Differential impact of synthetic antitumor lipid drugs on the membrane organization of phosphatidic acid and diacylglycerol monolayers
Graphical abstract
Introduction
Antitumor lipid (ATL) drugs, synthetic derivatives of lysophosphatidylcholine, have received considerable attention in recent years (van der Luit et al., 2007). These compounds have demonstrated both cytotoxic and cytostatic effects in cancer cells (Ruiter et al., 1999), as well as antimicrobial and antiparasitic activity (Widmer et al., 2006; Llull et al., 2007; Dorlo et al., 2012). ATLs have two key properties which make them desirable anticancer drugs: firstly, due to their amphiphilic nature, ATLs interact readily with membranes, while conventional chemotherapeutics interacting with DNA have the potential to be mutagenic (van der Luit et al., 2007). Thus ATLs alleviate many of the side effects associated with cancer treatment. Secondly, these drugs have been observed to selectively accumulate in tumor cells (Andreesen et al., 1979, 1978; Modolell et al., 1979), though the reason for cellular discrimination is not currently understood. The chemical structures of the three investigated ATL drugs are presented in Fig. 1.
The original drug, edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the archetype first generation ATL. Similar to lysophosphatidylcholine, edelfosine has a polar phosphocholine head group, a glycerol backbone, and a single hydrocarbon tail. The major difference is the presence of ether linkages connecting the C18 hydrocarbon tail at the sn1 position and methyl group at the sn2 position of edelfosine. The presence of the ether linkages in contrast to ester bonds in the native lipids makes edelfosine more metabolically stable. As the prototypic ATL drug, edelfosine is the most widely studied; however its only clinical application currently is for purging of bone marrow in acute leukemia (van Blitterswijk and Verheij, 2008). The second drug studied is miltefosine (hexadecylphosphocholine), which also has the phosphocholine headgroup which is directly conjugated to a C16 hydrocarbon tail, with no glycerol backbone present. Due to its demonstrated hemolysis, miltefosine is used mostly in topological treatments for skin metastases of breast cancer and cutaneous lymphomas (Terwogt et al., 1999). Additionally, miltefosine has been found quite effective in the treatment of leishmaniasis (Dorlo et al., 2012). Finally the third drug is perifosine (octadecyl-(1,1-dimethyl-4-piperidynio-4-yl)-phosphate). This drug is similar to miltefosine as it also lacks the glycerol backbone. However it differs in structure, due to the presence of a phosphopiperidine headgroup conjugated to its C18 hydrocarbon tail. This drug has been shown to enhance treatment when coupled with other chemotherapies (Dasmahapatra et al., 2004) and radiation (Vink et al., 2006a, b) and is utilized as an inhibitor of the PI3K/Akt pathway (Fensterle et al., 2014).
Though the mode of action of these ATL drugs is not yet fully elucidated, the current model for their mechanism includes biophysical changes in the lateral organization of biomembranes disrupting normal biochemical and metabolic processes in the cell, ultimately leading to cell death. Lateral organization refers to the assembly of lipids and membrane proteins within a single leaflet of a bilayer, whereby localized rigid regions can form platforms referred to as membrane domains. These domains have received considerable attention in biomimetic systems in vitro (van Meer et al., 2008; Rietveld and Simons, 1998; Prenner et al., 2007; Gröger et al., 2012; Nathoo et al., 2013; London, 2002; Veatch and Keller, 2005; Huang and Feigenson, 1999; Heberle et al., 2013; Feigenson, 2009; Betaneli et al., 2012; Bastos et al., 2012; Matsumori et al., 2011; Jiang et al., 2014; Graber et al., 2012; Pan et al., 2013; Vega Mercado et al., 2012; Collado et al., 2005), however the existence of discrete immiscible membrane phases in vivo (termed lipid rafts (Simons and Van Meer, 1988)) is more controversial, with the proposed model gaining both support (Brown and London, 1998a, b; Pike, 2003, 2009) and opposition (Edidin, 2003; Munro, 2003; Kraft, 2013). These regions of biomembranes are believed to be enriched in sphingolipids and sterols and act as platforms for the spatio-temporal organization of membrane proteins (Hąc-Wydro et al., 2011). The lipids within domains are in the more rigid liquid-ordered (Lo) phase, while the unsaturated glycerophospholipids that make up the remaining membrane are primarily in the more fluid liquid-disordered phase (Ld). A variety of membrane proteins have been found to be associated with these regions (Lingwood and Simons, 2010), and their lateral organization is necessary for a wide variety of cell trafficking and signaling pathways (van Meer et al., 2008). Many of these processes are both dysregulated and crucial to the viability of cancer cells, and as such, membrane domains provide an exciting target for cancer therapy.
