title: Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells.
abstract: Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom.
authors.forename: Wendy S
authors.initials: WS
authors.lastname: Smith
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom; Human Development and Health Academic Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.
authors.forename: Ella J
authors.initials: EJ
authors.lastname: Baker
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Holmes
authors.affiliation: NIHR Respiratory Biomedical Research Unit, UHS, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom; Clinical and Experimental Sciences, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: Grielof
authors.initials: G
authors.lastname: Koster
authors.affiliation: Clinical and Experimental Sciences, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: Alan N
authors.initials: AN
authors.lastname: Hunt
authors.affiliation: Biomedical Imaging Unit, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Johnston
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom.
authors.forename: Sopsamorn U
authors.initials: SU
authors.lastname: Flavell
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom. Electronic address: DavidF@leukaemiabusters.org.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Flavell
mesh_terms: D018941:Antigens, CD19 / Q000276:immunology*; D045744:Cell Line, Tumor; D008078:Cholesterol, LDL / Q000502:physiology*; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008223:Lymphoma / Q000737:chemistry / Q000188:drug therapy*; D008563:Membrane Lipids / Q000502:physiology*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000494:pharmacology*; D012503:Saponins / Q000494:pharmacology*; D000076985:Saporins; D014315:Triterpenes / Q000494:pharmacology*
chemical_list: D018941:Antigens, CD19; C000604811:CD19 molecule, human; D008078:Cholesterol, LDL; D000922:Immunotoxins; D008563:Membrane Lipids; D054789:Ribosome Inactivating Proteins, Type 1; D012503:Saponins; D014315:Triterpenes; D000076985:Saporins
keywords: Antibody; Cholesterol; Flow cytometry; Mass spectrometry (MS); Phospholipid; Saponin