Measurement of intracellular superoxide anion Superoxide anion production was evaluated by measuring changes in fluorescence resulting from intracellular probe oxida- tion. Dihydroethidium (DHE) assay was performed according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). Briefly, 1.06104 cells were cultivated on round glass coverslips and treated with 10 mM jacaranone for 3 h at 37uC. Analysis by fluorescence microscopy was carried out on Olympus BX-51 microscope using 60X oil immersion objective. Images were processed with ImageJ (http://rsb.info.nih.gov/ij/). As a positive control, cells were treated with 5 mM H2O2 at 37uC for 20 minutes. Determination of N-acetylcysteine (NAC) effects on jacaranone-induced cell death Murine melanoma B16F10-Nex2 cells (56103/well) were cultured in 96-well plates in the presence or absence of NAC at 1, 2 or 4 mM for 30 min. Subsequently, 20 mM jacaranone was added and incubated for 18 h at 37uC. After the incubation period, cell viability was obtained by Trypan Blue (Gibco, Grand Island, NY) dye exclusion test. Redox cycling induced by jacaranone To evaluate the jacaranone-catalyzed redox cycling ability, the nitroblue tetrazolium (NBT, Sigma Aldrich, St. Louis, MO)/ glycinate assay was performed as described previously [11]. NBT/glycinate assay is a colorimetric test based on the fact that quinones, at an alkaline pH, can oxidize glycine in an oxidative amine degradation reaction. The hydroquinones or semiquinones formed react with molecular oxygen, which is monitored by the reduction of NBT to formazan [12]. NBT/glycinate assay was performed in 96-well microtiter plates, using 60 ml jacaranone sample at different final concentrations of 20, 10, 5, 2.5, 1.5 or 0.65 mM ; 20 ml BSA (initial concentration of 50 mg/ml in 10 mM potassium phosphate, pH 7), and 200 ml NBT/ glycinate reagent (0.24 mM NBT in 2 M potassium glycinate, pH 10). After incubation at 37uC for 1 h, the absorbance was read at 605 nm. Cell lysate extracts and Western blotting For protein extraction, 1.06106 B16F10-Nex2 cells treated with 20 or 50 mM jacaranone for 24 h and untreated cells (control) were washed with PBS and lysed by adding 100 mL of 1x SDS sample buffer (62.5 mM Tris-HCl, pH 6.8 at 25uC, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Lysates were transferred to microcentrifuge tubes, and kept on ice. After sonication for 15 seconds, samples were heated to 95uC for 5 min and centrifuged at 1,500 rpm for 10 min. Total proteins from each cell lysate were separated in SDS gel electrophoresis and Western blotting was carried out as described elsewhere [13]. The following primary antibodies were used: rabbit anti-b-actin, rabbit anti-phospho-p38 (Thr180/Tyr182) or total-p38, rabbit anti-phospho Akt (Thr308) or total-Akt, all purchased from Cell Signaling Technology (Beverly, MA), and mouse anti-Bax (Merck, Darmstadt, Germany). b-actin was used as loading control. Secondary antibodies conjugated with IgG horseradish peroxidase were purchased from Sigma-Aldrich (St. Louis, MO) and immunoreactivity was detected using the Immobilon solution (Millipore, Billerica, MA). To evaluate the involvement of ROS in jacaranone-mediated effects, lysates from cells preincubated with 4 mM NAC and exposed to 20 or 50 mM jacaranone were obtained and subjected to immunoblottings as described above. Antitumor Activity of Jacaranone Animals and subcutaneous tumor growth Eight-week-old male C57BL/6 mice were obtained from the Center for Development of Experimental Models (CEDEME), Federal University of Sa˜o Paulo (UNIFESP). Animal studies were conducted in compliance with UNIFESP Ethics Committee for Animal Experimentation. The therapeutic efficacy of jacaranone was examined in syngeneic B16F10-Nex2 melanoma-bearing mice. C57BL/6 mice were inoculated subcutaneously (s.c.) with 56104 viable melanoma cells in the right flank. Two treatment groups of 5 mice were subjected to seven intraperitoneal (i.p.) doses of 0.8 mg/kg or 4 mg/kg of jacaranone (diluted in PBS). An untreated tumor control group received 100 mL of PBS. Treatment started one day after tumor cell implantation and was administered on alternate days in a period of 14 days. After administration of the therapeutic doses, the tumor size was measured daily with a caliper. Tumor volume (V) was calculated by the formula V = 1/26d26D, where d and D are the short and long diameters of the tumor, respectively. Mice were removed from the therapy study and 3 sacrificed when tumor size exceeded 3,000 mm . The protection experiment was repeated for confirmation with a group of 6 animals, using 4 mg/kg of jacaranone and doxorubicin (0.1 mg/kg, Sigma-Aldrich, St.Louis, MO) as a pos- itive control. Statistical analysis Unless otherwise specified, two independent experiments were performed in triplicate. Experimental values are expressed as mean value 6 standard deviation (S.D.). For significance analyses Student’s t-tests were calculated using GraphPad Prism 4.0 software (La Jolla, CA). p values ,0.05 were considered significant. Results Cytotoxic effects of jacaranone on cancer cells The cytotoxicity of jacaranone [Methyl (1-hydroxy-4-oxo-2,5- cyclohexandienyl) acetate] (Fig. 1) was determined on human cancer cell lines and the murine melanoma B16F10-Nex2 cell line. In all tested cell lines jacaranone induced cell death in vitro in a dose-dependent manner and showed different degrees of cytotoxicity. As observed in Table 1, IC50 values (the concentra- tion of jacaranone that reduced cell survival by 50%) varied from 9 to 145 mM for human cancer cells. The IC50 for murine melanoma B16F10-Nex2 was 17 mM. Figure 1. Chemical structure of jacaranone isolated from leaves of P. desiderabilis. doi:10.1371/journal.pone.0038698.g001 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38698