Chapter Three - Gamma-Glutamyl Transpeptidase: Redox Regulation and Drug Resistance

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Abstract

The expression of gamma-glutamyl transpeptidase (GGT) is essential to maintaining cysteine levels in the body. GGT is a cell surface enzyme that hydrolyzes the gamma-glutamyl bond of extracellular reduced and oxidized glutathione, initiating their cleavage into glutamate, cysteine (cystine), and glycine. GGT is normally expressed on the apical surface of ducts and glands, salvaging the amino acids from glutathione in the ductal fluids. GGT in tumors is expressed over the entire cell membrane and provides tumors with access to additional cysteine and cystine from reduced and oxidized glutathione in the blood and interstitial fluid. Cysteine is rate-limiting for glutathione synthesis in cells under oxidative stress. The induction of GGT is observed in tumors with elevated levels of intracellular glutathione. Studies in models of hepatocarcinogenesis show that GGT expression in foci of preneoplastic hepatocytes provides a selective advantage to the cells during tumor promotion with agents that deplete intracellular glutathione. Similarly, expression of GGT in tumors enables cells to maintain elevated levels of intracellular glutathione and to rapidly replenish glutathione during treatment with prooxidant anticancer therapy. In the clinic, the expression of GGT in tumors is correlated with drug resistance. The inhibitors of GGT block GGT-positive tumors from accessing the cysteine in extracellular glutathione. They also inhibit GGT activity in the kidney, which results in the excretion of GSH in the urine and a rapid decrease in blood cysteine levels, leading to depletion of intracellular GSH in both GGT-positive and GGT-negative tumors. GGT inhibitors are being developed for clinical use to sensitize tumors to chemotherapy.

Introduction

In 1985, gamma-glutamyl transpeptidase (GGT, aka gamma-glutamyl transferase) was first proposed to play a role in tumor formation (Hanigan & Pitot, 1985b). Preneoplastic liver foci in rats treated with chemical carcinogens were identified by their expression of GGT (Goldsworthy, Hanigan, & Pitot, 1986). We proposed that the expression of GGT provided the cells within the foci a selective growth advantage during the promotion phase of carcinogenesis (Hanigan & Pitot, 1985b). This hypothesis was based on the observation that the treatment regimens, which gave rise to GGT-positive liver foci, all included promoting compounds that depleted glutathione (GSH). The hypothesis was that GGT, which is localized to the cell surface, cleaved extracellular GSH, thereby providing the cell with the amino acids necessary for intracellular GSH synthesis. GGT activity enabled the cells to maintain their intracellular GSH levels, thus resisting the toxicity of the promoting compounds and enabling them to respond to the proliferative signals triggered by the carcinogenic regimen. Now, decades later, there is a great deal of new information about the enzyme that supports this hypothesis. These data will be reviewed in this chapter. Further, studies from many laboratories have demonstrated that this same mechanism, through which GGT was proposed to contribute to the preneoplastic cell's resistance to the toxicity of promoting agents, also confers GGT-positive tumors with resistance to prooxidant anticancer therapy. GGT enhances the tumor's access to cysteine, thereby increasing the intracellular GSH level. This enables the tumors to maintain their redox balance during the onslaught of reactive oxygen species (ROS) generated by the prooxidant anticancer therapies and to avoid death via the cell death pathways triggered by oxidative stress. Clinical studies have shown a correlation between GGT expression in human tumors and their resistance to therapy. Studies in cell culture and data from animal models have provided information on the signaling and regulatory pathways that underlie this correlation. To understand the relationship between GGT expression and drug resistance, it is necessary to first understand the role of GGT in normal physiology. This chapter will review the current information about GGT and its role in redox regulation, its expression in tumors, its induction by toxins including many of the most commonly used chemotherapy agents, and a strategy to use GGT inhibitors to overcome the resistance of both GGT-positive and GGT-negative tumors to prooxidant anticancer therapy.

