Role of Glutathione S-Transferases in the Resistance of Human Colon Cancer Cell Lines to Doxorubicin1

[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 Â¡P.0. B., M. J. M.. K. A., M. M. P., C. L, I). S. R.Â¡,ami Department of Medicine, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario M5C 2AÃ•9Â¡M.J. M. Â¡,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-dinitrobenzene; 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. (Â¡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 mÃ©thylÃ¨nebluestaining. Relative survival was human colon adenocarcinoma cell lines (HT-29, LoVo, SW620, and expressed as the number of colonies produced by drug-treated cells divided by Caco-2). We also examined MRP and Pgp expression in these cell the number of colonies produced by untreated control cells. Cytosolic Catalytic Activities. Cells in logarithmic growth were harvested lines at the protein level. Finally, we determined whether a series of by trypsinization and cytosol was prepared in HEDG buffer (25 mM HEPES. GSH analogues that function as GST class-selective inhibitors can 1.5 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.4) by differential sensitize these colon cancer cell lines to the cytotoxic effects of centrifugation as the 106,000 x g supernatant (43). The 106,000 x g pellet doxorubicin. was resuspended in 10 mM Tris. 20% glycerol. and 1 niM EDTA (pH 7.4) and denoted the "membrane pellet" fraction. Protein concentration was determined by the method of Bradford (44). Cytosol and membrane pellet fractions were MATERIALS AND METHODS frozen in liquid nitrogen and stored at â€”¿70Â°Cuntiluse. Source of Chemicals. Doxorubicin hydrochloride was obtained from Spectrophotometric assays for total GST catalytic activity (CDNB as sub Farmitalia Carlo Erba (Milan, Italy). The following GST class-selective inhib strate) as well as assays with GST class-selective substrates were conducted itors were supplied by Dr. Amy S. Morgan (Terrapin Technologies Inc.. South according to the method of Habig et al. (45). These reactions were performed San Francisco. CA): TER 117 [â€¢y-glutamyl-5-(benzyl)cysteinyl-Ã„(-)-phenyl- in 0.1 M potassium phosphate (pH 6.5) at 25Â°Cin a total volume of 1 ml. In glycine] and its diethyl ester TER 199: TER 143 |7-glutamyl-S-(octyl)cystein- all cases, the rate of the enzyme-catalyzed reaction was determined by sub yl-glycine] and its diethyl ester TER 183: and TER 211 [-y-glutamyl-S- tracting the rate of reaction occurring in the absence of cytosol. For total GST (naphthyl) cysteinyl-glycine] and its diethyl ester TER 206. tPBO was activity, cytosol (200 /xg) was incubated with CDNB (2 mM) and GSH (2 mM). purchased from Aldrich Chemical Co. (Milwaukee. WI) and EA. CHP. CDNB. and the time course of the reaction was monitored at 340 nm with â‚¬¿=9.6 mM"'cm~'. For GST-u.-selective activity, cytosol (400 jug) was incubated glutathione reducta.se. GSH. NADPH. HEPES. and MTT were purchased from Sigma Chemical Co. (St. Louis. MO). H2O, was obtained from Caledon with tPBO (0.05 mM) and GSH (0.25 mM). and the reaction was monitored at Laboratories Ltd. (Georgetown. Ontario. Canada). [y-'2P]ATP (specific activ 290 nm with e = 24.8 mM 'cm '. For GST-7r-selective activity, cytosol (200 ity, >5000 Ci/mmol: radiochemical purity, >95%) and enhanced chemilumi- tig) was incubated with EA (0.2 mM) and GSH (0.25 mM), and the reaction was monitored at 270 nm with â‚¬¿=5.0 mM~'cm~'. nescence detection reagents were purchased from Amersham (Arlington Heights, IL). T4 polynucleotide kinase was purchased from Pharmacia (Upp We also assessed cytosolic GSH peroxidase activity with an organic (CHP) sala, Sweden). Electrophoresis and blotting equipment and reagents were and inorganic (H,O,) peroxide substrate (46. 47). GSH-dependent reduction of purchased from Bio-Rad (Hercules. CA). Tri-Reagent was purchased from CHP reflects the contribution of the GST-a class and the selenium-dependent Molecular Research Center Inc. (Cincinnati. OH). GSH peroxidase, whereas GSH-dependent reduction of H,O, reflects the Cell Lines. The human colon adenocarcinoma cell lines HT-29, LoVo, activity of the selenium-dependent GSH peroxidase alone. These reactions SW620. and Caco-2 were obtained from the American Type Culture Collection were performed in 0.1 M potassium phosphate (pH 7.0) at 25Â°Cin a total (Rockville. MD). Cells were grown as monolayer cultures in a-MEM supple volume of 1 ml. Cytosol (200 ^g) was incubated with CHP or H,O, (1.5 mM). mented with 10% fetal bovine serum and maintained in an atmosphere of 5% GSH (1 mM). NADPH (0.1 mM). and glutathione reducÃase(0.3 units), and the CO2 and 95% air at 37Â°C. time course of NADPH disappearance was monitored at 340 nm with t = 6.2 mM~'cm~'. Cytotoxicity Assays. Sensitivity of the cell lines to doxorubicin was as sessed using a microplate colorimetrie assay, which measures the ability of To assess the in vitro potency of the GST inhibitors (TER 117 and TER viable cells to reduce a soluble tetrazolium salt (MTT) to an insoluble purple 143), CDNB conjugation by HT-29 and Caco-2 cytosol was measured in the forma/.an precipitate (42). Cells in the logarithmic phase of growth were plated presence of concentrations of the inhibitors ranging from 0.005 to 50 /XM.The at the appropriate density (10.000 cells per well for HT-29, LoVo, and SW620 inhibitors were prepared in DMSO. To assess the ability of the diethyl ester and 20.000 cells per well for Caco-2) in 96-weII microtiter plates. After a 24-h TER 199 to inhibit GST catalytic activity in intact HT-29 cells, cells were incubation, cells were exposed to various concentrations of doxorubicin (pre exposed to TER 199 (1-100 iiM) for 4 h. cytosol was isolated, and CDNB pared in culture medium) for an additional 4 h. After doxorubicin treatment, conjugation activity was measured. In all experiments with GST inhibitors, a the medium was removed and replaced with doxorubicin-free medium, and GSH concentration of 0.05 mM was used in the CDNB conjugation assay. plates were incubated for an additional 96 h. At this time, 50 id of MTT ( 1 Immunoblot Analysis. For immunodetection of specific GSTs in cytosols mg/ml) was added to the cells and 200 fil of medium in each culture well. After prepared from each cell line, equal amounts of cytosolic protein (varied from a 4-h incubation at 37Â°C,medium was removed, and the formazan crystals 6 to 20 u,g depending on experiment) were resolved by SDS-PAGE (48) and were solubilized by adding 150 /j.1 of DMSO and by mixing on an orbital transferred to nitrocellulose (Hybond-ECL. Amersham; Ref. 49). Rabbit poly- shaker for 5 min. Absorbance at 540 nm was measured on a Labsystems clonal antibodies raised against human GST-a, -/a, and -TTwere purchased Multiskan LS microplate reader. Fractional absorbance was expressed as a from Biotrin International Ltd. (Dublin. Ireland). The GST-a. -/j., and -IT percentage of the absorbance displayed by cells not exposed to doxorubicin. primary antibodies were used at dilutions of 1:1,000, 1:500, and 1:50.000. The IC5(, was determined by interpolation from a plot of fractional absorbance respectively. A second rabbit polyclonal antibody against human GST-a was versus log|,,[drug|. In experiments assessing the toxicity of the GST inhibitors provided by Dr. Kenneth D. Tew (Fox Chase Cancer Center. Philadelphia. PA) alone, various concentrations of the inhibitors (prepared in ethanol) were added and was used at a 1:500 dilution. On the GST-a immunoblots. purified human to the cells after the initial 24-h incubation period, and the cells were then GST proteins (0.01 u,g) obtained from the following sources were run as incubated for an additional 96 h in the presence of the GST inhibitor without standards: GST Al and A2. Dr. Alan J. Townsend (Bowman Gray School of 94X

