review glutathione 5-transferases: role in alkylating agent … · [cancer research 50. 6449-6454....

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[CANCER RESEARCH 50. 6449-6454. October 15. 1990] Review Glutathione 5-Transferases: Role in Alkylating Agent Resistance and Possible Target for Modulation Chemotherapy— A Review1 David J. Waxman2 Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 The effectiveness of many clinically useful anticancer drugs can be severely limited by drug resistance, which appears to be intrinsic to some tumors but can also arise during multiple courses of chemotherapy in the case of many others. Studies carried out using cultured tumor cell models and other systems have established that a variety of mechanisms can contribute to drug resistance (1-3). These include alterations in drug uptake or drug efflux from the cell [transporter defects, P-glycoprotein (Mdr) overexpression], changes in drug-metabolizing enzymes [e.g., aldehyde dehydrogenase (cyclophosphamide), folylpoly- glutamate synthetase (methotrexate), metallothionein (cispla- tin)], and changes in target enzymes [dihydrofolate reducÃ-ase (methotrexate), topoisomerase II (etoposide, teniposide), and enzymes of DNA repair [O6-methylguanine alkyltransferase]. Acquired resistance to chemotherapeutic drugs typically in volves several independent mechanisms (multifactorial resist ance) and can lead to cross-resistance to structurally unrelated drugs that exhibit distinct mechanisms of action. Recent studies have indicated that GSTs3 may play an important role in the resistance of cells and organisms not only to electrophilic herbicides, insecticides, and carcinogens (4, 5) but also to anti- cancer drugs as well. General aspects of the biochemistry and regulation of mammalian GST enzymes have been summarized in a number of excellent reviews and monographs (6-11). This article focuses on the interaction of GSTs with cancer chemo therapeutic drugs and evaluates our current understanding of the importance of GST-dependent drug conjugation reactions for alkylating agent resistance in tumor cells that overexpress one or more GSTs. Important gaps in our current knowledge about the interactions between GSTs and anticancer alkylating agents are identified, as are several possible ways in which GSTs might be targeted for modulation chemotherapy. Biochemistry of the GSH/GST System: Enzymatic Properties and Multiplicity of Isozymes GSTs catalyze conjugation of electrophilic compounds to glutathione, 7-glutamylcysteinylglycine, an intracellular cys- teine-containing tripeptide present at high concentrations (up to 5-10 HIM) in most mammalian cells. The GSTs are an integral part of the Phase I (oxidation)/Phase II (conjugation) system that metabolizes many lipophilic drugs and other foreign compounds, including anticancer drugs (12, 13). The overall result of metabolism by this system is the conversion of these lipophilic chemicals to more polar derivatives in a manner that Received 3/1/90; accepted 7/19/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Preparation of this article was supported in part by Grant CH-487 from the American Cancer Society and Grant CA-19589 from the NIH. 2To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, JF-525, 44 Binney Street, Boston, MA 02115. 3The abbreviations used are: GST, glutathione S-transferase; GSH, glutathi one; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; BSO, buthionine sulfoximine. can facilitate their inactivation and elimination. Alterations in the GSH/GST system are frequently found in alkylating agent resistance phenotypes. As detailed below, these include in creases in cellular GSH levels, elevation of GST activity, and changes in the expressed levels of one or more GST enzymes (isozymes). These observations have suggested that a more thorough understanding of the biochemistry of the GSH/GST system, as it relates to anticancer drug metabolism and inacti vation, may contribute to the elucidation of important mecha nisms of alkylating agent resistance. Ultimately this may lead to novel approaches to circumventing drug resistance through modulation of GSH/GST-dependent metabolic processes. GSTs utilize GSH in a broad range of reactions of cellular metabolism, including conjugation and peroxidase activities, as well as noncatalytic ligand binding reactions. (For general re views see Refs. 6-9.) Conjugation Reactions. These GST-catalyzed reactions in volve the direct coupling of GSH to electrophilic drugs, carcin ogens, and endogenous compounds, such as leukotriene A4, which is conjugated to yield the paracrine hormone leukotriene C4. The glutathionyl 5-conjugates that are thus formed are generally chemically stable but can be enzymatically metabo lized to mercapturates (/V-acetylcysteine derivatives), which are readily excreted. Metabolism of l-chloro-2,4-dinitrobenzene to its GSH conjugate is a prototypic GST-dependent conjugation reaction, and involves nucleophilic attack of a thiolate aniónof glutathione (i.e., GS~) (14) on the substrate's aromatic ring with displacement of chloride. Although reactions of this gen eral type (Fig. la) may occur nonenzymatically, particularly at high pH (>8-9), substantial rate enhancements can be achieved through GST catalysis. With alkylating agents such as nitrogen mustards, these reactions may occur by GSH displacement of chloride, perhaps via an aziridine intermediate, resulting in inactivation of the electrophilic mustard functionality (Fig. \b). Peroxidase Activity. GST enzymes can also catalyze a sele nium-independent GSH peroxidase activity which leads to the detoxification of lipid and nucleic acid hydroperoxides (Fig. 2). These peroxides not only form during the redox recycling of many drugs and other xenobiotics but can also be generated during normal metabolism as a by-product of cellular oxygen utilization. In the case of doxorubicin and other anticancer drugs that generate hydroperoxides or other reactive oxygen species via redox recycling, GST, as well as the selenium- dependent enzyme GSH peroxidase, can deactivate these me tabolites through peroxidative mechanisms, resulting in de creased cytotoxicity ( 15, 16). Ligand-binding Properties. Many GST enzymes exhibit a ligand binding ("ligandin") function, which involves the non- covalent binding of nonsubstrate hydrophobic ligands such as heme, bilirubin, various steroids, and conceivably some lipo philic anticancer drugs as well. GST-mediated ligand binding is often associated with inhibition of GST activity by the bound 6449 on May 26, 2020. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Page 1: Review Glutathione 5-Transferases: Role in Alkylating Agent … · [CANCER RESEARCH 50. 6449-6454. October 15. 1990] Review Glutathione 5-Transferases: Role in Alkylating Agent Resistance

