobayashi et al. [22] reported also that beta- to the membrane plays a role. Further studies on beta- glucan from Agaricus blazei Murill had cytotoxic effect glucan are required to find new biochemical and thera- against human ovarian cancer HRA cells, but not against mu- peutic properties of it have and assess their effects and rine Lewis lung cancer 3LL cells [22]. mechanisms of actions. High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 591 9. Hong JH, Jung HK (2014) Antioxidant and antitumor activities of Summingup,basedonthetest conductedonthreecell β-glucanrich. Exopolysaccharides with different molecular weight lines: human lung adenocarcinoma cancer, human multi- from Paenibacillus polymyxa JB115. Korean Soc Appl Biol Chem drug resistant small cell lung cancer and normal human 57:105–112 keratinocytes was found that 1–3, 1–4 –D-beta-glucan de- 10. 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High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung Cancer

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Abstract

Pathol. Oncol. Res. (2018) 24:583–592 DOI 10.1007/s12253-017-0278-3 ORIGINAL ARTICLE High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung Cancer 1 1 2 2 Anna Choromanska & Julita Kulbacka & Joanna Harasym & Remigiusz Oledzki & 3 1 Anna Szewczyk & Jolanta Saczko Received: 23 February 2016 /Accepted: 12 July 2017 /Published online: 29 July 2017 The Author(s) 2017. This article is an open access publication Abstract Beta-glucans are widely used in treatment, cos- Introduction metics, and the food industry. Glucans play a significant role in activation of the immune and antioxidant system Despite of the advances methods in medicine and the dynam- and inhibiting tumor proliferation. In the current study the ics of biochemical and biotechnological techniques, it is more antitumor activities of new high and low molecular weight often reached up to the sources of natural medicine. Herbal beta-glucan derived from oats were investigated in two medicine, principles of rational nutrition concerns of growing human lung cancer cell line (A549, H69AR) and normal as an alternative way of treatment and support for pharmaco- keratinocytes (HaCaT). The effect of high and low molec- logical therapy. In recent years interest in the use of plants for ular weight beta-glucan from oat was evaluated by cellular the production of compounds of pharmacological is increased. viability assessment, lipid peroxidation and manganese Additionally various studies have shown that components of superoxide dismutase evaluation and cytoskeleton visual- plant can prevent disease, especially cancer. Using analytical isation. Additionally the level of red blood cells hemolysis methods for pharmacodynamic and which are used to assess was performed. Our results indicate strong anti-tumor the therapeutic properties and their actions, we get more and properties of new beta-glucan from oat and at the same more data on the therapeutic efficiency of herbal supplements time no toxicity for normal cells. [1–3]. Recently, one of the active ingredients responsible for the anticancer therapy of many of these herbs was found to be a form of complex polysaccharides known as Bbeta-D- . . . Keywords Oat beta-glucan Viability Lung cancer glucan^. It is non cellulosic polymers, elevating of the cellular . . Keratinocytes Oxidative stress Cytoskeleton walls, of beta-glucose, which is glycoside in position beta (1– 3), (1–4) or beta (1–6). It occurs in the form of long-chain, three-dimensional molecule polysaccharide side chains of glu- cose molecules built [4]. Beta-glucans are isolated mainly Electronic supplementary material The online version of this article from different fungi. However there are obtained from other (doi:10.1007/s12253-017-0278-3) contains supplementary material, which is available to authorized users. sources, such as cereals, barley, bacteria or seaweeds. Beta- glucans, which are isolated from different sources character- * Anna Choromanska ized similar biological properties but different structures and anna.choromanska@umed.wroc.pl solubility in water or other solvents [5]. The variety of mate- rials from which beta-glucan is obtained, makes it possible to get a large number formulations of similar or different param- Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368 Wroclaw, Poland eters. Immunomodulatory