Growth-Inhibitory and Apoptosis-Inducing Effects of Punica Granatum L. Var. Spinosa (Apple Punice) on Fibrosarcoma Cell Lines

Punica granatum L. var. granatum (Pomegranate), an herbaceous plant found in Iran, The aim of this study was to investigate the cytotoxic effects, induction of apoptosis, and the mechanism of cell death of ethanol extract from Punica granatum L. var. spinosa on the mouse fibrosarcoma cell line, WEHI-164. Various parts of the herbs were extracted from fruit using ethanol as the solvent, and the cytotoxicity and cell viability of the ethanolic extract were determined by the MTT assay. To determine whether necrosis or apoptosis is the predominant cause of cell death, cell death detection was performed using the ELISA method. The induction of apoptosis was confirmed using the terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Moreover, a sensitive immunoblotting technique was used to examine the production of Caspase-3 and Bcl2 proteins. Our findings suggested that the ethalonic extract of Punica granatum L. var. spinosa altered cell morphology, decreased cell viability, suppressed cell proliferation and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 229.024μg/ml), when compared to a chemotherapeutic anticancer drug, Toxol (Vesper Pharmaceuticals), with increased nucleosome production from apoptotic cells. Induction of apoptosis by the plant extract was proved by the decrease of pro-Caspase-3 and Bcl2 proteins and quantitatively confirmed by Immunoblotting analysis. The results obtained from the present study have demonstrated the growth-inhibitory effect of Ethanol Extracts from Punica granatum L. var. spinosa, and clearly showed that apoptosis was the major mechanism of in-vitro cell death induced by the extract.