Monolayer studies are ideal for investigating lipid packing and stability as well as lateral domain organization (Brockman, 1999; Brown and Brockman, 2007), and observations made at surface pressures between 30−35 mN/m in monolayers represent lipid packing densities equivalent to bilayers (Marsh, 1996). Monolayer studies can be coupled with other techniques for studying film organization (Dynarowicz-Łątka et al., 2001) including Brewster angle microscopy (BAM) (Hönig and Möbius, 1992; Hénon and Meunier, 1991). BAM allows direct and label-free visualization of the lateral organization of simple and more complex biomimetic monolayers at the air – water interface (Daear et al., 2017). BAM has been previously used to investigate membrane extracts of lung surfactant (Discher et al., 1996; Schief et al., 2003; Ding et al., 2003; Winsel et al., 2003; Alonso et al., 2004; Taeusch et al., 2005; Nag et al., 2007; Pinheiro et al., 2013), myelin (Rosetti and Maggio, 2007; Rosetti et al., 2008, 2005), and tear film (Kaercher et al., 1993; Tsanova et al., 2014; Georgiev et al., 2011; Ivanova et al., 2015), as well as whole cell bacterial extracts (Zerrouk et al., 2008; Benamara et al., 2011), as well as cortical vesicles obtained from the oocytes of sea urchins (Strongylocentrotus purpuratus) (Mahadeo et al., 2015a). Previously, our group has characterized the effect of edelfosine treatment on the lateral membrane organization of Saccharomyces cerevisiae (Mahadeo et al., 2015b). In that study BAM imaging revealed that treatment with edelfosine resulted in a breakdown in domain structure in the membrane extracts. Here we postulated that the complementary geometries of the ATL and cholesterol helped facilitate the strong interactions between the molecules, and suggested that PA and DAG could have similar interactions due to their similar cone geometry (negative curvature). In this current work, monolayers of PA or DAG were prepared in the absence and presence of one of the ATL drugs. The packing and stability of these films were observed using surface pressure – area isotherms, and the lateral organization was imaged using BAM. In order to better understand the relationship between DAG, PA and the ATL, an equimolar mixed monolayer of PA and DAG was also investigated.
Section snippets
Materials
Dimyristoyl phosphatidic acid (DMPA) and dimyristoyl glycerol (DMG) were purchased as lyophilized powders from Avanti polar lipids (Alabaster, USA). Edelfosine was purchased from Medmark Pharma GmbH (Oberhaching, Germany), Miltefosine from Echelon Biosciences Inc. (Salt Lake City, USA) and Perifosine from Sigma Aldrich (Oakville, Canada). Monolayer experiments were all performed on highly purified water from a Millipore Synergy 185 System with resistivity of 18.2 MΩ.cm purchased from Thermo
Analysis of ATL monolayers
The surface pressure – area isotherms for the ATLs edelfosine, miltefosine and perifosine are presented in Fig. 2 (left panel). All three drugs formed stable monofilms, which initially exhibited a gas phase, characterized by very little intermolecular interactions, then transitioned to the fluid liquid expanded (LE) phase. While some lipids upon further compression will enter into the more rigid liquid condensed (LC) phase, the ATL drugs did not, and collapsed at pressures between 33−35 mN/m.
Discussion
In this work, monolayers comprised of DMPA, DMG or a 1:1 DMPA: DMG mixture, were analyzed using monolayers in the absence and presence of 10 mol % of edelfosine, miltefosine or perifosine. As mentioned before, these studies were of interest due to the complementary geometery both PA and DAG have (cone shaped, negative curvature) to the ATLs (inverted cone shaped, positive curvature). These presented experiments were performed to better understand the effect of the drugs on packing, stability
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Acknowledgements
This work was funded by NSERC DG grant to EJP and Queen Elizabeth II graduate scholarships to MM in 2015 and 2016.
References (85)
- et al.
More than a monolayer: relating lung surfactant structure and mechanics to composition
Biophys. J.
(2004) - et al.
Selective sensitivity of chronic myelogenous leukemia cell populations to alkyl-lysophospholipids
Blood
(1979) - et al.