and anti-cancer properties of beta- glucans result from their structure, molecular weight, degree Department of Food Biotechnology, Wroclaw University of Economics, Komandorska 118-120, 53-345 Wroclaw, Poland of branching, conformation and its behavior in gastrointestinal tract [6]. Moreover, these properties depend on an isolation Department of General Zoology, Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw, Poland procedure of beta-glucan. Beta-glucans belong to well-known 584 Choromanska A. et al. biologic modifiers, which play immunostimulatory function piling of cells. Even when cells are healthy and well-attached, against infections and cancer [7, 8]. This compound activated there are many viable floating cells. the response of adaptive immune cells such as CD4+ or/and All cell lines were grown in polystyrene flasks with 25 cm CD8+ T cells and in addition B cells. It is one of the possible cell culture surface (Eppendorf, Germany) as a monolayer in mechanisms of protective and anticancer action. However, the Dulbecco modified Eagle medium (DMEM, Sigma-Aldrich, mechanisms of anticancer activities of beta-glucan seem to be USA) for HaCaT and A549 cell line or RPMI-1640 Medium, multicomplex and still understood. Beta-glucan extracted (Gibco, USA) for H69AR cell line. Mediums were enhanced from cell wall of fungi is characterized by low purity and 10% (DMEM) or 20% (RPMI-1640) fetal bovine serum additionally causes unfavorable effect. Moreover, there are (FBS, Sigma-Aldrich, USA) and 50 μg/ml streptomycin water insoluble fraction [9]. (Sigma-Aldrich, USA). Cells were incubated at 37 °C in 5% Recently more remark is concentrated on beta-glucan from CO . Before the every experiments the cells were removed by cereals mainly from oat. This soluble fibre fraction is the 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, USA). greatest find of recent years [10]. Numerous researches con- firmed the significant anticancer activity of beta-glucan HMW and LMW Beta-Glucans Recovery Procedure [11–13]. In the treatment of breast cancer, and also in areas undergoing irradiation after amputation, more rapid healing In this examination beta-glucan from oats was derivedin form was obtained of inflammation. The use of beta-glucan in these of white powder. HMW and LMW beta-glucan was obtained appeared unaffected healthy tissue [14]. Still is well docu- at the University of Economics in Wroclaw. HMW oat beta- mented only the immune activation role of oat beta-glucan glucan was obtained due to procedure described elsewhere in killing cancer cells [15, 16]. We postulated that anticancer [17] with beta-glucanase inactivation during lipid removal activity of oat beta-glucan is more complicated and complex. step, alkaline extraction, protein removal in isoelectric point, Many of the compounds used in the methods of standard solution neutralization to pH = 7,0 and beta-glucan precipita- anticancer therapy activated oxidative stress in cancer cells, tion with ethanol. Low molecular oat 1–3, 1–4–D-beta-glucan which leads to cell death through lipid and thiol groups of was manufactured due to procedure described elsewhere [10] protein peroxidation. Moreover, beta-glucan can influence with multistep freeze-milling of raw materials (20% beta- on cells cytoskeleton and antioxidant preventing system. glucan fiber, Microstructure, Poland), fat removal with etha- The aim of the study was to evaluate the cytotoxicity and nol extraction, alkaline extraction (pH 8–10) of beta-glucan antitumor activities of high-molecular weight (HMW) and and oat proteins, protein precipitation and separation in iso- low-molecular weight of beta-glucan (LMW) derived from electric point, and finally beta-glucan precipitation with etha- oats, based on the two cell lines, normal and cancerous. nol in equilibrium. Beta-glucan preparations was then dried Additionally, the effect of the plant polysaccharides, such for 24 h. Purity was determined with according to AOAC the beta-glucan, on human red blood cell hemolysis as an 995.16 method with test kit (Megazyme, Ireland) and was indicator of membrane stability, was investigated. 