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- Ullio, Chiara, et al.
(författare)
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Sphingosine mediates TNF alpha-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma cells
- 2012
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Ingår i: Journal of Lipid Research. - : American Society for Biochemistry and Molecular Biology. - 0022-2275 .- 1539-7262. ; 53:6, s. 1134-1143
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Tidskriftsartikel (refereegranskat)abstract
- Normally, cell proliferation and death are carefully balanced in higher eukaryotes, but one of the most important regulatory mechanisms, apoptosis, is upset in many malignancies, including hepatocellular-derived ones. Therefore, reinforcing cell death often is mandatory in anticancer therapy. We previously reported that a combination of tumor necrosis factor-alpha (TNF) and cycloheximide (CHX) efficiently kill HTC cells, a rat hepatoma line, in an apoptosis-like mode. Death is actively mediated by the lysosomal compartment, although lysosomal ceramide was previously shown not to be directly implicated in this process. In the present study, we show that TNF/CHX increase lysosomal ceramide that is subsequently converted into sphingosine. Although ceramide accumulation does not significantly alter the acidic compartment, the sphingosine therein generated causes lysosomal membrane permeabilization (LMP) followed by relocation of lysosomal cathepsins to the cytoplasm. TNF/CHX-induced LMP is effectively abrogated by siRNAs targeting acid sphingomyelinase or acid ceramidase, which prevent both LMP and death induced by TNF/CHX. Taken together, our results demonstrate that lysosomal accumulation of ceramide is not detrimental per se, whereas its degradation product sphingosine, which has the capacity to induce LMP, appears responsible for the observed apoptotic-like death.-Ullio, C., J. Casas, U. T. Brunk, G. Sala, G. Fabrias, R. Ghidoni, G. Bonelli, F. M. Baccino, and R. Autelli. Sphingosine mediates TNF alpha-induced lysosomal membrane permeabilization and ensuing programmed cell death in hepatoma.
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| 2. |
- Autelli, Riccardo, et al.
(författare)
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Divergent pathways for TNF and C₂-ceramide toxicity in HTC hematoma cells
- 2009
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 1793, s. 1182-1190
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Tidskriftsartikel (refereegranskat)abstract
- We previously showed that, in the rat hepatoma cell line HTC, TNF brings about a non-caspase-dependent, apoptosis-like process requiring NADPH oxidase activity, an iron-mediated pro-oxidant status, and a functional acidic vacuolar compartment. This process may thus involve mechanisms such as autophagy or relocation of lysosomal enzymes, perhaps secondary to the formation of ceramide by acidic sphingomyelinase. Here we investigated whether ceramide formation contributes to the apoptogenic process. HTC cells were found to be sensitive to exogenous ceramide and significantly protected against TNF by desipramine, an inhibitor of lysosomal acid sphingomyelinase. However, Bcl-2 transfection and Bcl-x(L) upregulation by dexamethasone significantly diminished the apoptogenic effect of ceramide but not that of TNF, suggesting that ceramide is not directly involved in TNF toxicity. Moreover, Bcl-x(L) silencing precluded dexamethasone-induced protection against ceramide and, by itself, induced massive death, demonstrating the strict dependence of HTC cells on Bcl-x(L) for survival also under standard culture conditions.
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| 4. |
- Ullio, Chiara, et al.
(författare)
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Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells
- 2015
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Ingår i: Autophagy. - : TAYLOR & FRANCIS INC. - 1554-8627 .- 1554-8635. ; 11:12, s. 2184-2198
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Tidskriftsartikel (refereegranskat)abstract
- Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.
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