Antitumor Activity of Jacaranone Figure 2. Jacaranone suppressed cell proliferation and induced apoptosis in melanoma cells. (A) 56105 B16F10-Nex 2 cells were seeded onto 6-well plates and treated with 20 or 50 mM jacaranone at indicated times. The cells were incubated with a FITC-conjugated annexin V and PI solution, and then analyzed by flow cytometry. (B) B16F10-Nex2 cells were incubated with or without 100 mM jacaranone for 18 hours. The isolated DNA was electrophoresed and fragments were visualized by staining with ethidium bromide under UV light. (C) After treatment with 50 mM jacaranone (Jac) for 3 h, the cells were fixed and incubated using TUNEL reaction solution and then visualized under a fluorescence microscope as described in experimental procedures. (D) Cells were treated with or without jacaranone at 20 mM for different times, harvested, and cell lysates were tested for caspase-2, -8, -9 and -3 activities. Results are representative of at least two independent experiments, and shown as mean 6 S.D. *p #0.02 versus control conditions. doi:10.1371/journal.pone.0038698.g002 B16F10-Nex2 cells was shown by reduction in 51% of the accumulation of TMRE in the organelle (Fig. 4D) thus implying a decreased permeability threshold. Taken together, these data strongly suggest that jacaranone produces ROS through its redox cycling capacity, and ROS mediates jacaranone-induced apoptosis in melanoma cells. Downregulation of Akt, activation of p38 MAPK and induction of Bax expression by jacaranone Akt is a serine/threonine kinase that stimulates cell pro- liferation, inhibits apoptosis and has been associated with melanoma progression and prognosis [21]. In this context, Govindarajan et al. demonstrated that melanoma cells over- PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e38698