84%. Molecular weight of oat beta-glucan was measured with HPLC-SEC with guard column (OHpak SB-G, Shodex), a GPC column (SB-806 M HQ, Shodex) and was 69,650 g/ mol. Firstly the stock solution of beta-glucan was prepared. Material and Methods Two mg of beta-glucan was dissolved in 1 ml of sterile dis- tilled water and one drop of 10% NaOH was added. Then the Cell Culture stock solution was incubated at 37 °C for 24 h. The different concentrations of this compound were used to the studies Three human cell lines, (purchased from the American Type (5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, 50 μg/ml, 75 μg/ Culture Collection Cell-ATCC-American Type Culture ml, 100 μg/ml, 150 μg/ml and 200 μg/ml). Collection) were used in this investigations: HaCaT - normal, immortal cell line of the transformed phenotype in vitro, the Cellular Viability - MTT Assay first stable line of adult human keratinocytes presenting nor- mal differentiation. That is typical adherent cell line, growing The viability of cells was determined by MTT assay (Sigma- in monolayer. A549 - human epithelial lung cancer cell line, Aldrich, USA) after experiments with different concentrations derived from the 58-year-old Caucasian man. That is typical of beta-glucan. The MTT assay was used to estimation of mi- adherent cell line, growing in monolayer. H69AR - human tochondrial metabolic function through the measurement of multidrug resistant small cell lung cancer cell line, derived mitochondrial dehydrogenase. For the experiment the cells from the 55-year-old Caucasian man. H69AR cells begin to were seeded into 96-well micoculture plates at 1 × 10 cells/ grow as aggregates which attach as domed patches. Most cells well and grown overnight. After incubation with selected con- flatten into an epithelial monolayer, however some areas have centrations of LMW and HMW beta-glucan the experiments High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 585 were realized according to the manufacture’sprotocol. Those conventional bright-field microscopy (peroxidase-ABC label- cells were incubated 24 h. The absorbance was determined ling), the samples were incubated with a diaminobenzidine- using a multiwell scanning spectrophotometer at 570 nm H O mixture to visualize the peroxidase label and counter- 2 2 (Enspire Perkin Elmer Multiplatereader, USA). Mitochondrial stained with haematoxylin for 30 s. The samples were ana- metabolic function was expressed as a percentage of viable lyzed with the upright microscope (Olympus BX51, Japan). treated cells in relation to untreated control cells. Stained cells were determined by counting 100 cells in ran- domly selected fields. The result was judged positive if stain- Lipid Peroxidation ing was observed in more than 5% of cells. The intensity of immunohistochemical staining was evaluated as: (−) negative, The measurement of lipid peroxidation assay is based on the (+) weak, (++) moderate and (+++) strong. reaction of malondialdehyde with thiobarbituric acid (TBA). The cells were treated with selected concentrations of LMW Red Blood Cells Hemolysis and HMW beta-glucan. After 24 h incubation the cells were removed by trypsinization and suspended in phosphate buff- The level of hemolysis was determined by spectrophotometry. ered saline (PBS, Sigma Aldrich, USA). The final product of Red blood cell hemolysis was induced using sodium chloride lipid peroxidation, malondialdehyde (MDA), reacts with TBA solutions of various degrees of hypotonicity. The effect of to form a colored complex. The level of MDA was measured each type of beta-glucan (LMW and HMW beta-glucan frac- by the absorbance at a wavelength of 535 nm. The concentra- tion) on erythrocytes hemolysis was examined at two concen- tion of MDA was quantified spectrophotometrically based on trations (300 μg/ml and 400 μg/ml) and incubation periods of a set of MDA standards of known concentration [18]. 24 h. Immunofluorescencent Assessment of Cytoskeleton – Preparation of red Blood Cells Suspensions CLSM Study Blood samples from healthy volunteers were obtained in hep- The confocal laser scanning microscopy (CLMS) was used to arinized tubes. The red blood cells were separated from leu- assess the morphology of treated cells. A549, H69AR and kocytes by centrifugation (3000 rpm for 10 min.) and subse- HaCaT cells were prepared for immunofluorescence reaction. quently, washed three times with 0.9% sodium chloride solu- The cells were grown on coverslips, than fixed 4% parafor- tion (saline). Then prepared mixture of erythrocytes was fill- maldehyde (PFA, Sigma-Aldrich, USA) in PBS, perme- ing by saline until the correct hematocrit for an individual abilized with 0.5% Triton X-100 (Sigma-Aldrich, USA) in subjects (approximately 40% in healthy females and 43% in PBS (v/v) for 5 min. And blocked with 1% FBS in PBS (for healthy men) [19]. 