INVESTIGATION OF ANTICANCER EFFECTS OF EXOPOLYSACCHARIDES ISOLATED FROM PLEUROTUS OSTREATUS ON COLON AND LUNG CANCER CELLS LINES Miriş Dikmen 1,2 , Zerrin Canturk 3 , Elif Kaya Tilki 1 , Selin Engür 4 , Mustafa Güçlü Özarda 3 1 Department of Pharmacology, 2 Department of Clinical Pharmacy, 3 Department of Pharmaceutical Microbiology, 4 Institute of Health Sciences, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey. mirisd@anadolu.edu.tr A promising and important group of potential cancer preventive agents are those derived from natural products. Recent studies revealed various biological activities including the antitumor properties of exopolysaccharides produced by macrofungus. Edible mushrooms are a valuable source of biologically active compounds. Mushrooms demonstrate their efficiency against numerous diseases and metabolic disturbances as serious as cancer. The most significant medicinal effects of mushrooms and their metabolites that have attracted the attention of the public is their antitumor properties. The oyster mushroom (Pleurotus ostreatus) is one of the widely cultivated edible mushrooms. Lung and colon cancers continue to be the most common cancers. In this study, we aimed to determine the antiproliferative and antimetastatic effects of Pleurotus ostreatus ethanol extract on A549 human non-small lung cancer and Caco-2 colon cancer cells. INTRODUCTION METHODS Cell culture The A549 (CCL-185™) human lung adenocarcinoma and Caco-2 (HTB-37™) human colorectal adenocarcinoma cell lines were obtained from American Type Culture Collection (ATCC®, USA) and maintained in RPMI medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO 2 . Pleurotus ostreatus extract was dissolved in dimethyl sulfoxide (DMSO) at a stock solution and it was diluted to the required concentrations. A total of 70–80% confluent cells were treated with concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562 and 0.781 μg/mL) of P. ostreatus extract for 24h in the growth medium. Cell Viability Assay In order to obtain non-cytotoxic concentrations of the P. ostreatus extract, cell viability was determined by MTT assay as described previously [1]. In short, cells were grown in 96-well plates at a density of 3×10 3 cells per well and subjected to different P. ostreatus extract (400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562 and 0.781 μg/mL) concentrations. After 24h incubation period, MTT solution was added to reach a final concentration of 0.5 mg/mL. The cells were incubated for another 4 h. Then, current medium was removed from the wells and 100 µL of DMSO solution was added. The absorbance was measured at 540 nm using a Cytation 3 Cell Imaging Multi-Mode Reader (Bio-Tek). The measured absorbance directly correlates to the number of viable cells. Cell survival rates were expressed as the percentage of the DMSO (0.1%) solvent control. Graphics were drawn with GraphPad Prism version 6.0 software (San Diego, CA) and were statistically analyzed using one-way ANOVA and Tukey’s post hoc test. Results are expressed as mean ± standard deviation and the means of three independent experiments (n = 8), n.s.; p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001 and ****p < 0.0001 were considered to be significant compared to control group. IC 50 values of the P. ostreatus extract were calculated according to the MTT assay results for 24 hours. Cell proliferation assay with Real-Time Cell Analysis System (RTCA DP) Kinetics of cell proliferation were monitored with xCELLigence RTCA DP instrument which measures electrical impedance across interdigitated micro- electrodes incorporated on the bottom of tissue culture E-Plates. Electrical impedance indicated as cell index (CI) value and provides quantitative information about the condition of the cells, involving cell number, viability, and morphology [2]. Background of the E-plates (16 well) were measured in 100 µl medium in the RTCA DP station. Afterwards 100 µl of a A549 and Caco-2 cell suspensions was added (10.000 cells/well). Plates were incubated for 30 min at room temperature and E-plates were placed into the instrument and impedance was measured at 1 hour intervals. Approximately 24 hours