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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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Activation of caspases and cleavage of apoptotic substrates induced by aeroplysinin-1. (A) Western-blot detection of PARP cleavage in aeroplysinin-1-treated BAE cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western-blot detection of cleaved lamin-A in aeroplysinin-1-treated BAE cells with or without pre-incubation of cells with the pan-caspase inhibitor z-VAD 25 μM. (C) Effect of 10 µM aeroplysinin-1 in BAE cells caspase-2, -3, -8 and -9 activation. Activity values from treated cells are expressed as percentage of untreated (control) cells. Bars represent the standard deviation from duplicated samples in the same assay. Similar results were obtained in three independent experiments.
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marinedrugs-10-02033-f004: Activation of caspases and cleavage of apoptotic substrates induced by aeroplysinin-1. (A) Western-blot detection of PARP cleavage in aeroplysinin-1-treated BAE cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western-blot detection of cleaved lamin-A in aeroplysinin-1-treated BAE cells with or without pre-incubation of cells with the pan-caspase inhibitor z-VAD 25 μM. (C) Effect of 10 µM aeroplysinin-1 in BAE cells caspase-2, -3, -8 and -9 activation. Activity values from treated cells are expressed as percentage of untreated (control) cells. Bars represent the standard deviation from duplicated samples in the same assay. Similar results were obtained in three independent experiments.

Mentions: In order to provide biochemical evidence for the induction of apoptosis in BAE cells treated with aeroplysinin-1, the cleavage of poly (ADP-ribose) polymerase (PARP) and lamin-A, was studied. PARP cleavage by activated caspase-3 is a key event in the process of apoptosis and is used as an early marker for apoptosis induction [12]. As shown in Figure 4A, PARP was cleaved from 116 kDa intact form into 85 kDa fragment after BAE cell treatment with 10 μM of aeroplysinin-1. Lamin-A is a structural protein belonging to the intermediate filament family that, together with other lamin species, constitutes the scaffolding of the nuclear envelope. During apoptosis, lamin A is cleaved at Asp230 by caspase-6 [13] causing the characteristic collapse of the nucleus observed during apoptosis. Treatment of BAEC with 10 µM aeroplysinin-1 caused lamin A cleavage, generating a fragment of 28 kDa. Based on those results, we reasoned whether aeroplysinin-1 induced apoptosis could be mediated by caspases, and used a pharmacological inhibitor of caspases to gain insight into this question. As shown in Figure 4B, the addition of 25 µM of the pan-caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) inhibited lamin A cleavage, indicating that aeroplysinin-1 induces apoptosis in BAE cells through a caspase-dependent pathway.


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)

Activation of caspases and cleavage of apoptotic substrates induced by aeroplysinin-1. (A) Western-blot detection of PARP cleavage in aeroplysinin-1-treated BAE cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western-blot detection of cleaved lamin-A in aeroplysinin-1-treated BAE cells with or without pre-incubation of cells with the pan-caspase inhibitor z-VAD 25 μM. (C) Effect of 10 µM aeroplysinin-1 in BAE cells caspase-2, -3, -8 and -9 activation. Activity values from treated cells are expressed as percentage of untreated (control) cells. Bars represent the standard deviation from duplicated samples in the same assay. Similar results were obtained in three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3475271&req=5

marinedrugs-10-02033-f004: Activation of caspases and cleavage of apoptotic substrates induced by aeroplysinin-1. (A) Western-blot detection of PARP cleavage in aeroplysinin-1-treated BAE cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Western-blot detection of cleaved lamin-A in aeroplysinin-1-treated BAE cells with or without pre-incubation of cells with the pan-caspase inhibitor z-VAD 25 μM. (C) Effect of 10 µM aeroplysinin-1 in BAE cells caspase-2, -3, -8 and -9 activation. Activity values from treated cells are expressed as percentage of untreated (control) cells. Bars represent the standard deviation from duplicated samples in the same assay. Similar results were obtained in three independent experiments.
Mentions: In order to provide biochemical evidence for the induction of apoptosis in BAE cells treated with aeroplysinin-1, the cleavage of poly (ADP-ribose) polymerase (PARP) and lamin-A, was studied. PARP cleavage by activated caspase-3 is a key event in the process of apoptosis and is used as an early marker for apoptosis induction [12]. As shown in Figure 4A, PARP was cleaved from 116 kDa intact form into 85 kDa fragment after BAE cell treatment with 10 μM of aeroplysinin-1. Lamin-A is a structural protein belonging to the intermediate filament family that, together with other lamin species, constitutes the scaffolding of the nuclear envelope. During apoptosis, lamin A is cleaved at Asp230 by caspase-6 [13] causing the characteristic collapse of the nucleus observed during apoptosis. Treatment of BAEC with 10 µM aeroplysinin-1 caused lamin A cleavage, generating a fragment of 28 kDa. Based on those results, we reasoned whether aeroplysinin-1 induced apoptosis could be mediated by caspases, and used a pharmacological inhibitor of caspases to gain insight into this question. As shown in Figure 4B, the addition of 25 µM of the pan-caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) inhibited lamin A cleavage, indicating that aeroplysinin-1 induces apoptosis in BAE cells through a caspase-dependent pathway.

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