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. 2012;7(6):e38698.
doi: 10.1371/journal.pone.0038698. Epub 2012 Jun 6.

Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo

Mariana H Massaoka  1 , Alisson L Matsuo, Carlos R Figueiredo, Camyla F Farias, Natália Girola, Denise C Arruda, Jorge A B Scutti, Paulete Romoff, Oriana A Favero, Marcelo J P Ferreira, João H G Lago, Luiz R Travassos
Affiliations

Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo

Mariana H Massaoka et al. PLoS One. 2012.

Abstract

Background: Malignant melanoma is a deadly type of metastatic skin cancer with increased incidence over the past 30 years. Despite the advanced knowledge on the biology, immunobiology and molecular genetics of melanoma, the alternatives of treatment are limited with poor prognosis. On clinical trials, natural products and among them redox-active quinones have been tested in the attempt to control the growth of cancer cells. Recently, we isolated jacaranone from Pentacalia desiderabilis, a benzoquinone derivative that showed a broad antitumor activity and protective anti-melanoma effect in a syngeneic model. The purified substance is active at micromolar concentrations, is not hemolytic, and is not toxic in naïve mice.

Methodology/principal findings: The jacaranone antitumor activity was shown against several human cancer cell lines in vitro. Moreover, the induction of apoptosis in murine melanoma cells and jacaranone antitumor activity in vivo, in a melanoma experimental model, were also shown. Jacaranone renders antiproliferative and proapoptotic responses in tumor cells, by acting on Akt and p38 MAPK signaling pathways through generation of reactive oxygen species (ROS). The free radical scavenger N-acetyl-cysteine (NAC) was able to completely suppress cell death induced by jacaranone as it blocked Akt downregulation, p38 MAPK activation as well as upregulation of proapoptotic Bax. Notably, treatment of melanoma growing subcutaneously in mice with jacaranone significantly extended the mean survival times in a dose-dependent manner.

Conclusions/significance: The results provide evidence for the mechanisms of action of jacaranone and emphasize the potential use of this quinone for the treatment of melanoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Chemical structure of jacaranone isolated from leaves of P. desiderabilis .
Figure 2
Figure 2. Jacaranone suppressed cell proliferation and induced apoptosis in melanoma cells.
(A) 5×105 B16F10-Nex 2 cells were seeded onto 6-well plates and treated with 20 or 50 µM 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 µM 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 µM 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 µM 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 ± S.D. *p ≤0.02 versus control conditions.
Figure 3
Figure 3. Jacaranone induced apoptotic morphological alterations on melanoma cells.
(A) 1.0×104 B16F10-Nex2 cells were seeded on coverslips and treated with 20 or 50 µM jacaranone (Jac) for 3 h. State of chromatin was assessed using Hoechst 33342 (2 µM). 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 µM 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 µM jacaranone. Original magnification ×10,000 (a, b and c); bar = 2 µm. Original magnification ×20,000 (d); bar = 1µm.
Figure 4
Figure 4. ROS generation by jacaranone and loss of mitochondrial transmembrane potential (Δψ m) in jacaranone-treated cells.
(A) Redox cycling ability of jacaranone detected by NBT/glycinate assay. (B) Untreated melanoma cells (a) or cells treated with jacaranone at 50 µM for 3 h (b), were incubated with the oxidative fluorescent dye DHE (5 µM) for detection of superoxide anion production. Bars = 20 µm. (C) B16F10-Nex2 cells were pre-incubated with NAC at indicated concentrations for 30 min, and then jacaranone at 20 µM was added. Cell viability was assessed using Trypan Blue exclusion test 24 h after jacaranone addition. (D) B16F10-Nex2 cells were treated with 50 µM jacaranone for 24 h and then Δψm was determined using TMRE by flow cytometry.
Figure 5
Figure 5. Jacaranone treatment of B16F10-Nex2 cells inhibited Akt pathway activation through ROS generation.
Whole-cell extracts from B16F10-Nex2 exposed to 0 (control), 20 or 50 µM jacaranone (Jac) in the presence (A) or absence (B) of NAC were subjected to Western blotting and probed with phospho-p38, phospho-Akt, total Akt, total p38 and Bax antibodies. Protein levels were normalized to the actin level.
Figure 6
Figure 6. Jacaranone increased survival of B16F10-Nex2 tumor-bearing mice.
Animals (5 mice per group) were subcutaneously injected with 5×104 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 23.4±2.1 days for untreated tumor control group, 29±4.3 days (p = 0.05) for 0.8 mg/kg of jacaranone-treated group, and 34±3.6 days (*p<0.01) for 4 mg/kg jacaranone-treated group.
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