Cytotoxicity and mitochondrial-mediated apoptosis induced by Fenugreek seed oil in human hepatocellular carcinoma cells via reactive oxygen species generation

Abstract

Background: Cancer has been recognized as a major cause of death globally. Traditional medicines have been used to treat cancer for several periods, and herbal medicines are currently being used for the treatment of cancer worldwide. Fenugreek is an annual plant (family: Fabaceae) and is documented as a medicinal herb since olden days. Objective: The present study was aimed to investigate the anticancer activity of Fenugreek seed oil (FSO) against human hepatocellular carcinoma (HepG2) cell line. Materials and Methods: The HepG2 cells were exposed to 10–1000 μg/ml of FSO for 24 h to assess the cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test, neutral red uptake assay, and morphological changes. The cytotoxic concentrations of FSO were used to study the induction in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels. Further, the expression of pro-and antiapoptotic genes (p53, caspase-3, caspase-9, Bax and Bcl-2) were studied. Results: A concentration-dependent decrease in cell viability of HepG2 cells was observed after FSO exposure. Our results showed that FSO decreased cell viability of HepG2 cells in a concentration-dependent manner. FSO was also found to increase the ROS generation and decrease the MMP level in HepG2 cells. Our gene expression study indicates upregulation in apoptotic marker genes and downregulation in antiapoptotic gene. Conclusion: The results showed that FSO is capable to induce mitochondrial-mediated apoptosis through ROS generation in HepG2 cells and could be a useful resource in developing effective remedies for the treatment of hepatocellular carcinoma. Abbreviations used: FSO: Fenugreek seed oil; HepG2: Human hepatocellular carcinoma; ROS: Reactive oxygen species; NRU: Neutral red uptake; DMSO: Dimethyl sulfoxide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP: Mitochondrial membrane potential; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; DCFH-DA: 2,7-dichlorofluorescein diacetate; FBS: Fetal bovine serum; PBS: Phosphate-buffered saline; PCRq: quantitative real-time PCR; NaHCO3: Sodium bicarbonate; CO2: Carbon dioxide; DMEM: Dulbecco's modified eagle medium.