Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Two major issues which hamper the use of the anticancer drug cisplatin are the development of cancer cell resistance and its nephrotoxicity. One possible mechanism by which resistance is reported to develop is a reduction in drug uptake across the cell membrane. While the passive uptake of cisplatin has long been cited as an important contribution, far greater attention has been given to active modes of uptake, particularly in recent research. Using unilamellar lipid vesicles together with the stopped-flow kinetic method we show here that the permeability coefficient of cisplatin increases significantly with the chloride concentration of the medium. This supports the hypothesis that cisplatin can enter cells via passive permeation through the lipid phase of the membrane, but becomes trapped within the cytoplasm because dissociation of chloride ligands yields a membrane-impermeant positively-charged aqua derivative. This is important evidence for a major role of passive membrane diffusion in the uptake of cisplatin, and suggests that reduced cell uptake is unlikely to be a significant mechanism leading to the development of drug resistance. Studies of rubidium ion uptake into the cytoplasm of Xenopus oocytes via the Na+,K+-ATPase show significant inhibition of this ion pump when cisplatin is present in the cytoplasm. Because Na+,K+-ATPase activity is essential to the survival of all animal cells, e.g. via maintenance of cell volume, and the Na+,K+-ATPase is expressed at particularly high levels within the membranes of kidney tubules where it plays a crucial role in nutrient reabsorption, these results suggest that cisplatin-induced inhibition of the Na+,K+-ATPase is a likely contributing cause for the nephrotoxicity of cisplatin.DFG, EXC 314, Unifying Concepts in Catalysi

Membrane accessibility of glutathione

Regulation of the ion pumping activity of the Na+,K+-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane was found. Therefore, the most likely mechanism whereby the cysteine residue could become glutathionylated is via a loosening of the -β subunit association, creating a hydrophilic passageway between them to allow access of glutathione to the cysteine residue. By such a mechanism, glutathionylation of the protein would be expected to anchor the modified cysteine residue in a hydrophilic environment, inhibiting further motion of the β-subunit during the enzyme’s catalytic cycle and suppressing enzymatic activity, as has been experimentally observed. The results obtained, therefore, suggest a possible structural mechanism of how the Na+,K+-ATPase could be regulated by glutathione.Australian Research Council, National Health and Medical Research Counci