Medicine, Wake Forest University, Winston-Salem. NC); and GST PI, Dr. 120-, David J. Meyer (University College London, London. UK). The secondary antibody was a donkey anti-rabbit immunoglobulin-horseradish peroxidase conjugate (Amersham) and was used at the following dilutions: 1:1.(XX) 8 80 (GST-a, Biotrin and K. D. Tew), 1:2.()00 (GST-ju), and 1:5,000 (GST-ir). .o For immunodetection of MRP in membrane pellet fractions prepared from each cell line, equal amounts of protein (24 /ig) were analyzed. A mouse monoclonal antibody raised against human MRP (MRPmo) was purchased 40- from Kamiya BiomÃ©dicalCo. (Tukwila, WA) and was used at a 1:20 dilution. The secondary antibody was a sheep anti-mouse immunoglobulin-horseradish 20- peroxidase conjugate (Amersham) and was used at a 1:1.(XX)dilution. 0 An enhanced chemiluminescence system (ECL. Amersham) was used for protein detection, and films were scanned by laser densitometry for relative 10-2 10-1 10" 101 10* GST-TT quantitation (LKB 2222-020 UltroScan XL Laser Densitometer). [Doxorubicin] uM Northern Blot Analysis. Total RNA was isolated from cells in logarithmic growth by the acid guanidinium thiocyanate-phenol-chloroform extraction Fig. I. Concentration-response curves for four human colon cancer celt lines after method (50) using Tri-Reagent. RNA yield and purity was assessed by deter (reatmenl wilh doxorubicin. The cells (HT-29. LoVo. SW620. and Caco-2) were Irealed for 4 h with the concentrations of doxorubicin indicated on the abscissa and then mining the A2ht,:A2m ratio. A dilution series was prepared for each RNA incubated in the absence of doxorubicin for an additional 96 h. Cytoloxicity was assessed sample such that 5-, 10-, and 15-/xg quantities of total RNA were loaded onto using a microplate MTT colorimetrie assay. Fractional absorbance is expressed as a agarose gels containing 2.2 M formaldehyde (51 ). RNA integrity was assessed percentage of the absorbance displayed by cells not exposed to doxorubicin. Each poinl represents the mean Â±SD (fcun) of three to five independenl experiments, each per by comparing the relative intensities of the 28S and 18S rRNA bands as formed on duplicate 96-well microplates with sextuplÃcale determinations per plate. visualized on ethidium bromide-stained gels. After electrophoresis. RNA was transferred by capillary action to a nylon membrane (Genescreen: Dupont Co.. Wilmington, DE). Sequences for specific oligonucleotide probes for human icity assays were performed using cell densities that yielded a linear GSTs were provided by Dr. David J. Waxman (Boston University, Boston, relationship between absorbance and time of culture. We also exam MA). The following probe sequences were used: GST Al (5'-AAGTTCTT- GGCCTCCATGACTGCGT-3',4 nucleotides 738-762 of cDNA; Ref. 52); ined the sensitivities of the four cell lines under conditions in which GST A2 (5'-ATGTTCTTGACCTCTATGGCTGGTT-3',1 nucleotides 738- the doxorubicin was added to the cells and then left for the entire 96-h 762 of cDNA: Ref. 53); GST Ml (5'-TCAGCCACTGGCTTCTGTCA-3'; incubation period. The same rank order of resistance was observed; Ref. 54; nucleotides 138-157 of cDNA; Ref. 55); GST M2 (5'-AATCT- however, the concentration-response curve for the Caco-2 cells was GGGTCATAGCAGAGTTT-3'/ section from exon 5 of cDNA: Ref. 56); shifted to the left by an order of magnitude, as reflected by the GST M3 (S'-GTCAGAGCTGTAACAGAGCCTTATC-J',3 nucleotides 362- following IC5() values: Caco-2, 6.7 /AM;HT-29, 1.5 /AM;LoVo, 1.3 386 of cDNA; Ref. 57); GST PI (5'-GTTCTGGGACAGCAGGGTCT-3'; /AM: and SW620, 0.1 /Ã•.M.Thus, Caco-2 cells demonstrated time- Ref. 54; nucleotides 398-417 of cDNA; Ref. 58); and a-tubulin (5'-GA- dependent enhanced doxorubicin cytotoxicity, a response character CATCTTTGGGGACCACATCACCACG-3'; Ref. 59: nucleotides 39-66 of istic of topoisomerase II poisons. However, the other cell lines dis rat a-tubulin 3'-end cDNA; Ref. 60). The oligonucleotides were synthesized played similar sensitivity to doxorubicin after a 4- or 96-h exposure, by General Synthesis and Diagnostics (Toronto, Ontario. Canada) and labeled suggesting that the time-dependent nature of doxorubicin-induced cell at the 5'-end using T4 polynucleotide kinase and |-y-'2P]ATP. Hybridization killing may be influenced by factors that differ among cell lines. and wash conditions followed the procedures described previously (54, 59). The catalytic activities of the cytosolic fractions prepared from the For relative quantitative analysis, the intensity of individual bands on Northern cell lines toward various substrates are shown in Table 1. Caco-2 cells blots was determined by laser densitometry, and results for GST PI mRNA expression were normalized for the level of tubulin mRNA expression in each showed a higher total cytosolic GST activity than the other three cell sample. lines, as reflected by CDNB conjugation. Conjugation of EA and Analysis of Pgp Levels. Cells in logarithmic growth were analyzed for Pgp tPBO have been reported to reflect mainly GST-TT and -/Â¿catalytic- expression at the protein level in the laboratory of Dr. David W. Hedley activity, respectively (61 ). The activities of all cell lines toward tPBO (Ontario Cancer Institute/Princess Margaret Hospital, Toronto. Ontario. Can were very low, reflecting low levels of GST-/X expression in these ada). A quantitative immunofluorescence assay with the MRK-16 antibody cells. No significant differences in EA or tPBO conjugation were was used, and the assay was standardized using two human T-lymphoblast cell observed among the four cell lines. We have examined the GSH- lines: CCRF-CEM parental cell line (negative control) and the vinblastine- dependent reduction of CHP as a selective catalytic marker for GST-a resistant subline CCRF-CEM VLB O.I (positive control). forms (61), recognizing that the selenium-dependent GSH peroxidase Statistical Analysis. Where appropriate, data are presented as mean Â±SD. can also contribute to this reaction (46). LoVo and especially Caco-2 Comparisons of the four cell lines were carried out by randomized design one-way ANOVA followed by Newman-Keuls test. A result was considered to cytosols were deficient in this enzyme activity. We also assessed the be statistically significant at P < 0.05. GSH-dependent reduction of H2O2, because this is a substrate that can be metabolized by the selenium-dependent GSH peroxidase but not RESULTS GSTs (46). The cell lines showed the same rank order of activity toward both CHP and H2O2, and the catalytic activity toward H2O2 We first examined the relationship between doxorubicin resistance ranged from 65 to 85% of the activity toward CHP. and expression of GST-a, -ja, and -ir classes at the catalytic activity, To determine whether GST catalytic activity differences among the protein, and mRNA levels in four human colon adenocarcinoma cell cell lines were reflected by differences in GST protein expression, we lines (HT-29, LoVo, SW620, and Caco-2). The sensitivities of the performed immunoblot analyses with polyclonal antibodies specific four cell lines to a 4-h doxorubicin exposure, based on the microplate for members of the human GST-a. -JLL.and -IT classes. The immuno colorimetrie assay, are shown in Fig. 1. The following rank order of blot shown in Fig. 2 demonstrates that all four cell lines express resistance was observed with the IC50 value indicated: Caco-2, 78 /IM; GST-TT protein (â€”25kDa): however, there was no significant differ HT-29, 1.0 /IM; LoVo, 0.86 /AM;and SW620, 0.13 JUM.The four cell ence among the cell lines in GST-ir immunoreactivity as determined lines displayed doubling times between 32 and 39 h, and the cytotox- by densitometry. Fig. 3 shows the results of immunoblot analysis with the GST-a antibody from Biotrin. Caco-2 cells showed a unique, 4 D. Waxman, unpublished sequence. highly expressed GST-a-immunoreactive band, co-migrating with 949

