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From: Cancer Drug Discovery and Development: Cancer Drug Resistance Edited by: B. Teicher © Humana Press Inc., Totowa, NJ
Glutathione and Glutathione
S-Transferases in Drug Resistance
Victoria J. Findlay, P h D , Danyelle M. Townsend, P h D , and Kenneth D. Tew, P h D
C
ONTENTSGENERAL INTRODUCTION
GLUTATHIONE GSHIN SIGNALING
GLUTATHIONES-TRANSFERASE
GSHAND GSTS AS THERAPEUTIC AGENTS
CONCLUDING REMARKS REFERENCES
SUMMARY
The major roles of glutathione (GSH) and glutathione S-transferases (GSTs) in the detoxification of xenobiotics predicts their important role in drug resistance. As such, both GSH and GSTs have been manipulated as targets in the design of novel chemo- therapeutic drugs. The discovery that GSTs have additional roles in the cell as regula- tory molecules in the mitogen-activated protein kinase pathways together with the more recent discovery of GSH as a regulatory posttranslational modification lend further weight to their already important roles in the anticancer drug resistance response. These findings highlight the importance of these targets in the creation of future novel anti- cancer drugs. This chapter gives a brief overview of the importance of both GSH and GST in the response to anticancer drug resistance, and highlights some of the anticancer drugs currently being investigated at various stages in the process from lab to clinic.
Key Words: Glutathione; glutathione S-transferase; drug resistance; cancer; MAPK pathway.
1. GENERAL INTRODUCTION
Reactive oxygen species (ROS) are generated as a result of normal cellular metabo- lism, which is critical for the generation of energy in biological systems. Although low amounts of ROS are easily tolerated by the cell, abnormally high levels of ROS induce
oxidative stress (OS), leading to cellular damage. In fact, ROS are implicated in a wide variety of diseases including Parkinson’s, Alzheimer’s, and cancer (1). ROS are also produced after exposure to ionizing radiation, selected chemotherapeutic agents, hyper- thermia, inhibition of antioxidant enzymes, or depletion of cellular reductants such as NADPH and glutathione (GSH). Consequently, cells have evolved protective mecha- nisms including antioxidants that detoxify ROS, and tolerable levels are maintained because of a complex redox buffering system.
The sensitivity of cells to OS depends on their intrinsic antioxidant systems, in particu- lar, the levels of GSH within the cell. When GSH levels are low, the cellular environment will be oxidizing and the functioning of enzymes, particularly those with thiol groups, will be altered. A caveat to this complex defense system is the fact that the production of ROS is a mechanism shared by many chemotherapeutic agents. The ability of cells to detoxify exogenous substrates means that components of the cellular redox system may be targeted to enhance cell killing in the case of tumors.
2. GLUTATHIONE
GSH homeostasis is maintained in cells by a complex series of balanced pathways. De novo synthesis can occur through the γ-glutamyl cycle, where the three constituent amino acids (Glu-Cys-Gly) are combined with rate-limiting catalysis through γ-glutamylcysteine synthetase. Salvage of GSH can occur through the cleavage activity of the membrane associatedγ-glutamyl transpeptidase, which can recycle constituents of the molecule.
Whereas intracellular concentrations of GSH may vary considerably, 0.1–10 mM are not uncommonly found in mammalian cells (10–30 μM in plasma). Glutathione can occur in reduced (GSH), oxidized (GSSG), or in mixed disulfide forms, and its ubiquitous abun- dance is testament to its biological importance. The GSH:GSSG ratio is the major cellular redox sensor and determines the antioxidative capacity of the cell, although it can be affected by other redox sensors within the cell. As such, intracellular GSH contributes toward redox balance, and the variety of pathways that synthesize or use GSH influence this homeostasis. Owing to its reactivity and high intracellular concentrations, GSH has been implicated in resistance to several chemotherapeutic agents. Included among these are platinum-containing compounds, alkylating agents such as melphalan, anthracyclines including doxorubicin, as well as arsenic.
GSH participates in many cellular reactions directly as a free radical and ROS scav- enger and indirectly as a cofactor in enzymatic reactions. During these processes, GSH is oxidized to GSSG. To restore homeostasis, GSSG is subsequently reduced by the NADPH-dependent glutathione reductase. GSH also reacts with exogenous substrates such as the aforementioned drugs that are subsequently removed from the cellular milieu via efflux through the multidrug resistance-associated protein, a member of the ATP- binding cassette transporter superfamily. In this capacity, GSH has a major role in the cell’s survival to commonly used chemotherapeutic agents.
