The degradation of platelet-activating factor and related lipids: Susceptibility to phospholipases C and D

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) is an ether-linked lipid that exhibits selective cytotoxicity toward several types of tumor cells and is relatively inactive toward normal cells under the same conditions of treatment. The mechanism of this selective cytotoxicity is unknown. We conducted studies to determine whether this compound is metabolized by phospholipases C and D and, if so, whether sensitive and resistant cells differ in their ability to degrade ET-18-OCH₃ by these enzymes. We have examined the metabolism of the L-isomer of ET-18-OCH₃, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH₃), by lysophospholipase D of rat liver microsomes and by a phospholipase D from the marine bacteriumVibrio damsela. The metabolism of L-ET-18-OCH₃ was also examined in cell culture using Madin-Darby canine kidney cells, human promyelocytic leukemia cells and human myelocytic leukemia cells. In these studies, L-ET-18-OCH₃ and related 1-O-alkyl-linked phosphocholine analogs radiolabeled with³H in the 1-O-alkyl chain were used. L-ET-18-OCH₃ was not hydrolyzed by lysophospholipase D from rat liver microsomes under conditions where cleavage of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine was observed. However, phospholipase D from the marine bacteriumV. damsela readily hydrolyzed L-ET-18-OCH₃ to 1-O-³H]octadecyl-2-O-methyl-sn-glycero-3-phosphate, demonstrating that L-ET-18-OCH₃ can be degraded by a phospholipase D. Platelet-activating factor (PAF) and lyso-PAF were also substrates for the bacterial phospholipase D. When intact cells were incubated with radiolabeled L-ET-18-OCH₃ a product was formed that was identified as 1-O-³H]octadecyl-2-O-methyl-sn-glycerol. There are two mechanisms that could account for the appearance of this product. The first involves cleavage of the compound by a phospholipase C, resulting in direct release of the diglyceride. The second possible mechanism involves cleavage by a phospholipase D to form the phosphatidic acid analog with subsequent hydrolysis to the diglyceride by a phosphohydrolase. Preliminary data support the phospholipase C-type mechanism. Regardless of which mechanism operates in intact cells, the metabolic degradation of L-ET-18-OCH₃ does not appear to be a significant factor in the selective cytotoxicity of this antitumor agent.