[CANCER RESEARCH 50. 6449-6454. October 15. 1990]

Review

Glutathione 5-Transferases: Role in Alkylating Agent Resistance and PossibleTarget for Modulation Chemotherapy— A Review1

David J. Waxman2

Department of Biological Chemistry and Molecular Pharmacology and Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

The effectiveness of many clinically useful anticancer drugscan be severely limited by drug resistance, which appears to beintrinsic to some tumors but can also arise during multiplecourses of chemotherapy in the case of many others. Studiescarried out using cultured tumor cell models and other systemshave established that a variety of mechanisms can contribute todrug resistance (1-3). These include alterations in drug uptakeor drug efflux from the cell [transporter defects, P-glycoprotein(Mdr) overexpression], changes in drug-metabolizing enzymes[e.g., aldehyde dehydrogenase (cyclophosphamide), folylpoly-glutamate synthetase (methotrexate), metallothionein (cispla-

tin)], and changes in target enzymes [dihydrofolate reducíase(methotrexate), topoisomerase II (etoposide, teniposide), andenzymes of DNA repair [O6-methylguanine alkyltransferase].

Acquired resistance to chemotherapeutic drugs typically involves several independent mechanisms (multifactorial resistance) and can lead to cross-resistance to structurally unrelateddrugs that exhibit distinct mechanisms of action. Recent studieshave indicated that GSTs3 may play an important role in the

resistance of cells and organisms not only to electrophilicherbicides, insecticides, and carcinogens (4, 5) but also to anti-cancer drugs as well. General aspects of the biochemistry andregulation of mammalian GST enzymes have been summarizedin a number of excellent reviews and monographs (6-11). Thisarticle focuses on the interaction of GSTs with cancer chemotherapeutic drugs and evaluates our current understanding ofthe importance of GST-dependent drug conjugation reactionsfor alkylating agent resistance in tumor cells that overexpressone or more GSTs. Important gaps in our current knowledgeabout the interactions between GSTs and anticancer alkylatingagents are identified, as are several possible ways in whichGSTs might be targeted for modulation chemotherapy.

