Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells
Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells
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.
Cholesterol, phospholipid membranes, Saponin, mass spectrometry (MS), antibody therapy, Flow Cytometry
993-1007
Smith, Wendy S.
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Baker, Ella J.
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Holmes, Suzanne E.
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Koster, Grielof
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Hunt, Alan N.
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Johnston, David A.
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Flavell, Sopsamorn U.
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Flavell, David J.
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28 February 2017
Smith, Wendy S.
83c13f47-f5f9-47dc-9dc8-ee4e486030da
Baker, Ella J.
7cd5b762-d7d7-4584-b9a7-dba555085440
Holmes, Suzanne E.
df2f1eed-45a4-4caf-ac64-542d91558bd1
Koster, Grielof
e404c38a-6f48-430a-adf0-5208228cb9e7
Hunt, Alan N.
95a3e223-da96-40e7-b47d-27dce014e305
Johnston, David A.
b41163c9-b9d2-425c-af99-2a357204014e
Flavell, Sopsamorn U.
fa2b4670-1836-42e2-b68a-5d646899d711
Flavell, David J.
3a0f7124-7d44-42bc-b6f6-6fb12552fbd6
Smith, Wendy S., Baker, Ella J., Holmes, Suzanne E., Koster, Grielof, Hunt, Alan N., Johnston, David A., Flavell, Sopsamorn U. and Flavell, David J.
(2017)
Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1859 (5), .
(doi:10.1016/j.bbamem.2017.02.013).
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.
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Accepted/In Press date: 20 February 2017
e-pub ahead of print date: 21 February 2017
Published date: 28 February 2017
Keywords:
Cholesterol, phospholipid membranes, Saponin, mass spectrometry (MS), antibody therapy, Flow Cytometry
Organisations:
Clinical & Experimental Sciences
Identifiers
Local EPrints ID: 406398
URI: http://eprints.soton.ac.uk/id/eprint/406398
ISSN: 0304-4165
PURE UUID: e62f4907-a8e1-4433-a53a-0069f07bb5c1
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Date deposited: 10 Mar 2017 10:46
Last modified: 16 Mar 2024 05:03
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Contributors
Author:
Wendy S. Smith
Author:
Ella J. Baker
Author:
Suzanne E. Holmes
Author:
Grielof Koster
Author:
Alan N. Hunt
Author:
David A. Johnston
Author:
Sopsamorn U. Flavell
Author:
David J. Flavell
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