An active endocytosis pathway is required for the cytotoxic effects of glycosylated antitumor ether lipids

Glycosylated antitumor ether lipids (GAELs) kill cells by an apoptosis-independent pathway. A hallmark of this pathway is the formation of large acidic vacuoles; however, very little is known about the process. We examined the hypothesis that 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-β-D-glucopyranosyl)-sn-glycerol (Gln), a potent GAEL, diffuses across cell membranes into lysosomes, where protonation of the amine leads to its accumulation and generation of the vacuoles.

N-Benzylamine analogs with similar pKa values, but with greater hydrophobicity than the parental Gln were synthesized and their activities against epithelial cancer cell lines were compared. The role of endocytosis in Gln action was investigated by inhibiting endocytosis with methyl-β-cyclodextrin (MCD), and inhibiting the maturation of the endocytic vesicles by low temperature incubation and analyzing their effects on Gln activity.

The N-benzylamines were either inactive or less active than Gln, indicating that activity was unrelated to diffusion or protonation. Toxicity was only observed with analogs that generated vacuoles. The incubation of cells with MCD inhibited the generation of the vacuoles and the toxic effects of Gln. The toxic effect of Gln was inhibited when cells were incubated with the drug at 20°C, a temperature that inhibits the maturation of early endosomes.

The results of the study show that GAELs are taken up by endocytosis and an active endocytic pathway is required for the formation of large acidic vacuoles by GAELs and manifestation of their cytotoxic effects.