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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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Inhibition of tumor growth and metastasis dorsal treated with jara toxins and jari. C57/Bl6J mice were administered via subcutaneous with B16F10 melanoma cells pretreated with 0.8 μM jara, 0.2 μM jari, and with untreated cells (control group). The growth curve (A) and macroscopic aspect of the melanoma dorsal tumor of the control and treated groups (B) are shown, as well as the Kaplan-Meier survival curve (C), metastasis multiplicity (D) and cell cycle phase of lung metastasis (E). Pre-treatment of tumor cells in vitro with jara or jari reduced the dorsal tumor volume of B16F10 melanoma cells, with statistically significant increase in survival rates with p < 0.05 (Log rank p < 0.018). The number of nodules in lung parenchyma was reduced on jara and jari groups. The distribution of cell cycle phase showed increase of tumor cells in sub-G1, fragmented DNA and decreased number of cells in proliferative response arrest in G2/M, caused by jara and jari on metastasis lung.
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Fig8: Inhibition of tumor growth and metastasis dorsal treated with jara toxins and jari. C57/Bl6J mice were administered via subcutaneous with B16F10 melanoma cells pretreated with 0.8 μM jara, 0.2 μM jari, and with untreated cells (control group). The growth curve (A) and macroscopic aspect of the melanoma dorsal tumor of the control and treated groups (B) are shown, as well as the Kaplan-Meier survival curve (C), metastasis multiplicity (D) and cell cycle phase of lung metastasis (E). Pre-treatment of tumor cells in vitro with jara or jari reduced the dorsal tumor volume of B16F10 melanoma cells, with statistically significant increase in survival rates with p < 0.05 (Log rank p < 0.018). The number of nodules in lung parenchyma was reduced on jara and jari groups. The distribution of cell cycle phase showed increase of tumor cells in sub-G1, fragmented DNA and decreased number of cells in proliferative response arrest in G2/M, caused by jara and jari on metastasis lung.

Mentions: B16F10 cells treated with 0.8 μM jara and 0.2 μM jari were injected s.c. (5 × 104 cells/mice) in C57BL/6 J mice. Untreated tumor cells were injected in the control group. Dorsal tumors were visualized in the injection site in all animal groups (100%) after the tenth day. The mean volume of dorsal tumors was significantly decreased in animals injected with cells pretreated with jara or jari (p = 0.0016) (Figure 8A). Dorsal tumor incidence was smaller in animals injected with cells pretreated with 0.8 μM jara (80%), but was 100% both in controls and the 0.2 μM jari group. After 40 days, the mean volume of dorsal tumors in controls was 17.8 ± 4.5 mm3 for control group; 5.8 ± 1.2 mm3 for 0.8 μM jara (p < 0.01), and 3.4 ± 0.7 mm3 for 0.2 μM Jari (p < 0.001), i.e., it was demonstrated a reduction in tumor volume of 67.5% (jara) and 86.5% (jari) compared with tumors developed by untreated B16F10 cells (Figure 8A, B). The survival probability (Kaplan-Meier curve) was significantly increased for animals injected with both jara and jari treated cells (log rank p = 0.00218), (Figure 8C). The long-rank test was used to test whether the difference between survival times between two groups: control X jara; control X jari and jara X jari is statistically different. Histopathological analysis of dorsal tumors induced by B16F10 cells pretreated with jara showed leukocytes infiltration, areas of regression-like fibrosis, and few blood vessels, as compared with controls (not shown).Figure 8


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)

Inhibition of tumor growth and metastasis dorsal treated with jara toxins and jari. C57/Bl6J mice were administered via subcutaneous with B16F10 melanoma cells pretreated with 0.8 μM jara, 0.2 μM jari, and with untreated cells (control group). The growth curve (A) and macroscopic aspect of the melanoma dorsal tumor of the control and treated groups (B) are shown, as well as the Kaplan-Meier survival curve (C), metastasis multiplicity (D) and cell cycle phase of lung metastasis (E). Pre-treatment of tumor cells in vitro with jara or jari reduced the dorsal tumor volume of B16F10 melanoma cells, with statistically significant increase in survival rates with p < 0.05 (Log rank p < 0.018). The number of nodules in lung parenchyma was reduced on jara and jari groups. The distribution of cell cycle phase showed increase of tumor cells in sub-G1, fragmented DNA and decreased number of cells in proliferative response arrest in G2/M, caused by jara and jari on metastasis lung.
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Related In: Results  -  Collection

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Fig8: Inhibition of tumor growth and metastasis dorsal treated with jara toxins and jari. C57/Bl6J mice were administered via subcutaneous with B16F10 melanoma cells pretreated with 0.8 μM jara, 0.2 μM jari, and with untreated cells (control group). The growth curve (A) and macroscopic aspect of the melanoma dorsal tumor of the control and treated groups (B) are shown, as well as the Kaplan-Meier survival curve (C), metastasis multiplicity (D) and cell cycle phase of lung metastasis (E). Pre-treatment of tumor cells in vitro with jara or jari reduced the dorsal tumor volume of B16F10 melanoma cells, with statistically significant increase in survival rates with p < 0.05 (Log rank p < 0.018). The number of nodules in lung parenchyma was reduced on jara and jari groups. The distribution of cell cycle phase showed increase of tumor cells in sub-G1, fragmented DNA and decreased number of cells in proliferative response arrest in G2/M, caused by jara and jari on metastasis lung.
Mentions: B16F10 cells treated with 0.8 μM jara and 0.2 μM jari were injected s.c. (5 × 104 cells/mice) in C57BL/6 J mice. Untreated tumor cells were injected in the control group. Dorsal tumors were visualized in the injection site in all animal groups (100%) after the tenth day. The mean volume of dorsal tumors was significantly decreased in animals injected with cells pretreated with jara or jari (p = 0.0016) (Figure 8A). Dorsal tumor incidence was smaller in animals injected with cells pretreated with 0.8 μM jara (80%), but was 100% both in controls and the 0.2 μM jari group. After 40 days, the mean volume of dorsal tumors in controls was 17.8 ± 4.5 mm3 for control group; 5.8 ± 1.2 mm3 for 0.8 μM jara (p < 0.01), and 3.4 ± 0.7 mm3 for 0.2 μM Jari (p < 0.001), i.e., it was demonstrated a reduction in tumor volume of 67.5% (jara) and 86.5% (jari) compared with tumors developed by untreated B16F10 cells (Figure 8A, B). The survival probability (Kaplan-Meier curve) was significantly increased for animals injected with both jara and jari treated cells (log rank p = 0.00218), (Figure 8C). The long-rank test was used to test whether the difference between survival times between two groups: control X jara; control X jari and jara X jari is statistically different. Histopathological analysis of dorsal tumors induced by B16F10 cells pretreated with jara showed leukocytes infiltration, areas of regression-like fibrosis, and few blood vessels, as compared with controls (not shown).Figure 8

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