Figure 3. Jacaranone induced apoptotic morphological altera- tions on melanoma cells. (A) 1.06104 B16F10-Nex2 cells were seeded on coverslips and treated with 20 or 50 mM jacaranone (Jac) for 3 h. State of chromatin was assessed using Hoechst 33342 (2 mM). Positive cells for Hoechst staining were visualized under a fluorescence microscope and expressed as percentage of total cell counts of control cells. Representative images of cells treated without (Control) or with 20 mM jacaranone. (B) Transmission electron micrographs revealed mitochondrial shrinkage (arrows), plasma membrane blebbing (black triangles) and disruption of nuclear membrane (asterisk) after treatment with (b, c, and d) or without (a) 50 mM jacaranone. Original magnification 610,000 (a, b and c); bar = 2 mm. Original magnification 620,000 (d); bar = 1mm. doi:10.1371/journal.pone.0038698.g003 expressing Akt led to rapidly growing tumors in vivo [22]. To determine whether downregulation of Akt is involved in jacaranone-induced apoptosis in B16F10-Nex2, we evaluated the phosphorylation status of Akt using Western blotting analysis. As shown (Fig. 5A), phosphorylated Akt (p-Akt) was detected in lysates of non-treated cells and treatment with jacaranone reduced the level of p-Akt in a dose-dependent manner. Interestingly, the expression of total Akt was abolished in cells treated with 50 mM jacaranone, suggesting protein degradation at this concentration. The p38 MAPK is preferentially activated by cell stress- inducing signals (e.g. oxidative stress) and leads to apoptosis in response to several cellular injuries [23]. As p38 is partly downregulated due to Akt activation in human cancer [24,25] we investigated whether p38 activation could contribute to jacaranone-induced apoptosis. High levels of phosphorylated p38 (p-p38) were observed in whole-cell lysates of B16F10-Nex2 exposed to 20 or 50 mM jacaranone, but not in non-treated cells (Fig. 5A). Antitumor Activity of Jacaranone Because proapoptotic members of Bcl-2 family are implicated in mitochondria-dependent cell death, we examined the effect of jacaranone on enhancing expression of Bax. In line with our results, expression of the proapoptotic Bax protein was consistently upregulated in B16F10-Nex2 after treatment with 20 and 50 mM jacaranone (Fig. 5A). The role of ROS in the signaling pathways induced by jacaranone was elucidated by pre-incubation of B16F10-Nex2 cells with NAC before treatment with 20 or 50 mM jacaranone. In this experiment, both concentratios were sublethal due to the high number of cells. As shown in Fig. 5B, preincubation with NAC at 4 mM did not result in a decrease of p-Akt protein level, thus restored Akt function in contrast with the cells that were exposed to jacaranone alone. The presence of the antioxidant NAC prevented the induction of active p38 MAPK expression after 24 h of 20 or 50 mM jacaranone treatment. Moreover, alteration in Bax level was not detected upon NAC incubation (data not shown). These results indicated that the modulation of Akt and p38 MAPK cascades, as well as their downstream target Bax, is ROS- dependent and may contribute to regulation of apoptosis in response to jacaranone. Jacaranone improves survival of melanoma-bearing mice To determine the effect of jacaranone on tumor development in vivo, we used a well-established syngeneic murine melanoma model. B16F10-Nex2 cells were injected subcutaneously into the right flanks of C57BL/6 mice and tumor growth was measured daily. Two treatment groups of 5 mice received 0.8 and 4 mg/kg of jacaranone intraperitoneally every other day for 14 days. A control group received 100 mL of normal saline solution. Mice were removed from the therapy study and sacrificed when tumor size exceeded 3,000 mm3. The survival rate (percent value of the number of mice with tumor growth and the total number of mice inoculated in the group) was monitored every day. Treatment of melanoma-bearing mice with 0.8 and 4 mg/kg of jacaranone resulted in a dose-dependent protective effect (Fig. 6). The mean survival times of tumor mice in the 0.8 and 4 mg/kg jacaranone treatment groups were extended to 2964.3 days (p = 0.05) and 3463.6 days (*p ,0.01), respectively, compared with the 23.462.1 days of the control group. Moreover, we noted that jacaranone-treated animals maintained healthy physical appear- ance, normal activity levels and healthy normal-weight throughout the study period. The result shown with 4 mg/kg of jacaranone was confirmed by a second experiment with a group of 6 animals. A positive control with doxorubicin was included which rendered 84% survival of challenged animals (data not shown). Further- more, no hemolytic activity was detected with jacaranone treatment as described previously [7]. Discussion The present work shows that jacaranone is a promising antitumor drug that acts by inducing apoptosis in tumor cells and is especially active against melanoma. Malignant melanoma is the leading cause of skin cancer-related death worldwide [26] and exhibits poor prognosis owing to the lack of efficient treatment. Most chemotherapeutic drugs act through induction of apoptosis [27], and their reduced efficacy in melanoma likely is related to the high resistance of melanoma cells compared to other malignant cell types [28]. Although several therapy strategies have been tested, beneficial effects remain limited, inducing satisfactory responses in a minority of patients [29]. Therefore, novel therapeutic approaches are needed. In this context, plant-derived PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38698