Table I Ctitiilwic tli'livitie* Inward various substrates determined in c\tosolic fractions prepared frani human coltm cancer celi Unes Cytosol was isolated from the four cell lines (HT-29, LoVo. SW620, and Caco-2) and assayed for catalytic activity toward five substrates: l-chloro-2.4-dinitrobenzene (CDNB), ethacrynic acid (EAI. fra;i.v-4-phenyl-3-butcn-2-one (tPBO). eumene hydroperoxide (CHP), and hydrogen peroxide (H2O2). All data are expressed as mean Â±SD of triplicate determinations from either three or four independent cytosol preparations for each cell line. CDNB EA tPBO CHP Cell line (nmol/min/mg cytosolic protein) HT-29LoVoSW620Caco-2229 -1-26278 +8.212.9 +0.070.13 -"-14.41 1 +4.08.8 1.0 Â±1.7"24.0 Â±\.f'b16.4 Â±33245 Â±3.06.1 Â±0.080.04 +42412 +2.215.6 Â±4.84.0 Â±0.9"2.6 Â±0.040.13 Â±0.8'' Â±36'13.1 Â±5.20.14 Â±0.0330.6 Â±1.6"-*26. " Significantly different (P ^ 0.05) from HT-29, based on randomized design one-way ANOVA and post-hoc Newman-Keuls test. ''Significantly different (P ^ 0.05) from SW620. based on randomized design one-way ANOVA and post-hoc Newman-Keuls test. 'Significantly different (P Â£0.05) from all other cell lines, based on randomized design one-way ANOVA and post-hoc Newman-Keuls test.

