Therefore, elevated ROS levels are increasingly accepted as a valuable therapeutic target in anticancer drug discovery [14, 15]. this context. We found that hederagenin effectively induced apoptosis in cisplatin-resistant HNC cells and by targeting the Nrf2-ARE antioxidant pathway. 2. Materials and Methods 2.1. Cell Lines We evaluated HNC cell lines (AMC-HN2C10), previously established at our institute, as well as SNU-1041, SNU-1066, and SNU-1076 cell lines (Korea Cell Line Lender, Seoul, Republic of Korea). All of the cell lines were authenticated using short tandem repeat-based DNA fingerprinting and multiplex polymerase chain reaction (PCR). The cells were cultured ACT-335827 in Eagle’s minimum essential medium or Roswell Park Memorial Institute 1640 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum. The cells were maintained at 37C in a humidified atmosphere with 5% CO2. Normal human oral keratinocytes and normal human fibroblasts (HOF) obtained from patients undergoing oral medical procedures were used for cell viability assays. The cisplatin-resistant HNC cell lines (HN3-cisR, HN4-cisR, and HN9-cisR) were generated by prolonged exposure of the cisplatin-sensitive parental cell lines (HN3, HN4, and HN9 cells, resp.) to increase concentrations of cisplatin (Sigma-Aldrich, St. Louis, MO, USA). The half maximal inhibitory concentration (IC50) of cisplatin, as decided using cell viability assays, was 2.2C3.5?(Nrf2) expression, cisplatin-resistant HN4-cisR cells were seeded and transfected 24? h later with 10?nmol/L small interfering RNA (siRNA) targeting human or or with a scrambled control siRNA (Integrated DNA Technologies, Coralville, IA, USA). siRNA-induced gene silencing was confirmed using reverse transcription-quantitative PCR (RT-qPCR) analysis of 1-2?overexpression was confirmed using RT-qPCR and Western blotting. 2.5. ACT-335827 Western Blot Assays The cells were plated, grown to 70% confluence, and subsequently treated with the indicated reagents. The cells were lysed at 4C in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo ACT-335827 Fisher Scientific). A total of 50?test or Csta analysis of variance with Bonferroni post hoc test. The data were analyzed using SPSS version 23.0 (IBM, Armonk, NY, USA). Statistical significance was defined as a two-sided value <0.05. 3. Results and Discussion 3.1. Hederagenin Induces Apoptosis in Cisplatin-Sensitive and Cisplatin-Resistant HNC Cells The molecular weight of hederagenin is usually 472.7?g/mol (Physique 1(a)). Hederagenin decreased the viability of cisplatin-sensitive and cisplatin-resistant cancer cells in a dose-dependent manner (Figures 1(b) and 1(c)). The viability decreased by up to 50% in cells treated with 20?< 0.05) (Figure 2(b)). Hederagenin induced apoptotic cell death in all cisplatin-resistant HNC cell lines evaluated. This effect was observed as early as 24?h after treatment, and it increased in a time-dependent treatment manner (Figures 2(c) and 2(d)). Open in a separate window Physique 2 Hederagenin induces apoptotic ACT-335827 cell death in cisplatin-resistant HNC cells. (a, b) Changes in cell number and colony-forming ability in cisplatin-resistant HNC cells exposed to hederagenin (Hdg) with or without pretreatment with the antioxidant trolox (0.5?mM). NT: control cells not treated with hederagenin. (c) FACS analyses of Annexin V and propidium iodide staining of cisplatin-resistant HNC cells treated with 100?< 0.05, ??< 0.01 relative to the control or between groups. Previous studies exhibited that hederagenin induced cell death in human cancer cells by activating intrinsic apoptotic pathways [16C18]. A hederagenin saponin, macranthoside B, ACT-335827 extracted from the Chinese herb also selectively exerted cytotoxic effects in breast and lung cancer cells [17]. Hederagenin induces mitochondria-driven apoptosis and anti-inflammatory effects by suppressing the NF-< 0.05) (Figures 3(a) and 3(b)), and these effects were significantly inhibited by pretreatment with 0.5?mM trolox (< 0.05). Cisplatin treatment alone did not affect the cellular levels of GSH and ROS. In addition, hederagenin induced changes in < 0.05 relative to control, ??< 0.05 between groups. Our study focused on the effect of hederagenin on.