3. GSH IN SIGNALING
One of the more interesting conundrums to emerge from the completion of the genome project is the realization that humans are a composite of <30,000 genes, and yet complex- ity of protein structure/function seems distinctly more layered. In the burgeoning era of proteomics, it becomes clear that the central dogma of genetic determinism can be influ-
enced by a number of processes that include, polymorphic variants, gene splicing events, exon shuffling, protein domain rearrangements, and the large number of posttranslational modifications that contribute to alterations in tertiary and quaternary protein structure.
Amongst these, phosphorylation, glycosylation, methylation, and acetylation can account for a large proportion of modifications. More recently, however, addition of GSH to available Cys residues (glutathionylation) has been shown to be of consequence (Fig. 1).
The importance of modifying Cys residues is not necessarily restricted to redox regula- tion, but now seems to be a plausible event that can lead to changes in protein function and thereby signaling processes, particularly in response to a divergent number of stresses (2).
By adding GSH to a target protein, an additional negative charge is introduced (as a consequence of the Glu residue), and a change in protein conformation is made likely. The implication from this somewhat terse analysis is that cells actively participate in the stochastic production of multiple protein building blocks with the intent of realizing functional nonredundancy. Adding a further layer of complexity is the understanding that proteins do not act in isolation in a cellular milieu. Rather, essential protein:protein inter- actions govern how cellular events unfold. This process has proved to be significant to the regulation of JNK (c-Jun N-terminal kinase) signaling by GST-π (3,4). This same para- digm seems to hold for thioredoxin and GST-μ with respect to the apoptosis signal- Fig. 1. Possible mechanisms of reactive oxygen species-induced protein glutathionylation. Reac- tive oxygen species may induce glutathionylation of protein thiols by many different routes. Those highlighted here include the direct oxidation of protein cysteines to generate a reactive protein thiol intermediate such as the reactive cysteinyl radical or sulfenic acid which further reacts with glutathione (GSH) to form a mixed disulfide. Alternatively, a mixed disulfide is formed through reaction with oxidized forms of GSH, i.e., GS-OH or GS(O)SG.
regulating kinase, ASK1 (5), implying the possible existence of a general regulatory mechanism for kinases that may involve GSH and associated pathways (6).
Emergent literature suggests that direct glutathionylation of critical signaling mol- ecules may serve as a trigger for cellular events that are influenced by oxidative stress (7,8). More specifically, Cross and Templeton (8) identified that site-specific glutathiony- lation of the ATP-binding domain of mitogen-activated protein kinase (MAPK) kinase kinase (MEKK1) functions as an inhibitory regulator of the MAPK pathway in response to oxidative stress. In this capacity it serves to distinguish between ASK1, which pro- motes an apoptotic signal, and MEKK1 which promotes a cell survival signal, toward MAPK kinase 4 and stress-activated protein kinase/JNK1 (8). In addition, this inhibitory modification appears to be “dominant” over activation of the kinase by phosphorylation.
The small GTPase Ras modulates diverse signaling pathways and modification, by nitrosation, of its critical Cys-118 in the GTP-binding region has been shown to lead to an increase in Ras activity and to downstream signaling. However, more recent studies show glutathionylation of Ras at Cys-118 is a critical step in the redox-sensitive signaling leading to the activation of p38 and Akt, events that contribute to hypertrophic signaling induced by angiotensin II (AII) (7). AII increases production of ROS from NAD(P)H oxidase that activates downstream kinases p38 and Akt, a response that contributes to vascular dystrophy.
NE-F2 related factor (Nrf2) is a redox-sensitive transcription factor that has been implicated in cellular responses to OS. Nrf2 regulates numerous genes through the an- tioxidant response element, such as GSH synthesis enzymes (9,10). Generation of ROS leads to the dissociation of Nrf2 from its cytoplasmic anchor Kelch-like ECH-associating protein 1, which allows Nrf2 to relocate to the nucleus where antioxidant response ele- ment responsive genes become actively transcribed (11). This dissociation is largely because of modification of key cysteine residues in Kelch-like ECH-associating protein 1. Recent studies now implicate GSH in the dissociation/nuclear translocation of Nrf2, through a type I (thiylation) redox switch, which is distinct from the transcription factor binding to DNA regulated by thioredoxin (12).
Glutathionylation is emerging as a significant posttranslational modification that af- fects protein function and cellular response. The relevance of glutathionylation with respect to disease state, and the question of whether or not it is protective or detrimental in nature is an ongoing “hot spot” in research. The future promises to hold many poten- tially interesting insights into the significance of this modification and the importance of GSH within the cell will continue to grow.
4. GLUTATHIONE S-TRANSFERASE
GSTs (EC 2.5.1.18) are a family of phase II detoxification enzymes that promote the conjugation of GSH to an electrophilic center of endogenous and exogenous compounds, resulting in the formation of the corresponding GSS conjugates (13). The mechanism by which GSTs increase the rate of GSH conjugation involves deprotonation of GSH to GS by a tyrosine residue, which functions as a base catalyst. GST isoenzymes have been divided into at least seven classes based on amino acid sequence similarity, five of which are cytosolic (designated α, μ, π, θ, and κ), and two are membrane-bound. Several isoenzymes, including those from μ, π, and θ, have been shown to be polymorphic in humans (for a review, see ref. 14).
