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

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.