purified GST Al and A2 proteins (â€”26kDa). that was not detected in followed by a 4-h exposure to doxorubicin with the GST inhibitor, the other lines. An additional band of slightly lower Mr was observed followed by an additional 96-h incubation in the presence of the GST inhibitor alone (i.e., "chronic" GST inhibitor and "acute" doxorubi in all cell lines with this antibody: however, when this experiment was repeated with a second GST-a polyclonal antibody (provided by Dr. cin). We also observed no evidence for potentiation or synergism in Kenneth Tew), only the prominent band overexpressed in Caco-2 cells experiments that used a chronic GST inhibitor and chronic doxorubi was observed. No specific GST-/n-immunoreactive band was detected cin or acute GST inhibitor and acute doxorubicin exposure protocol. in any of the four cell lines. We further investigated the combination of doxorubicin with TER 199 We performed Northern blot analysis to determine whether the in HT-29 cells using a clonogenic assay, which can detect cytotoxicity patterns of GST protein expression in the cell lines were reflected by over a broader range of cell killing. Fig. 6ÃŸdemonstrates that a 4-h similar patterns of expression at the mRNA level. The results in Fig. incubation with TER 199 followed by a 4-h incubation with doxoru 4 show that all cell lines express GST PI mRNA (-0.75 kb); based bicin and TER 199 does not potentiate the cytotoxic effects of doxo on densitometry with normalization for levels of a-tubulin mRNA rubicin. Two important differences between the results from the expression, the four cell lines demonstrated less than 2-fold variation clonogenic and microplate colorimetrie assays were noted: HT-29 in GST PI mRNA levels. We also detected a very weak hybridization cells show greater sensitivity to doxorubicin in the clonogenic assay, signal (â€”0.9kb) with the GST A2 oligonucleotide in the Caco-2 lane and TER 199 shows little cytotoxicity on its own in the clonogenic loaded with 15 /xg of total RNA. Hybridization with oligonucleotides assay, even at concentrations up to 30 /AM. for GST Al, Ml, M2, and M3 did not yield a specific signal with any In the absence of any evidence for potentiation of doxorubicin of the cell lines. cytotoxicity by the GST inhibitors, it was important to confirm that A major focus of this work was to determine whether a series of GSH analogues that function as GST class-selective inhibitors can sensitize colon cancer cell lines to the cytotoxic effects of doxorubi- 34.9 cin. We first examined the toxicity of the GST inhibitors alone in the 28.7- four cell lines using the microplate colorimetrie assay. In these ex periments, the GST inhibitor was added to the cells and then left for 20.5 the entire 96-h incubation period. The results in Fig. 5 show the sensitivity of HT-29 cells to EA, a prototype GST inhibitor, as well as SW620 HT-29 Caco-2 LoVo the three GSH analogues. TER 183, TER 199, and TER 206. It is 10.1 Â±1.5 12.5 + 3.5 13.613.3 8.1 Â±1.3 important to note that TER 183, TER 206, and TER 199 are cell GST Pi Â¡mmunoreactivity(arbitraryunits) membrane-traversing diethyl ester derivatives of GSH analogues that Fig. 2. Comparison of GST-Tr protein expression in four human colon cancer cell lines. function as selective competitive inhibitors of members of the human Immunoblot analysis of cytosolic protein (6 /ig) prepared from the cell lines (HT-29. GST-a, -pi, and -TTclasses, respectively (28-30). EA produced min LoVo. SW620. and Caco-2) using a polyclonal antibody directed against human GST-Tr. imal toxicity to HT-29 cells at concentrations up to 100 /AM.The TER Each lane represents an independent cytosol preparation. Numbers on the Â¡eftindicate the size of molecular mass markers in kilodaltons. The GST-TT immunoreactivity (in arbitrary compounds caused marked toxicity only at concentrations of greater units) is shown as the mean Â±SD of densitometry determinations from three or four than 10 JAM.The following IC5(, values were determined for each GSH independent cytosol preparations. analogue with each cell line: SW620 (TER 183. 10 AIM;TER 206, 20 IIM; and TER 199. 26 /AM);LoVo (TER 183. 8 /AM;TER 206, 16 /AM; and TER 199, 26 /AM):HT-29 (TER 183, 13 /AM;TER 206, 20 /AM;and 31 TER 199, 20 /AM);and Caco-2 (TER 183, 20 /AM;TER 206, 40 /AM; and TER 199, 28 /AM). Because of the lack of GST-/A expression in the colon cancer cell lines, we focused on TER 183 and TER 199 as selective GST-a and 21.5- -77 inhibitors, respectively, in combination studies with doxorubicin. HT-29 Caco- Results of a representative experiment are shown in Fig. 6A. In HT-29 SW620 LoVo cells, the GST-TTinhibitor TER 199 (concentrations of 1.1, 3.3, and 10 /AM)increased the toxicity observed in the presence of doxorubicin in A1 A2 P1 an additive manner: there was no evidence of potentiation or syner- Fig. 3. Comparison of GST-a protein expression in four human colon cancer cell lines. gism with this drug combination. Similar results were obtained with Immunoblot analysis of cytosolic protein (20 ng) prepared from the cell lines (HT-29, EA, TER 183, and TER 199 in all four cell lines (data not shown). The LoVo. SW620. and Caco-2l using a polyclonal antibody directed against human GST-a experiment shown in Fig. M was performed with a 4-h exposure to (Biotrin) is shown. Each lane represents an independent cytosol preparation. Numbers on the left indicate the size of molecular mass markers in kilodaltons. The lanes on the right the GST inhibitor (to allow for hydrolysis of the diethyl esters). were loaded with O.OI /j.g of the purified human proteins GST Al. A2. and PI. 950

