Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and mitochondria

Servais, Hélène; Smissen, Patrick Van Der; Thirion, Gaëtan; Essen, Gauthier Van Der; Bambeke, Françoise Van; Tulkens, Paul M.; Mingeot‐Leclercq, Marie‐Paule

doi:10.1016/j.taap.2004.11.024

Cited by 124 publications

(78 citation statements)

References 61 publications

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“…The diagrams were constructed based on data from This contrasts with observations made for other drugs that accumulate in lysosomes, like chloroquine (53) or aminoglycosides (54,55), which neutralize the pH of lysosomes or increase the permeability of their membranes.…”

Section: Figmentioning

confidence: 99%

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Lemaire

Mingeot‐Leclercq

Tulkens

et al. 2014

Antimicrob Agents Chemother

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Oritavancin, a lipoglycopeptide antibiotic in development, accumulates to high levels in the lysosomes of eukaryotic cells. We examined specific functions of macrophages (phagocytic capacity, lysosomal integrity, metabolic activity, and production of reactive oxygen species [ROS]) in correlation with the cellular accumulation of the drug, using J774 mouse macrophages and THP-1 human monocytes differentiated into macrophages using phorbol 12-myristate 13-acetate. Oritavancin did not affect Pseudomonas aeruginosa phagocytosis, lysosomal integrity, or metabolic activity in cells incubated for 3 h with extracellular concentrations ranging from 5 to 50 g/ml. At extracellular concentrations of >25 g/ml, oritavancin reduced latex bead phagocytosis by approximately 50% and doubled ROS production in J774 macrophages only. This may result from the fact that the cellular accumulation of oritavancin was 15 times higher in J774 cells than in activated THP-1 cells at 3 h. Human pharmacokinetic studies estimate that the concentration of oritavancin in alveolar macrophages could reach approximately 560 g/ml after administration of a cumulative dose of 4 g, which is below the cellular concentration needed in the present study to impair latex bead phagocytosis (1,180 g/ml) or to stimulate ROS production (15,000 g/ml) by J774 cells. The data, therefore, suggest that, in spite of its substantial cellular accumulation, oritavancin is unlikely to markedly affect macrophage functions under the conditions of use investigated in current phase III trials (a single dose of 1,200 mg).

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Section: Figmentioning

confidence: 99%

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Lemaire

Mingeot‐Leclercq

Tulkens

et al. 2014

Antimicrob Agents Chemother

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“…61 Aminoglycoside nephrotoxicity is an example of lysosomal participation in apoptosis ( Figure 4C). 87 Lysosomal accumulation of gentamicin may initially prevent its more toxic cytosolic localization. Eventually, permeabilization of the lysosomal membrane releases free gentamicin to the cytosol and/or releases other lysosomal components that trigger a Bax-mediated mitochondrial pathway of apoptosis.…”

Section: Nephrotoxins Illustrate Intracellular Death Pathwaysmentioning

confidence: 99%

“…Eventually, permeabilization of the lysosomal membrane releases free gentamicin to the cytosol and/or releases other lysosomal components that trigger a Bax-mediated mitochondrial pathway of apoptosis. [87][88][89] The proapoptotic role of p53 has been characterized in cisplatin nephrotoxicity ( Figure 4D). Cisplatin damages genomic DNA and markedly induces the expression and phosphorylation of p53.…”

Section: Nephrotoxins Illustrate Intracellular Death Pathwaysmentioning

confidence: 99%

Mechanisms of Renal Apoptosis in Health and Disease

Sanz

Santamaría²,

Ruíz-Ortega³

et al. 2008

Journal of the American Society of Nephrology

236

192

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“…These, however, must be exposed to high concentrations of gentamicin because of poorly efficient drug uptake (12). Using LLC-PK 1 cells, an accepted model of renal proximal tubular cells for the study of aminoglycoside toxicity (24,64,66,67,72), we observed that gentamicin causes an early permeabilization of lysosomes before activation of the mitochondrial pathway of apoptosis can be detected (61). In parallel, recent data indicate that a minor but probably significant proportion of the gentamicin accumulated by cells can reach the cytosol and the mitochondria by trafficking through the Golgi apparatus and the endoplasmic reticulum (56).…”

mentioning

confidence: 93%

Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK ₁ Cells Subjected to Electroporation

Servais¹,

Jossin

Bambeke³

et al. 2006

Antimicrob Agents Chemother

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Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can be reproduced with cultured renal cells, but only at high extracellular concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK 1 cells involves a rapid (2-h) permeabilization of lysosomes and activation of the mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery of gentamicin to the cytosol by electroporation would sensitize LLC-PK 1 cells to apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square waves) in the presence of gentamicin (3 M to 3 mM; 1.2 mg/liter to 1.2 g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax (a marker of mitochondrial pathway activation) contents by Western blotting and competitive reverse transcriptase PCR and at 24 h for apoptosis by 4,6-diamidino-2-phenylindole staining (confirmed by electron microscopy) and for necrosis (by determination of lactate dehydrogenase release). Nonelectroporated cells were incubated with gentamicin for 8 and 24 h. Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the Bax level was not associated with an increase in the level of its mRNA but was associated with the accumulation of ubiquitinated forms (probably as a result of impairment of its degradation by the proteasome). Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubated cells. The data indicate that cytosolic gentamicin triggers apoptosis. Sequestration of gentamicin in lysosomes would, to some extent, protect against apoptosis.

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Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and mitochondria

Cited by 124 publications

References 61 publications

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Mechanisms of Renal Apoptosis in Health and Disease

Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK ₁ Cells Subjected to Electroporation

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Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and mitochondria

Cited by 124 publications

References 61 publications

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Study of Macrophage Functions in Murine J774 Cells and Human Activated THP-1 Cells Exposed to Oritavancin, a Lipoglycopeptide with High Cellular Accumulation

Mechanisms of Renal Apoptosis in Health and Disease

Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK 1 Cells Subjected to Electroporation

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Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK ₁ Cells Subjected to Electroporation