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.

No abstract

We describe metabolically inert l-glucosamine-based glycosylated antitumor ether lipids (L-GAELs) that retain the cytotoxic effects of the D-GAELs including the ability to kill BT-474 breast cancer stem cells (CSCs). When compared to adriamycin, cisplatin, and the anti-CSC agent salinomycin, L-GAELs display superior activity to kill cancer stem cells (CSCs). Mode of action studies indicate that L-GAELs like the D-GAELs kill cells via an apoptosis-independent mechanism that was not due to membranolytic effects.

Two ether glucosyl diglyceride analogs were synthesized, and their antiproliferative activity against four epithelial cancer cell lines was evaluated. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-beta-D- glucopyranosyl)-sn-glycerol (4) was synthesized by reaction of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl chloride with 1-O-hexadecyl-2-O-methyl-sn-glycerol followed by deacetylation by methanolic hydrolysis. The N-acetyl group of 4 was removed by hydrolysis with ethanolic potassium hydroxide to form 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-beta-D-glucopyranosyl)- sn-glycerol (5). Compounds 4 and 5 inhibited the proliferation of MCF-7, A549, A427, and T84 cancer cell lines. The IC(50) values for 5 ranged from 6.5 to 12.2 microM, whereas 4 was more effective against A549 cells (IC(50) 9 microM) than against MCF-7 (IC(50) 17 microM) and A427 (IC(50) 25 microM) cells and was inactive against T84 cells. Under identical incubation conditions, compounds 4 and 5 were potent inhibitors of the proliferation of OVCAR-3 cells with IC(50) values of 12 and 4 microM, respectively, whereas ET-18-OCH(3), hexadecylphosphocholine, and erucylphosphocholine had IC(50) values of 24, >30, and >30 microM, respectively. The cell-inhibitory profile of these ether-linked glucosyl diglycerides strengthens the hypothesis that such glycolipids represent a distinct group of antitumor ether lipids, having antineoplastic activities that differ from the well-known alkylphosphocholines and alkyllysophospholipids.

[formula: see text] A novel methodology has been developed, employing the Ramberg-Bäcklund rearrangement and ionic hydrogenation to synthesize C-glycosides with high stereoselectivity at the anomeric center. The C-glycolipid 14b exhibits antiproliferative properties similar to those of O-glycoside analogue 14a.

The potent antitumor activity of 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-β-D-glucopyranosyl)-sn-glycerol (1) was previously shown to arise through an apoptosis-independent pathway. Here, a systematic structure-activity study in which the effects of the anomeric linkage, the cationic charge and the glycero moiety on the antitumor activity is described. Eight analogues of 1 were synthesized, and their antitumor activity against breast (JIMT1 and BT549), pancreas (MiaPaCa2) and prostate (DU145, PC3) cancer was determined. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-α-D-glucopyranosyl)-sn-glycerol (2) consistently displayed the most potent activity against all five cell lines with CC(50) values in the range of 6-10 μM. However, replacement of the O-glycosidic linkage by a thioglycosidic linkage or replacement of the amino group by an azide or guanidino group leads to a threefold or greater decrease in potency. The glycero moiety also contributes to the overall activity of 1 and 2 but its effects are of lesser importance. Investigation into the mode of action of this class of compounds revealed that, in agreement with previous findings, the cytotoxic effects arise through induction of large acid vacuoles.

[figure: see text] 2-Amino C-glycerolipid 1b was synthesized by using the Ramberg-Bäcklund rearrangement as the key step. beta-C-Glycerolipid 1b exhibits in vitro antiproliferative effects strikingly similar to those of O-glycoside analogue 1a.

Glycosylated antitumor ether lipids (GAELs) are a class of amphiphilic antitumor agents that kill cancer cells by a non-apoptotic pathway.