[CANCER RESEARCH 58. 947-955. March 1. 1998] Role of Glutathione S-Transferases in the Resistance of Human Colon Cancer Cell Lines to Doxorubicin1 Peter O. Beaumont, Malcolm J. Moore, Kamran Ahmad, Michelle M. Payne, Chunja Lee, and David S. Riddick2 Department of Pharmacology. Medical Sciences Biiildinn, University of Toronto, Toronto, Ontario M5S IAS ¡P.0. B., M. J. M.. K. A., M. M. P., C. L, I). S. R.¡,ami Department of Medicine, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario M5C 2AÕ9¡M.J. M. ¡,Cernada ABSTRACT The GSTs (EC 2.5.1.18) constitute an important family of detoxi fication enzymes, catalyzing the conjugation of electrophilic sub The anthracycline doxorubicin has little activity against colorectal stances with the ubiquitous nucleophile GSH and the non-selenium- cancers. It is hypothesized that this is attributable to a multifactorial resistance mechanism in which the glutathione S-transferases (GST) may dependent reduction of organic hydroperoxides (5, 6). Humans play a role. We studied the relationship between GST expression and possess four classes of cytosolic GSTs and two membrane-bound doxorubicin resistance in four human colon adenocarcinoma cell lines GST enzymes. The cytosolic enzymes belong to the a. ;u,. TT,and 0 (HT-29, LoVo, SW620, and Caco-2), with the goal of modulating GST classes, and these forms have been studied most extensively with activity to overcome resistance. Caco-2 cells were the most resistant to respect to their involvement in antineoplastic drug resistance. Within doxorubicin, showing an ICSOvalue approximately 80- to 90-fold higher each GST class there can be multiple protein subunits, each encoded than HT-29 or LoVo and 600-fold higher than SVV620. Total GST catalytic by a distinct gene; each functional GST enzyme exists as a ho- activity was significantly higher in Caco-2 cells compared with the other lines. All four cell lines expressed GST-n- at the catalytic activity, protein, modimer or heterodimer formed from two protein subunits from within the same class. The human GST-w class contains a single gene and mRNA levels; however, no significant differences were observed among the cell lines. GST-fi expression was not detectable at the protein encoding the GST PI protein, whereas there are currently six putative and mRNA levels, and the four cell lines displayed very low catalytic a class genes encoding subunits GST Al, A2, A3, A4, 9.9, and u>.The activity toward a GST-ju-selective substrate. Caco-2 cells showed a H class contains five genes encoding subunits GST M1, M2, M3, M4, unique, highly expressed GST-a-immunoreactive band that was not de and M5, and the 0 class consists of two genes encoding the GST Tl tected in the other lines; however, the glutathione peroxidase activity of and T2 proteins (6). Caco-2 cells was the lowest among the four cell lines. Neither ethacrynic There are six major lines of evidence that suggest that GSTs may acid nor glutathione analogues that function as GST class-selective inhib play a role in the resistance of tumor cells (including colon) to itors were able to potentiate the cytotoxic effects of doxorubicin in these doxorubicin and other antineoplastic agents. First, colon tumors often colon cancer cell lines, as demonstrated in both microplate colorimetrie and clonogenic assays. The multidrug resistance-associated protein and show elevated levels of GST expression compared with normal mu P-glycoprotein were either not detectable or expressed at such low levels cosa (9-11), and doxorubicin-resistant colon cancer cell lines often that they are not likely to contribute to the differences in doxorubicin show elevated GST expression (12-15). GST-Tr is the form most sensitivity observed among these cell lines. commonly overexpressed in colon tumors and cell lines. Second, transfection of cDNAs encoding GST-TT and -a forms into drug- sensitive cell lines can result in increased resistance to doxorubicin INTRODUCTION (16. 17): however, other transfection studies have demonstrated no The anthracycline antibiotic doxorubicin is one of the most useful effect (18). Third, numerous studies have demonstrated that depletion antineoplastic agents, displaying a broad range of clinical activity of GSH by treatment with buthionine sulfoximine sensitizes cells to a against several solid tumors as well as lymphoid and myelogenous variety of antineoplastics including doxorubicin (12, 13, 19, 20). Fourth, reduction of GST-77 expression in colon cancer cells via tumors (1). However, doxorubicin is generally inactive in the treat ment of cancers of the colon and rectum. This inactivity is generally antisense technology results in increased sensitivity to doxorubicin attributed to intrinsic resistance to the cytotoxic effects of doxorubicin (21). Fifth, inhibition of GST activity in cancer cell lines can increase displayed by colon cancer cells. In vitro studies have shown that there sensitivity to alkylating agents and anthracyclines. Most investiga tions to date have used EA as a nonselective GST inhibitor (22-24); are many mechanisms of