A glycosylated antitumor ether lipid kills cells via paraptosis-like cell death

Abstract

Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3-green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.

1-O-Hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-∃-D-glucopyranosyl)-sn-glycerol (Gln) induces cell death with more autophagosomes which is autophagy-independent

“…Here we have shown with a range of assays, including of LC3-II synthesis studies (in the presence of bafilomycin A1), studies with GFP-RFP-LC3, and assays of both endogenous and exogenous autophagy substrate accumulation/degradation, that Gln primarily acts to decrease autophagic flux by impairing lysosomal acidification. However, our data showing that Gln-induced cell death is independent of autophagy and caspases are compatible with those of Sammader et al 26 Here, we have advanced the understanding of the cell death mechanism by revealing that this is due to leakage of cathepsins from lysosomes. Thus, this cell death is independent of autophagy and caspases, but appears to be due to leakage of cathepsins from lysosomes.…”

“…26 Indeed, this situation has some similarities to what was previously noted by Overmeyer et al 29 and represents a form of cell death mediated by lysosomes. 30 It has been previously suggested that some lysosomotropic amines can act as detergents, once they reach a high enough concentration in the lysosomes.…”

“…Indeed caspase inhibition did not influence the toxicity of Gln. 26 The nonapoptotic nature of the cell death caused by Gln was supported by the absence of a loss of mitochondrial membrane potential or leakage of cytochrome c into the cytoplasm (Fig. 1E and F).…”

“…14 To further investigate this effect on acidification, wild-type MEFs were stained with acridine orange for 15 min and then treated for 4 hours with 7.5 μM Gln or with 400 nM bafilomycin as a control. For this aim, acridine orange dye represents an interesting tool, in that it emits fluorescence around 525 nm (green) when cytosolic or in the nucleoli, upon excitation by blue light, but can also act a lysosomotropic dye, i.e., upon protonation fibroblasts and HeLa cells), 26 thus its effects may be fairly generic. Sammader et al 26 interpreted their data showing increased numbers of autophagosomes caused by Gln to be due to increased synthesis.…”

“…For this aim, acridine orange dye represents an interesting tool, in that it emits fluorescence around 525 nm (green) when cytosolic or in the nucleoli, upon excitation by blue light, but can also act a lysosomotropic dye, i.e., upon protonation fibroblasts and HeLa cells), 26 thus its effects may be fairly generic. Sammader et al 26 interpreted their data showing increased numbers of autophagosomes caused by Gln to be due to increased synthesis. Here we have shown with a range of assays, including of LC3-II synthesis studies (in the presence of bafilomycin A1), studies with GFP-RFP-LC3, and assays of both endogenous and exogenous autophagy substrate accumulation/degradation, that Gln primarily acts to decrease autophagic flux by impairing lysosomal acidification.…”