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The antiangiogenic compound aeroplysinin-1 induces apoptosis in endothelial cells by activating the mitochondrial pathway.

Martínez-Poveda B, Rodríguez-Nieto S, García-Caballero M, Medina MÁ, Quesada AR - Mar Drugs (2012)

Bottom Line: Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo.Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism.Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, Málaga E-29071, Spain. bmpoveda@gmail.com

ABSTRACT
Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound.

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Aeroplysinin-1 potentiates apoptosis in endothelial cells. (A) Nuclear morphology of endothelial and tumor cells after treatment with aeroplysinin-1, assessed under a fluorescence microscope. (B) Cell cycle distribution of endothelial and tumor cells after treatment with aeroplysinin-1, analyzed by FACS. M1 indicates the subG1 population. (C) Determination of endothelial cell (BAEC) apoptosis by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining. (D) 7AAD−/PE-Annexin V−, 7AAD−/PE-Annexin V+, 7AAD+/PE-Annexin V+ and 7AAD+/PE-Annexin V− populations, corresponding to viable (Q3), early apoptotic (Q4), late apoptotic (Q2) and necrotic (Q1) cells respectively, were evaluated as in (C). Values are expressed as means ± SD of three independent experiments. * p < 0.05; ** p < 0.005 versus control.
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marinedrugs-10-02033-f003: Aeroplysinin-1 potentiates apoptosis in endothelial cells. (A) Nuclear morphology of endothelial and tumor cells after treatment with aeroplysinin-1, assessed under a fluorescence microscope. (B) Cell cycle distribution of endothelial and tumor cells after treatment with aeroplysinin-1, analyzed by FACS. M1 indicates the subG1 population. (C) Determination of endothelial cell (BAEC) apoptosis by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining. (D) 7AAD−/PE-Annexin V−, 7AAD−/PE-Annexin V+, 7AAD+/PE-Annexin V+ and 7AAD+/PE-Annexin V− populations, corresponding to viable (Q3), early apoptotic (Q4), late apoptotic (Q2) and necrotic (Q1) cells respectively, were evaluated as in (C). Values are expressed as means ± SD of three independent experiments. * p < 0.05; ** p < 0.005 versus control.

Mentions: As a first approach to study the effect of aeroplysinin-1 in endothelial and tumor cells, the nuclear morphology of BAE, HT1080 and HCT116 cells was analyzed by Hoechst staining after 14 h treatment with this compound. As shown in Figure 3A, treatment with 10 μM aeroplysinin-1 induced chromatin condensation and nuclear fragmentation in endothelial (BAE) cells, but not in colon carcinoma (HCT-116) or fibrosarcoma (HT-1080) cells. Nuclei of BAE cells treated with 3 μM aeroplysinin-1 did not show morphological changes compared to nuclei of non-treated cells, showing a dose dependence of this effect that is in agreement with that observed in the cell growth assay, where 10 μM aeroplysinin-1 was required to completely inhibit proliferation of BAEC. To confirm these results, the cell cycle distribution of propidium iodide-stained cells was analyzed by flow-cytometric analysis. Figure 3B shows that a significant increase (6-fold) in the sub-G1 population was observed in BAE cells treated with 10 μM aeroplysinin-1 when compared to untreated cells. Nevertheless, no significant increases of sub-diploid population were observed in either endothelial cells treated with 3 μM aeroplysinin-1, or in HCT-116 or HT-1080 cells treated with 10 μM aeroplysinin-1 (Figure 3B). These results suggested that aeroplysinin-1 might be a selective apoptosis trigger in endothelial cells. Endothelial cell apoptosis induced by aeroplysinin-1 was also confirmed by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining (Figure 3C,D), showing that 10 µM aeroplysinin-1 induced a significant increase in the percentage of cells in both early and late apoptotic cell subpopulations.


The antiangiogenic compound aeroplysinin-1 induces apoptosis in endothelial cells by activating the mitochondrial pathway.