Biochemistry of the GSH/GST System: Enzymatic Propertiesand Multiplicity of Isozymes

GSTs catalyze conjugation of electrophilic compounds toglutathione, 7-glutamylcysteinylglycine, an intracellular cys-teine-containing tripeptide present at high concentrations (upto 5-10 HIM) in most mammalian cells. The GSTs are anintegral part of the Phase I (oxidation)/Phase II (conjugation)system that metabolizes many lipophilic drugs and other foreigncompounds, including anticancer drugs (12, 13). The overallresult of metabolism by this system is the conversion of theselipophilic chemicals to more polar derivatives in a manner that

Received 3/1/90; accepted 7/19/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Preparation of this article was supported in part by Grant CH-487 from theAmerican Cancer Society and Grant CA-19589 from the NIH.

2To whom requests for reprints should be addressed, at Dana-Farber CancerInstitute, JF-525, 44 Binney Street, Boston, MA 02115.

3The abbreviations used are: GST, glutathione S-transferase; GSH, glutathione; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; BSO, buthionine sulfoximine.

can facilitate their inactivation and elimination. Alterations inthe GSH/GST system are frequently found in alkylating agentresistance phenotypes. As detailed below, these include increases in cellular GSH levels, elevation of GST activity, andchanges in the expressed levels of one or more GST enzymes(isozymes). These observations have suggested that a morethorough understanding of the biochemistry of the GSH/GSTsystem, as it relates to anticancer drug metabolism and inactivation, may contribute to the elucidation of important mechanisms of alkylating agent resistance. Ultimately this may leadto novel approaches to circumventing drug resistance throughmodulation of GSH/GST-dependent metabolic processes.

GSTs utilize GSH in a broad range of reactions of cellularmetabolism, including conjugation and peroxidase activities, aswell as noncatalytic ligand binding reactions. (For general reviews see Refs. 6-9.)

Conjugation Reactions. These GST-catalyzed reactions in

volve the direct coupling of GSH to electrophilic drugs, carcinogens, and endogenous compounds, such as leukotriene A4,which is conjugated to yield the paracrine hormone leukotrieneC4. The glutathionyl 5-conjugates that are thus formed aregenerally chemically stable but can be enzymatically metabolized to mercapturates (/V-acetylcysteine derivatives), which arereadily excreted. Metabolism of l-chloro-2,4-dinitrobenzene toits GSH conjugate is a prototypic GST-dependent conjugationreaction, and involves nucleophilic attack of a thiolate aniónofglutathione (i.e., GS~) (14) on the substrate's aromatic ring

with displacement of chloride. Although reactions of this general type (Fig. la) may occur nonenzymatically, particularly athigh pH (>8-9), substantial rate enhancements can be achievedthrough GST catalysis. With alkylating agents such as nitrogenmustards, these reactions may occur by GSH displacement ofchloride, perhaps via an aziridine intermediate, resulting ininactivation of the electrophilic mustard functionality (Fig. \b).

Peroxidase Activity. GST enzymes can also catalyze a selenium-independent GSH peroxidase activity which leads to thedetoxification of lipid and nucleic acid hydroperoxides (Fig. 2).These peroxides not only form during the redox recycling ofmany drugs and other xenobiotics but can also be generatedduring normal metabolism as a by-product of cellular oxygenutilization. In the case of doxorubicin and other anticancerdrugs that generate hydroperoxides or other reactive oxygenspecies via redox recycling, GST, as well as the selenium-dependent enzyme GSH peroxidase, can deactivate these metabolites through peroxidative mechanisms, resulting in decreased cytotoxicity ( 15, 16).