1.4-

Fig. 4. Comparison of GST PI mRNA expres Â» GST P1 sion in four human colon cancer cell lines. North- I em hlol analysis of lotal RNA (5. 10, and 15 ng) isolalo! from the cell lines (HT-29, LoVo. SW620. and Caco-2) using gene-specific oligonucleotide probes for GST PI mRNA (topi and a-tubulin 2.4 mRNA (fiottoni) is shown. Each lane represents a different amount of total RNA from a single RNA â€¢¿tubulin isolation for each cell line. Numbers on the left indicate the si/e of molecular mass markers in 1.4- kilobases. The blot was initially probed for GST PI and then stripped and sequentially probed for a-tu bulin and other GST mRNAs. The level of GST PI mRNA in each cell line, normali/ed for the level of a-tubulin mRNA. is shown in arbitrary densitom- 10 15 10 15 10 15 5 10 15 |tg etry units. Caco-2 LoVo SW620 HT-29

0.54 0.39 0.29 0.42 GST P1 /tubulin mRNA (arbitrary units)

140-, the TER compounds were able to inhibit GST catalytic activity in these colon cancer cell lines. The results in Fig. 7 show that CDNB Â§120-1 conjugation by cytosol from HT-29 (Fig. 1A) and Caco-2 (Fig. IB) m | 100- cells was inhibited in a concentration-dependent manner by TER 143 and TER 117, selective GST-a and -TTinhibitors, respectively. The 1 80-1 following IC50 values were determined for each GSH analogue with each cell line: HT-29 (TER 143, 2.3 /MM:and TER 117, 1.0 /AM)and Caco-2 (TER 143, 2.1 /UM;and TER 117, 2.0 JAM).Additional in vitro 8 40J experiments demonstrated that TER 143, TER 211. and TER 117, 20- selective GST-a. -p.. and -TTinhibitors, respectively, did not inhibit GSH-dependent reduction of CHP by LoVo cytosol in a concentra 0- tion-dependent manner (data not shown). Finally, we examined the 10-3 10-2 10-' 10" 101 102 103 ability of the diethyl ester TER 199 to inhibit CDNB conjugation [GST inhibitor] uM when added to HT-29 cells in culture. After exposure of HT-29 cells Fig. 5. Concentration-response curves for HT-29 cells after treatment with GST to TER 199 ( 1, 10, and 100 /J.M)for 4 h, we detected no inhibition of inhibitors. HT-29 cells were exposed to the concentrations of TER 206, TER 183. TER cytosolic CDNB conjugation activity. 199, and EA indicated on the Ã•//MCÃ•.V.VÃ•;andthen incubated for an additional 96 h in the We supplemented our studies of GST expression in the four colon presence of the GST inhibitor without a change of medium. Cytotoxicity was assessed using a microplate MTT colorimetrie assay. Fractional absorbance is expressed as a cancer cell lines by examining the levels of two transport proteins that percentage of the absorbance displayed by cells not exposed to a GST inhibitor. Each are involved in MDR phenotypes. MRP and Pgp. MRP protein was point represents Ihe mean Â±SD (bars) of sextuplÃcate determinations from a single not detected in membrane pellet fractions from any of the cell lines by experiment. immunoblot analysis (data not shown). Expression of Pgp was as-

