7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide

Abstract

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 μM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

Fig. 1

Scheme 1. Predicted reduction of NBF-SPh from the 7-nitro to a 7-amine

Fig. 2

Effects of BFZ derivatives and protectants on the survival of EMT6 cells. a Cultured EMT6 tumor cells were incubated with increasing concentrations of NBF-SPh (filled circles) and SBF-SPh (open circles) for 2 h. b Prior to a 30-min treatment with 50 µM NBF-SPh, cultured EMT6 tumor cells were first incubated for 1 h with various antioxidant protectants (light bars): sodium pyruvate (5 mM), uric acid (500 µM), and thioglycerol (5 mM). Parallel studies were also conducted without thioglycerol (dark bars). For both experiments, the treated cells were then grown for 7–10 days as described in the “Materials and methods”. Percent survival was measured by colony formation, using cells treated with solvent vehicle as a reference. Each point represents the average of four separate experiments, and the associated bars represent SE. For rescue experiments, one-way analysis of variance (ANOVA) confirmed the significance between the two sets of data (P < 0.0001)

Fig. 3

Initial rates of O₂ consumption for redox-cycling compounds. The redox-cycling activity exhibited by 25 µM of each of six quinones or nitroaromatic redox-cycling compounds was compared against NBF-SPh. O₂ consumption was monitored using 0.26 U/mL of P450Red to couple electron transfer from 2 mM NADPH. Each point represents the average of three separate experiments, and the associated bars represent standard error. According to one-way analysis of variance (ANOVA), the mean values are significantly different (P < 0.0001). As indicated by (double asterisks) for P < 0.01 and (triple asterisks) for P < 0.001, Dunnett’s post-test shows O₂ consumption is statistically different between NBF-SPh and juglone (P < 0.01), and also between NBF-SPh and each of metronidazole, paraquat, and nitrofurazone (P < 0.001)

Fig. 4

Redox-cycling of NBF-SPh and production of reactive oxygen species. a O₂ consumption was monitored with increasing concentrations of NBF-SPh (0, 2, 5, 10, and 25 µM), using 0.26 U/mL of P450Red to couple electron transfer from 100 µM NADPH. b O₂ consumption by 50 µM NBF-SPh (black line) or 50 µM SBF-SPh (gray line) was monitored, using a coupled assay with 1 mM G6P and 5 U/mL of G6PDH to maintain reduced NADPH (100 µM). Addition of 0.26 U/mL of P450Red initiated the reaction. The control assay with 10 mM glucose, 2 U/mL of glucose oxidase, and 10,000 U/mL of catalase is also presented (dotted line). Arrows indicate injection of P450Red or catalase. c A coupled assay with 1 mM G6P, 5 U/mL of G6PDH, 25 µM NADP⁺, 0.26 U/mL of P450Red, and 25 µM NBF-SPh was used to oxidize 250 µM l-adrenaline. The accumulation of adrenochrome was followed for 5 min at 451 nm (thick line), and control experiments were conducted with 10,000 U/mL of superoxide dismutase (thin line) or without 25 µlM NBF-SPh (dotted line)

Fig. 5

Lipid peroxidation by NBF-SPh in EMT6 cells. The 2-thio-barbituric acid-reactive substances (TBARS) were quantified with fluorescence (λ_ex = 532 nm, λ_em = 550 nm), after 1 h treatment with NBF-SPh (10, 25, and 50 µM) or FeCl₃-EDTA (50 and 200 µM). Values are normalized to the sample protein content and expressed as the increase of TBARS from control samples. Data are mean ± SE of 10 replicates. According to one-way analysis of variance (ANOVA), the mean values are significantly different (P = 0.0002). As indicated by (double asterisks) for P < 0.01 and (triple asterisks) for P < 0.001, Dunnett’s post-test shows TBARS were statistically different between treatments of 50 and 200 µM FeCl₃-EDTA (P < 0.001) and between treatments of 10 and 50 µM NBF-SPh (P < 0.01)

Fig. 6

DP polarography of NBF-SPh and SBF-SPh. The electroactive species of 25 µM NBF-SPh (black) or SBF-SPh (gray) were examined at a scan rate of 2 mV/s. NBF-SPh exhibited 3 peaks (1_a, 1_b, and 1_c), while SBF-SPh exhibited only 1 peak (2_a). The predicted products are shown. Arrows indicate the observed reversible or irreversible electrochemical transitions. SHE standard hydrogen electrode

Fig. 7

Cyclic voltammetry of BFZ derivatives. Scan rates of 10–400 mV/s were used to investigate 25 µM NBF-SPh (peaks 1_a, 1_b, and 1_c from Fig. 6) or 25 µM SBF-SPh (peak 2_a from Fig. 6). a Voltammetric analysis of 1_a revealed a reversible electron transfer step with cathodic (1_cath.) and anodic (1_anod.) peaks. b Analysis of 1b and 1c revealed irreversible electron transfer steps. c Analysis of 2_a revealed an irreversible electron transfer step. Ag/AgCl was a saturated Ag/AgCl reference electrode

Fig. 8

Investigating the proton/electron ratios. Plotting peak potentials versus pH revealed pK_a values at a pH = 5.5 for 25 µM NBF-SPh (1_anod., 1_cath., 1_b, 1_c) or b pH = 6.5 for 25 µM SBF-SPh (2_a). All peaks became more electronegative at increasing pH values, and the slopes are described in the text. Ag/AgCl was a saturated Ag/AgCl reference electrode

Fig. 9

Scheme 2. Observed conversion of NBF-SPh to the 1,2,3-triamine