Ligand-binding Properties. Many GST enzymes exhibit aligand binding ("ligandin") function, which involves the non-

covalent binding of nonsubstrate hydrophobic ligands such asheme, bilirubin, various steroids, and conceivably some lipophilic anticancer drugs as well. GST-mediated ligand bindingis often associated with inhibition of GST activity by the bound

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GLUTATHIONE S-TRANSFERASES AND DRUG RESISTANCE

& R-X + GSH

R-NR'-CH2CH2-C1 + GSH

R-SG + H-X

R-NR'-CH2CH2-SG HC1

Fig. 1. Conjugation reactions catalyzed by GSTs. Shown is a generalizedconjugation of GSH with electrophilic substrate R—X (a), and a more specificexample of GSH conjugation to a nitrogen mustard (*).

R-OOII -> R-OH + GSSG + H2O

Fig. 2. GSH peroxidase reaction catalyzed by GSTs. Reduction of the hydro-peroxide R—OOH consumes 2 mole of GSH and proceeds without incorporationof GSH into the final product R—OH.

ligand and appears to facilitate the intracellular transport ofthese lipophilic compounds (10).

Although GSTs are generally viewed as playing a protectiverole in foreign compound metabolism, they can also catalyzereactions that lead to toxification. Examples include the GST-dependent metabolism of 1,2-dibromoethane and related hal-oalkanes and probably also metabolism of the 6-thiopurineprodrug azathioprin (11, 17-19). Similarly, the cytotoxicity ofthe polypeptide antibiotic neocarzinostatin is greatly enhancedby thiols such as GSH, although in this case there is no apparentrequirement for GST catalysis (20). Compounds such as thesethat are metabolically activated by GSH or GSH/GST mightconceivably have utility as cytotoxic agents that are preferentially directed towards drug-resistant tumor cells with elevatedGST activity levels (see below).

The diverse biological functions of the GSH/GST systemoutlined above are mediated by multiple GST enzymes. Theseenzymes generally exhibit broad and overlapping substrate specificities and functions and are differentially inducible by exposure to diverse drugs, carcinogens, and other xenobiotics (7,11). Mammalian GSTs can presently be grouped into fourclasses (corresponding to four distinct gene families) on thebasis of their immunochemical properties and primary structurerelatedness (21, 22). Members of one class are bound to themembrane of the endoplasmic reticulum. These microsomalGSTs are functional trimers with molecular weights of~17,000/subunit and are subject to activation (up to 10-fold ormore) by endogenous disulfides and by various reactive intermediates that form during the metabolism of drugs and otherforeign compounds (22). Enzymes that belong to the otherthree GST classes, designated a, n, and TT,are localized in thecytosol and include both homo- and heterodimeric complexesformed from GST subunits that range in size from M, —23,000-28,000 (21, 23). In humans, but not other species, the majorcytosolic GSTs belonging to each class exhibit similar isoelec-tric points, which can be described as basic (a-class), near-neutral (//-class), and acidic (-¡r-class).As a group, the cytosolic

GSTs are abundant and together may constitute up to about5% of soluble cellular protein.

Whereas only one GSTn- gene appears to be expressed in

mammalian species, including humans (Refs. 24 and 25; see,however, Ref. 26), multiple GSTa-class (27, 28) and GST/i-class genes (29, 78) are expressed in the same individual. Aswith the rodent GSTs, unique patterns of tissue-specific geneexpression characterize the human GST enzymes. Of particularinterest is the tumor-associated expression of GST^r, whichserves as a marker for hepatocarcinogenesis in rodent systems(30) and is present at elevated levels in many tumor tissuesrelative to matched normal tissue (31-33). These findings haveled to the suggestion that GST?r may contribute to the intrinsicresistance associated with some of these tumors. Also significant, and of potential relevance to cancer chemotherapy, is agenetic polymorphism that characterizes the expression of one

