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Coibamide A induces mTOR-independent autophagy and cell death in human glioblastoma cells.

Hau AM, Greenwood JA, Löhr CV, Serrill JD, Proteau PJ, Ganley IG, McPhail KL, Ishmael JE - PLoS ONE (2013)

Bottom Line: Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and "COMPARE-negative" profile indicative of a unique mechanism of action.Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type.Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and "COMPARE-negative" profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1- MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5- MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

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Coibamide A does not block ligand-induced degradation of EGFR.(A) Immunoblot analysis of EGFR expression up to 4 h after the addition of ligand (EGF) in the presence of vehicle (control), coibamide A (30 nM) or bafilomycin A1 (100 nM). On the day of the experiment U87-MG cells were starved of serum, to promote membrane localization of EGFR, and at t = 0 were chased with EGF (100 ng/mL) and cycloheximide (25 µg/mL), with or without coibamide A, or bafilomycin A1. Cells were lysed at the times indicated and processed for immunoblot analysis. (B) Quantitation of ligand-induced EGFR degradation from three independent experiments as described in A. Graph represents % EGFR expression, following the addition of EGF, as a function of incubation time in the presence of vehicle, coibamide A or bafilomycin A1. EGFR intensity was normalized to tubulin and determined relative to EGFR level at t = 0.
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pone-0065250-g008: Coibamide A does not block ligand-induced degradation of EGFR.(A) Immunoblot analysis of EGFR expression up to 4 h after the addition of ligand (EGF) in the presence of vehicle (control), coibamide A (30 nM) or bafilomycin A1 (100 nM). On the day of the experiment U87-MG cells were starved of serum, to promote membrane localization of EGFR, and at t = 0 were chased with EGF (100 ng/mL) and cycloheximide (25 µg/mL), with or without coibamide A, or bafilomycin A1. Cells were lysed at the times indicated and processed for immunoblot analysis. (B) Quantitation of ligand-induced EGFR degradation from three independent experiments as described in A. Graph represents % EGFR expression, following the addition of EGF, as a function of incubation time in the presence of vehicle, coibamide A or bafilomycin A1. EGFR intensity was normalized to tubulin and determined relative to EGFR level at t = 0.

Mentions: Autophagy requires a coordinated series of specific endomembrane fusion events to traffic the contents of autophagosomes to the lysosomes for degradation. We therefore assessed the potential of coibamide A to interfere with a major endocytic-lysosomal pathway by studying turnover of endogenous EGFR. For these studies, U87-MG cells were serum-starved to promote localization of EGFR to the plasma membrane and were then chased in the presence of EGF (100 ng/mL), to induce internalization of EGFR, plus cycloheximide (25 µg/mL) to inhibit protein synthesis. In the presence of coibamide A, ligand-induced EGFR degradation progressed at a rate that could not be distinguished from vehicle-treated control cells (Fig. 8A and 8B). This finding was in contrast to U87-MG cells cultured in the presence of bafilomycin A1. Bafilomycin A1 dramatically inhibited endocytosis-mediated degradation of EGFR over the time course of these experiments (Fig. 8A and B), in a manner consistent with its ability to block lysosomal degradation of EGFR. Taken together our findings indicate that the autophagic-lysosomal pathway remains functional in U87-MG cells in the presence of coibamide A and that coibamide A does not produce a global block in endocytosis.


Coibamide A induces mTOR-independent autophagy and cell death in human glioblastoma cells.

Hau AM, Greenwood JA, Löhr CV, Serrill JD, Proteau PJ, Ganley IG, McPhail KL, Ishmael JE - PLoS ONE (2013)

Coibamide A does not block ligand-induced degradation of EGFR.(A) Immunoblot analysis of EGFR expression up to 4 h after the addition of ligand (EGF) in the presence of vehicle (control), coibamide A (30 nM) or bafilomycin A1 (100 nM). On the day of the experiment U87-MG cells were starved of serum, to promote membrane localization of EGFR, and at t = 0 were chased with EGF (100 ng/mL) and cycloheximide (25 µg/mL), with or without coibamide A, or bafilomycin A1. Cells were lysed at the times indicated and processed for immunoblot analysis. (B) Quantitation of ligand-induced EGFR degradation from three independent experiments as described in A. Graph represents % EGFR expression, following the addition of EGF, as a function of incubation time in the presence of vehicle, coibamide A or bafilomycin A1. EGFR intensity was normalized to tubulin and determined relative to EGFR level at t = 0.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675158&req=5

pone-0065250-g008: Coibamide A does not block ligand-induced degradation of EGFR.(A) Immunoblot analysis of EGFR expression up to 4 h after the addition of ligand (EGF) in the presence of vehicle (control), coibamide A (30 nM) or bafilomycin A1 (100 nM). On the day of the experiment U87-MG cells were starved of serum, to promote membrane localization of EGFR, and at t = 0 were chased with EGF (100 ng/mL) and cycloheximide (25 µg/mL), with or without coibamide A, or bafilomycin A1. Cells were lysed at the times indicated and processed for immunoblot analysis. (B) Quantitation of ligand-induced EGFR degradation from three independent experiments as described in A. Graph represents % EGFR expression, following the addition of EGF, as a function of incubation time in the presence of vehicle, coibamide A or bafilomycin A1. EGFR intensity was normalized to tubulin and determined relative to EGFR level at t = 0.
Mentions: Autophagy requires a coordinated series of specific endomembrane fusion events to traffic the contents of autophagosomes to the lysosomes for degradation. We therefore assessed the potential of coibamide A to interfere with a major endocytic-lysosomal pathway by studying turnover of endogenous EGFR. For these studies, U87-MG cells were serum-starved to promote localization of EGFR to the plasma membrane and were then chased in the presence of EGF (100 ng/mL), to induce internalization of EGFR, plus cycloheximide (25 µg/mL) to inhibit protein synthesis. In the presence of coibamide A, ligand-induced EGFR degradation progressed at a rate that could not be distinguished from vehicle-treated control cells (Fig. 8A and 8B). This finding was in contrast to U87-MG cells cultured in the presence of bafilomycin A1. Bafilomycin A1 dramatically inhibited endocytosis-mediated degradation of EGFR over the time course of these experiments (Fig. 8A and B), in a manner consistent with its ability to block lysosomal degradation of EGFR. Taken together our findings indicate that the autophagic-lysosomal pathway remains functional in U87-MG cells in the presence of coibamide A and that coibamide A does not produce a global block in endocytosis.

Bottom Line: Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and "COMPARE-negative" profile indicative of a unique mechanism of action.Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type.Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and "COMPARE-negative" profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1- MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5- MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

Show MeSH
Related in: MedlinePlus