Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione

Abstract

Summary The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1 -mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-a-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two-to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.

Role of Multidrug Resistance Protein 1 (MRP1) and Glutathione S-Transferase A1-1 in Alkylating Agent Resistance

“…Whereas, the monoglutathionyl conjugate of CHB, CHB-SG, is known to inhibit Alpha-class GST (12), again no direct comparison between inhibition by CHB-SG and the corresponding conjugate of MLP, MLP-SG, has been reported. Finally, previous studies on MRP1-mediated MLP-SG and CHB-SG transport did not report kinetic constants for the transport of these two compounds (18).…”

“…This was surprising because both of these structurally similar drugs ( Fig. 1) are reportedly substrates of Alpha-class GST (12, 14 -17), and because their monoglutathionyl conjugates are transported by MRP1 (18). To * This work was supported by National Institutes of Health Grants CA70338 and CA64579.…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…Most of these discrepancies were resolved by the findings that several drugs are transported by MRP1 as glutathione or glucuronide conjugates or require GSH for their transport. The former is exemplified by chlorambucil-GS, melphalan-GS, or etoposide-glucuronide [9,60], while the latter is represented by vincristine and daunorubicin [86,87,133]. Overexpression of MRP1 has been found in numerous drug-selected cell lines derived from various tumors, including lung cancers, breast cancer, gastric and colon carcinomas, prostate cancer, melanoma, neuroblastoma and glioma, fibrosarcoma and epidermoid carcinoma, as well as diverse types of leukemias.…”

2,2'-Dihydroxychalcone, a glutathione transferase inhibitor, sensitises human colon adenocarcinoma cells to chlorambucil and melphalan, but not to actinomycin D

“…Previous studies have indicated that plant polyphenols sensitise human tumour cells to chlorambucil by inhibiting GST activity (8), glutathione reductase (GSH-RD) (9) and the transport of GSH conjugates (8,10). Increased expression of glutathione S-transferase (GST) (11) and GS-X pump (12) was also observed in melphalan-resistant tumour cells. Moreover, the inhibition of GST by ethacrynic acid (13)(14)(15) and the depletion of GSH by buthionine sulphoximine (16) potentiated melphalan cytotoxicity in tumour cells.…”