30 min. at room temperature). The cells were washed in PBS on the every steps of procedure. The following antibodies Preparation of Beta-Glucan Solutions were used: primary antibody monoclonal anti-F-actin anti- body produced in mouse (overnight incubation at 4 °C; The solutions of beta-glucan (LMW and HMW beta-glucan 1:100; Sigma-Aldrich, USA); secondary antibody goat anti- fraction) were prepared by dissolving the requisite amount of mouse IgG FITC conjugated (for 60 min. at room tempera- solute in mixture of erythrocytes to obtain the desired concen- ture; 1:50; Sigma-Aldrich, USA). DNA was stained with trations (300 μg/ml and 400 μg/ml). DAPI (4,6-diamidino-2-phenylindole; 0.2 μg/ml in mounting Each of the prepared test mixtures of red blood cells with medium with Mowiol-0.1% and DABCO). For imaging, beta-glucan (0.02 mL) was introduced into a tube with respec- Olympus FluoView FV1000 confocal laser scanning micro- tive concentration of hypotonic sodium chloride solution. scope (Olympus, Japan) was used. The images were recorded Then the tubes were incubated for 0.5 h at 23 °C (at room by employing a Plan-Apochromat 60× oil-immersion temperature) and then centrifuged (2000 rpm for 5 min.). The objective. absorbance of the supernatant solution was measured spectro- photometrically at 540 nm using a microplate reader (EnSpire, Immunocytochemical MnSOD Evaluation PerkinElmer, USA) [20]. Immunocytochemistry was performed using the ABC meth- Statistical Analysis od. The cultures were fixed and dehydrated using 4% PFA during 10 min. The samples were then permeabilized and Data are reported as mean ± standard deviation (SD). Analysis blocked by incubation with 0.1% Triton X-100 in PBS. The utilized the one-way repeated measures analysis of variance enzymes expression were visualized with polyclonal antibody (ANOVA). As a post-hoc test when results of the above test (1:100, anti-MnSOD; SOD 2; Santa Cruz, USA). For were significant Fisher’s Least Significant Difference (LSD), 586 Choromanska A. et al. Tukey’s Honestly Significant Difference (HSD) and Scheffe’stest wereapplied, where Scheffe’s is the most con- servative of the three. A level of P < 0.05 was considered to be statistically significant. The results from analysis are pre- sented in supplementary material (SM). Results Cellular Viability The MTTassay showed that LMW beta glucan derived from oats hasn’t demonstrated cytotoxic effect against normal HaCaT cells, while in A549 and H69AR cells it caused slightly decreased in cells viability (to about 80%) (Fig. 1a). In turn HMW beta-glucan hasn’t demonstrated cytotoxic effect against normal cells (Fig. 1b), but for can- cerous it cytotoxic. The cytotoxicity was increased with ris- ing concentration of beta-glucan. The greatest decrease of Fig. 2 The effect of and LMW (panel A) and HMW (panel B) beta- cells viability was observed for 200 μg/ml and reached glucan on the MDA level in cells. Error bars shown are means ±SD for about 60% for A549 cells and 70% for H69AR. That cells n ≥ 3. *P ≤ 0.05 were more sensitive to HMW beta-glucan from oats (Fig. 1b). cell line this increase was almost the same in all HMW beta- glucan concentrations. In H69AR cell line MDA level was Lipid Peroxidation increasing with increase of HMW beta-glucan concentrations, while for HaCaT cells MDA level was increasing also, but only LMW beta glucan didn’t cause significant changes in MDA to 50 μg/ml concentration of HMW beta-glucan. Above that level in tested cells (Fig. 2a), where HMW beta glucan clearly concentration the level of MDA was decreasing (Fig. 2b). caused increase of MDA level in all tested cell lines. In A549 Assessment of Changes in the Cell Cytoskeleton A low (50 μg/ml) as well as high (200 and 400 μg/ml) con- centrations of LMW beta-glucan cause dramatic changes in cell morphology manifested by nucleus perturbations such as nuclear blebbing and abnormal chromatin condensation in A549 and HaCaT cell lines (Fig. 3). In H69AR cells LMW beta-glucan did not cause significant changes in the cytoskel- eton (Fig. 3). In case of a HMW beta-glucan abnormalities in actin fibers arrangement were observed at each tested concen- trations in H69AR and HaCaT cell line (Fig. 4). MnSOD Expression LMW and HMW beta glucan increased the expression of MnSOD compared to control cells in all tested cell lines, but the highest expression was visible in A549 cells after incuba- tion with LMW beta-glucan for concentration 50 μg/ml, 400 μg/ml (90 and 100% respectively) and HMW beta- glucan for 400 μg/ml (100%). HaCaT and H69AR cells dem- Fig. 1 The effect of and LMW (panel A) and HMW (panel B) beta- onstrated lower expression than A549 cells for experimental glucan on the cells viability. Error bars shown are means ±SD for n ≥ 3.