As expected, frugoside significantly decreased colony formation compared with vehicle-treated M14 and A375 cells (Figure 6A). various human cancer cells, including non-small cell lung cancer, glioblastoma, and prostate cancer cell lines [15,16,17,18]. However, the biological effect of frugoside on melanoma cells has not been evaluated. Reactive oxygen species (ROS), known as secondary messengers in intracellular signaling, contribute to cancer progression and development at low levels; however, at high levels, ROS can act as an anti-tumor species by inducing cell senescence and apoptosis. In fact, cancer cells do exhibit an abnormal redox status followed by increased basal ROS production, and they thus cannot tolerate higher levels of free radicals . Indeed, recent studies have shown that ROS, generated through redox dysregulation, contribute to the malignant transformation and progression of melanoma by altering cellular signaling Rabbit Polyclonal to MBTPS2 and survival pathways . Therefore, a compound that hinders redox regulation and selectively targets tumors might be a promising treatment, especially for melanoma. Recent data showed that antioxidant proteins protect several types of cancer cells from oxidative stress. The enzymes include catalase, glutathione peroxidase (GPx), and peroxiredoxins (Prxs). The predominant enzymes responsible for the elimination of H2O2 in cells are Prxs and catalase. Tolrestat Catalase is exclusively localized in peroxisomes and eliminates H2O2 when it is present at a high concentration compared with other antioxidant proteins. Kinetic and structural analyses have revealed that Prxs possess an active-site pocket that gives rise to a high-affinity peroxide binding site that is absent in catalase and GPxs [20,21,22]. As a consequence, Prxs are the major cellular antioxidants that scavenge peroxides and mediate H2O2-induced intracellular signaling. Prxs comprise three subfamilies: 2-Cys (PrxI to PrxIV), Tolrestat atypical 2-Cys, and 1-Cys [23,24]. The 2-Cys Prxs exist as homodimers and contain two conserved cysteine residues. The N-terminal Cys-SH is first oxidized by peroxides to Cys-SOH, and Tolrestat it then forms a disulfide bond together with the C-terminal Cys-SH of the other subunits. This disulfide is specifically reduced by thioredoxin, whose oxidized thioredoxin Tolrestat is then reduced by thioredoxin reductase. The sulfenic intermediates (Cys-SOH) are occasionally further oxidized to cysteine sulfinic acid (Cys-SO2H), which causes the inactivation of peroxidase that cannot be reduced by thioredoxin . Sulfiredoxin (Srx) is an important enzyme that protects against oxidative damage of host cells through the reduction of hyperoxidized peroxiredoxin (Prx-SO2H), a type of cellular antioxidant [26,27,28]. However, the importance of Srx in the pathogenesis of human diseases, including cancer, is poorly understood. Recent reports indicate that Srx is overexpressed in a variety of cancers and may promote tumorigenesis in a Prx-dependent or independent manner [26,27,28]. It is therefore important to address Srx regulation. In the present study, we reported that frugoside induces oxidative mitochondrial damage and mitochondria-mediated apoptotic cell death by inhibiting Srx expression and delaying the reduction of hyperoxidized Prx in melanoma cells. Our results suggest that frugoside might be a potential therapeutic agent for melanoma treatment. 2. Results 2.1. Frugoside Leads to Attenuated Srx Expression and Subsequently Delays Reduction of Hyperoxidized Prxs in Melanoma Cells Srx is crucial for cellular redox homeostasis and cancer progression. Additionally, redox dysregulation is very important for malignant transformation and progression in melanoma. Therefore, we first examined the expression of Srx in various melanoma cells. As shown in Figures S1A,B and S8, Srx was highly expressed in melanoma cells. From these data and recent reports , we verified Srx as a drug target to develop anti-cancer drug treatments against melanoma. One hundred compounds screened from the in-house library using the western blot assay with the Prxs-SO2 antibody to determine Srx inhibitors. The screening identified the inhibitor frugoside and its chemical structure (Figure S1C). To confirm the influence of frugoside, we examined its dose- and time-dependent effects on Srx expression. As shown in Figure 1A,B, the expression of Srx decreased in time- and dose-dependent manners in M14 and A375 human melanoma cells. In contrast, other antioxidant proteins, such as Prx2 and Prx3, were not decreased by frugoside treatment. To further confirm Tolrestat whether decreased Srx expression affects hyperoxidized Prxs, we examined the reduction of sulfinic Prxs in melanoma cells cultured in the presence of frugoside. Consequently, the decreased Srx expression.