Enhancement of glucuronosyl etoposide transport by glutathione in multidrug resistance-associated protein-overexpressing cells
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.).
References (29)
- et al.
Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells
Cell
(1986) - et al.
The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates
J. Biol. Chem.
(1994) - et al.
Hepatobiliary elimination of the peroxisome proliferator nafenopin by conjugation and subsequent ATP-dependent transport across the canalicular membrane
Biochem. Pharmacol.
(1994) - et al.
Correlation between glutathione and stimulation of the pentose phosphate cycle in situ in Chinese hamster ovary cells exposed to hydrogen peroxide
Arch. Biochem. Biophys.
(1996) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles
J. Biol. Chem.
(1996) - et al.
Posttranscriptional regulation of MRP/GS-X pump and γ-glutamylcysteine synthetase expression by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea and by cycloheximide in human glioma cells
Biochem. Biophys. Res. Commun.
(1997) - et al.
Coordinated induction of MRP/GS-X pump and γ-glutamylcysteine synthetase by heavy metals in human leukemia cells
J. Biol. Chem.
(1996) - et al.
Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies
Proc. Natl. Acad. Sci. USA
(1986) - et al.
Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line
Science
(1992)