Table 1. Cytotoxic activity of jacaranone on human cancer cells growing in vitro. Cell line A2058 SK-MEL-28 B16F10-Nex2 HCT-8 LS160 SiHa HL-60 SK-BR-3 MDA IC50 (mM)a 30 23 17 24 36 145 9 30 77 aIC50 is the concentration that kills 50% of viable cells in a dose-dependent survival curve. doi:10.1371/journal.pone.0038698.t001 Jacaranone induces apoptosis in melanoma cells To evaluate the cytotoxic activity, we investigated whether cell death induced by jacaranone was associated with biochemical features of apoptosis [14]. Treatment of melanoma cells with jacaranone elicited a concentration-dependent phosphatidyl serine (PS) translocation (Fig. 2A) as determined by annexin-V assay. The early apoptotic cells (regarded as annexin V positive and PI negative cells) increased as compared with the non-treated cells. A very low percentage of annexin-V positive cells (3.45%) was observed in the non-treated population. Jacaranone at 20 and 50 mM induced apoptosis in 13.8 and 21.5% of cells, respectively. In this experiment, both concentrations were sublethal due to the high number of cells. On looking for DNA fragmentation, a ladder pattern of internucleosomal fragmentation was observed after treatment with jacaranone at 100 mM for 18 h (Fig. 2B). In addition, DNA strand breaks of apoptotic cell death were visualized by TUNEL assay under fluorescence microscope (Fig. 2C). Since caspases play a major role in the execution of apoptosis, we therefore assessed the activity of initiator caspases -2, -8 and -9 and effector caspase-3. After treatment of B16F10-Nex2 cells for different times (2 h, 3 h, 6 h, 12 h and 24 h), caspase activation was assessed. The levels of caspase-2, -8, and -3 activities were 2-fold, 1.3-fold, and 2-fold higher, respectively, in 20 mM jacaranone-treated cells for 24 h compared with the control, all of which were significant (*p ,0.05) (Fig. 2D). Activation of caspase-9 occurred 6 h after treatment and its activity was 2.5-fold higher when compared to untreated cells. Melanoma cells treated with jacaranone exhibit morphological alterations of apoptosis In order to study the details of apoptotic morphological changes [14] displayed by jacaranone in B16F10-Nex2 cells, Hoechst staining and transmission electron microscopy were used. The density of nuclear chromatin was analyzed by culturing cells on cover slips in the presence of 20 or 50 mM jacaranone for 3 h. Hoechst 33342 dye was added and its fluorescence was captured in the 4,6-diamidino-2-phenylindole channel. Fluorescence observa- tion confirmed that cells treated with jacaranone exhibited condensation as in apoptotic nuclei (Fig. 3A). The percentage of apoptotic nuclei was 25.5% and 39.5% in melanoma cells incubated with 20 and 50 mM jacaranone, respectively. For TEM, approximately 56104 cells were incubated with 50 mM jacaranone for 3 h. Then, sections were made, as described in Antitumor Activity of Jacaranone experimental procedures. Micrographs showed that jacaranone caused mitochondrial shrinkage (arrows), blebbing of plasma membrane (asterisks), and nuclear membrane disruption (black triangles) (Fig. 3B). These results, collectively with biochemical features [14,15] demonstrated on Fig. 3, confirmed that jacaranone-treated cells undergo apoptosis. Jacaranone induces programmed cell death through oxidative stress and causes mitochondrial depolarization Previously, it was found that jacaranone exhibits weak scavenging effect on 1,1-diphenyl-2-picryl- hydrazyl (DPPH) radical [16]. To better characterize the molecule, we further investigated whether jacaranone exhibited redox-cycling ability, a well-known chemical property of many quinoid compounds. Using the NBT/glycinate assay we demonstrated for the first time that jacaranone is markedly efficient in redox cycling (Fig. 4A). The cycling reaction of jacaranone reduces nitroblue tetrazolium to formazan, resulting in increased optical density measured at 605 nm. It has been reported that increased liberation of reactive oxygen species (ROS) can trigger a cascade of events leading to apoptosis [17]. Moreover, previous studies demonstrated that quinones induce tumor cells death through redox-mediated mechanisms [18,19]. We have further investigated whether the production of intracellular ROS, as induced by jacaranone, could mediate apoptosis in melanoma cells (Fig. 4B). After 3 h treatment with 50 mM jacaranone, cells were stained with dihydroethidium (DHE) for 30 minutes at 37uC and its conversion to ethidium by oxidation was assessed under a fluorescence microscope. As observed, treatment with jacaranone resulted in highly increased ethidium fluorescence intensity as compared to non-treated cells. In this experiment, the jacaranone concentration was sublethal by itself due to the high number of cells. Growing evidence suggests that cancer cells, relative to normal cells, have increased generation of ROS and an altered redox status [20]. To determine whether oxidative stress mediated by jacaranone contributed to the differential susceptibility of mela- noma cells to ROS generation in comparison with normal cells, the oxidation of DHE was also measured in murine melanoma cells (B16F10-Nex2), murine melanocytes (melan-A) and primary culture of bone marrow macrophages from C57BL/6 mice after treatment with 50 mM jacaranone for 3 h. In melanoma cells ROS production increased by 50–57%, whereas in normal murine melanocytes and primary macrophages it increased only 18– 19.5% and 4–5% respectively (data not shown). When B16F10-Nex2 cells were pre-incubated with N-acetyl cysteine (NAC), a ROS scavenger, at 2 or 4 mM for 30 min before jacaranone treatment, cell death was completely abrogated (Fig. 4C). A nuclear magnetic resonance analysis excluded any direct interaction between jacaranone and NAC (data not shown). The involvement of caspases-2 and -9 in the jacaranone- induced apoptotic death of melanoma cells and the increased ROS production suggested that jacaranone treatment might act by disrupting the mitochondrial membrane. We therefore performed a time-course analysis of the mitochondrial transmembrane potential using the lipophilic cationic dye TMRE to evaluate the effect of jacaranone on melanoma cells. A 0.5–1 h exposure to 50 mM jacaranone was found to induce an initial hyperpolariza- tion of the mitochondrial membrane reflected by increase in fluorescence intensity as compared to untreated cells (data not shown). However, after 2 and 4 h of treatment, fluorescence intensity decreased significantly, suggesting that jacaranone treatment resulted in dissipation of DYm. After 24 h of treatment, the mitochondrial damage during jacaranone-induced apoptosis in PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38698