after seeding, when the cells were in the log growth phase, the instrument was paused and the cells were treated with 100 µl of medium containing IC 50 concentrations of P. ostreatus extract (21,6 µg/mL for Caco-2 and 38,7 µg/mL for A549 cells) and impedance monitoring continued for another 24 hours. The electrical impedance was analysed by the RTCA-connected software of the RTCA DP system as a dimensionless parameter called cell index. Cell migration and invasion assays with Real-Time Cell Analysis System (RTCA DP) Cell migration and invasion assays were conducted with RTCA DP by using CIM-16 plates that contains microelectronic sensors connected onto the underside of the plate [3]. For invasion assay, top part wells of the CIM-16 plate were coated with 20 μL of 20% matrigel and incubated at 37 °C, 5% CO 2 for 4 hours before the experiment. And then for both migration and invasion assays, the wells of the bottom chamber were filled with 160 μl of 10% serum containing growth RPMI medium and the top and bottom parts of the CIM-16 plates were assembled together. After the addition of 20 μl serum-free RPMI medium to the top chamber wells, the assembled CIM-Plates 16 were allowed to stabilize for 1 h at the incubator. After the incubation, A549 and Caco-2 cells were seeded at a 2x10 4 density per well onto the top chambers of CIM-16 plates in 80 μl of serum-free media, then 100 µl of IC 50 concentrations of P. ostreatus extract were added. After stabilizing CIM-plates for 30 minutes at room temperature, the chamber was loaded in the RTCA DP machine and software was set to collect impendence data, reported as CI values, once every 10 minutes for 24 hours [2]. REFERENCES 1. Hansen, M. B., Nielsen, S. E., & Berg, K. (1989). Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. Journal of immunological methods, 119(2), 203-210. 2. Bird, C., & Kirstein, S. (2009). Real-time, label-free monitoring of cellular invasion and migration with the xCELLigence system. Nature methods, 6(8). 3. Limame, R., Wouters, A., Pauwels, B., Fransen, E., Peeters, M., Lardon, F., ... & Pauwels, P. (2012). Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays. PloS one, 7(10), e46536. RESULTS Figure 1. Effects of P. ostreatus extract on A549 (A) and Caco-2 (B) cell viability. The results are the means of 3 independent experiments. The error bars represent the standard deviations ((n = 8), p>0.05 not significant, *p<0.05, *p<0.01 *** p<0.001 and **** p<0.0001 compared to control group). A B A B Figure 2. Real-time monitoring of antiproliferative effects of P. ostreatus extract on A549 (A) and Caco-2 (B) cell lines using RTCA-DP System (n = 8) (left) and the slope graph was drawn according to these cell index values (right). A B Figure 3. Migration assay results were obtained with the xCELLigence RTCA DP system (left). The slope of the migration curves were calculated using the RTCA 1.2.1 Software (right) (n = 4). P. ostreatus extract were decreased A549 (A) and Caco-2 (B) cells migration levels significantly. A B Figure 3. Invasion assay results were obtained with the xCELLigence RTCA DP system (left). The slope of the invasion curves were calculated using the RTCA 1.2.1 Software (right) (n = 4). P. ostreatus extract were decreased A549 (A) and Caco-2 (B) cells invasion levels significantly. CONCLUSIONS While fruits and vegetables are recommended for prevention of cancer and other diseases, their active ingredients (at the molecular level) and their mechanisms of action less well understood. According to our results, the cytotoxic effects of different concentrations of the Pleurotus ostreatus extract were investigated on both cancer cells and then IC50 values were calculated. Cell proliferation assay was performed with IC50 concentrations of the extract (21.6 μg/mL for Caco-2 and 38.7 μg/mL for A549 cells). In conclusion, extract of Pleurotus ostreatus was induced cell death and antimetastatic activities of both cancer cells, especially A549 lung cancer. Therefore, by identifying the active molecule in P. ostreatus extract and the molecular targets modulated by it may be the basis for how these dietary agent not only prevent but also treat colon and lung cancer.