*P ≤ 0.05 conditions (Fig. 5 ant Table 1). High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 587 Fig. 3 The cytoskeleton visualization in A549, H69AR and HaCaT cell line after treatment with LMW beta-glucan. Red fluorescence: F-actin fibers, blue fluorescence: cell nucleus Red Blood Cells Hemolysis mechanisms by which beta-glucan can destroy cancer cells are very complex and still not fully understand. Some results Our results showed that the inhibition of hemolysis in the suggest that immunomodulatory and anti-cancer features of b- presence of beta-glucan and the inhibition of hemolysis was glucans consequence from their structure, molecular weight, concentration dependent (Figs. 6 and 7). The highest level of degree of branching and conformation. At present most inves- inhibition of hemolysis was also observed at the higher of the tigation has been focus on cereals’ extract due to its good two tested concentrations of HMW beta-glucan (Figs. 6 and water-solubility. It is commonly known that beta-glucan is 7). The red blood cells hemolysis in water free of sodium useful in adjuvant or supplementation therapy but not as a chloride (in distilled water) was inhibited by 36,95%, standard recurrent treatment. Management of the standard 49,5%, 38,18% and 51,64% at 300 μg/mL concentrations of cancer treatment protocol is still required [14, 21]. LMW beta-glucan, 300 μg/ml concentrations of HMW beta- Even though there is no complete evidence that beta-glucan glucan, 400 μg/ml concentrations of LMW beta-glucan, and can be effectively used as anti-cancer factors, there are many 400 μg/ml concentrations of HMW beta-glucan, respectively. interesting investigations confirming its usefulness to affect Results were considered statistically significant at p >0.05. cancer cells in vitro and in vivo [22–24]. Our data showed The concentrations of chloride solution needed to cause he- that the high molecular weight beta-glucan similar to low molysis of red blood cell in the presence of beta-glucan are didn’t affect the normal human keratinocytes in concentration shown in Table 2. 50 to 200 μg/ml. In both tested lung cancer cell line we observed the propor- tional decrease of cell viability after incubation with HMW Discussion beta-glucan. The LMW beta-glucan causes only a slight cyto- toxicity in these cells. The research of Hong et al. [9]iscon- Beta-glucan is frequently a natural products used in medicine, centrated on the antitumor effect of glucan from microorgan- cosmetics, and the food industry. Glucans can activate the isms (Paenibacillus polymyxa JB115) on four different cancer lines (A549, Hela, Hep3B, Sarcoma 180) [9]. Significant immune system and demonstrate antitumor effects. The 588 Choromanska A. et al. Fig. 4 The cytoskeleton visualization in A549, H69AR and HaCaT cell line after treatment with HMW beta-glucan. F-actin fibers, blue fluorescence: cell nucleus cytotoxicity was observed in Hela and Sarcoma 180 cells. The cancer cells is dependent on the applied dose of beta-glucan. cytotoxicity of beta-glucan was confirmed by Kim et al. [21]. They tested with MTT assay usage indicated that 200 μg/ml They studied colon cancer cells and postulated that viability of dose caused decreasing of viability of cancer cells about 50% CONTROL LMW beta-glucan LMW beta-glucan HMW beta-glucan HMW beta-glucan 50μg/ml 400μg/ml 50μg/ml 400μg/ml Fig. 5 MnSOD expression in A549, H69AR and HaCaT cell line after treatment with LMW and HMW beta-glucan (×400) HaCaT H69AR A549 High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 589 Table 1 The The intensity of % of positively immunocytochemistry analysis of immunocytochemical staining stained cells MnSOD expression in normal HaCaT and lung cancer