Cancer chemotherapy and pharmacology. melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR. transcript was elevated in 1205Lu and MeWo compared with HEMn (Suppl. Figure 1B). Subsequently, we determined the overall cell redox status. Under balanced cellular redox conditions, reduced glutathione (GSH) makes up approximately 90% of total glutathione and is constantly converted from the oxidized form (GSSG). Therefore, oxidized glutathione FICZ levels are indicative of oxidative stress. We assessed oxidized (GSSG) glutathione levels using a luminescence-based assay. Compared with melanocytes, melanoma cell lines had elevated oxidized glutathione (nmol/mg protein), with the WM793B melanoma cells showing the highest GSSG level (Figure ?(Figure1D).1D). Overall, oxidized glutathione was significantly elevated (4 to 6 6 fold) in all melanoma cells compared with melanocytes (Figure ?(Figure1D).1D). Examination of protein carbonylation, a well-established marker of severe oxidative protein damage showed that all melanoma cells had higher endogenous protein carbonyls compared with melanocytes (Figure ?(Figure1E).1E). Lastly, we evaluated basal mitochondrial membrane potentials, which reflect intracellular redox homeostasis. In healthy, polarized mitochondria, accumulation of potentiometric dye TMRM can be seen, whereas depolarized mitochondria do not retain the dye and leakage of TMRM is diffused in the cytosol. Confocal imaging of mitochondria allows for quantification of the TMRM fluorescent intensity and used in a Nernst equation derivative, which allows for cellular voltage calculation. We found that all melanoma cell FICZ lines had significantly lower basal mitochondrial membrane potentials compared with HEMn cells, indicated by a more positive voltage, which is interpreted as more uncoupled mitochondrial membranes (Figure ?(Figure1F).1F). Collectively, the data presented CDKN1A in Figure ?Figure11 indicate that basal oxidative stress is higher in melanoma cells compared with melanocytes, a feature that might enhance their survival. Therefore, we hypothesized that when melanocytes and melanoma cells are challenged with oxidative stress-inducing agent, the former would exhibit an antioxidant response while the latter would not producing opposite outcomes. To test this hypothesis we used NexrutineR as the oxidative stress-inducing agent. Open in a separate window Figure 1 Basal ROS and oxidative stress markers in melanoma cells and melanocytes(A) Fluorescent micrographs showing total intracellular ROS by carboxy-H2DCFDA, nuclear counterstain by Hoechst, and merged image in melanocytes (HEMn) and melanoma cells (WM793B, 1205Lu, MeWo) at 10X magnification. (B) Evaluation of basal H2O2-specific ROS by Peroxy Orange 1 (PO-1), 20X magnification. (C) Basal protein levels of PGC1 and NRF2 by western blotting. Quantification FICZ of band densitometry is shown below, relative to -actin loading control. (D) Basal level of oxidized intracellular glutathione (GSSG; nmol/mg protein) determined using luminescence-based assay. (E) Intracellular protein carbonylation used as a measure of protein damage, determined by ELISA. (F) Mitochondrial membrane potentials () were determined using Nernst equation derivative. Data are presented as means of three independent experiments. Statistical analysis was performed using Student’s 0.05; and ***indicates 0.001. NexrutineR increases oxidative stress in melanoma cells Recent work from our laboratory suggests that NexrutineR modulates ROS in pancreatic cancer cells [22]. As such, we evaluated the potential of NexrutineR to disrupt the oxidative stress threshold in melanoma cells. Using fluorescence microscopy we found that total ROS levels (carboxydichlorofluorescein) increased in a dose-dependent manner after treatment with NexrutineR (Figure ?(Figure2A).2A). Quantification of the imaging data showed an increase in the percentage of ROS-positive cells in all three melanoma cell lines (Suppl. Figure 2A). Validation of ROS levels using flow cytometry showed a higher percentage of ROS-positive cells after 10 and 20 g/ml NexrutineR treatment for 3 h compared with control cells (data not shown). To evaluate the source of ROS from NexrutineR treatment, we determined mitochondrial superoxide production using the Mitosox red fluorescent indicator. We found that NexrutineR treatment (10, 20 g/ml; 3 h) resulted in a significant increase in mitochondrial superoxide production in all the melanoma cells that appeared to saturate at 10 g/ml NexrutineR (Figure ?(Figure2B).2B). Interestingly, we did not observe increased superoxide levels in the melanocyte.