High levels of exogenous C2-ceramide promote morphological and biochemical evidences of necrotic features in thyroid follicular cells.
CD95 and ceramide are known to be involved in the apoptotic mechanism. The triggering of CD95 induces a cascade of metabolic events that progressively and dramatically modifies the cell shape by intense membrane blebbing, leading to apoptotic bodies production. Although the CD95 pathway has been abundantly described in normal thyrocytes, the effects of cell permeable synthetic ceramide at morphological and biochemical levels are not fully known. In the present study, we show that thyroid follicular cells (TFC) exposed to 20 microM of C(2)-ceramide for 4 h are characterized by morphological features of necrosis, such as electron-lucent cytoplasm, mitochondrial swelling, and loss of plasma membrane integrity without drastic morphological changes in the nuclei. By contrast, TFC treated with 2 microM of C(2)-ceramide for 4 h are able to accumulate GD3, activate caspases cascade, and induce apoptosis. Furthermore, we provide evidence that 20 microM of C(2)-ceramide determine the destruction of mitochondria and are not able to induce PARP cleavage and internucleosomal DNA fragmentation, suggesting that the apoptotic program is not activated during the death process and nuclear DNA is randomly cleaved as the consequence of cellular degeneration. Pretreatment with 30 microM of zVAD-fmk rescued TFC from 2 microM of C(2)-ceramide-induced apoptosis, whereas, 20 microM of C(2)-ceramide exposure induced necrotic features. Deltapsi(m) was obviously altered in cells treated with 20 microM of C(2)-ceramide for 4 h (75% +/- 3.5%) compared with the low percentage (12.5% +/- 0.4%) of cells with altered Deltapsi(m) exposed to 2 microM of C(2)-ceramide. Whereas, only 20% +/- 1.1% of cells treated with anti-CD95 for 1 h showed altered Deltapsi(m). Additionally, Bax and Bak, two pro-apoptotic members, seem to be not oligomerized in the mitochondrial membrane following ceramide exposure. These results imply that high levels of exogenous ceramide contribute to the necrotic process in TFC, and may provide key molecular basis to the understanding of thyroid signaling pathways that might promote the apoptotic mechanism in thyroid tumoral cells.
Todaro
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M
Department of Surgical and Oncological Sciences, University of Palermo, Via del Vespro 129, 90127 Palermo, Italy.
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Stassi
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D017209:Apoptosis / Q000187:drug effects; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000473:pathology; D009336:Necrosis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D013110:Sphingosine / Q000008:administration & dosage* / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000633:toxicity; D013961:Thyroid Gland / Q000187:drug effects* / Q000473:pathology* / Q000648:ultrastructure; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000378:metabolism
C494772:BAK1 protein, human; C494826:BAX protein, human; D008565:Membrane Proteins; C064769:N-acetylsphingosine; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D011065:Poly(ADP-ribose) Polymerases; D013110:Sphingosine