Diacylglycerol and the promotion of lamellar-hexagonal and lamellar-isotropic phase transitions in lipids: implications for membrane fusion
Biophys. J.
(1996) - et al.
Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes
Chem. Phys. Lipids
(2012) - et al.
Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aeruginosa
Biochim. Biophys. Acta
(2011) - et al.
Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV)
Chem. Phys. Lipids
(2012) Lipid monolayers: why use half a membrane to characterize protein-membrane interactions?
Curr. Opin. Struct. Biol.
(1999)- et al.
Diacylglycerol, when simplicity becomes complex
Trends Biochem. Sci.
(2007) - et al.
Applications of Brewster angle microscopy from biological materials to biological systems
Biochimica et Biophysica Acta (BBA)-Biomembranes
(2017) - et al.
Lateral phase separation in interfacial films of pulmonary surfactant
Biophys. J.
(1996)
Modern physicochemical research on Langmuir monolayers
Adv. Colloid Interface Sci.
Streptomyces chromofuscus phospholipase D interaction with lipidic activators at the air–water interface
Biochim. Biophys. Acta (BBA) – Biomembr.
Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures
Biochim. Biophys. Acta (BBA) – Biomembr.
Diacylglycerols, multivalent membrane modulators
Chem. Phys. Lipids
Structure and functional properties of diacylglycerols in membranes
Prog. Lipid Res.
Phosphatidylinositol-4,5-bisphosphate ionization and domain formation in the presence of lipids with hydrogen bond donor capabilities
Chem. Phys. Lipids
Real-time imaging of lipid domains and distinct coexisting membrane protein clusters
Chem. Phys. Lipids
Edelfosine disturbs the sphingomyelin–cholesterol model membrane system in a cholesterol-dependent way – the Langmuir monolayer study
Colloids Surf. B Biointerfaces
Reflectometry at the Brewster angle and Brewster angle microscopy at the air-water interface
Thin Solid Films
A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers
Biophys. J.
Insights into lipid raft structure and formation from experiments in model membranes
Curr. Opin. Struct. Biol.
Secretory vesicle cholesterol: correlating lipid domain organization and Ca2+ triggered fusion
Biochim. Biophys. Acta
Disruption of lipid domain organization in monolayers of complex yeast lipid extracts induced by the lysophosphatidylcholine analogue edelfosine in vivo
Chem. Phys. Lipids
Lateral pressure in membranes
Biochim. Biophys. Acta (BBA)-Rev. Biomembr.
Fluorinated cholesterol retains domain-forming activity in sphingomyelin bilayers
Chem. Phys. Lipids
Lipid rafts: elusive or illusive?
Cell
Visualizing a multidrug resistance protein, EmrE, with major bacterial lipids using Brewster angle microscopy
Chem. Phys. Lipids
Edelfosine—a new antineoplastic drug based on a phospholipid-like structure: the Langmuir monolayer study
Colloids Surf. A Physicochem. Eng. Asp.
Using small-angle neutron scattering to detect nanoscopic lipid domains
Chem. Phys. Lipids
Lipid rafts bringing order to chaos
J. Lipid Res.
The challenge of lipid rafts
J. Lipid Res.
Interplay of mycolic acids, antimycobacterial compounds and pulmonary surfactant membrane: a biophysical approach to disease
Biochim. Biophys. Acta
Imaging of the domain organization in sphingomyelin and phosphatidylcholine monolayers
Chem. Phys. Lipids
The differential miscibility of lipids as the basis for the formation of functional membrane rafts
Biochi. Biophys. Acta (BBA) – Rev. Biomembr.
Protein-induced surface structuring in myelin membrane monolayers
Biophys. J.
The Folch-Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers
Biochim. Biophys. Acta
The self-organization of lipids and proteins of myelin at the membrane interface
Biochim Biophys Acta.
Liquid-crystalline collapse of pulmonary surfactant monolayers
Biophys. J.
Inactivation of pulmonary surfactant due to serum-inhibited adsorption and reversal by hydrophilic polymers: experimental
Biophys. J.
Seeing spots: complex phase behavior in simple membranes
Biochim. Biophys. Acta (BBA) – Mol. Cell Res.
Modulation of the domain topography of biphasic monolayers of stearic acid and dimyristoyl phosphatidylcholine
Chem. Phys. Lipids
Phase I and pharmacokinetic study of combined treatment with perifosine and radiation in patients with advanced solid tumours
Radiother. Oncol.
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