ns 100-

g 80-

o 10-1- 8 4(H o 20- i 5Â« O-l 10-2-

0 10-2 10-' 10Â° 101 102 10Â° 10' [Doxorubicin] [Doxorubicin] uM Fig. 6. Concentration-response curves for HT-29 cells after treatment with doxoruhicin in the absence and presence of the GST-rr inhibitor TER !99. A. alter a 4-h exposure of HT-29 cells to either vehicle or TER 199 alone (I.I. 3.3, and 10 JIM), cells were then exposed to the concentrations of doxoruhicin indicated on the abscissa in the presence of vehicle or TER 199 for an additional 4 h. Culture medium was then removed and replaced with medium containing vehicle or TER 199 alone at the original concentration, and the cells were then incubated for an additional 96 h. Cytotoxicity was assessed using a microplate MTT colorimetrie assay. Fractional ubsorbancc is expressed as a percentage of the ah.sorbaiiee displayed by untreated control cells. Each point represents the mean Â±SD (hars) of duplicate 96-welI microplates with se.xtuplicate determinations per plate. H. after a 4-h exposure of HT-29 cells to either vehicle or TER 199 alone (3 and 30 /IM), cells were then exposed to the concentrations of doxorubicin indicated on the abscissa in the presence of vehicle or TER 199 for an additional 4 h. Medium was removed, and cells were harvested by trypsini/.alion and plated at various densities in six-well culture plates. Colonies of -*4()cells were counted after incubation for 14 days by mÃ©thylÃ¨nebluestaining. Relative survival is expressed as the number of colonies produced by drug-treated cells divided by the number of colonies produced by untreated control cells. Each point represents the mean Â±SD (bars} of triplicate to sextuplÃcate determinations from a single experiment. 951

Fig. 7. Concentration-dependent in vilni inhibi B tion of CDNB conjugation activity by TER 117 and 100- TER 143 in cytosol prepared from HT-29 cells (A) 100- C o and Caco-2 cells (ÃŸ).Cytosol (200 /ig) was incu- o _ s ^ a o 80- buted in the presence of either vehicle or the con 5? 80 o>i = e centrations of TER 117 and TER 143 indicated on n ~EO60- the abscissa and assayed for CDNB conjugation 60 o u Â§8"5 using 2 niM CDNB and 0.05 HIMGSH. Enzyme 40- 40- activity is expressed as a percentage of the activity observed in the absence of a GST inhibitor. Control 20- Â¡I20- activities (100%) were as follows: HT-29, 47.5 U nmol/min/mg protein; Caco-2, 47.0 nmol/min/mg 0- 0- protein. Each point represents the mean of duplicate determinations from a single cytosol preparation for 10-3 10-2 10-1 10Â° 101 102 IO-' 10-2 10-1 10Â° 101 102 each cell line. [GST inhibitor] uM [GST inhibitor] uM sessed using a quantitative immunofluorescence assay. With immu- 600-fold range in sensitivities to doxorubicin. Our inability to detect nofluorescence from the negative control CCRF-CEM parental cell GST-/J, expression in these cell lines at the protein and mRNA levels, line defined as zero, the following measures of Pgp (in arbitrary units) along with the very low catalytic activities toward tPBO, agree with were obtained for the various cell lines: Caco-2, -I: HT-29, +1; recent work showing that a variety of colon cancer cell lines (includ SW620, + 1; LoVo, +6; and the positive control vinblastine-resistant ing HT-29 and SW620) either do not express or express very low subline CCRF-CEM VLB 0.1, +96. levels of GST-jit protein and/or mRNA (15, 30). An interesting finding from our study is that Caco-2 cells showed DISCUSSION a significantly higher total GST catalytic activity than the other cell lines and also displayed a unique GST-a-immunoreactive protein that Several sources of evidence have implicated the GST enzymes in was not present in the other cell lines. Previous studies have shown an the resistance of cancer cells to alkylating agents and anthracyclines elevated level of total GST catalytic activity and GST-a-immunore (5, 6). In the present study, we investigated the relationship between active protein in Caco-2 cells compared with the doxorubicin-sensi- doxorubicin resistance and expression of GST-a, -p., and -TTclasses at tive SW1116 line (14), and GST-a catalytic activity was detected in the catalytic activity, protein, and mRNA levels in four human colon SW620 cells (12). More recently, HT-29 and SW620 cells were adenocarcinoma cell lines (HT-29, LoVo, SW620, and Caco-2). We shown to be negative for GST A1 at the protein level and GST-a at