Interaction of the anti-cancer drug cisplatin with phosphatidylserine in intact and semi-intact cells.

The anti-cancer drug cisplatin (cis-diamminedichloroplatinum(II)) forms a stable coordination complex with phosphatidylserine (PS) in model membrane systems (Speelmans et al., Biochemistry 36 (1997) 10545-10550). Because a similar interaction in vivo would be expected to have important physiological implications we studied cisplatin-PS interaction in human erythrocytes and tumor cell lines. Although cisplatin was efficiently taken up by intact erythrocytes, a cisplatin-PS complex was only detected in cells which had lysed as a result of prolonged storage or hypotonic shock. Despite the use of highly sensitive detection methods, and despite efficient cellular uptake of cisplatin, a complex could also not be detected in four human tumor cell lines, unless cells were permeabilized. In experiments in which cisplatin was incubated with PS-containing liposomes in the presence of an alternative cellular substrate, such as reduced glutathione, the relative affinity of cisplatin for PS was found to be low. Moreover, loading erythrocyte ghosts with physiological concentrations of glutathione strongly reduced cisplatin-PS complexation. Thus, in intact (tumor) cells a complex is not detected, most likely, because of the presence of higher affinity substrates. Though a transient complexation of cisplatin to PS cannot be excluded, our data suggest that cisplatin-PS does not play a direct role in the cellular (cyto)toxicity of cisplatin.

Cisplatin complexes with phosphatidylserine in membranes.

Upon incubation of the anticancer drug cisplatin [cis-diamminedichloroplatinum(II)] with model membranes composed of phosphatidylserine (PS), a stable product is formed that has been isolated after chloroform/methanol extraction of the sample. The product formation is specific for PS and does not occur with other major membrane phospholipids. The rate and extent of product formation is dependent on the pH, chloride ion concentration, and temperature, with the highest rate at pH 6.0, in the absence of Cl- and at 37 degrees C, indicating that positively charged aquated cisplatin is the reactive species. Over 80% of PS is converted within 15 h under these conditions with a halftime of 5 h. PS can be regenerated by an excess of glutathione. Mass spectrometry experiments demonstrate that interaction of cisplatin with PS involves a loss of two chloride ions and coordination of platinum to the amine and carboxyl group of the serine moiety. Cisplatin forms complexes specifically with PS not only in model membranes but also in the plasma membrane of human erythrocytes. Since PS is essential in several cellular processes, its interaction with cisplatin may have important physiological implications.

Transport of the anti-cancer drug doxorubicin across cytoplasmic membranes and membranes composed of phospholipids derived from Escherichia coli occurs via a similar mechanism.

An assay was developed to measure and directly compare transport of doxorubicin across right-side-out cytoplasmic membrane vesicles (ROV) and across model membranes (LUVET) composed of pure phospholipids, isolated from the corresponding cells. Escherichia coli was used as a model organism, since mutants are available which differ in phospholipid composition. Both in LUVET and ROV only passive diffusion across the bilayer is involved, because effects of drug concentration, pH, divalent cations, the phospholipid composition, and the active transport inhibitor verapamil were comparable. Permeability coefficients were about 2-3-times higher in ROV compared to LUVET. Furthermore, in LUVET an average activation energy of 87 kJ/mol and in ROV of 50 kJ/mol was observed. These differences are suggested to result from differences in membrane order between LUVET and ROV and differences in the temperature dependence of membrane order in LUVET and ROV, respectively. Because no background carrier-facilitated doxorubicin transport seems to be present, ROV are an excellent model system to study the effect of phospholipid composition on drug transport after expression of a multidrug resistance-conferring protein. Furthermore, data of passive diffusion of doxorubicin obtained with LUVET are representative for more complex, biologically relevant membrane systems.

The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration.

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.

Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin.

It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux. However, less information is available about effects of verapamil on drug-phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs. Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes. An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil. Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism. Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids. It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane.

Transport studies of doxorubicin in model membranes indicate a difference in passive diffusion across and binding at the outer and inner leaflets of the plasma membrane.

The kinetics of passive transport of the anticancer drug doxorubicin were analyzed in relation to membrane composition in large unilamellar vesicles in which DNA was enclosed. Special attention was paid to lipids that are typical for the inner and outer leaflet of the plasma membrane of mammalian cells: Phosphatidylethanolamine and anionic phosphatidylserine versus phosphatidylcholine, sphingomyelin, and cholesterol, respectively. The presence of anionic phospholipids results in a highly efficient incorporation of the drug into biological and model membranes [de Wolf, F. A., et al. (1993) Biochemistry 32, 6688-6695]. Therefore, the effect of drug binding on the amount of free, transportable drug was explicitly taken into account. However, even after correction for binding the permeability coefficient was about 35% lower in membranes containing 50 mol % of the anionic phosphatidylserine than in membranes consisting only of zwitterionic phospholipids (0.71-0.79 versus 1.18-1.25 microns s-1). This shows that drug binding and insertion also affect the intrinsic transport characteristics of the membranes. As compared to pure phosphatidylcholine, binding was not influenced by the incorporation of sphingomyelin or cholesterol, but equimolar amounts of sphingomyelin and cholesterol in phosphatidylcholine membranes decreased the rate of doxorubicin transport by 60% and 80%, respectively. The inhibitory effect of these two lipids is probably due to a closer packing of the membranes. In accordance, after the acyl chain order was decreased by adding the anaesthetic-like phenethyl alcohol (0.5% v/v), transport was stimulated more than 4-fold. The implications of our findings for the functioning and rate of drug pumping by the multidrug resistance-conferring P-glycoprotein in cancer cells are discussed.

Role of anionic phospholipids in the interaction of doxorubicin and plasma membrane vesicles: drug binding and structural consequences in bacterial systems.

Anthracycline-membrane interactions play a role in the transport, the cytoplasmic distribution, and possibly also the activity of anthracyclines. Previous work on model membranes has shown that the widely-applied anticancer drug doxorubicin interacts specifically with anionic phospholipids [de Wolf, F. A., et al. (1991) Biochim. Biophys. Acta 106, 67-80]. We have now been able to investigate these interactions, and their selectivity for anionic phospholipids, directly in plasma membranes. Because of the recent availability of Escherichia coli mutants in which the anionic phospholipid content ranges from only 10% to as much as 100% of the total phospholipid content, we used this bacterium as a source of plasma membranes. We compared the interactions of the cationic anthracycline doxorubicin with (1) plasma membranes of different mutant strains, (2) total lipid extracts of these membranes, and (3) synthetic phospholipid mixtures in which a comparable fraction of the phospholipids was negatively charged. The results show that anionic phospholipids are important determinants of doxorubicin binding, not only in model membranes but also in plasma membrane systems. Only in plasma membranes with a very low anionic lipid content was the binding to the anionic phospholipid masked by other factors. Using an unsaturated fatty acid auxotroph grown on [11,11-2H2]oleic acid, it appeared from 2H-NMR data that doxorubicin induces a disordering of acyl chains in bacterial plasma membranes and their total lipid extracts. This indicates that the binding is not purely electrostatic but involves the insertion of drug molecules into the lipid matrix, probably due to hydrophobic interactions.