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Cancer is one of the life-threatening diseases and a leading cause of death worldwide. Herbal medicine has a potential of treating many diseases. Nigella sativa L. is a widely used plant in traditional systems of medicine. The cytotoxic potential of Nigella sativa seed oil (NSO) was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), neutral red uptake (NRU) assays and morphological alterations in HepG2, MCF-7, A-549 and HEK293 cell lines. Further, the influence of cytotoxic concentrations (50-250 mu g/ml) of NSO on oxidative stress markers (GSH and LPO), reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) and mRNA expression of apoptotic marker genes (p53, caspase-3, caspase-9, Bax, Bcl-2) were studied. The results exhibited significant decrease in the percentage cell viability of HepG2, MCF-7 and A-549 cells in a concentration-dependent manner. However, NSO showed higher cytotoxic response in HepG2 cells and less in HEK293 cells. Therefore, HepG2 cells were selected to further investigate the underlying mechanism(s) responsible for the cytotoxic response. NSO was found to induce oxidative stress in a concentration-dependent manner, which was indicated by induction of ROS and LPO along with decrease in reduction in GSH and MMP. Quantitative real-time PCR data showed that following the exposure of HepG2 cells to NSO, the level of mRNA expression of apoptotic marker genes (p53, caspase-3, caspase-9 and bax) was upregulated whereas, anti-apoptotic gene bcl-2 was down-regulated. The results demonstrated that NSO induced cytotoxicity and apoptosis in HepG2 cells via ROS generation, which is likely mediated through the p53 pathway. (C) 2017 SAAB. Published by Elsevier B.V. All rights reserved.