Cytotoxicity and Metabolism of Alkyl Phospholipid Analogues in Neoplastic Cells1

The cytotoxic response of several types of neoplastic cells to analogues of unnatural alkyl phospholipids (e.g., roc-l-hexadecyl-2-methoxy-glycero-3-phosphocholine) has been partially attributed to their accumulation as a result of the low activity of the alkyl cleavage enzyme (a tetrahydropteridine-dependent monooxygenase) in tumor cells. We tested this possibility by comparing the alkyl cleavage enzyme activity in cells that exhibit differences in sensitivity toward the cytotoxic effects of the rac\ -hexadecyl^-methoxy-glycero-S-phosphocholine. Human promyelocytic leukemia cells (III.-60), a cell line highly sensitive to the cytotoxic alkyl phospholipid analogue, possessed an alkyl cleavage enzyme activity (0.25 pmol/min/«igprotein) similar to that found in three cell types known to be relatively resistant to the cytotoxic activity of the analogue: imma ture human promyeloblastic leukemia cells (K562) (0.22 pmol/min/^g protein), human polymorphonuclear neutrophils (0.34 pmol/min/fig pro tein), and Madin-Darby canine kidney cells (0.37 pmol/min/Mg protein). Moreover, our results indicate that the cytotoxic rac-l-octadecyl-2-methoxy-glycero-3-phosphocholine analogue is not a substrate for the alkyl cleavage enzyme with an active microsomal preparation of the enzyme from rat liver; cleavage of this analogue was 200-fold less than the rate obtained with 1-octadecylglycerol as substrate. In cultures of either sensitive or resistant type cells, approximately 90% of the added rac-t|9',IO'-3H)octadecyl-2-methoxy-glycero-3-phosphocholine was not me tabolized during a 24-h incubation. The amount of radiolabel in fatty acids, a major product of alkyl cleavage activity, was small, and essen tially identical amounts were produced in all four cell types (3.1 ±0.2% (SD)|. These data indicate that differences in the cellular activities of the alkyl cleavage enzyme are not responsible for the differential cytotoxic responses between normal and specific types of neoplastic cells toward rac-l-octadecyl-2-methoxy-glycero-3-phosphocholine. On the other hand, the cellular uptake of the alkyl phospholipids could be a factor in explaining the cytotoxic response of certain tumor cells, since more radiolabeled l-octadecyl-2-methoxy-glycero-3-phosphocholine was as sociated with the susceptible III.-60 cells than with the resistant cell types. Autoradiography revealed that the radiolabeled 2-methoxy analogue accumulates at the periphery of 111.-60 leukemia cells, whereas the label was more uniformly distributed in polymorphonuclear neutrophils and KS62 cells. In contrast, the relatively nontoxic naturally occurring 1|l',2'-3H|alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor), an important cellular mediator involved in hypersensitivity and cardiovascular responses, is rapidly metabolized by III.-60 cells and appears evenly distributed throughout the leukemia cells as judged by autoradiography. Data from these experiments indicate that the unnatural alkyl phospholipids accumulate at the surface membrane of "sensitive" cancer cells, the site where their cytotoxic action appears to be elicited.