A549 and A549 control − 0 H69AR cell lines after exposure to the LMW and HMW beta- LMW beta-glucan 50 μg/ml + 90 glucan LMW beta-glucan 400 μg/ml ++ 100 HMW beta-glucan 50 μg/ml ++ 40 HMW beta-glucan 400 μg/ml ++/+++ 100 H69AR control − 0 LMW beta-glucan 50 μg/ml + 30 LMW beta-glucan 400 μg/ml + 30 HMW beta-glucan 50 μg/ml +/++ 50 HMW beta-glucan 400 μg/ml +/++ 50 HaCaT control − 0 LMW beta-glucan 50 μg/ml + 40 LMW beta-glucan 400 μg/ml + 80 HMW beta-glucan 50 μg/ml +/++ 50 HMW beta-glucan 400 μg/ml + 50 Results were presented as the percentage of stained cells. The evaluation of stained reaction: (−) negative, no reaction, (+) weak, (++) moderate and (+++) strong. Result present as a mean of cells number counted from 3 fields with ± SD (×400) [21]. Other studies showed in contrast the inhibition rate of We found some straight cytotoxic effects of beta-glucan in beta-(1–3) glucan isolated from Poria cocos mycelia on A549 and H69AR cell line in opposite to other investigation Sarcoma 180 as less than 10% [25, 26]. Moreover Zhang in which any direct decrease of tumor cells proliferation was et al. [27] used water-soluble beta-glucan including mainly initiated [6, 28]. We examined the oxidative markers such 1 → 3and1 → 4 linkages obtained from the mycelia of lipid peroxidation, expression of mitochondrial superoxide Poria cocos (PCM3-II). The dose effect of PCM3-II on dismutase MnSOD and cytoskeletal changes. In contrast to MCF-7 cell line was studied by incubating these cells with normal human keratinocytes the level of MDA was increased 12.5–400 μg/ml of the glucan for 72 h. In this case the MTT both in human adenocarcinoma lung cell line and in multidrug examination showed that PCM3-II reduced proliferation and resistant small cell lung cancer cell line in every concentration. viability of the MCF-7 cells dose-dependently, so that the Yamamoto et al. [29] described that beta-glucan from mush- cancer-cell growth was reduced by 50% of the control level room activated suppression of angiogenesis and metastasis in at 400 μg/ml of the beta-glucan [27]. orally controlled model. Also it is well documented that beta- Fig. 6 Inhibition of hypotonic sodium chloride solution-induced Fig. 7 Inhibition of hypotonic sodium chloride solution-induced hemolysis of human erythrocytes by beta-glucan (300 μg/ml). On the hemolysis of human erythrocytes by beta-glucan (400 μg/ml). On the graph marked course of hemolysis with LMW beta glucan (dash line) graph marked course of hemolysis with LMW beta glucan (dash line) and HMW beta glucan (small dots line) after 24 h of incubation. Course and HMW beta glucan (small dots line) after 24 h of incubation. Course of hemolysis without beta-glucan (control) marked on the graph solid line of hemolysis without beta-glucan (control) marked on the graph solid line 590 Choromanska A. et al. Table 2 The percent hemolysis of human erythrocytes treated with Bone marrow hemopoietic suppression and decrease of exemplary hypotonic sodium chloride solution. The rate of hemolysis blood cell populations represent major damaging conse- decreased with increasing concentrations of the protective agent – oat quences in anticancer chemotherapy. Therefore, we beta-glucan wanted to evaluate the effect of beta-glucan not only on Form and concentration Concentration of % hemolysis cancer cells but also on normal human red blood cells. Our of beta-glucan [μg/ml] hypotonic sodium of human erythrocytes studies have also showed that the protective effects of chloride solution [g/L] beta-glucan against hemolysis increased in a dose- LMW 300 0 63,05 dependent manner. The test performed has allowed to HMW 300 0 50,50 demonstrate that relatively low concentrations of beta-- LMW 400 0 61,82 glucan (300 μg/ml) have been shown to efficiently inhibit the hemolytic action of hypotonic sodium chloride solu- HMW 400 0 48,36 LMW 300 1 61,45 tion and distilled water. It can suggest that hemolysis of the red blood cells can be blocked by virtue of reversible HMW 300 1 42,36 binding of the beta-glucan to the erythrocyte membrane. LMW 400 1 55,64 Hemolysis by hypotonic solution of sodium chloride ap- HMW 400 1 44,55 peared to be inhibited by beta-glucan, presumably by a LMW 300 3 26,73 mechanical enhancement mechanism of erythrocyte HMW 300 3 7,26 phospholipid. LMW 400 3 29,09 The effect of beta-glucan is consistent with our suggestion