Section snippets

Expression of GGT and Drug Resistance in Human Tumors

Clinical studies show a strong correlation between the expression of GGT in tumors and poor survival. Our study of 451 human tumors prior to treatment showed that GGT is induced during the development of many tumors (Hanigan, Frierson, Swanson, & De Young, 1999). Tumors derived from ductal epithelial cells that normally express GGT were generally strongly GGT-positive (Hanigan & Frierson, 1996). These include liver, renal, prostatic, pancreatic, and breast carcinomas (Table 3.1). Due to the

Structure of GGT

In eukaryotes, GGT is a cell surface glycoprotein. It is anchored in the cell membrane via a single N-terminus transmembrane domain. All of the catalytic activity is within the extracellular domain of the protein (Ikeda, Fujii, Taniguchi, & Meister, 1995). Human GGT is synthesized as a 569 amino acid propeptide (Rajpert-De Meyts, Heisterkamp, & Groffen, 1988). The propeptide is enzymatically inactive, but is activated by autocleavage into two subunits (West et al., 2010, West et al., 2011). The

Biochemistry of GGT-Catalyzed Reactions

GGT is localized to the cell surface and only cleaves extracellular substrates. GSH and oxidized GSH (GSSG) are the most abundant physiological substrates, although GGT cleaves any gamma-glutamyl substrate including GSH S-conjugates (Wickham, West, Cook, & Hanigan, 2011). The substrate glutamate moiety must be unrestricted except for the gamma-glutamyl bond (Fig. 3.2). GGT will not cleave gamma-glutamyl bonds formed with glutamate that is within a peptide or with glutamate bound to any other

In normal tissues and in tumors

GGT is expressed on the luminal surface of excretive and absorptive cells that line glands and ducts throughout the body, with the highest level of GGT activity in the kidney (Hanigan & Frierson, 1996). The development of strains of GGT knockout mice revealed the role of GGT in the distribution of cysteine throughout the body (Harding et al., 1997, Lieberman et al., 1996, Yamada et al., 2013). GGT knockout mice excrete 2500-fold more GSH in their urine than wild-type mice (Lieberman et al., 1996

GSH and intracellular redox regulation

GSH is the most abundant nonprotein thiol in mammalian systems and the major redox buffer in the cell (Moriarty-Craige & Jones, 2004). GSH protects cellular components from oxidative damage of ROS, such as hydrogen peroxide and organic peroxides via GSH peroxidases, and also detoxifies the electrophilic metabolites of toxins, including chemotherapy drugs (Moriarty-Craige & Jones, 2004) (Fig. 3.4). The ratio of oxidized GSH (GSSG) to 2GSH can be used as a measure of the intracellular redox

The Role of GGT in Enhancing Cysteine Availability and Drug Resistance

A dramatic shift in the availability of cysteine occurs when GGT-positive cells are depolarized or GGT is induced in GGT-negative cells. Under these conditions, GGT is no longer restricted to substrates present in the fluids within ducts and glands as it is in normal tissues, but now can cleave both oxidized and reduced GSH in interstitial fluid, providing the cell access to the cystine and cysteine therein. Cameron et al. reported that in rats with neoplastic liver nodules and carcinomas, GSH

Redox Regulation of GGT

Expression of GGT and its role in redox regulation have been studied extensively in rats, mice, and humans. However, there are differences in the expression and regulation of GGT among these species that are often not recognized, but provide insight into GGT regulation and function. Therefore, we will consider the data from each species separately.

Overcoming Resistance to Prooxidant Anticancer Therapy by Inhibiting GGT

Reducing the intracellular GSH concentration sensitizes tumors to many diverse chemotherapy drugs (Butturini et al., 2013, Calvert et al., 1998, Estrela et al., 2006, Hamaguchi et al., 1993, Maciag et al., 2013, Ortega et al., 2011, Ruoso and Hedley, 2004, Traverso et al., 2013) (Crook, Souhami, Whyman, & McLean, 1986). To date, two different approaches have been evaluated clinically to try to reduce the synthesis of intracellular GSH. Sulfasalazine, a drug that blocks the cystine transporter

Summary

Expression of GGT is essential in maintaining the cysteine levels in the body. Induction of GGT expression in response to redox stress provides the cell with access to additional cysteine, which becomes rate-limiting for intracellular GSH synthesis. Our understanding of the role of GGT in redox regulation and its induction in cells under redox stress will aid in the development of new anticancer therapies. GGT first came to the attention of cancer researchers as a biomarker for preneoplastic

Acknowledgments

We gratefully acknowledge Dr. Stephanie Wickham's assistance in preparing Fig. 3.3. This work was supported in part by the National Institutes of Health Grants P20GM103640 (an Institutional Development Award (IDeA)).

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