pro-apoptotic Bax. We also evidenced the antitumor potential of jacaranone as a therapeutic anticancer drug using several human cancer cells in vitro. In particular, we showed the protective effect of this quinone in a melanoma syngeneic model in vivo, using B16F10-Nex2 cells grafted subcutaneously in mice. The antitumor and protective effects of jacaranone are highly significant at low therapeutic doses. Materials and Methods Ethics statement All necessary permits were obtained for the described field studies. The State of Sa˜o Paulo Research Support Foundation (FAPESP), Brazil, and the Brazilian National Research Council (CNPq) provided permits to collect plant material. The site of plant collection is privately owned by Mackenzie Presbyterian University, where the authors carried out the plant collection work. We ensure that the procedures involving plant material were applied in accordance with label guideline and the field studies did not involve endangered or protected species. Cell lines were originally obtained from Prof. Luiz Fernando Lima Reis, Ludwig Institute for Cancer Research, Sa˜o Paulo, Brazil. These are long established cell lines, acquired from public culture collections or transferred from Ludwig Institute in New York, and maintained in appropriate conditions to serve as standard tumor cell lines for local studies and collaborative research. We confirm that animal experiments were carried out using protocols approved by the Ethics Committee for Animal Experimentation of Federal University of Sa˜o Paulo, Brazil and the specific Project presented by the Experimental Oncology Unit, including the animal experiments herein reported, has been approved via doc CEP 1234/2011. Plant material Leaves from Pentacalia desiderabilis (Vell.) Cuatrec. were collected in Campos do Jorda˜o, Sa˜o Paulo, SP, in August 2008. A voucher specimen was deposited at Herbarium of D. Bento Pickel – Instituto Florestal under number SPSF 37596. General Procedures 1H (300 MHz) and 13C (75 MHz) NMR spectra were obtained on a Bruker (Billerica, MA) model DPX-300 spectrometer with sample dissolved in CDCl3 containing 1% of tetramethylsilane (TMS, TediaBrazil, Rio de Janeiro, Brazil). The LREIMS (low resolution electron impact mass spectrum) was obtained at 70 eV on Finnigan-Mat INCOS50 quadrupole spectrometer. Chromato- graphic separation procedures were performed using silica gel (230–400 mesh; Merck, Darmstadt, Germany) for CC and silica gel 60 PF254 (Merck, Darmstadt, Germany) for analytical TLC (0.25 mm). Isolation of jacaranone from leaves of P. desiderabilis Dried and powdered leaves of P. desiderabilis (232 g) were defatted with hexane and exhaustively extracted with methanol. After solvent evaporation under reduced pressure, the crude methanol extract (13.8 g) was dissolved in methanol : H2O 1:2 and partitioned sequentially using hexane and CH2Cl2. After solvent evaporation under reduced pressure, the following yields were obtained: 0.41 g /hexane and 2.50 g /CH2Cl2 phases. Part of CH2Cl2 phase (0.4 g) was subjected to silica gel column chromatography eluted with CH2Cl2 containing increasing amounts of ethyl acetate (up to 100%) and ethyl acetate containing increasing amount of methanol (up to 100%), to give twelve fractions (A1 – A12). Jacaranone was isolated as colourless needles Antitumor Activity of Jacaranone (132.5 mg) from fraction A4. Jacaranone [Methyl (1-hydroxy-4- oxo-2,5-cyclohexandienyl) acetate]. Colourless needles; 1H NMR (CDCl3, 300 MHz): d 6.97 (d, J = 10.2 Hz, H-29/H-69), 6.21 (d, J = 10.2 Hz, H-39/H-59), 3.75 (s, OCH3), 2.72 (s, H-2). 13C NMR (CDCl3, 75 MHz): 171.0 (C-1), 43.4 (C-2), 67.3 (C-19), 149.0 (C- 29/C-69), 128.2 (C-39/C-59), 185.0 (C-49), 52.2 (OCH3). LREIMS m/z (rel. int.): 182 [M+] (3), 166 (2), 150 (19), 122 (16), 109 (84), 94 (8), 81 (44), 74 (100), 69 (7), 59 (19), 53 (36). Cell lines and culture conditions Murine melanoma subline B16F10-Nex2 was established at the Experimental Oncology Unit (UNONEX), Federal University of Sa˜o Paulo, UNIFESP, as described previously [9]. The human melanoma cell lines A2058 and SK-MEL-28, human colon carcinoma cell lines HCT-8 and LS160, human cervical carcinoma cell line SiHa, human myeloid leukemia cells HL-60, human breast cancer cells MDA and SK-BR-3, and the murine melanocytes melan-A cell line were provided by the Ludwig Institute for Cancer Research, Sa˜o Paulo, Brazil. These cell lines were maintained in complete medium consisting in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10 mM N-2- hydroxyethylpiperazine-N2 ethanesulphonic acid (HEPES; Sig- ma-Aldrich, St. Louis, MO), 24 mM sodium bicarbonate, 40 mg/l gentamicin (Hipolabor, Minas Gerais, Brazil), pH 7.2, and 10% fetal bovine serum (Gibco, Grand Island, NY) at 37uC in a humidified atmosphere with 5% CO2. Preparation of murine bone marrow cells and macrophage differentiation Fresh bone marrow cells were used to generate macrophages using L929-cell supernatant conditioned medium (LCCM) as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). The cells were resuspended in 10 ml of bone marrow differentiation medium, which consists of RPMI1640 (Gibco, Grand Island, NY) supplemented with 20% fetal bovine serum (Gibco, Grand Island, NY), 30% LCCM, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Cells were seeded in non-tissue culture treated Optilux Petri dishes (BD Biosciences, Franklin Lakes, NJ) and incubated at 37uC in a 5% CO2 atmosphere. After 4 days, 10 ml of fresh medium was added per plate and incubated for additional 3 days. To obtain the macrophages, the supernatants were discarded and the attached cells were washed with 15 ml of sterile PBS. Macrophages were detached gently using a cell scraper and PBS. The cells were centrifuged at 200 g for 5 minutes and resuspended in 10 ml of RPMI 1640 (Gibco, Grand Island, NY). The cells were counted, seeded and cultivated in tissue culture plates for 12 h. Cell viability assay Viable cells were quantified using the MTT (3-[4,5-di- methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Al- drich, St. Louis, MO) assay. Human tumor cell lines or B16F10- Nex2 (1.06104 cells/well) were cultured on 96-well plates in RPMI supplemented with 10% serum, and jacaranone (1.0, 2.5, 5.0, 10.0, 20.0 or 50.0 mM) or fresh medium (for control cells) was added 8 h after the tumor cell inoculum. After 24 h, MTT solution (5 mg/ml) in 16 phosphate-buffered saline (PBS) was directly added to the cells, followed by incubation for 4 h at 37uC. Absorbance was measured with an automated spectrophotometric plate reader (SpectraMax-M2, Molecular Devices Software Pro 5.4, Sunnyvale, CA) at a wavelength of 570 nm. Cell viability was expressed as percent values in comparison with untreated cells. PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38698