HMW 400 3 10,00 that this plant polysaccharide prevent hemolysis by binding loosely to the erythrocyte, presumably to the erythrocyte surface. Clarification of this issue would require carrying glucan from mushrooms has reduced pulmonary metastasis out microscopic examination to show whether the beta- and inhibited the growth of metastatic cancer in the lung [29]. glucan is present in the form of one larger conglomerate or Possibly beta-glucan can induces oxidative stress into the more discrete molecules physically associated with erythro- tumor cells. The high expression of mitochondrial superoxide cyte plasma membranes. Possibly in these instances, single dismutase and significant changes in cytoskeleton of A549 beta-glucan molecules appear to encompass the plasma and H69AR lung cancer cell line confirm our suggestion. membrane of erythrocytes in the form of network. The mor- Some research demonstrated that apoptosis is activated in phology analysis of the red blood cells surface would pro- cancer cells by beta-glucan through an increase the expression vide confirmation of this thesis that the beta-glucan can cre- ate insoluble linkages with the red blood cell membrane of caspase-3 enzyme. Additionally beta-glucan can lead to changes morphology and of the expression of proapoptotic constituents. It can be seen that HMW beta-glucan was more gene [21]. The apoptosis death pathways can be activated effective in inhibition of hemolysis. Probably this fraction of multifactorial. One of the ways of inducing apoptosis in tumor beta-glucan has greater avidity for erythrocyte membrane cells is oxidative stress. Some studies show that bioactive phospholipids. beta-glucan polysaccharide of the Maitake mushroom has cy- Presumably the protective effect of the beta-glucan in totoxic outcome probably through oxidative stress on prostatic part was dependent also by virtue of impact against lipid cancer cells, which lead to apoptosis. peroxidation [32, 33]. Beta-glucan is known to have pro- To explore more effective treatment for hormone-refractory tective effects on antioxidant status and peroxidation of prostate cancer, they investigated the potential antitumor ef- phospholipids. Several studies have shown that oral con- fect of beta-glucan, on prostatic cancer cells in vitro. sumption of beta-glucan in human and animals can be Enhancement of cytotoxic effect of glucan by vitamin C and effective in preventing oxidative stress which damages carmustine can also have clinical application [30]. Previous other relevant blood components such as platelets [34]. results show that beta-glucan can induced apoptosis by inter- Obtained results allow to suggest that plant polysac- nal pathway, due to modulation of Bcl-2 family and activation charides as beta-glucan may afford beneficial effects in of caspase 3 expression [21]. Soluble beta-glucan from preventing oxidative damage to membranes of erythro- Candida albicans induced significantly apoptosis and oxida- cytes. These observations may be useful in preventing tive stress, enhanced the formation of 8-OHdG and HO-1in or treating various disease conditions relating to erythro- the lung isolated from mice, which is associated with lung cytes in which lipid peroxidation and mechanical damage injury [31]. Kobayashi et al. [22] reported also that beta- to the membrane plays a role. Further studies on beta- glucan from Agaricus blazei Murill had cytotoxic effect glucan are required to find new biochemical and thera- against human ovarian cancer HRA cells, but not against mu- peutic properties of it have and assess their effects and rine Lewis lung cancer 3LL cells [22]. mechanisms of actions. High- and low-Molecular Weight oat Beta-Glucan Reveals Antitumor Activity in Human Epithelial Lung... 591 9. Hong JH, Jung HK (2014) Antioxidant and antitumor activities of Summingup,basedonthetest conductedonthreecell β-glucanrich. Exopolysaccharides with different molecular weight lines: human lung adenocarcinoma cancer, human multi- from Paenibacillus polymyxa JB115. Korean Soc Appl Biol Chem drug resistant small cell lung cancer and normal human 57:105–112 keratinocytes was found that 1–3, 1–4 –D-beta-glucan de- 10. 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Pathology & Oncology ResearchSpringer Journals

Published: Jul 29, 2017

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