of the human GST/¿genes. About 50% of the human populationis phenotypically deficient with respect to expression of thecorresponding GST/u enzyme, which actively conjugates (andthereby detoxifies) carcinogenic polycyclic aromatic hydrocarbon epoxides that are formed during the course of cytochromeP-450-dependent Phase I bioactivation reactions (29, 34). Conceivably, expression of this GST/x could influence the pharma-cokinetics and possibly also the toxicity of electrophilic alkyl-ating agents (such as nitrosoureas; see below) that may bepreferentially metabolized by this particular GST enzyme. Correlative data suggest that the expression of this polymorphicGST¿(enzyme (which can readily be assayed in human lymphocyte preparations) may impart to lung tissue some measure ofprotection from the carcinogenic insult of cigarette smoke (35).However, the higher activity of GSTjr with some polycyclicaromatic epoxide substrates (9) and its greater abundance,compared to ( ¡ST/j.in many human tissues, including lung(33), suggests that GST^r may also play an important role inthe detoxification of lung carcinogens.

Possible Contribution of GSH Conjugation Reactions to DrugResistance

As noted above, in vitro studies of alkylating agent-resistantcells carried out in a number of laboratories have demonstratedthat elevation of cellular GST levels (as well as cellular GSHlevels) may occur in tumor cells in response to anticancer drugselection pressure. This can lead to stable overexpression ofindividual GST enzymes, in some cases involving gene amplification as a mechanism (36). The overexpression of GSTs inthese tumor cells is associated with drug resistance phenotypesand has been proposed to contribute directly to the observedresistance of these cells to alkylating agents (e.g., Refs. 36-38).

At the present time, several mechanisms whereby the observed elevations in cellular GST and GSH levels might contribute to tumor cell resistance seems plausible. These include:(a) enhanced inactivation of electrophilic alkylating agents,such as melphalan (39), by direct conjugation to GSH (Fig. 3);(b) GSH-dependent denitrosation of nitrosoureas (Fig. 4), areaction that is preferentially catalyzed by one of the rat liverGST/i enzymes (GST form 4-4) in the case of BCNU (45) andwhich leads to a decrease in nitrosourea cytotoxicity (46); (c)scavenging for reactive organic peroxides, a process that also iscatalyzed by the selenium-dependent enzyme GSH peroxidase(49); (d) quenching of chloroethylated-DNA monoadducts (50),platinum-DNA adducts (51), and DNA hydroperoxides (52).These latter activities would presumably be catalyzed by GSTsassociated with the nucleus (52).

Correlative observations in wild type versus drug-resistanttumor cells notwithstanding (e.g., Refs. 36-38, 45, and 53-55),clear evidence for a direct metabolic role of GSTs in tumor cellresistance is still lacking. Although it is widely assumed thatelectrophilic anticancer drugs, such as nitrogen mustards, nitrosoureas, and perhaps also platinum-containing drugs (56, 57),are inactivated intracellularly through GSH/GST-dependentmetabolism, this has not been proved. For instance, althoughnitrogen mustard resistance in a Chinese hamster ovary cellline is associated with large overexpression of a class a GST(36), direct evidence for the participation of that GST in intracellular drug inactivation is absent. Even in cases where conjugation of GSH to an anticancer drug has been observed in vitro[e.g., conversion of melphalan to its mono- and di-GSH conjugates catalyzed by Sepharose-bound liver cytosolic protein (39)

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Fig. 3. Metabolism of melphalan by GSH/GST system. Metabolism may proceed by displacement of a nitrogen mustard moiety withformation of 4-(glutathionyl)phenylalanine(Reaction 1; Ref. 40), or by direct conjugationof GSH to the mustard group with formationof mono- and di-GSH adducts (Reactions 2and 3; Ref. 39). These reactions presumablycompete with hydrolysis of the chloro groups(Reactions 4 and 5), and may proceed via azir-idine intermediates.