also determined whether a series of GSH analogues that function as the mRNA level (15). It is tempting to speculate that the overexpres- GST class-selective inhibitors can sensitize these colon cancer cell sion of a GST-a form may contribute to the resistance of Caco-2 cells lines to the cytotoxic effects of doxorubicin. Our results show that the to doxorubicin; however, our GST-a immunoblot results contrast with level of expression of GST-Tr, the predominant GST class expressed our finding that Caco-2 cells have the lowest catalytic activity toward in these colon cell lines, does not predict the degree of resistance to CHP, a putative GST-a-selective substrate. Caco-2 cells were shown doxorubicin. Caco-2 cells were the most resistant to doxorubicin. previously to contain low levels of selenium-dependent and non- showed the highest level of total GST catalytic activity, and also selenium-dependent GSH peroxidase activity compared with SW1116 expressed a unique GST-a-immunoreactive protein that was not cells (14). We believe that the assay for GSH-dependent reduction of found in the other cell lines; these findings suggest that members of CHP reflects largely a contribution from the selenium-dependent GSH the GST-a class may contribute to the resistance of this cell line to doxorubicin. GST class-selective inhibitors did not potentiate the peroxidase in these cell lines for two reasons. First, catalytic activity toward H-,0-, (which reflects selenium-dependent GSH peroxidase) cytotoxic effects of doxorubicin in these colon cancer cell lines. Taken was 65-85% of the catalytic activity toward CHP (which reflects both together, our results suggest that the GSTs do not function as major selenium-dependent GSH peroxidase and GST-a). Second, the GST determinants of doxorubicin resistance in the four colon cancer cell a-selective inhibitor TER 143 was ineffective (at concentrations up to lines studied. HT-29, LoVo, SW620, and Caco-2 cells were chosen because of 500 /XM)at inhibiting the peroxidase activity in LoVo cytosol with CHP as substrate. It appears that Caco-2 cells overexpress a GST-a the previously reported broad range of sensitivities to doxorubicin (12-14). Based on our results, Caco-2 cells were the most resistant to protein that co-migrates with GST Al and A2 on immunoblot analysis doxorubicin, showing an ICS(I value approximately 80- to 90-fold but has low catalytic activity toward CHP. Although we do not know higher than HT-29 or LoVo and 600-fold higher than SW620. A the identity of this GST-a-immunoreactive protein, we suggest that previous study (14) found that Caco-2 cells were approximately GST A4 may be an important protein for future study. Human GST 30-fold resistant to doxorubicin compared with both HT-29 and A4 has been shown to display catalytic activity toward CDNB but SW620 cells. With respect to GST expression in these cell lines, our very low activity toward CHP (6). In addition, mouse GST A4 has work demonstrates that GST-Tr is the major form expressed at both the been shown to catalyze the conjugation of hydroxyalkenals, toxic protein and mRNA levels. High levels of GST-Tr protein and mRNA products of lipid peroxidation, and to confer resistance to doxorubicin were previously found in Caco-2 cells (14), and SW620 cells were in Chinese hamster ovary cells (17). shown to express GST-Tr at the mRNA level (12, 13). A recent In addition to the GST enzymes, we also examined the expression characterization of GSH-associated enzymes in human cell lines of of MRP and Pgp in the four colon cancer cell lines. We examined the National Cancer Institute drug screening program (15) showed MRP because it is a protein that contributes to anthracycline resist that HT-29 and SW620 cells expressed comparable levels of GST-Tr ance (36) and appears to function in a GSH-dependent manner (40, protein and mRNA. Although GST-Tr appears to be the major form 41). We found that MRP was not detectable in the four colon cancer expressed in these colon cancer cell lines, our data suggest that the cell lines by immunoblot. A recent immunohistochemical and immu degree of variation in expression of this enzyme at the catalytic noblot study with the same MRP monoclonal antibody used in our activity, protein, and mRNA levels is not sufficient to account for the study found that 15 of 16 colon carcinoma samples were negative for 952

