A. C. Olson

Comparison of selective cytotoxicity of alkyl lysophospholipids.

Alkyl lysophospholipids have been shown to be cytooxic to a number of neoplastic tissues. One, ET-18-OCH3, has been used to selectively purge leukemic cells from mixtures with normal marrow progenitor cells,in vitro andin vivo. We have measured the 50% inhibitory (IC50) effect of a series of ether, lipids (EL) on leukemic cells (HL60, K562, Daudi, KG-1, KG-1a) and normal marrow progenitor cells. Cells were incubated with varying concentrations of EL for 4 hr and assayed for viability, [3H]thymidine incorporation and clonogenicity in semi-solid media. The effect on protein kinase C (PKC) activity was assayed for each compound. Compounds tested included three glycerophosphocholine analogs-ET-18-OCH3, ET-16-NHCOCH3, and BM 41.440. In addition, a lipoidal amine, CP 46665, an ethyleneglycolphospholipid, AEPL, and four single chain alkylphosphocholine analogs, HePC2, HePC3, HePC4 and HePC6 were also tested. During the period of incubation, the cells remained viable (>70%) as judged by trypan blue dye exclusion. The glycerophosphocholines were the most active and showed the highest therapeutic index. The lipoidal amine was active, but toxic to normal marrow progenitor cells. The ethyleneglycolphospholipid was active against HL60, but not against the other cell lines. The single chain alkylphosphocholine analogs were less active. All of the compounds inhibited PKC activity; however, the glycerophosphocholines were the most inhibitory.

Experimental studies on the role of alkyl lysophospholipids in autologous bone marrow transplatation

The selective cytocidal effect of alkyl lysophospholipids against neoplastic cells while sparing normal cells make these ideal candidates for purging leukemic cells from bone marrows obtained during remission. To test the feasibility of such an approach, a murine model and an in vitro human cell model were developed. In the murine system a mixture of normal bone marrow cells and WEHI IIIB myelomonocytic leukemic cells was incubated with varying doses of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-Me) for 24 hr before being injected into tail veins of lethally irradiated Balb/c mice. At doses of 20 and 100 μg/ml, long-term survivors were noted. The additional steps of freezing and thawing following incubation resulted in significantly longer survival with doses of 10 to 50 μg/ml, but were toxic to marrow stem cells at 100 μg/ml.In the in vitro model, normal marrow progenitor cells and leukemic cells (the promyelocytic cell line HL60) were exposed to varying concentrations of ET-Me for 1 and 4 hr alone or mixed, and clonogenicity was assayed by colony formation in semisolid medium during 7–14 days’ incubation. At doses up to 100 μg/ml exposed for 4 hr normal progenitor cells were spared and HL60 colonies eliminated. Other phospholipids analogues were less effective in eliminating leukemic cells, but spared normal progenitor cells.A survey of fresh leukemic cells found varying degrees of sensitivity to ET-Me, indicating the need for testing a variety of compounds.These studies clearly indicated the potential usefulness of alkyl lysophospholipid compounds in selectively purging leukemic cells from remission marrows for autologous bone marrow transplantation.

Structure-function relationships of alkyl-lysophospholipid analogs in selective antitumor activity

This investigation was initiated in order to delineate the structure-function relationship of the anticancer alkyllysophospholipids and assess their degree of selective cytotoxicity toward neoplastic cells. A series of glycerol phosphocholine analogs with varying substitutions in thesn-1 andsn-2 position were tested for their inhibitory activity as measured by thymidine incorporation, clonogenic assays and effects on protein kinase C activity against a series of human leukemic cell lines and healthy bone marrow progenitor cells. The IC50 was determined for each of the compounds in each cell line and healthy bone marrow cells following a 4-h incubation. The data indicated that a 16–18 carbon chain at thesn-1 coupled with a short substitution atsn-2 had the broadest antitumor activity and was the least toxic to normal bone marrow cells. The results provide a number of useful leads toward the design and development of potentially more active phospholipid compounds.

Scanning Electron Microscope Study of the Effect of Alkyl-Lysophospholipid on Bone Marrow and Leukemic Cells

Alkyl-lysophospholipids are antileukemic agents which selectively kill leukemic cells and spare normal bone marrow cells. Thus, they would be optimal agents for purging leukemic remission marrows prior to autotransplantation. Their site of action appears to be the cell membrane, but the exact mechanism is unknown. The sensitive cell line HL60 and a resistant cell line, K562, as well as normal bone marrow cells were incubated with or without 1-octadecyl-2-0-methyl-rac-glycero-3-phosphocholine for 0, 2 and 4 hours at previously determined IC50 clonogenic doses and then prepared for scanning electron microscopy. The compound exerted no significant morphological changes on the resistant cell and on the normal bone marrow cells, but resulted in loss of villi and rugae and a tendency for cell fusion in the sensitive HL60 cell line as early as a two-hour exposure. These results indicate that cell surface changes occur early after exposure to the phospholipid analog and suggests that subsequent events are mediated through initial changes in the cell membrane.

Anticancer Activity and Effect on Protein Kinase C (PKC) of Synthetic Phospholipid Analogs

The discovery that alkyl-lysophospholipids (ALP), ether analogs of naturally occurring lysophosphatidylcholine (LPC) have antitumor activity has prompted the synthesis of numerous compounds which are cytotoxic to cells by acting on components of membranes (1). These glycerol phospholipids have been shown to have antitumor activity in vitro and in vivo and to be selectively cytotoxic to neoplastic tissues while sparing normal hematopoietic progenitor cells (2–5). Although the exact mechanism of action is unknown and may be multiple, the following observations have been made.