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Antiproliferative effect of the jararhagin toxin on B16F10 murine melanoma.

Maria DA, da Silva MG, Correia Junior MC, Ruiz IR - BMC Complement Altern Med (2014)

Bottom Line: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin.Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins.In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, Av, Vital Brasil 1500, CEP 05503-900 Sao Paulo, SP, Brazil. durvanei.maria@butantan.gov.br.

ABSTRACT

Background: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin. Jararhagin toxin, a metalloproteinase isolated from Bothrops jararaca snake venom acts upon several biological processes, as inflammation, pain, platelet aggregation, proliferation and apoptosis, though not yet approved for use, may one day be employed to treat tumors.

Methods: B16F10 murine melanoma cells were treated with jararhagin (jara), a disintegrin-like metalloproteinase isolated from Bothrops jararaca snake venom, and jari (catalytic domain inactivated with 1,10-phenanthroline). Viability and adhesion cells were evaluated by MTT assay. The expression of caspase-3 active, phases of the cell cycle and apoptosis were assessed by flow cytometry. We analyze in vivo the effects of jararhagin on melanoma growth, apoptosis and metastasis.

Results: The tumor cells acquired round shapes, lost cytoplasmic expansions, formed clusters in suspension and decreased viability. Jari was almost 20 times more potent toxin than jara based on IC50 values and on morphological changes of the cells, also observed by scanning electron microscopy. Flow cytometry analysis showed 48.3% decrease in the proliferation rate of cells and 47.2% increase in apoptosis (jara) and necrosis (jari), following 1.2 μM jara and 0.1 μM jari treatments. Caspase-3 activity was increased whereas G0/G1 cell cycle phase was on the decline. Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins.

Conclusions: In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity. This data strengthens the potential use jararhagin as an anti-neoplastic drug.

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Analysis of apoptosis by flow cytometry. Induction of apoptosis in B16F10 cells by jara (0.4-0.8 μM) and jari (0.2-0.4 μM) for 24 h, as shown through Annexin-V/PI double staining by flow cytometry. The representative dotplot adquisitions showed apoptotic cells and necrosis areas (A). The apoptosis rate was estatistically significant for the percentage of early/late apoptotic cells after jara treatment, while jari showed significant increase in the percentage of necrotic cells (B). Data represent mean ± SD of three independent experiments. *p < 0.05 and ***p < 0.001.
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Fig6: Analysis of apoptosis by flow cytometry. Induction of apoptosis in B16F10 cells by jara (0.4-0.8 μM) and jari (0.2-0.4 μM) for 24 h, as shown through Annexin-V/PI double staining by flow cytometry. The representative dotplot adquisitions showed apoptotic cells and necrosis areas (A). The apoptosis rate was estatistically significant for the percentage of early/late apoptotic cells after jara treatment, while jari showed significant increase in the percentage of necrotic cells (B). Data represent mean ± SD of three independent experiments. *p < 0.05 and ***p < 0.001.

Mentions: Jara significantly increased early and late apoptosis of B16F10 cells. As shown in Figure 6A and B the number of jara treated cells in apoptosis was significantly increased (***p < 0.001) in a dose-dependent manner (0.4 and 0.8 μM), when compared to control cells. Contrarily, lower apoptosis and higher necrosis levels were observed (***p < 0.001) on jari treated cells (0.4 μM).Figure 6


Antiproliferative effect of the jararhagin toxin on B16F10 murine melanoma.

Maria DA, da Silva MG, Correia Junior MC, Ruiz IR - BMC Complement Altern Med (2014)

Analysis of apoptosis by flow cytometry. Induction of apoptosis in B16F10 cells by jara (0.4-0.8 μM) and jari (0.2-0.4 μM) for 24 h, as shown through Annexin-V/PI double staining by flow cytometry. The representative dotplot adquisitions showed apoptotic cells and necrosis areas (A). The apoptosis rate was estatistically significant for the percentage of early/late apoptotic cells after jara treatment, while jari showed significant increase in the percentage of necrotic cells (B). Data represent mean ± SD of three independent experiments. *p < 0.05 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289281&req=5

Fig6: Analysis of apoptosis by flow cytometry. Induction of apoptosis in B16F10 cells by jara (0.4-0.8 μM) and jari (0.2-0.4 μM) for 24 h, as shown through Annexin-V/PI double staining by flow cytometry. The representative dotplot adquisitions showed apoptotic cells and necrosis areas (A). The apoptosis rate was estatistically significant for the percentage of early/late apoptotic cells after jara treatment, while jari showed significant increase in the percentage of necrotic cells (B). Data represent mean ± SD of three independent experiments. *p < 0.05 and ***p < 0.001.
Mentions: Jara significantly increased early and late apoptosis of B16F10 cells. As shown in Figure 6A and B the number of jara treated cells in apoptosis was significantly increased (***p < 0.001) in a dose-dependent manner (0.4 and 0.8 μM), when compared to control cells. Contrarily, lower apoptosis and higher necrosis levels were observed (***p < 0.001) on jari treated cells (0.4 μM).Figure 6

Bottom Line: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin.Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins.In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Biophysics Laboratory, Butantan Institute, Av, Vital Brasil 1500, CEP 05503-900 Sao Paulo, SP, Brazil. durvanei.maria@butantan.gov.br.

ABSTRACT

Background: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin. Jararhagin toxin, a metalloproteinase isolated from Bothrops jararaca snake venom acts upon several biological processes, as inflammation, pain, platelet aggregation, proliferation and apoptosis, though not yet approved for use, may one day be employed to treat tumors.

Methods: B16F10 murine melanoma cells were treated with jararhagin (jara), a disintegrin-like metalloproteinase isolated from Bothrops jararaca snake venom, and jari (catalytic domain inactivated with 1,10-phenanthroline). Viability and adhesion cells were evaluated by MTT assay. The expression of caspase-3 active, phases of the cell cycle and apoptosis were assessed by flow cytometry. We analyze in vivo the effects of jararhagin on melanoma growth, apoptosis and metastasis.

Results: The tumor cells acquired round shapes, lost cytoplasmic expansions, formed clusters in suspension and decreased viability. Jari was almost 20 times more potent toxin than jara based on IC50 values and on morphological changes of the cells, also observed by scanning electron microscopy. Flow cytometry analysis showed 48.3% decrease in the proliferation rate of cells and 47.2% increase in apoptosis (jara) and necrosis (jari), following 1.2 μM jara and 0.1 μM jari treatments. Caspase-3 activity was increased whereas G0/G1 cell cycle phase was on the decline. Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins.

Conclusions: In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity. This data strengthens the potential use jararhagin as an anti-neoplastic drug.

Show MeSH
Related in: MedlinePlus