Lysosome-dependent cell death and deregulated autophagy induced by amine-modified polystyrene nanoparticles

Abstract

Nanoparticles (NPs) typically accumulate in lysosomes. However, their impact on lysosomal function, as well as autophagy, a lysosomal degradative pathway, is still not well known. We have previously reported in the 1321N1 cell line that amine-modified polystyrene (NH2-PS) NPs induce apoptosis through damage initiated in the lysosomes leading ultimately to release of lysosomal content in the cytosol, followed by apoptosis. Here, by using a combination of biochemical and cell biological approaches, we have characterized in a mouse embryonic fibroblast cell line that the lysosomal alterations induced by NH2-PS NPs is progressive, initiating from mild lysosomal membrane permeabilization (LMP), to expansion of lysosomal volume and intensive LMP before the summit of cell death. Though the cells initially seem to induce autophagy as a surviving mechanism, the damage of NH2-PS NPs to lysosomes probably results in lysosomal dysfunctions, leading to blockage of autophagic flux at the level of lysosomes and the eventual cell death.

Lysosomal membrane permeabilization and cell death

“…By contrast, cathepsins released during LMP produce a diffuse fluorescence pattern throughout the cell . Cytosolic release of cathepsins can also be detected by Western blot of the cytosolic fractions after cell fractionation . This method can be used to estimate the extent of LMP by simultaneously determining the levels of several cathepsins of different sizes over time .…”

“…Cytosolic release of cathepsins can also be detected by Western blot of the cytosolic fractions after cell fractionation . This method can be used to estimate the extent of LMP by simultaneously determining the levels of several cathepsins of different sizes over time . Alternatively, cathepsins in cytosolic extracts can be detected using cathepsin‐specific substrates (eg, Magic Red) and a fluorescence plate reader.…”

“…Repnik et al have suggested that lysosomal enlargement by proton trapping is not sufficient to trigger membrane lysis, whereas other authors have proposed that large lysosomes are more prone to rupture . Moreover, the same lysosomotropic agents have been shown to induce both lysosomal enlargement and LMP . Further research will be necessary to better understand the characteristics of enlarged lysosomes (eg, pH, lysosomal membrane composition, content) and their role, if any, in autophagy and LMP.…”

“…Smaller fluorescein isothiocyanate (FITC)‐dextran molecules (10 and 40 kDa) are released from the lysosome in response to apoptosis‐associated LMP, while larger dextran molecules (70 and 250 kDa) are retained . Moreover, cationic nanoparticles first induce the release from the lysosome of smaller cleaved‐cathepsin D (~27 kDa), followed later by the larger cathepsin B (~37 kDa) . A recent study reported that the lysosomotropic compound l ‐leucyl‐ l ‐leucine O ‐methyl ester renders lysosomes permeable to protons prior to the release of preloaded dextrans, and that ESCRT machinery is initiated to repair the damaged membrane .…”

Control of the Endo-Lysosome Homeostasis by the Paracaspase MALT1 regulates Glioma Cell Survival

“…Impairment in autophagic flux reduces a cell's ability for bulk degradation 36 . Others have shown that lysosomal dysfunction, such as LMP, can impede upon autophagic flux and eventually lead to cell death 49,50 . Because of this, we infer that reduced autophagic flux is a downstream consequence of LMP, and ultimately contributes to LCD in our cells.…”