Cancer Letters

Cancer Letters

Volume 135, Issue 1, 11 December 1998, Pages 113-119
Cancer Letters

Enhancement of glucuronosyl etoposide transport by glutathione in multidrug resistance-associated protein-overexpressing cells

https://doi.org/10.1016/S0304-3835(98)00285-7Get rights and content

Abstract

Multidrug resistance-associated protein (MRP) has been shown to transport glutathione (GSH) S-conjugates such as leukotriene C4 (LTC4) and S-(2,4-dinitrophenyl)-glutathione (DNP-SG). On the other hand, it has while it has been reported that MRP-overexpressing cells exhibit decreased sensitivity to drugs which do not form GSH S-conjugates. In this study, we found that GSH affects the transport of glucuronosyl etoposide as a major metabolite of etoposide in MRP-overexpressing KB/VP-4 cells. The relative resistance level of KB/VP-4 cells to etoposide was 70-fold that of wild-type KB cells. Membrane vesicles prepared from KB/VP-4 cells exhibited markedly enhanced ATP-dependent transport of glucuronosyl etoposide as well as LTC4. Transport of glucuronosyl etoposide was augmented in the presence of GSH. Treatment of KB/VP-4 cells with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, resulted in about 75% depletion of cellular GSH levels, a four-fold increase of the sensitivity to etoposide and depression of glucuronosyl etoposide efflux. These results suggest that GSH plays a role in the enhancement of MRP-mediated glucuronosyl etoposide transport.

Introduction

The development of multidrug resistance to anticancer agents is a major problem in cancer chemotherapy. Multidrug resistance involves simultaneous resistance to a spectrum of functionally and structurally distinct anticancer agents. One form of multidrug resistance results from overexpression of the multidrug resistance protein such as P-glycoprotein and multidrug resistance-associated protein (MRP) [1], [2], [3], [4], [5]. Recently, MRP has been shown to transport glutathione (GSH) S-conjugates such as the endogenous GSH S-conjugated leukotriene C4 (LTC4) and S-(2,4-dinitrophenyl)-glutathione (DNP-SG) [4], [5]. On the other hand, it has been reported that MRP-overexpressing cells exhibit decreased sensitivity to drugs which do not form GSH S-conjugates, such as doxorubicin, vinblastine and etoposide [6], [7]. Thus, it is possible that some conjugates and compounds in addition to GSH S-conjugates are substrates for MRP.

GSH plays roles not only in the protection of cells against active oxygen species, but also in xenobiotic metabolism and transport for conjugate formation. Several studies have shown that the treatment of cells with buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (γ-GCS), results in the depletion of intracellular GSH and an increase in the sensitivity to several anticancer agents of cells independent of overexpression of the MRP gene [8], [9], [10], [11]. Thus, the intracellular GSH concentration affects the sensitivity of cells to anticancer agents.

Etoposide has a wide spectrum of activities in the treatment of small cell lung cancer and various lymphomas and leukemias [12]. A major metabolite of etoposide was identified as glucuronosyl etoposide [13] and the urinary excretion of glucuronosyl etoposide accounts for roughly 9% of an administered etoposide dose [14], [15].

It has been shown that an etoposide-selected multidrug-resistant human epidermoid cell line (KB/VP-4) overexpresses the MRP gene [16]. In this study, we clarified whether MRP mediates glucuronosyl etoposide transport out of cells as well as into membrane vesicles. In addition, we report that GSH affects MRP-mediated glucuronosyl etoposide transport.

Section snippets

Chemicals

[14,15,19,20-3H]LTC4 (165 Ci/mmol) was purchased from DuPont New England Nuclear (Boston, MA) and [3H]etoposide (1.3 Ci/mmol) was from Moravek Biochemicals (Brea, CA). Creatine phosphate (CP), creatine kinase (CK), ATP and AMP were from Oriental Yeast (Tokyo, Japan). Uridine-5′-diphosphoglucuronic acid (UDPGlcA) was from Nacalai Tesque (Kyoto, Japan). Buthionine sulfoximine (BSO), UDP β-d-glucuronosyltransferase and β-glucuronidase were purchased from Sigma (St. Louis, MO). Etoposide was kindly

Transport of LTC4 and glucuronosyl etoposide in membrane vesicles

Membrane vesicles were prepared from KB and KB/VP-4 cells and used in drug uptake studies. The MRP is known to function in the ATP-dependent transport of LTC4, an endogenous GSH S-conjugate. The vesicles were first incubated with 10 nM [3H]LTC4 in the presence of 4 mM ATP or AMP as a blank to confirm the function of the membrane vesicles prepared. As shown in Fig. 1, ATP-dependent [3H]LTC4 uptake was detected for KB/VP-4 membrane vesicles (0.86 pmol/mg protein) but little was detected for the

Discussion

MRP belongs to the ATP-binding cassette transmembrane transporter superfamily and is involved in the transport processes of many drug metabolites. Several groups have shown that MRP is a GSH S-conjugated pump [4], [5]. However, Jedlitschky et al. [18] recently reported that MRP functions in the ATP-dependent transport of not only GSH S-conjugates but also other conjugates into membrane vesicles prepared from MRP-transfected cells. In this study, we clarified whether MRP mediates glucuronosyl

Acknowledgements

The authors would like to thank Professor Michihiko Kuwano of Kyushu University for providing the KB/VP-4 cell line and Nippon Kayaku for providing etoposide. This work was supported in part by a Grant-in-Aid for Scientific Research in Japan from the Ministry of Education, Science, Sports and Culture (to T.A.).

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