p38 activation in endothelial cells. Similarly in our system, ROS generation was responsible for jacaranone-induced p38 MAPK activation and proapoptotic Bax expression. Consistent with the effect on Akt downregulation, pretreatment with NAC blocked jacaranone-induced Akt inhibition compared with cells treated with jacaranone alone. Taken together, our data suggest that ROS mediate the downregulation of Akt and enables the activation of p38 MAPK, thus stimulates apoptosis in jacaranone-treated melanoma cells. P38-triggered apoptosis is mediated by transcriptional and post- translational mechanisms, which affect death receptors, cell survival pathways, caspases or Bcl-2 family proteins [56]. Besides being well known that Bax is phosphorylated by stress-activated p38 kinase and that phosphorylation of Bax leads to translocation from the cytosol to mitochondria prior to apoptosis [57], there is evidence that p38 may also upregulate Bax expression, thus contributing to apoptosis [58,59]. Similarly, we found that jacaranone is capable to increase Bax expression in B16F10- Nex2 cells at 20 or 50 mM. Given that Akt can suppress apoptosis through phosphorylation and inactivation of many proapoptotic proteins, such as Bax and Bad [57,60], Akt downregulation and the increased expression induced by jacaranone may facilitate Bax activation and consequent translocation to the mitochondria, where it forms homo and heterodimers (along with Bak) and creates pores within the outer mitochondrial membrane [17], resulting in release of cytochrome C and activating mitochondria- dependent downstream cascades. References 1. Demain AL, Vaishnav P (2011) Natural products for cancer chemotherapy. Microb Biotechnol 4: 687–699. 2. Woldemichael GM, Turbyville TJ, Linehan WM, McMahon JB (2011) Carminomycin I is an apoptosis inducer that targets the Golgi complex in clear cell renal carcinoma cells. Cancer Res 71: 134–142. 3. Powis G (1989) Free radical formation by antitumor quinones. Free Radic Biol Med 6: 63–101. 4. Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS- mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8: 579–591. 5. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 160: 1–40. 6. Verrax J, Beck R, Dejeans N, Glorieux C, Sid B, et al. (2011) Redox-active quinones and ascorbate: an innovative cancer therapy that exploits the vulnerability of cancer cells to oxidative stress. Anticancer Agents Med Chem 11: 213–221. 7. Morais TR, Romoff P, Favero OA, Reimao JQ, Lourenco WC, et al. (2012) Anti-malarial, anti-trypanosomal, and anti-leishmanial activities of jacaranone isolated from Pentacalia desiderabilis (Vell.) Cuatrec. (Asteraceae). Parasitol Res 110: 95–101. 8. Loizzo MR, Tundis R, Statti GA, Menichini F, Houghton PJ (2005) In-vitro antiproliferative effects on human tumour cell lines of extracts and jacaranone from Senecio leucanthemifolius Poiret. J Pharm Pharmacol 57: 897–901. 9. Matsuo AL, Figueiredo CR, Arruda DC, Pereira FV, Scutti JA, et al. (2011) alpha-Pinene isolated from Schinus terebinthifolius Raddi (Anacardiaceae) induces apoptosis and confers antimetastatic protection in a melanoma model. Biochem Biophys Res Commun 411: 449–454. 10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. 11. Paz MA, Fluckiger R, Boak A, Kagan HM, Gallop PM (1991) Specific detection of quinoproteins by redox-cycling staining. J Biol Chem 266: 689–692. 12. Fluckiger R, Paz MA, Gallop PM (1995) Redox-cycling detection of dialyzable pyrroloquinoline quinone and quinoproteins. Methods Enzymol 258: 140–149. 13. Xavier CP, Lima CF, Preto A, Seruca R, Fernandes-Ferreira M, et al. (2009) Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells. Cancer Lett 281: 162–170. 14. Saraste A, Pulkki K (2000) Morphologic and biochemical hallmarks of apoptosis. Cardiovasc Res 45: 528–537. 15. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646–674. Antitumor Activity of Jacaranone Most importantly, consistent with our findings in vitro, jacaranone was able to delay the tumor development in a sub- cutaneous model with a dose response effect. Based on Akt role as a key regulator of cellular survival and its association with tumorigenesis and melanoma progression, perturbations on Akt signaling pathway may be an important approach to treat this human malignancy. Furthermore, activation of p38 and in- activation of Akt by jacaranone, and its mitochondria-targeted cytotoxicity through ROS may implicate in tumor suppression and sensitization to apoptosis. It should be noted that a smaller effect on ROS generation was observed in normal cells (murine melanocytes and primary cultures of bone marrow macrophages from C57BL/6 mice), suggesting that jacaranone may be employed as a selective cytotoxic agent for the eradication of cancer cells. In conclusion, our results indicate that jacaranone is a promising cancer therapeutic agent potentially able to eliminate tumor cells including those, which are resistant to conventional therapies. Author Contributions Conceived and designed the experiments: MHM ALM JHL LRT. Performed the experiments: MHM ALM CRF CFF NG DCA JAS. Analyzed the data: MHM ALM. Contributed reagents/materials/analysis tools: PR OAF MJF JHG. Wrote the paper: MHM JAS ALM LRT JHG. 16. Jo Y, Suh J, Shin MH, Jung JH, Im KS (2005) Jacaranone and related compounds from the fresh fruits of Ternstroemia japonica and their antioxidative activity. Arch Pharm Res 28: 885–888. 17. Fulda S, Kroemer G (2011) Mitochondria as therapeutic targets for the treatment of malignant disease. Antioxid Redox Signal 15: 2937–2949. 18. Chaudhuri L, Sarsour EH, Goswami PC (2010) 2-(4-Chlorophenyl)benzo-1,4- quinone induced ROS-signaling inhibits proliferation in human non-malignant prostate epithelial cells. Environ Int 36: 924–930. 19. Yu D, Berlin JA, Penning TM, Field J (2002) Reactive oxygen species generated by PAH o-quinones cause change-in-function mutations in p53. Chem Res Toxicol 15: 832–842. 20. Pelicano H, Carney D, Huang P (2004) ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7: 97–110. 21. Dai DL, Martinka M, Li G (2005) Prognostic significance of activated Akt expression in melanoma: a clinicopathologic study of 292 cases. J Clin Oncol 23: 1473–1482. 22. Govindarajan B, Sligh JE, Vincent BJ, Li M, Canter JA, et al. (2007) Overexpression of Akt converts radial growth melanoma to vertical growth melanoma. J Clin Invest 117: 719–729. 23. Cai B, Chang SH, Becker EB, Bonni A, Xia Z (2006) p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65. J Biol Chem 281: 25215–25222. 24. Liao Y, Hung MC (2003) Regulation of the activity of p38 mitogen-activated protein kinase by Akt in cancer and adenoviral protein E1A-mediated sensitization to apoptosis. Mol Cell Biol 23: 6836–6848. 25. Lin CF, Chen CL, Chang WT, Jan MS, Hsu LJ, et al. (2004) Sequential caspase- 2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis. J Biol Chem 279: 40755–40761. 26. Garbe C, Leiter U (2009) Melanoma epidemiology and trends. Clin Dermatol 27: 3–9. 27. Jerant AF, Johnson JT, Sheridan CD, Caffrey TJ (2000) Early detection and treatment of skin cancer. Am Fam Physician 62: 357–6, 381. 28. Grossman D, Altieri DC (2001) Drug resistance in melanoma: mechanisms, apoptosis, and new potential therapeutic targets. Cancer Metastasis Rev 20: 3–11. 29. Gogas HJ, Kirkwood JM, Sondak VK (2007) Chemotherapy for metastatic melanoma: time for a change? Cancer 109: 455–464. 30. Pfisterer PH, Wolber G, Efferth T, Rollinger JM, Stuppner H (2010) Natural products in structure-assisted design of molecular cancer therapeutics. Curr Pharm Des 16: 1718–1741. 31. Doughan AK, Dikalov SI (2007) Mitochondrial redox cycling of mitoquinone leads to superoxide production and cellular apoptosis. Antioxid Redox Signal 9: 1825–1836. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e38698