EVALUATION OF CITOTOXICITY, CELLULAR PROLIFERATION AND APOPTOSIS IN HTC CELLS TREATED WITH β-GLUCAN EXTRACTED FROM BARLEY

BM05

Simone Cristine Semprebon (1) sc.semprebon@gmail.com, Juliana Cristina Macarini (1), Marcela Stefanini Ferreira Tsuboy (1), Mário Sérgio Mantovani (1)

(1) Universidade Estadual de Londrina - UEL

Introduction: The β-glucans (βG) are polysaccharides produced by several plants (such as oats, barley and algae) and constitute the cell wall of pathogenic bacterias and fungi. Some of its benefits are: the stimulation of the immune system, of haematopoiesis, the prevention of mutagenic effects, antigenotoxic capacity against DNA damage inducing agents, such as hydrogen peroxide, reduction of growth of the mammary carcinoma and melanoma, anticlastogenic potential, and others. Objectives: evaluate citotoxicity of five concentrations of βG extracted from barley (Sigma) through the cytotoxicity assay (MTT); observe whether βG changes the cellular cycle, accelerating or delaying; and whether occurs cellular death, through Cellular Proliferation Kinetics Analysis; evaluate apoptosis inductor effects on cells culture exposed to βG through Apoptosis detection in situ assay with Acridine Orange. All of these assays were made in hepatic lineage of Rattus novergicus (HTC)- cellular cycle of 24 hours. Materials and Methods: The cells were cultivated in DMEM-F12 medium. The cytotoxicity analysis in five concentrations (10, 50, 100, 200 e 400 µg/mL) was made through MTT assay in the times 24, 48, 72h. The Cellular Proliferation Kinetics Analysis was made in eight treatments: Mitomicina-C: 0,3 µg/mL, control (medium + 1% of DMSO), three concentrations of βG (10, 50 e 100 µg/mL) and these same concentrations of βG associated to chemotherapic agent Mitomicine C. The analysis was made in neubauer plate after 24, 48, 72 and 96 hours of the treatment to form a growth curve. In these treatments, the cell viability was also evaluated through Trypan Blue method. The Apoptosis detection in situ assay analyzed the apoptosis rate of the HTC cells exposed to the same three concentrations of βG. Cysplatine was used as damage induction control for apoptosis and medium with 1% of DMSO as control. The analysis was made in fluorescent microscope after 24 hours of treatment. Results: The concentrations 10, 50 and 100 µg/mL of βG did not show cytotoxicity. In the Cellular Proliferation Kinetics Analysis, the cycle was amended only in the cells exposed to Mitomicine C, though the treatments with βG didn’t significantly changes the cellular cycle (p<0,05). The results of Apoptosis detection in situ assay with Acridine Orange were analyzed by Chi-square test (p<0,05) and there wasn’t significant difference between treatments. Conclusion: The data obtained in the experimental tested conditions, didn’t show that β-glucan of the barley induces apoptosis or changes the cellular cycle.