Figure 6. Jacaranone increased survival of B16F10-Nex2 tumor-bearing mice. Animals (5 mice per group) were subcutane- ously injected with 56104 cells and treated with PBS, 0.8 mg/kg or 4 mg/kg of jacaranone on alternate days. Intraperitoneal therapy began one day after inoculation of melanoma cells and was extended for 14 days. Mean survival times were 23.462.1 days for untreated tumor control group, 2964.3 days (p = 0.05) for 0.8 mg/kg of jacaranone- treated group, and 3463.6 days (*p,0.01) for 4 mg/kg jacaranone- treated group. doi:10.1371/journal.pone.0038698.g006 radicals (.OH) [31,32]. In addition, we showed that jacaranone induces Akt inactivation, p38 MAPK phosphorylation and expression of proapoptotic protein Bax. Lastly, jacaranone not only showed a remarkable antitumor effect in vitro against a number of human cancer cell lines but also acted in vivo increasing survival of melanoma-bearing mice in the B16F10-Nex2 syngeneic melanoma model. As a naturally occurring benzoquinone, jacaranone generates ROS causing an oxidative stress, with enhanced activity of the antioxidant defense system and consequent mitochondrial damage [5,33]. Interestingly, our data using fluorescent dye TMRE revealed that jacaranone first caused hyperpolarization of the mitochondrial membrane, which is recorded to be one of the earlier key event in apoptosis [34–36]. It has been proposed that mitochondrial hyperpolarization may result in a more reduced state of electron carriers responsible for single-electron transfer to oxygen producing primarily the superoxide anion radical, [36] a precursor of most other ROS, also involved in the propagation of oxidative chain reactions [37]. The breakdown of the mitochon- drial membrane potential after 2–24 h incubation with jacaranone was detected in melanoma cells. Mitochondrial permeabilization as well as the leakage of proteins normally confined to mitochondria may increase ROS release and determine the catabolic features of cell death [17]. Besides being predominantly implicated in damaging cellular structures, ROS also play an important role in various physiolog- ical aspects of intracellular signaling and regulation. It has been established that ROS interfere with the expression of a number of genes and signal transduction pathways [5]. As shown, the apoptotic effect of jacaranone involves caspase activation. Caspase-9, which was found to be activated at early times in jacaranone-induced cell death process, is an upstream caspase in the mitochondria-dependent pathway [38]. Mitochon- drial dysfunction, characterized by loss of mitochondrial trans- membrane potential and opening of mitochondrial permeability transition pores, is an effect caused by many apoptotic stimuli either directly or indirectly [39]. In consequence, these organelles release various important proteins to the cytosol to terminate the apoptotic process, including cytochrome c, which is responsible (along with Apaf-1) for the activation of caspase-9. Caspase-2 activation is another mechanism by which apoptotic stimuli may affect mitochondrial function. It has been shown that Antitumor Activity of Jacaranone caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c and other mitochondrial apoptogenic factors into the cell cytoplasm [40]. Caspase-8 is a major activator of caspase-3 and amplifies the apoptotic signal by directly activating downstream caspases and cleaving BH3 domain-only members of the Bcl-2 family, such as proapoptotic Bid [41]. It has been demonstrated that activation of caspase-8 can occur in death receptor-independent apoptosis [25] and Caspase-2 may act upstream of mitochondria to promote apoptosis [40]. Kim and Hong [42] have reported that Inhibition of caspase-2 suppressed caspase-8 activation by Saikosaponin- a (SSa), but knockdown of caspase-8 did not prevent SSa-induced caspase-2 activation, suggesting that sequential activation of caspase-2 and caspase-8 may occur. It has been clearly demon- strated [43,44] that ROS triggers activation of caspases during the apoptotic process, including caspase-2 and -8, and facilitates caspase-9 activation via disulfide-mediated interaction with Apaf-1 [45]. Consistent with these previous studies, we detected a significant increase of caspase-2, -8, -9 and -3 activities in B16F10-Nex2 cells after jacaranone treatment. Akt is a downstream target of the phosphatidylinositol 3-kinase (PI3K) signaling pathway that is activated through phosphoryla- tion of Ser-473/474 and Thr-308/309 [24] and positively regulates cell growth and survival [21]. It has been suggested that Akt is a key regulator of melanoma progression [46]. Indeed, elevated Akt expression in severely dysplastic nevi and melanoma samples, but not in normal and slightly dysplastic nevi of samples from patients with pigmented skin lesions has already been detected, as well as constitutive activation of Akt in several melanoma cell lines [47]. Recent works have reported that oxidative stress can modulate Akt activation [47–49]. To elucidate the link between Akt signaling and ROS generation, we investigated the expression of active Akt in B16F10 melanoma cells after treatment with jacaranone. Immunoblotting analysis revealed a high expression of phospho-Akt in non-treated melanoma cells that was downregulated after treatment with jacaranone. It should also be noted that jacaranone treatment at 50 mM reduced the overall expression of Akt. This result raises the possibility that Akt inactivation might also represent a consequence of engagement of the caspase cascade, since Akt degradation by caspases has been reported as a survival signal that allows apoptosis to occur [50]. Another intracellular signaling molecule sensitive to redox modulation is the p38 MAPK, which belongs to the stress- activated protein kinase (SAPK) family [51]. The p38 is an ubiquitous and highly conserved proline-directed serine/threonine kinase considered important in regulation of cell survival, differentiation and apoptosis [52]. The p38 pathway is activated upon cellular stress and often engages signals that suppress proliferation or promote apoptosis, thus it has been referred as a tumor suppressor. Activation of p38 MAPK by ROS has been investigated in a wide variety of experimental models and a number of studies reported that the serine/threonine kinases of the MAPK family can be regulated by oxidants [5]. Particularly in skin cancer, there is evidence that supports a tumor-suppressive role of p38 and most clinically used chemotherapeutics have an anti-proliferative effect on melanoma cells mediated by activation of p38 [53]. In agreement with these observations, we found a marked increase in the expression of p-p38 in B16F10-Nex2 cells after treatment with 20 or 50 mM jacaranone. A crosstalk between the p38 MAPK and PI3K/Akt pathways has been the focus of several studies [54,55]. Gratton et al. [54] reported that the blockade of Akt signaling stimulates p38-dependent apoptosis, whereas overexpression of constitutively active Akt downregulates PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e38698