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

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 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.
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pone-0038698-g003: 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.

Mentions: In order to study the details of apoptotic morphological changes [14] displayed by jacaranone in B16F10-Nex2 cells, Hoechst staining and transmission electron microscopy were used. The density of nuclear chromatin was analyzed by culturing cells on cover slips in the presence of 20 or 50 µM jacaranone for 3 h. Hoechst 33342 dye was added and its fluorescence was captured in the 4,6-diamidino-2-phenylindole channel. Fluorescence observation confirmed that cells treated with jacaranone exhibited condensation as in apoptotic nuclei (Fig. 3A). The percentage of apoptotic nuclei was 25.5% and 39.5% in melanoma cells incubated with 20 and 50 µM jacaranone, respectively. For TEM, approximately 5×104 cells were incubated with 50 µM jacaranone for 3 h. Then, sections were made, as described in experimental procedures. Micrographs showed that jacaranone caused mitochondrial shrinkage (arrows), blebbing of plasma membrane (asterisks), and nuclear membrane disruption (black triangles) (Fig. 3B). These results, collectively with biochemical features [14], [15] demonstrated on Fig. 3, confirmed that jacaranone-treated cells undergo apoptosis.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368838&req=5

pone-0038698-g003: 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.
Mentions: In order to study the details of apoptotic morphological changes [14] displayed by jacaranone in B16F10-Nex2 cells, Hoechst staining and transmission electron microscopy were used. The density of nuclear chromatin was analyzed by culturing cells on cover slips in the presence of 20 or 50 µM jacaranone for 3 h. Hoechst 33342 dye was added and its fluorescence was captured in the 4,6-diamidino-2-phenylindole channel. Fluorescence observation confirmed that cells treated with jacaranone exhibited condensation as in apoptotic nuclei (Fig. 3A). The percentage of apoptotic nuclei was 25.5% and 39.5% in melanoma cells incubated with 20 and 50 µM jacaranone, respectively. For TEM, approximately 5×104 cells were incubated with 50 µM jacaranone for 3 h. Then, sections were made, as described in experimental procedures. Micrographs showed that jacaranone caused mitochondrial shrinkage (arrows), blebbing of plasma membrane (asterisks), and nuclear membrane disruption (black triangles) (Fig. 3B). These results, collectively with biochemical features [14], [15] demonstrated on Fig. 3, confirmed that jacaranone-treated cells undergo apoptosis.

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

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