Molecular analysis of the massive GSH transport mechanism mediated by the human Multidrug Resistant Protein 1/ABCC1

Rachad Nasr,Ahcène Boumendjel,Basile Pérès,Ruttiros Khonkarn,Doriane Lorendeau,Jean-Claude Cortay,Vincent Chaptal,Pierre Falson,Lauriane Dury,Hélène Baubichon-Cortay

doi:10.1038/s41598-020-64400-x

Abstract

The transporter Multidrug Resistance Protein 1 (MRP1, ABCC1) is implicated in multidrug resistant (MDR) phenotype of cancer cells. Glutathione (GSH) plays a key role in MRP1 transport activities. In addition, a ligand-stimulated GSH transport which triggers the death of cells overexpressing MRP1, by collateral sensitivity (CS), has been described. This CS could be a way to overcome the poor prognosis for patients suffering from a chemoresistant cancer. The molecular mechanism of such massive GSH transport and its connection to the other transport activities of MRP1 are unknown. In this context, we generated MRP1/MRP2 chimeras covering different regions, MRP2 being a close homolog that does not trigger CS. The one encompassing helices 16 and 17 led to the loss of CS and MDR phenotype without altering basal GSH transport. Within this region, the sole restoration of the original G1228 (D1236 in MRP2) close to the extracellular loop between the two helices fully rescued the CS (massive GSH efflux and cell death) but not the MDR phenotype. The flexibility of that loop and the binding of a CS agent like verapamil could favor a particular conformation for the massive transport of GSH, not related to other transport activities of MRP1.

Highlights

Cancer cells develop resistance to chemotherapeutic treatments through different mechanisms
We used a strategy based on MRP1/MRP2 chimeras to screen for regions and residues of MRP1 that are essential for the Collateral Sensitivity (CS) agents-mediated stimulation of GSH efflux and attempted to discriminate these regions from that involved in drug transport
One of the most promising strategies to overcome this multi-drug resistance, MRP1-mediated CS, relies on the ability of certain ligands of MRP1 to induce a massive MRP1-dependent GSH efflux, that depletes the cells of their anti-oxidant defense, leading to their selective cell death[15]

Summary

Introduction

Cancer cells develop resistance to chemotherapeutic treatments through different mechanisms. We have shown that the stimulation of GSH efflux by verapamil, a MRP1 ligand[11], triggers the selective cell death of BHK21 (Baby Hamster Kidney 21) cells overexpressing MRP1 following a rapid and massive depletion of cellular GSH12 This led us to propose and develop a strategy that was first discovered in 1952 by Szybalski and Bryson[13]; it is called “Collateral Sensitivity (CS)”[14]. In polarized cells, MRP2 is able of anti-cancer drugs transport, its specificity and affinities are generally different from MRP128–30 Taken together, this suggests that the structural determinants of substrate transport, notably GSH and drugs are different in MRP1 and MRP2. In the light of these results, we proposed a mechanistic hypothesis to explain the strong efflux of glutathione observed in the presence of our CS ligands

Methods

Results

Conclusion