Development of drug resistance is a key element in the failure of chemotherapy treat- ment. Exposure to anticancer agents leads to the induction and expression of gene prod-
ucts that protect the cell. GSTs have been implicated in the development of resistance toward chemotherapy agents (15). It is plausible that GSTs serve two distinct roles in the development of drug resistance via direct detoxification as well as acting as an inhibitor of the MAPK pathway. Hence, it is not surprising that high levels of GSTs have been reported in a large number of tumors types (15).
The connection between GST and their role in the regulation of MAPK pathways is relatively recent. GST-π plays a key role in the regulation of the MAPK pathway through a protein:protein interaction with JNK, a kinase involved in stress response (3,16). In nonstressed cells, JNK activity is low and is located in the cytoplasm bound to GST-π.
Under conditions of OS, more specifically UV irradiation and H2O2 treatment, oligomer- ization of GST-π occurs together with the release and phosphorylation of JNK. Phospho- rylated JNK is the active form, which then translocates to the nucleus, activating downstream transcription factors involved in gene expression and/or the induction of apoptosis. The precise mechanism of the disruption of the complex is unknown; however, oligomerization of the GST monomers implicates intermolecular disulfide bridge forma- tion between available Cys residues. Furthermore, the lack of catalytic activity for the regulation of the JNK pathway, shown by the mutation of the essential Tyr (Tyr-7) residue in the active site of the enzyme, suggests a novel nonenzymatic role for this enzyme (3). Upstream regulation of the MAPK pathways by GST is also observed, as demonstrated by the GST-μ:ASK1 complex. ASK1 is a MAP kinase kinase kinase (MAPKKK) that activates JNK and p38 pathways leading to cytokine- and stress-in- duced apoptosis (17). ASK1 is activated in response to OS and heat shock. Like JNK, the activity of ASK1 is low in nonstressed cells because of its sequestration via protein:protein interactions with GST-μ and or thioredoxin (5,18). The mechanism by which ASK1 is released from and activated by either of these proteins is distinct. GST-μ is responsive to heat shock, whereas thioredoxin responds to OS. The discovery of the involvement of GSTs in the regulation of these MAPK pathways, together with the known involvement of other small redox-regulated proteins adds an extra layer of complexity to these MAPK pathways with respect to signaling towards cell survival or cell death.
Other recent studies have broadened the role of GSTs. Small redox active protein families such as peroxiredoxin (Prx) have the potential to heterodimerize with GST-π.
Studies have shown that full activation of PrxVI requires heterodimerization of the oxidized protein with GST-π, followed by glutathionylation of its conserved Cys (Cys- 47) in a sterically protected region (19). Dissociation from GST-π, followed by sponta- neous reduction of glutathionylated protein by GSH, results in catalytically active protein.
Whereas PrxVI contains a single Cys residue, six other mammalian Prxs have been identified that all contain two conserved cysteine residues (20). This observation broad- ens the functional importance of GST-π into yet another arena.
5. GSH AND GST
SAS THERAPEUTIC AGENTS
GSTs are upregulated in a number of human tumors and as such, are promising thera- peutic targets in research. A number of potential anticancer agents have been designed with this in mind using several different approaches. The first approach was to design inhibitors of GST exploiting its role as a detoxifying enzyme. Another approach was to find inhibitors of the protein:protein interaction of GST with kinases from the stress- activated protein kinase pathways. A third strategy involved the exploitation of the eleva- tion of GSTs in tumors, with particular emphasis of the π isoform, through design of GST-activated prodrugs.
In the past, modulation of GSH and GST has been attempted as a means to improve response to cancer drugs. Lowering GSH levels in order to increase drug response being the ultimate goal. Use of, for example, buthionine sulfoximine and ethacrynic acid, whereas effective in their experimental effects on each system, was not successful enough in the clinic to merit continued development (21,22). One consequence of these ap- proaches was the conceptual design of a peptidomimetic inhibitor of GST-π, γ-glutamyl-S-(benzyl)cysteinyl-R-–phenyl glycine diethyl ester (TLK199). It was shown to potentiate the toxicity of numerous anticancer agents in different tumor cell lines. In addition, TLK199 was shown to be an inhibitor of multidrug resistance-associated pro- tein, which is a known multidrug efflux transporter (23). Preclinical and mechanism of action studies with this agent revealed an unexpected effect in animals, namely that the drug possessed myeloproliferative activity through disruption of the GST-π:JNK com- plex (24,25). As an extension of these data, the company has sponsored a phase I/II trial of TLK199 (now named Telintra™) in patients with myelodysplastic syndrome.
