Multiparametric flow cytometry of the modulation of tumor cell membrane permeability by developmental antitumor ether lipid SRI 62-834 in EMT6 mouse mammary tumor and HL-60 human promyelocytic leukemia cells.
(+-)-2-[Hydroxy[tetrahydro-2-(octadecyloxy)methylfuran-2- yl]methoxyl]phosphinyloxy-N,N,N-trimethylethaniminium hydroxide, inner salt (SRI 62-834) is a tetrahydrofuran analogue of platelet activating factor (PAF) that is currently entering clinical trial. Like other ether lipids it is of interest as a membrane-active antitumor agent. Here, we have used two-color multiparameter flow cytometry to study simultaneously its effects on cell membrane permeability, intracellular pH, and cell size/structure of EMT6 mouse mammary tumor cells and HL-60 human promyelocytic leukemia cells in vitro. Concentrations as low as 1 microM up to 100 microM SRI 62-834 caused a rapid, dose-dependent increase in membrane permeability, initially towards outward efflux of the preloaded fluorescein probe bis(carboxyethyl)carboxyfluorescein (green fluorescence) and then towards influx of extracellular propidium (red fluorescence). At the same time, median cell size from light scatter was reduced with an increased coefficient of variation, and the proportion of cell debris was elevated. In vitro antitumor activity was seen over the same concentration range, as measured by tetrazolium dye reduction and cell growth curves. Neither low concentrations of PAF (50 nM) nor the potent PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]tria- zolo[4,3a][1,4]diazepin-2-yl]-1-(4-morpholinyl)-1-propanone (0.5-100 microM) had any influence on the membrane effects of SRI 62-834, and at higher concentrations (1-200 microM) PAF mimicked the behavior of SRI 62-834. In addition, the PAF antagonist did not modulate the cytotoxicity of SRI 62-834 or PAF. HL-60 cells were more sensitive to SRI 62-834 than were EMT6 cells in terms of both cytotoxicity and membrane permeability. However, PAF was more potent than SRI 62-834 in causing membrane permeabilization with both cell lines, whereas PAF was less active than SRI 62-834 in cytotoxicity assays. The results support a membrane-damaging role in the cytotoxicity of SRI 62-834 but suggest that additional factors are also involved. Membrane permeabilization may be related to its reported effects on protein kinase C-dependent intracellular calcium signaling but apparently does not involve a conventional PAF receptor in HL-60 or EMT6 cells.
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Medical Research Council Clinical Oncology and Radiotherapeutics Unit, Medical Research Council Centre, Cambridge, United Kingdom.
Watson
J V
JV
Workman
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D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001381:Azepines / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects*; D005434:Flow Cytometry / Q000379:methods*; D005663:Furans / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000473:pathology; D015674:Mammary Neoplasms, Animal / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D010742:Phospholipid Ethers / Q000494:pharmacology*; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014230:Triazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000970:Antineoplastic Agents; D001381:Azepines; D005663:Furans; D010742:Phospholipid Ethers; D010972:Platelet Activating Factor; D014230:Triazoles; C052518:WEB 2086; C055462:SRI 62-834; C026613:1-0-octadecyl 2-0-acetyl sn-glycero-3-phosphorylcholine