Validation of an in vitro screen for phospholipidosis using a high-content biology platform

Morelli, James K.; Buehrle, M.; Philippe, François; Barone, Linda; Fieles, William; Ciaccio, Paul J.

doi:10.1007/s10565-006-0176-z

Cited by 104 publications

(99 citation statements)

References 34 publications

(20 reference statements)

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“…30 To challenge this hypothesis we tested whether siramesine treatment resulted in the accumulation of fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). 29 In order to visualize the possible localization of NBD-PE in lysosomes, we used siramesine sensitive (Fig. 4A) U2OS osteosarcoma cells expressing a fusion protein consisting of a monomeric red fluorescent protein (mDsRed) and lysosomal integrating membrane protein-1 (LIMP-1).…”

Section: Resultsmentioning

confidence: 99%

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Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

Ostenfeld¹,

Høyer-Hansen²,

Bastholm³

et al. 2008

Autophagy

140

120

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A σ-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6-and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.

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Section: Resultsmentioning

confidence: 99%

“…24 Phospholipidosis was visualized essentially as described previously. 29 Briefly, the cells stained with 90 μg/ml NBD-PE (Invitrogen) for 2 h at 37°C and washed with PBS were examined and photographed in an inverted Olympus IX70 fluorescent microscope (Filter O-MWU 330-385 nm) connected to an Olympus C-3030 digital camera.…”

Section: Methodsmentioning

confidence: 99%

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

Ostenfeld¹,

Høyer-Hansen²,

Bastholm³

et al. 2008

Autophagy

140

120

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“…Therefore, the accumulation of NBD-PE is used as a surrogate for PLD (Miyamoto et al, 2009;Morelli et al, 2006). We tested whether LipidTox positive results for acetaminophen and menadione can be reproduced in the NBD-PD assay using propranolol as a positive control.…”

Section: Pld Detection By Nbd-pe Assaysmentioning

confidence: 99%

<i>In vitro</i> validation of drug-induced phospholipidosis

2012

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-Intracellular accumulation of phospholipids with lamellar bodies is a hallmark of druginduced phospholipidosis (PLD) which is caused by impaired phospholipid metabolism of the lysosome. Although it remains uncertain whether PLD is associated with the adverse effects, sponsors generally terminate the development of a candidate drug when PLD is observed in an organ. For drugs that are used without serious adverse events, there should be labels indicating that the drug can induce PLD. We conducted LipidTox and NBD-PE assays for detecting PLD to compare and validate the methods. In the case of contrary results in both assays, electron microscopy was performed to confirm the data. We selected 12 chemicals and divided them into 4 categories: P+S+, PLD and steatosis positive; P+/S-, PLD positive and steatosis negative; P-S+, PLD negative and steatosis positive; P-/S-, PLD and steatosis negative. In general, results showed very good agreement with the known information with some minor discrepancies. LipidTox assay is proven to be a very sensitive method. Considering the contrary results of acetaminophen and menadione in LipidTox and the NBD-PE assay, the combination of two methods using different phospholipids is advantageous to reduce false positives. The finding that acetaminophen was positive in LipidTos assay and increased the frequency of lamellar body implies that acetaminophen is a weak inducer of PLD.

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“…These compounds showed positive responses in various in vitro models of PLD (Morelli et al, 2006;van de Water et al, 2011;Zhou et al, 2011). The ability for predicting drug-induced PLD has been reported in various cellbased assays.…”

Section: Discussionmentioning

confidence: 87%

A cell-based assay using HepaRG cells for predicting drug-induced phospholipidosis

2017

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-The utility of HepaRG cells as an in vitro cell-based assay system for predicting druginduced phospholipidosis (PLD) was investigated. In experiment 1, 10 PLD-positive compounds and 11 PLD-negative compounds were selected. HepaRG cells were treated with each compound for 48 hr. In experiment 2, loratadine and desloratadine, a major metabolite of loratadine, were used to assess metabolic activation for PLD. HepaRG cells were treated with loratadine and desloratadine in the presence or absence of 500 μM 1-aminobenzotriazole (ABT), a broad CYP inhibitor, for 48 hr. After treatment with compounds in experiments 1 and 2, the relative fluorescence intensity (RFI) was measured using LYSO-ID Red dye to assess the PLD induction. In experiment 1, our cell-based assay system using HepaRG cells exhibited 100% sensitivity and 100% specificity for predicting drug-induced PLD. In experiment 2, loratadine increased the RFI in the PLD assay. However, the increase in the RFI was not observed in co-treatment with loratadine and ABT. In addition, desloratadine increased the RFI in the presence and absence of ABT. These results suggested that metabolic activation of loratadine may contribute to PLD in HepaRG cells. We newly demonstrated that HepaRG cells have a high ability for predicting drug-induced PLD. In addition, we newly showed that HepaRG cells may predict drug-induced PLD mediated by metabolic activation of loratadine. Thus, a cell-based assay system using HepaRG cells is a useful model for predicting drug-induced PLD.

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Validation of an in vitro screen for phospholipidosis using a high-content biology platform

Cited by 104 publications

References 34 publications

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation

<i>In vitro</i> validation of drug-induced phospholipidosis

A cell-based assay using HepaRG cells for predicting drug-induced phospholipidosis

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