Limits...
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.

Show MeSH

Related in: MedlinePlus

Proliferative rate of B16F10 melanoma cells. Toxins inhibit the proliferation and induce cell cycle arrest in B16F10 cells. Proliferation rates in B16F10 melanoma cells treated with jara and jari toxins for 24, 48 and 72 h determined using the ModFitLT 2.0 software. (A) The means proliferative rate using CFSE-DA assay of jara group; (B) CFSE-DA proliferation jari group; (C) CFSE-DA proliferation lymphocytes positive control (inset – dot plot representative); (D) Histograms represent the flow cytometric of CFSE-DA proliferative assay analyzes the proliferation of B16F10 melanoma cells after exposed to jara and jari toxins. All the experiments were repeated three times. Data are expressed as means ± SD. The statistical difference was obtained between control group non treated and toxins groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Proliferative rate of B16F10 melanoma cells. Toxins inhibit the proliferation and induce cell cycle arrest in B16F10 cells. Proliferation rates in B16F10 melanoma cells treated with jara and jari toxins for 24, 48 and 72 h determined using the ModFitLT 2.0 software. (A) The means proliferative rate using CFSE-DA assay of jara group; (B) CFSE-DA proliferation jari group; (C) CFSE-DA proliferation lymphocytes positive control (inset – dot plot representative); (D) Histograms represent the flow cytometric of CFSE-DA proliferative assay analyzes the proliferation of B16F10 melanoma cells after exposed to jara and jari toxins. All the experiments were repeated three times. Data are expressed as means ± SD. The statistical difference was obtained between control group non treated and toxins groups.

Mentions: Melanoma cells were labelled with CFSE-DA to be used as control groups and were cultured for 24, 48 and 72 h; after culture, cells were harvested and analysed by flow cytometry. Cell division is characterized by sequential halving of CFSE fluorescence, generating equally spaced peaks on a logarithmic scale; peaks indicate the division cycle number. Similar results of MTT colorimetric assay were obtained using CSFE methodology, which accurately confirmed rate of proliferation of melanoma cells, in different periods of treatment with Jara and Jari. Figure 3 presents histograms acquired using ModFitLT 2.0 software and comparison of significant differences is shown in Figure 3A, B, C and D. Proliferation index showed a significant decrease in the population of melanoma cells at all concentrations of both toxins.Figure 3


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)

Proliferative rate of B16F10 melanoma cells. Toxins inhibit the proliferation and induce cell cycle arrest in B16F10 cells. Proliferation rates in B16F10 melanoma cells treated with jara and jari toxins for 24, 48 and 72 h determined using the ModFitLT 2.0 software. (A) The means proliferative rate using CFSE-DA assay of jara group; (B) CFSE-DA proliferation jari group; (C) CFSE-DA proliferation lymphocytes positive control (inset – dot plot representative); (D) Histograms represent the flow cytometric of CFSE-DA proliferative assay analyzes the proliferation of B16F10 melanoma cells after exposed to jara and jari toxins. All the experiments were repeated three times. Data are expressed as means ± SD. The statistical difference was obtained between control group non treated and toxins groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Proliferative rate of B16F10 melanoma cells. Toxins inhibit the proliferation and induce cell cycle arrest in B16F10 cells. Proliferation rates in B16F10 melanoma cells treated with jara and jari toxins for 24, 48 and 72 h determined using the ModFitLT 2.0 software. (A) The means proliferative rate using CFSE-DA assay of jara group; (B) CFSE-DA proliferation jari group; (C) CFSE-DA proliferation lymphocytes positive control (inset – dot plot representative); (D) Histograms represent the flow cytometric of CFSE-DA proliferative assay analyzes the proliferation of B16F10 melanoma cells after exposed to jara and jari toxins. All the experiments were repeated three times. Data are expressed as means ± SD. The statistical difference was obtained between control group non treated and toxins groups.
Mentions: Melanoma cells were labelled with CFSE-DA to be used as control groups and were cultured for 24, 48 and 72 h; after culture, cells were harvested and analysed by flow cytometry. Cell division is characterized by sequential halving of CFSE fluorescence, generating equally spaced peaks on a logarithmic scale; peaks indicate the division cycle number. Similar results of MTT colorimetric assay were obtained using CSFE methodology, which accurately confirmed rate of proliferation of melanoma cells, in different periods of treatment with Jara and Jari. Figure 3 presents histograms acquired using ModFitLT 2.0 software and comparison of significant differences is shown in Figure 3A, B, C and D. Proliferation index showed a significant decrease in the population of melanoma cells at all concentrations of both toxins.Figure 3

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