Elsevier

Biochemical Pharmacology

Volume 73, Issue 11, 1 June 2007, Pages 1727-1737
Biochemical Pharmacology

Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress

https://doi.org/10.1016/j.bcp.2007.02.005Get rights and content

Abstract

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC4) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels.

Introduction

Drug resistance is a major cause of treatment failure in clinical oncology [1]. Multidrug resistance (MDR) involves cross-resistance to a range of chemically unrelated agents with different cellular targets. The over-expression of ATP binding cassette (ABC) membrane proteins as causative proteins of MDR have been studied intensely over the past two decades [1]. These ABC proteins are, the P-glycoprotein 1 (P-gp1 or ABCB1), the breast cancer resistance protein (BCRP or ABCG2), and the multidrug resistance protein 1 (MRP1 or ABCC1) [2]. Although the molecular mechanism by which the latter ABC proteins mediate the transport of structurally diverse drugs remains elusive, ABC proteins have been shown to bind and transport various ligands, including anti-cancer drugs, in an ATP-dependent manner.

ABCC1 was first isolated from a doxorubicin-resistant small lung cancer line H69AR [3], [4], [5], [6]. The protein consists of 1531 amino acids, and when fully glycosylated, has a molecular mass of 190 kDa. ABCC1 is ubiquitously expressed in most normal tissues, with higher levels in lung, kidney, testis and blood mononuclear cells [6], [7]. ABCC1 causes resistance to a broad spectrum of compounds including natural product drugs such as epipodophyllotoxins, vinca alkaloids, and certain anthracyclines [8], [9], [10]. Furthermore, it transports a variety of substrates that are conjugated to glucoronide, sulfate, or glutathione [11], [12], [13], [14]. Leukotriene C4 (LTC4), a conjugate of glutathione is the highest affinity substrate of ABCC1 [11], [12]. Moreover, ABCC1 transports both reduced (GSH) and oxidized (GSSG) glutathione. GSH transport was observed in ABCC1-expressing H69AR cells, consistent with an earlier report by Cole et al. [15] which have shown that ABCC1-expressing H69AR cells have reduced cellular GSH levels compared to parental H69 cells. Later studies showed that GSH affinity for ABCC1 increases (from Km 10–20 mM to 10–100 μM) in the presence of certain drugs such as daunorubicin and vincristine [16], [17]. This increase in GSH transport in the presence of these drugs appears to be mediated by co-transport mechanism [18]. The importance of GSH in ABCC1 activity is illustrated by the reduction in transport of many substrates when GSH production is inhibited with l-buthionine (S,R)-sulfoximine (BSO) [18], [19], [20]. BSO is the most specific and least toxic inhibitor of γ-glutamylcysteine synthetase (γ-GCS), thereby preventing de novo synthesis of GSH [21]. Several other compounds not transported by ABCC1 have been shown to stimulate ABCC1-mediated GSH export. Two such molecules that stimulate GSH transport are the calcium channel blocker verapamil (VRP) and the flavonoid apigenin (API) [22], [23].

All aerobic organisms are subject to physiological oxidant stress as a consequence of aerobic metabolism. The production of ATP through oxidative phosphorylation leads to the formation of superoxide radicals (O2radical dot). This radical can then form other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and the hydroxyl radicals (OHradical dot) [24]. These reactive compounds cause lipid peroxidation and can disrupt metabolic processes. GSH is the predominant defense against these toxic products of oxygen, particularly in mitochondria [25]. GSH is a major non-protein intracellular thiol that participates in redox reactions maintaining a reducing environment in the cell. Consequently, low GSH levels are sometimes associated with mitochondrial dysfunction and induction of apoptosis [26], [27].

The present study explores some of the cellular consequences of GSH modulation in ABCC1 expressing cells. Our findings show that ABCC1-expressing tumor cell lines (H69AR or HelaABCC1 transfectant) are hypersensitive to a reduction in intracellular GSH. ABCC1-mediated drop in GSH levels appears to trigger apoptosis and is preceded by a burst of reactive oxygen species. This effect was triggered either by reducing GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. These observations demonstrate the mechanism by which ABCC1 expressing cells are hypersensitive to reductions in cellular GSH levels that can lead to avenues of targeting ABCC1 positive tumor cells.

Section snippets

Materials

Protein-A Sepharose CL-4B and carrier-free Na125Iodine (100 mCi/mL) were purchased from Amersham Biosciences (Baie d’Urfe, Quebec). The ABCC1 polyclonal Ab was produced as describe below; polyclonal anti-actin [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33] was purchased from Sigma (St. Louis, MO). NHS-ASA and ImmunoPure immobilized protein G were purchased from PIERCE (Rockford, IL). RPMI 1640 and α-MEM media were purchase from Gibco-BRL. Verapamil, apigenin,

Results

The initial characterization of the drug resistant H69AR cell line demonstrated that these cells displayed collateral sensitivity to BSO relative to their parental H69 cells. In light of more recent evidence for the role of ABCC1 in GSH transport, these findings implied that the over-expression of ABCC1 may reduce endogenous GSH levels rendering the cells hypersensitive or collaterally sensitive to BSO. The results in Fig. 1A shows that BSO has no effect on the H69 parental cells up to a

Discussion

ABCC1 has been shown to mediate the transport of endogenous cell metabolites and anti-cancer drugs via an ATP-dependent efflux mechanism [43]. One such endogenous substrate of ABCC1 is the tripeptide GSH, a major cellular detoxifying compound [44], [45]. In this report we demonstrate that over-expression of ABCC1 contributes to cell death by oxidative stress through drug enhanced GSH efflux. Several studies have now demonstrated that GSH plays an important role in ABCC1 functions. In one

Acknowledgements

The authors would like to thank Dr. S. Cole at the Cancer Research Laboratory of Queen's University for her kind gift of SCLC cell lines (H69, H69AR and H69PR). The HeLaABCC1 cells were a kind gift from Dr. P. Gros at the Department of Biochemistry of McGill University. This work was supported by funds from the Natural Sciences and Engineering Research Council of Canada (NSERC) to EG. Research at the Institute of Parasitology is supported by an FQRNT Center Grant.

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