GLUTATHIONE S-TRANSFERASES AND DRUG RESISTANCE

CH —COOH

HO 0-©-MELPHALAN

GSH/GST

di-OH

mono -GSH adduct di-GSH adduct

4-lGSH)Phe

GS-NO

S-nitroso

glutathione

NOi

Nitrite

1- chloroethyI

3-ethylglutathionyl urea

Fig. 4. Enzymatic denitrosation of BCNU. Deactivation of BCNU by GSH-dependent denitrosation may proceed (a) by a direct pathway that yields denitro-sated drug and 5-nitroso-GSH. This latter compound is a GST inhibitor (41 ) andmay decompose to yield nitric oxide (NO) plus glutathione disulfide (GSSG) (42).Further reaction of the denitrosated drug with GSH could yield the conjugate 1-chloroethyl-3-ethyl-GSH-urea. The analogous reaction pathway occurs duringthe metabolism of l -methyl-2-nitroso-1 -nitroguanidine by GSH/GST (43). Alternatively, (b) sulfur aniónattack at the chloro-bearing carbon might yield the drug-GSH conjugate directly and result in nitroso group departure with its eventualappearance as nitrite (NO2~); this latter route is suggested to be catalyzed by

rodent liver GSTs (44, 45). Another possible pathway (not shown) involves theformation of S-(2-chloroethyl)-GSH [i.e., GS-CH2CH¡CI] by reaction of GSHwith C1-CH2CH2+ formed during decomposition of BCNU (46). Finally, BCNUhas been shown to undergo a one electron reductive denitrosation catalyzed bythe microsomal enzyme NADPH cytochrome P-450 reducíaseto yield denitrosated drug and nitric oxide (47, 48).

(Fig. 3)], the roles played by individual GST isozymes areunknown, as is the extent to which these reactions with GSTmight proceed in the absence of GST catalysis. Moreover,results obtained with rodent GSTs cannot automatically beextrapolated to human GSTs of the same class, since markedlydifferent substrate specificities can characterize GSTs that exhibit as high as 93% overall amino acid sequence identity (58).Thus, the observed denitrosation of BCNU by a rat GST^isozyme (45) does not imply that the same detoxification iscatalyzed by human GST/i, despite the ~80% amino acid sequence identity between these protein families (29).

Transfection studies with GST?r carried out in MCF7 humanbreast cancer cells have indicated that increased expression ofthis GST can provide a modest degree of protection from thecytotoxic effects of some lipophilic carcinogens but does notconfer significant resistance to melphalan or cisplatin (59).Similarly, tranfection of GST;r into NIH-3T3 cells leads toincreased resistance to ethacrynic acid (a GST?r substrate) but

not melphalan, chlorambucil, or cisplatin (60). Thus, GSTîrisunlikely to catalyze the intracellular conjugation of GSH tothese anticancer alkylating agents at rates that impact on drugcytotoxicity. Other recent studies, however, have describedsmall but apparently significant increases in resistance to alkylating agents and anticancer drugs following stable transfectionof complementary DNAs encoding GST?r and other GST enzymes into yeast (61) or mammalian cells (62). Although theincrease in resistance obtained in these transfection experimentsis of the same magnitude as resistance occurring in the clinic,the question arises as to whether the observed small changes indrug sensitivity reflect clonal variability rather than expressionof the transfected GST gene per se. This problem has beenaddressed, however, by a study of transient GST expression ina population of COS-M6 monkey kidney cells; expression inthese cells of human GSTir, rat GST Ya (class a), or rat GSTYbl (class ft) imparts a small but significant increase in resistance (up to 1.5-fold) to both nitrogen mustards and cisplatin(62). These experiments were carried out using a sorted population of positive transfectants, which eliminates the likelihoodof interclonal variation in factors other than the recombinantGST as a cause for the elevated drug resistance. Moreover,reversion of GST expression in this system correlates with lossof drug resistance (62). Although the increase in drug resistancein these experiments is modest and perhaps not perfectly correlated with the fold increase in GST expression, this latterdiscrepancy may, in part, reflect the presence of high endogenous GST activity in the recipient COS cells.

