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

Massaoka MH, Matsuo AL, Figueiredo CR, Farias CF, Girola N, Arruda DC, Scutti JA, Romoff P, Favero OA, Ferreira MJ, Lago JH, Travassos LR - PLoS ONE (2012)

Bottom Line: The jacaranone antitumor activity was shown against several human cancer cell lines in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Unidade de Oncologia Experimental, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.

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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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.
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pone-0038698-g002: 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.

Mentions: To evaluate the cytotoxic activity, we investigated whether cell death induced by jacaranone was associated with biochemical features of apoptosis [14]. Treatment of melanoma cells with jacaranone elicited a concentration-dependent phosphatidyl serine (PS) translocation (Fig. 2A) as determined by annexin-V assay. The early apoptotic cells (regarded as annexin V positive and PI negative cells) increased as compared with the non-treated cells. A very low percentage of annexin-V positive cells (3.45%) was observed in the non-treated population. Jacaranone at 20 and 50 µM induced apoptosis in 13.8 and 21.5% of cells, respectively. In this experiment, both concentrations were sublethal due to the high number of cells. On looking for DNA fragmentation, a ladder pattern of internucleosomal fragmentation was observed after treatment with jacaranone at 100 µM for 18 h (Fig. 2B). In addition, DNA strand breaks of apoptotic cell death were visualized by TUNEL assay under fluorescence microscope (Fig. 2C). Since caspases play a major role in the execution of apoptosis, we therefore assessed the activity of initiator caspases -2, -8 and -9 and effector caspase-3. After treatment of B16F10-Nex2 cells for different times (2 h, 3 h, 6 h, 12 h and 24 h), caspase activation was assessed. The levels of caspase-2, -8, and -3 activities were 2-fold, 1.3-fold, and 2-fold higher, respectively, in 20 µM jacaranone-treated cells for 24 h compared with the control, all of which were significant (*p <0.05) (Fig. 2D). Activation of caspase-9 occurred 6 h after treatment and its activity was 2.5-fold higher when compared to untreated cells.


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

Massaoka MH, Matsuo AL, Figueiredo CR, Farias CF, Girola N, Arruda DC, Scutti JA, Romoff P, Favero OA, Ferreira MJ, Lago JH, Travassos LR - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368838&req=5

pone-0038698-g002: 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.
Mentions: To evaluate the cytotoxic activity, we investigated whether cell death induced by jacaranone was associated with biochemical features of apoptosis [14]. Treatment of melanoma cells with jacaranone elicited a concentration-dependent phosphatidyl serine (PS) translocation (Fig. 2A) as determined by annexin-V assay. The early apoptotic cells (regarded as annexin V positive and PI negative cells) increased as compared with the non-treated cells. A very low percentage of annexin-V positive cells (3.45%) was observed in the non-treated population. Jacaranone at 20 and 50 µM induced apoptosis in 13.8 and 21.5% of cells, respectively. In this experiment, both concentrations were sublethal due to the high number of cells. On looking for DNA fragmentation, a ladder pattern of internucleosomal fragmentation was observed after treatment with jacaranone at 100 µM for 18 h (Fig. 2B). In addition, DNA strand breaks of apoptotic cell death were visualized by TUNEL assay under fluorescence microscope (Fig. 2C). Since caspases play a major role in the execution of apoptosis, we therefore assessed the activity of initiator caspases -2, -8 and -9 and effector caspase-3. After treatment of B16F10-Nex2 cells for different times (2 h, 3 h, 6 h, 12 h and 24 h), caspase activation was assessed. The levels of caspase-2, -8, and -3 activities were 2-fold, 1.3-fold, and 2-fold higher, respectively, in 20 µM jacaranone-treated cells for 24 h compared with the control, all of which were significant (*p <0.05) (Fig. 2D). Activation of caspase-9 occurred 6 h after treatment and its activity was 2.5-fold higher when compared to untreated cells.

Bottom Line: The jacaranone antitumor activity was shown against several human cancer cell lines in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Unidade de Oncologia Experimental, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.

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.

Show MeSH
Related in: MedlinePlus