Antitumor Activity of Jacaranone Figure 4. ROS generation by jacaranone and loss of mitochondrial transmembrane potential (Dym) in jacaranone-treated cells. (A) Redox cycling ability of jacaranone detected by NBT/glycinate assay. (B) Untreated melanoma cells (a) or cells treated with jacaranone at 50 mM for 3 h (b), were incubated with the oxidative fluorescent dye DHE (5 mM) for detection of superoxide anion production. Bars = 20 mm. (C) B16F10-Nex2 cells were pre-incubated with NAC at indicated concentrations for 30 min, and then jacaranone at 20 mM was added. Cell viability was assessed using Trypan Blue exclusion test 24 h after jacaranone addition. (D) B16F10-Nex2 cells were treated with 50 mM jacaranone for 24 h and then Dym was determined using TMRE by flow cytometry. doi:10.1371/journal.pone.0038698.g004 compounds have played an important role in the development of promising new anti-cancer agents [30]. Jacaranone exhibits potent cytotoxic activity against several cancer cell lines (Table 1) and we demonstrate that this compound induces apoptosis in melanoma cells. The effect of jacaranone was confirmed by detection of surface exposed phosphatidyl serine, DNA fragmentation, loss of mitochondrial Dym, and activation of caspases in B16F10 cells. Also, in agreement with these results, jacaranone treatment rendered tumor cells with nuclear con- densed chromatin and nuclear membrane alterations, as well as plasma membrane blebbing. Jacaranone-induced apoptosis in- volves ROS generation, since concomitant incubation with the antioxidant NAC prevented cell death. We associated production of ROS by jacaranone with the well-known redox cycling capability of quinone-containing substances. During redox cycling, quinoid substances in their reduced states (semiquinones) can yield its extra electron to oxygen with the formation of superoxide anions, beginning a cascade that generates H2O2 and hydroxyl Figure 5. Jacaranone treatment of B16F10-Nex2 cells inhibited Akt pathway activation through ROS generation. Whole-cell extracts from B16F10-Nex2 exposed to 0 (control), 20 or 50 mM jacaranone (Jac) in the presence (A) or absence (B) of NAC were subjected to Western blotting and probed with phospho-p38, phospho-Akt, total Akt, total p38 and Bax antibodies. Protein levels were normalized to the actin level. doi:10.1371/journal.pone.0038698.g005 PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38698