Clearly, a more detailed and direct analysis of the chemo-therapeutic drug specificity profile of individual human GSTs,including the tumor-associated GSTir, would complement thesetransfection studies and would broaden the base of our knowledge from which a more definitive evaluation of the roles ofthese enzymes in tumor metabolism of anticancer drugs mightbe carried out. Moreover, a better understanding of the preciserole of GST;/ in alkylating agent metabolism would facilitatethe identification of patients who may be at greater risk to hosttissue toxicity as a consequence of the genetically determinedGST^i deficiency that occurs in about one-half of the generalpopulation. Further study of the pathways of alkylating agentmetabolism catalyzed by human GSTs is thus crucial and islikely to provide a better understanding of the contributionsthat GST enzyme overexpression can make to drug resistancephenotypes as well as to the protection of GST-expressing hosttissues. A better understanding of GST substrate specificitieswith anticancer alkylating agents may also help explain alkylating agent cross-resistance patterns (63) and may eventually

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GLUTATHIONE .V-TRANSFERASES AND DRUG RESISTANCE

be useful in detecting cross-resistant phenotypes in individualpatients on the basis of GST assays of tumor biopsy specimens.

with high dose therapies involving cyclophosphamide or BCNUwithout compromising antitumor activity (71).

Experimental Approaches to Modulation of GSH/GST Activity ¿Iterations of GST Activity

The current gaps in our knowledge about the enzymology ofGST-dependent alkylating agent metabolism notwithstanding,it seems probable that alterations in anticancer drug conjugation, and hence modulation of alkylating agent activity, can beachieved through the pharmacological modulation of cellularGSH and GST levels. In vitro and preclinical studies suggestthat this modulation can be accomplished through alterationsin either GSH pools (buthionine sulfoximine, GSH-monoethylester) or GST activity levels (GST-inducing agents, GSH analogues, quinone-based inactivators, competitive substrates).Modulation chemotherapy based on the GSH/GST systemcould be directed towards decreasing host toxicity or, alternatively, enhancing drug sensitivity of tumors that are otherwiseresistant to classical chemotherapeutic treatment regimens.However, because of the multifactorial nature of cellular resistance to alkylating agents (63), it is conceivable that in somesystems modulators could have a substantial biochemical effecton GST activity and yet provide only limited useful modulationbecause the GST/GSH system represents only one of a numberof determinants of drug sensitivity and resistance.

Several possible experimental approaches to the modulationof cellular GST activity are outlined below.

Alterations in GSH Pools

A reduction in cellular GSH levels can be achieved by treatment with BSO. This irreversible inhibitor (64) of -y-glutamyl-

cysteine synthetase, an enzyme required for GSH biosynthesis,has been used in studies carried out both in cell culture and invivo and can reduce cellular GSH levels by as much as 95%(65). Modulation of cellular GSH in this manner can lead to asubstantial reversal of alkylating agent resistance in drug-resistant tumor cells, while increasing the sensitivity of somealkylating agent-sensitive cell lines. In preclinical studies, BSOhas been shown to enhance the toxicity of melphalan towardsovarian cancer without increasing the toxicity of the drugtoward critical host tissues. This selectivity may be attributableto the preferential depletion by BSO of tumor cell GSH (<5%residual GSH) as compared to normal tissues, including bonemarrow (~20% residual GSH) (66). This partial sparing ofbone marrow contrasts with the increase in renal toxicity thatis observed when BSO pretreatment is combined with agentsthat have a tendency towards nephrotoxicity, such as methyl-1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (67). Thus,while BSO can favorably increase drug responses in someexperimental tumor systems, or with other chemotherapeuticagents, BSO may simultaneously potentiate drug toxicity tohost tissue.

