Yamogenin-Induced Cell Cycle Arrest, Oxidative Stress, and Apoptosis in Human Ovarian Cancer Cell Line

Abstract

Steroidal saponins are a group of compounds with complex structures and biological activities. They have anti-inflammatory, antimicrobial, fungicidal, and antitumor properties. Yamogenin is one of the spirostane saponins and occurs in Trigonella foenum-graecum, Asparagus officinalis, and Dioscorea collettii. It is a stereoisomer of diosgenin—a well-known compound whose activity and mechanisms of action in cancer cells are determined. However, the antitumor effect of yamogenin is still little known, and the mechanism of action has not been determined. In this study, we evaluated the effect of yamogenin on human ovarian cancer SKOV-3 cells in vitro by determining the cellular factors that trigger cell death. The viability of the cells was assessed with a Real-Time xCELLigence system and the cell cycle arrest with flow cytometry. The activity of initiator and executioner caspases (-8, -9, and -3/7) was estimated with luminometry and flow cytometry, respectively. The mitochondrial membrane depolarization, the level of oxidative stress, and DNA damage in the yamogenin-treated cells were also evaluated by flow cytometry. Genes expression analysis at the mRNA level was conducted with Real-Time PCR. Bid activation and chromatin condensation were estimated with fluorescent microscopy. The obtained results indicate that yamogenin has cytotoxic activity in SKOV-3 cells with an IC50 value of 23.90 ± 1.48 µg/mL and strongly inhibits the cell cycle in the sub-G1 phase. The compound also triggers cell death with a significant decrease in mitochondrial membrane potential, an increase in the level of oxidative stress (over two times higher in comparison to the control), and activation of caspase-8, -9, -3/7, as well as Bid. The results of genes expression indicate that the Tumor Necrosis Factor (TNF) Receptor Superfamily Members (TNF, TNFRSF10, TNFRSF10B, TNFRSF1B, and TNFRSF25), Fas Associated via Death Domain (FADD), and Death Effector Domain Containing 2 (DEDD2) were significantly upregulated and their relative expression was at least two times higher than in the control. Our work shows that yamogenin induces apoptosis in ovarian cancer cells, and both the extrinsic and mitochondrial—intrinsic pathways are involved in this process.

New Chalcone Derivatives Containing 2,4-Dichlorobenzenesulfonamide Moiety with Anticancer and Antioxidant Properties

“…The AGS cells were seeded in a 12-well plate (1 × 10 5 cells/well) and incubated with compound 5 at concentrations of 1, 5, and 10 µg/mL; oxaliplatin at the concentration of 40 µg/mL; and DMSO (0.2%, v / v ). After 5 h of exposure, the cells were stained with the Muse MitoPotential Assay Kit (Merck Millipore, Burlington, MA, USA) and analyzed with the Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA) [ 68 ]. The percentage of depolarized live and dead cells was estimated in the experiments, which were independently repeated three times.…”

“…The AGS cells were seeded in a 6-well plate (5 × 10 5 cells/well) and incubated with compound 5 at concentrations of 1, 5, and 10 µg/mL for 48 h; oxaliplatin at the concentration of 40 µg/mL; and DMSO (0.2%, v / v ). After treatment, the cells were stained with propidium iodide from the Muse Cell Cycle Assay Kit (Merck Millipore, Burlington, MA, USA), and the amount of cells in each phase of the cell cycle was determined using the Muse Cell Analyzer (Merck Millipore, Burlington, MA, USA) [ 68 ]. The experiment was independently repeated three times.…”

“…The AGS cells were seeded in a 96-well plate (5 × 10 3 cells/well) and treated with compound 5 at concentrations of 1, 5, 7, and 10 µg/mL for 24 h; oxaliplatin at the concentration of 20 µg/mL; and DMSO (1%, v / v ). The cells were then treated with reagents from Caspase-Glo 8 Assay or Caspase-Glo 9 Assay (Promega, Madison, WI, USA) and measured with the luminometer Glomax Multi + Detection System (Promega, Madison, WI, USA) [ 68 ]. The experiments were performed in two independent repeats in three repetitions.…”