resistance to anthracyclines and that resist ance is often multifactorial (2). The major cellular mechanisms of however, because of the lack of specificity of EA action (25) and resistance to doxorubicin include the Pgp'-mediated MDR phenotype limitations associated with the clinical use of EA as a drug resistance (3), MRP (4), increased detoxification via GSH-dependent enzymes modulator (26), more recent studies have begun to examine various (5, 6), alterations in topoisomerase II (7), increased DNA repair (8). GSH analogues that have been designed to function as GST class- and distal mechanisms of resistance involving alterations in apoptotic selective inhibitors (27-30). Sixth, several antineoplastic agents form pathways. GSH conjugates in reactions catalyzed by GSTs (31, 32). Interest ingly, GSH conjugates are not believed to be formed from doxorubi cin or its primary metabolites; however, anthracyclines are quinone- Received 8/22/97; accepted 1/5/98. The costs of publication of this article were defrayed in part by the payment of page containing compounds that undergo redox cycling leading to the charges. This article must therefore be hereby marked advertisement in accordance with formation of reactive oxygen species and subsequent lipid peroxida- 18 U.S.C. Section 1734 solely to indicate this faci. 1This work was supported in part by a Research Career Award in Health Science from tion (33). The GSTs may contribute to doxorubicin resistance by the Pharmaceutical Manufacturers Association of Canada-Health Research Foundation/ catalyzing the detoxification of two types of toxic lipid peroxidaiion Medical Research Council of Canada (to D. S. R.). 2 To whom requests for reprints should be addressed, at Department of Pharmacology, products; lipid hydroperoxides are reduced via a non-selenium-depen Medical Sciences Building. University of Toronto. Toronto, Ontario, Canada M5S 1A8. dent GSH peroxidase reaction catalyzed preferentially by members of Phone: (416) 978-0813; Fax: (416) 978-6395; E-mail: [email protected]. ' The abbreviations used are: Pgp, P-glycoprotein; MDR. multidrug resistance; MRP. the GST-a class (34), and hydroxyalkenals are conjugated with GSH multidrug resistance-associated protein; GSH. reduced glutathione: GST. glutathione in a reaction catalyzed by GSTs (17, 35). S-transfera.se: EA, ethacrynic acid; TER. Terrapin Technologies Inc.: tPBO. lrans-4- There also seems to be a connection between GSH and the ATP- phenyl-3-buten-2-one; CHP. eumene hydroperoxide; CDNB, l-chloro-2.4-dinitroben- zene; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra/olium bromide; H2O2, hydro binding cassette family of transport proteins. MRP confers resistance gen peroxide; DOX, doxorubicin. to anthracyclines, epipodophyllotoxins, and Vinca alkaloids (36); 947 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. (¡i.iTATHioNi:.V-TRANSH-:RA.SI-:SANDIXJXORUBICINRESISTANCE however, the question of whether MRP mediates the direct transport a change of medium. In experiments combining doxorubicin with a GST of these unmodified antineoplastic agents has remained controversial inhibitor, the inhibitor was added to the cells after the initial 24-h incubation (37-39). MRP transports GSH conjugates of endogenous substances period. After a 4-h exposure to the GST inhibitor alone, cells were then (e.g., leukotriene C4; Ref. 40), and GSH stimulates the MRP-mediated exposed to various concentrations of doxorubicin in the presence of the transport of vincristine (41 ). These recent findings raise the interesting inhibitor for an additional 4 h. Culture medium was then removed and replaced possibility that MRP may cooperate with the GSH-GST system to with medium containing the GST inhibitor alone at the original concentration, and the cells were then incubated for an additional 96 h. confer resistance to certain antineoplastic agents. Experiments combining doxorubicin with GST inhibitors were repeated We hypothesize that GSTs contribute to the multifactorial resist using a clonogenic cytotoxicity assay. Cells in the logarithmic phase of growth ance of colon cancer cells to doxorubicin, and that inhibition of GST were plated at a density of 1 X IO5 cells per flask in 25-cnr culture flasks. activity sensitizes such cells to the cytotoxic effects of doxorubicin. If After a 48-h incubation, cells were exposed to a GST inhibitor alone for 4 h, this hypothesis is correct, then a combination of doxorubicin with a followed by an additional 4-h incubation in the presence of the inhibitor and GST inhibitor should be examined in clinical studies in colorectal various concentrations of doxorubicin. Medium was removed, and cells were harvested by trypsinization and plated at various densities (1 X 102-1 X 10s cancer. In the present study, we have examined the relationship between doxorubicin resistance and expression of GST-a, -/x, and -TT cells per well) in six-well culture plates. Colonies of >40 cells were counted classes at the catalytic activity, protein, and mRNA levels in four after incubation for 14 days by méthylènebluestaining.