Martínez-Poveda B, Rodríguez-Nieto S, García-Caballero M, Medina MÁ, Quesada AR - Mar Drugs (2012)

Aeroplysinin-1 potentiates apoptosis in endothelial cells. (A) Nuclear morphology of endothelial and tumor cells after treatment with aeroplysinin-1, assessed under a fluorescence microscope. (B) Cell cycle distribution of endothelial and tumor cells after treatment with aeroplysinin-1, analyzed by FACS. M1 indicates the subG1 population. (C) Determination of endothelial cell (BAEC) apoptosis by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining. (D) 7AAD−/PE-Annexin V−, 7AAD−/PE-Annexin V+, 7AAD+/PE-Annexin V+ and 7AAD+/PE-Annexin V− populations, corresponding to viable (Q3), early apoptotic (Q4), late apoptotic (Q2) and necrotic (Q1) cells respectively, were evaluated as in (C). Values are expressed as means ± SD of three independent experiments. * p < 0.05; ** p < 0.005 versus control.
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Related In: Results  -  Collection

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marinedrugs-10-02033-f003: Aeroplysinin-1 potentiates apoptosis in endothelial cells. (A) Nuclear morphology of endothelial and tumor cells after treatment with aeroplysinin-1, assessed under a fluorescence microscope. (B) Cell cycle distribution of endothelial and tumor cells after treatment with aeroplysinin-1, analyzed by FACS. M1 indicates the subG1 population. (C) Determination of endothelial cell (BAEC) apoptosis by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining. (D) 7AAD−/PE-Annexin V−, 7AAD−/PE-Annexin V+, 7AAD+/PE-Annexin V+ and 7AAD+/PE-Annexin V− populations, corresponding to viable (Q3), early apoptotic (Q4), late apoptotic (Q2) and necrotic (Q1) cells respectively, were evaluated as in (C). Values are expressed as means ± SD of three independent experiments. * p < 0.05; ** p < 0.005 versus control.
Mentions: As a first approach to study the effect of aeroplysinin-1 in endothelial and tumor cells, the nuclear morphology of BAE, HT1080 and HCT116 cells was analyzed by Hoechst staining after 14 h treatment with this compound. As shown in Figure 3A, treatment with 10 μM aeroplysinin-1 induced chromatin condensation and nuclear fragmentation in endothelial (BAE) cells, but not in colon carcinoma (HCT-116) or fibrosarcoma (HT-1080) cells. Nuclei of BAE cells treated with 3 μM aeroplysinin-1 did not show morphological changes compared to nuclei of non-treated cells, showing a dose dependence of this effect that is in agreement with that observed in the cell growth assay, where 10 μM aeroplysinin-1 was required to completely inhibit proliferation of BAEC. To confirm these results, the cell cycle distribution of propidium iodide-stained cells was analyzed by flow-cytometric analysis. Figure 3B shows that a significant increase (6-fold) in the sub-G1 population was observed in BAE cells treated with 10 μM aeroplysinin-1 when compared to untreated cells. Nevertheless, no significant increases of sub-diploid population were observed in either endothelial cells treated with 3 μM aeroplysinin-1, or in HCT-116 or HT-1080 cells treated with 10 μM aeroplysinin-1 (Figure 3B). These results suggested that aeroplysinin-1 might be a selective apoptosis trigger in endothelial cells. Endothelial cell apoptosis induced by aeroplysinin-1 was also confirmed by flow cytometry analysis after PE-Annexin V and 7-aminoactinomycin D (7AAD) staining (Figure 3C,D), showing that 10 µM aeroplysinin-1 induced a significant increase in the percentage of cells in both early and late apoptotic cell subpopulations.

Bottom Line: Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo.Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism.Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of Málaga, Málaga E-29071, Spain. bmpoveda@gmail.com

ABSTRACT
Aeroplysinin-1 is a brominated metabolite extracted from the marine sponge Aplysina aerophoba that has been previously characterized by our group as a potent antiangiogenic compound in vitro and in vivo. In this work, we provide evidence of a selective induction of apoptosis by aeroplysinin-1 in endothelial cells. Studies on the nuclear morphology of treated cells revealed that aeroplysinin-1 induces chromatin condensation and nuclear fragmentation, and it increases the percentage of cells with sub-diploid DNA content in endothelial, but not in HCT-116, human colon carcinoma and HT-1080 human fibrosarcoma cells. Treatment of endothelial cells with aeroplysinin-1 induces activation of caspases-2, -3, -8 and -9, as well as the cleavage of apoptotic substrates, such as poly (ADP-ribose) polymerase and lamin-A in a caspase-dependent mechanism. Our data indicate a relevant role of the mitochondria in the apoptogenic activity of this compound. The observation that aeroplysinin-1 prevents the phosphorylation of Bad relates to the mitochondria-mediated induction of apoptosis by this compound.

Show MeSH
Related in: MedlinePlus