Another novel GSH peptidomimetic anticancer agent, NOV-002, is a platinum coor- dination complex of oxidized glutathione. This drug has undergone significant clinical testing in Russia, and evidence of efficacy has been reported in 340 patients with diseases such as non-small cell lung cancer, colorectal, pancreatic, and breast cancer (26). These trials are now being repeated in the United States. Of interest, the drug also acted on the bone marrow, with increases in circulating lymphocyte, monocytes, T-cell, and NK cell counts.
Examples of GSH-activated prodrugs, which ultimately takes advantage of the el- evated levels of GSH, include the novel thiopurine prodrugs cis-6-(2-acetylvinylthio) purine (cis-AVTP) and trans-6-(2-acetylvinylthio)guanine (trans-AVTG), which are α,β-unsaturated conjugates of the thiopurines 6-mercaptopurine and 6-thioguanine, re- spectively. These prodrugs have been shown to react rapidly with cellular thiols (like GSH) to yield the respective thiopurines as the major metabolites (27). As already men- tioned, these drugs take advantage of the elevated levels of GSH observed in tumor cells, and the upregulated levels of GSH associated with chemotherapeutic drug resistance.
Indeed, less bone marrow and intestinal toxicity was observed in mice after multiple treatments with the prodrugs than after equivalent treatments with 6-thioguanine (28).
More recently, cytotoxicity analysis using the National Cancer Institutes’ anticancer screening program showed the prodrugs to have enhanced in vitro cytotoxicity when compared with the parent thiopurines (29).
Many efforts are focused on GST-targeted agents. The rationale for such efforts lies with accumulated observations about GST expression in tumor and normal tissues. In particular, the association between high levels of expression of GST isozymes and malignancy and drug resistance (30) provided an ideal rationale for the design of GST- π activated prodrugs. In many instances, the GST-π isozyme can accumulate to levels that make it one of the more prevalent cytosolic proteins. In addition, even when the selecting drug is not a substrate for GST-π, its expression is most readily enhanced in drug resistant cells.
Such data complicated interpretation of the connection between GST-π and drug resistance in cell culture (31) and in clinical trials (26). Largely because of the connection between GST-π, JNK, and apoptosis pathways (16), there is now a clearer understanding of why increased GST-π is associated with so many divergent acquired drug resistant situations.
Exploitation of elevated levels of GSTs to preferentially activate drugs led to the development of γ-glutamyl-α-amino-β(2-ethyl-N,N,N',N'-tetrakis (2-chloroethyl) phosphorodiamidate)-sulfonyl)-propionyl-(R)-–phenylglycine (TLK286) and O2-[2,4-
dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen- 1-ium-1,2-diolate (PABA/NO). The early rationale for design, synthesis and testing of TLK286 incorporated the principle that enhanced tumor GST-π levels would preferen- tially activate more of the toxic phosphorodiamidate alkylating species (Fig. 2) with a commensurate advantage in therapeutic index (32,33). Drug sensitivity is correlated with increased levels of GST-π both in vitro and in vivo. TLK286 is also under active testing in phase III settings for a number of disease states including non-small cell, ovarian, and colon cancers.
Fig. 2. Structure of γ-glutamyl-α-amino-β(2-ethyl-N,N,N',N'-tetrakis (2-chloroethyl) phosphoro- diamidate)-sulfonyl)-propionyl-(R)-–phenylglycine (TLK286) and its activation by glutathione S-transferase-π (GST-π).
Another more recent example of a GST-activated prodrug is PABA/NO, a novel nitric oxide-releasing agent (34). Studies show that cells lacking GST-π in vitro are less sen- sitive to the cytotoxic effects of PABA/NO. The activation of JNK and p38 also appears to be important for the cytotoxic effects of PABA/NO, as the inhibition of these pathways led to a reduction in cell death. In vivo antitumor data suggest PABA/NO as a good lead compound for further structure activity and drug discovery efforts.
6. CONCLUDING REMARKS
The traditional view of ROS is that they have a negative effect on cell function and viability, and therefore, substances that inhibit their reactivity (i.e., antioxidants) must be beneficial to cells. The increasing recognition of roles of ROS in cell signaling and modification of gene expression has forced a reevaluation of this simplistic view (35). It has been demonstrated that GSH and GSTs have roles that extend much further than simple detoxification reactions. Indeed, it is not unreasonable to predict that glutathionyla- tion may provide regulatory control complementary to other well-studied and established posttranslational modifications. Future studies will shed an advanced knowledge of the proteins involved in the cells response to “stress” and the interplay of proteins within the cell, with not only themselves in an enzymatic manner, but with other proteins in a regulatory fashion.
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