Agents that have been reported to increase cellular GSHcontent include GSH-monoethyl ester (68) and L-2-oxothiazo-lidine-4-carboxylate (69). The former drug provides GSH inthe form of a membrane-permeable ethyl ester that is susceptible to intracellular hydrolysis. The latter compound is alsoreadily transported into cells but must be enzymatically metabolized to L-cysteine, which enhances the synthesis of GSH (70).In preclinical studies, GSH-monoethyl ester has been shown toeffectively protect normal host tissue from toxicities associated

Modulation of GST activity can likewise be considered fromthe perspective of host tissue protection. One approach couldinvolve the elevation of host cell GST enzyme levels. Thismight be accomplished, for instance, by administration of theantioxidant butylated hydroxytoluene (72) or other nontoxicGST-inducing agents. Given the tissue-specific responses toGST inducers, such a treatment would not necessarily elevatetumor cell GST levels as well. On the other hand, if inhibitionor inactivation (irreversible destruction) of tumor cell GSTenzymes is the goal, one or more of following possibilities couldbe considered.

Use of Inhibitory Peptide Analogues of GSH. One such compound is the aspartate GSH analogue 7-glutamylaspartylgly-cine, which exhibits a K-,of ~1 pM with respect to GST inhibition (73). Conceivably, peptide inhibitors could be delivered totumor cells as ethyl esters and might be used in combinationwith BSO to maximize the inhibitory effect.

Quinone Inactivators of GSTs. Tetrachloro-l,4-benzoqui-none, its GSH conjugates, and related compounds (74) appearto inactivate GSTs through covalent modification of an essential amino acid, probably a cysteine residue situated at the activesite of the enzyme (75). Although the utility of these compoundsfor GST modulation has not been fully explored in cellular orin vivo systems, the possible chemotherapeutic application ofsuch GST inactivators warrants consideration.

Other GST Inhibitors. Many structurally diverse chemicalsare effective inhibitors of one or more GST enzymes (9). Twocompounds of particular interest in the present context are thediuretic agent ethacrynic acid and the prostaglandin I, analoguepiriprost. Sensitization of cultured tumor cells to alkylatingagents such as chlorambucil is achieved when these relativelynontoxic GST inhibitors are added to the culture medium inthe low ¿(Mrange (76). It is probable that these compounds actby one or more of the following mechanisms: (a) competitionwith the alkylating agent for metabolism at the GST active site;(¿>)depletion of intracellular GSH; and (c) inactivation ofcellular GSTs, perhaps including those involved in prostaglandin and/or leukotriene biosynthesis (76), by covalent modification of the transferase itself. Use of these compounds inconjunction with BSO might enhance the overall effectivenessof a modulation strategy by counteracting the increases in GSTexpression that can result from BSO treatment (77).

In conclusion, although much remains to be learned aboutthe biochemistry of the GSH/GST system as it relates toanticancer drug metabolism, numerous studies point to thelikelihood that alterations in GSH and GST levels, and hencecellular GST activity, contribute in an important way to theresistance of tumor cells to anticancer drugs of the alkylatingagent class. Several promising strategies to overcome this resistance are emerging from basic and preclinical studies andseem likely to lead to improved therapeutic regimens based onmodulation or multimodulation chemotherapy. These andother ongoing studies may lead to the resolution of severaloutstanding questions, including: (a) the importance of GSTincreases as compared to other mechanisms of alkylating agentresistance; (b) whether the GSH/GST system is, in fact, a usefultarget for clinically effective modulation chemotherapy; (c) theextent to which therapeutic responsiveness is affected by inter-

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GLUTATHIONE S-TRANSFERASES AM) DRl'G RISISI AM C

individual differences in GST isozyme expression, arising fromgenetic factors or due to prior drug exposure; (d) the factorsthat determine which GST isozymes become elevated; and (e)the mechanisms responsible for the observed changes in GSTexpression following exposure of cells to drugs and other formsof toxic insult, and the extent to which investigation of theseprocesses can lead to a further understanding of basic cancercell biology.

References

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1990;50:6449-6454. Cancer Res   David J. Waxman  A Review

−−Resistance and Possible Target for Modulation Chemotherapy -Transferases: Role in Alkylating AgentSGlutathione

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