Effects of barakol on P19 embryonal carcinoma cells

Abstract

The cytotoxicity of barakol, extracted from fresh young leaves of Cassia siamea Lam., was investigated on P19 embryonal carcinoma cells (P19 cells) using biochemical and morphological method. The cytotoxic assay of barakol demonstrated that barakol induced a concentration and time-dependent decrease in the viable cells with 95 % cell death observed at the concentrations of is less than or equal to 0.8 mM following 72 h of exposure, as determined by the XTT assay. Trypan blue dye exclusion assay revealed that barakol at the concentrations of 0.8 and 1.0 mM showed significant cytotoxicity to P19 cells; the viable cell number was lower than the control approximately 63 % - 76 % at 36-72 h of exposure. The alteration of cell morphology was observable after 48 h of exposure with barakol at the concentration of 0.8 mM and appeared to be shrinkage with spherical shape but was still viable. The cytotoxic effect of barakol at the concentrations of is more than or equal to 0.8 mM for 72 h of exposure, was reversible upon replacing culture medium with barakol-free medium. However, most viable cells showed enlarged and swollen morphology. The pattern of cell death in undifferentiated P19 cells demonstrated to be apoptosis, as assessed by agarose gel electrophoresis. After the differentiation of P19 cells to P19 neurons, the cytotoxic assay of barakol on P19 neurons showed that barakol at the concentrations of 0.8 and 1.0 mM exerted slightly cytotoxic effect, which the cell viability was greater than 74 % after 72 h of exposure, as determined by the XTT assay. The viable cell morphology was observable with large cytoplasmic vacuolation, less and short neurites. The analysis of DNA fragmentation on agarose gel electrophoresis showed DNA ladders, which indicated apoptotic cell death. The assay of AChE activity in P19 neurons revealed that barakol at the concentrations ranging from 0.05-1.0 mM did not show significant reduction in the intracellular soluble and membrane-bound AChE activity at any exposure times.