MRP (62). In contrast, other immunohistochemical investigations and type GST inhibitor, and EA has been shown clearly to potentiate the studies of MRP mRNA expression have generally found MRP to be cytotoxic effects of the alkylating agent chlorambucil in a variety of expressed in colon tumors and cell lines at low and variable levels cell lines (23). With respect to doxorubicin. EA has been shown to (63-66). Most relevant to our study, recent work showed that HT-29, produce a 2- to 4-fold enhancement in doxorubicin toxicity in primary LoVo, and SW620 cells expressed only low levels of MRP mRNA, as cultures of lymphatic malignancies and normal peripheral blood lym detected by a highly sensitive reverse transcriptase-PCR assay, and phocytes (22). Recent work with an EA-sensitive subclone of HT-29 with 10 gastric and 10 colon cancer cell lines, the expression of MRP (HT4-1) showed that TER 199 (25 /LIM)enhanced chlorambucil mRNA did not correlate with sensitivity to doxorubicin (67). With toxicity by a factor of 2.39 but only enhanced doxorubicin toxicity by respect to Pgp, our quantitative immunofluorescence assay indicated a factor of 1.20, based on a clonogenic assay (30). Our work estab that Pgp is either not detectable or expressed at very low levels in all lishes that the lack of potentiation of doxorubicin toxicity by both four colon cancer cell lines. Normal human colon mucosa expresses TER 199 and TER 183 is consistent in four colon cancer cell lines. high levels of Pgp (68-70): expression in colon tumors is generally Most of our cytotoxicity experiments have used a microplate col lower than in adjacent normal mucosa, and the level of Pgp expression orimetrie assay to assess sensitivity of cell lines to doxorubicin and is related to the degree of differentiation of the tumor or cell line (69, GST inhibitors. Clonogenic assays may offer a more sensitive indi 71 ). Six of nine colon cancer cell lines in the National Cancer Institute cator of cell survival and have the advantage of being able to detect drug-screening program have detectable MDRI mRNA expression several orders of magnitude of cell killing, whereas the microplate (72), and Caco-2 cells have been shown to express Pgp protein (14) colorimetrie assay provides a convenient and rapid screening tool for with the level of expression varying with the age of the monolayer looking at multiple drug combinations and several cell lines. In our culture (73). HT-29 and SW620 cells were recently shown to be studies, the TER compounds were more toxic on their own when negative for MDRI mRNA, whereas LoVo expressed only low levels assessed using the microplate colorimetrie assay than by clonogenic of MDRI mRNA (67). Our data, together with results from other assay. Part of this difference may be attributable to the simple fact that investigators, suggest that MRP and Pgp do not contribute to the large in most of our colorimetrie assays cells were exposed to TER com range of doxorubicin sensitivities observed with the four colon cancer pounds for greater than 96 h. whereas in the clonogenic assay this cell lines. exposure period was only 8 h. Although the mechanisms by which the Inhibition of GST activity is considered a potentially useful ap TER compounds produce cytotoxicity are not known, it is possible proach to modulate resistance to certain antineoplustic agents (74, 75). that these compounds may impair mitochondria! function in such a Our in vitro GST inhibition studies focused on TER 143 and TER 117. way that MTT metabolism by mitochondria! succinate dehydrogenase GST-a and -Tr-selective inhibitors, respectively (28-30). The follow (76) is compromised without causing effects on clonogenicity. ing KÂ¡values have been reported for these agents with respect to in Resistance to anthracyclines appears to be multifactorial, with Pgp. hibition of CDNB conjugation by recombinant human GST isozymes: MRP. topoisomerase II. and the GSTs being contributors to the TER 143 (GST Al, 0.27 /IM; GST M la, 1.2/LIM;and GST PI, 1.9 /IM) resistance phenotype. The results of this study contribute to our and TER 117 (GST Al, 20 /LIM;GST Mia, 25 JAM;GST M2, 31 /UM; understanding of the cellular factors that determine the sensitivity of and GST PI, 0.4 /Â¿M)(30). Thus, TER 117 is approximately 5-fold colon cancer cell lines to doxorubicin. With respect to the four human more potent than TER 143 as a GST PI inhibitor, and we found that colon cancer cell lines examined, it appears that differences in Pgp. TER 117 was more than 2-fold more potent than TER 143 at inhib MRP, and GST-TT expression cannot account for the large range of iting CDNB conjugation in HT-29 cytosol. which contains predomi doxorubicin sensitivities observed. The most resistant cell line, nantly members of the GST-7T class. TER 143 was equipotent at Caco-2, showed the highest GST catalytic activity toward CDNB and inhibiting CDNB conjugation in both HT-29 and Caco-2 cytosols, overexpressed a novel GST-a form; the role of this protein in the whereas TER 117 was 2-fold more potent as an inhibitor in HT-29 resistance of Caco-2 cells to doxorubicin will be an interesting area cytosol than Caco-2 cytosol. These results suggest that GST enzymes for investigation. In the four colon cancer cell lines studied, GSH other than GST PI may make a greater contribution to CDNB con analogues that function as GST class-selective inhibitors did not jugation in Caco-2 cytosol than HT-29 cytosol, and we speculate that potentiate doxorubicin cytotoxicity. Our findings suggest that GST this may be attributable to a member of the GST-a class present in inhibition may not be an effective means to modulate doxorubicin Caco-2 cells that is distinct from the GST Al protein against which cytotoxicity in colon cancer, and that future studies with the TER TER 143 was developed as a specific inhibitor. compounds and other GST inhibitors should be targeted toward al A recent study (30) demonstrated that the diethyl ester forms of the kylating agents that are known to yield GSH conjugates in reactions GST inhibitors (e.g., TER 199) are toxic to HT-29 cells, with IC50 catalyzed by GSTs. values in the 20-50 /UMrange, whereas the unesterified forms are not The present study has not addressed the contribution of topoisomer toxic. This provides evidence that the diethyl esters diffuse through ase II to the doxorubicin sensitivity of the four colon cancer cell lines. the plasma membrane and produce biological effects in cultured cells; Although multiple mechanisms appear to contribute to the cytotoxic however, these compounds have not been shown to cause inhibition of effects of doxorubicin, topoisomerase II is a key target for anthracy- GST catalytic activity in intact cells. We failed to observe inhibition cline action (7). It will be important to examine the levels of topoi of CDNB conjugation in cytosol prepared from HT-29 cells exposed somerase II in these cell lines and to determine whether the levels of for 4 h to very high concentrations (up to 100 /UM)of TER 199. The this critical target enzyme predict the degree of doxorubicin sensitiv GSH analogues are reversible competitive inhibitors of GST with ity. In addition, it will be informative to determine whether the failure respect to GSH (28). Although we modified our CDNB conjugation of the GST inhibitors to potentiate doxorubicin cytotoxicity is accom assay to enhance the likelihood of observing inhibition by using a low panied by a failure of these modulators to alter the levels of DNA- GSH concentration (0.05 mM), we believe that the lack of GST topoisomerase II-drug complexes. inhibition is attributable to the dilution of the concentration of the GSH analogue during cytosol preparation. ACKNOWLEDGMENTS In the present study, EA and the TER compounds were unable to potentiate the cytotoxic effects of doxorubicin in four colon cancer We lhank the following individuals for their contributions lo Ibis work: cell lines. Most work on GST modulation has used EA as the proto Annaghan Afsar and Jennifer Lok for performing initial experiments lhat

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. (ÃŒUTA1HIOM-: .V-TKANSM-RASES ASM) IXIXORl BICIN RESISTANCE stimulated (his study: Peter McPherson and Pat Firby for assistance with 24. Xu. B. H.. and Singh. S. V. Effect of buthionine sulfoximine and ethacrynic acid on cytotoxicity assays; Dr. David Hedley and Trudey Nicklee for performing the cytotoxic activity of mitomycin C analogues BMY 25282 and BMY 25067. Cancer Res., 52: 6666-6670, 1992. Pgp quantitation: Drs. Gerald Goldenberg. David Hedley. and Ted Inaba for 25. Giaccio, P. J., Shen. H. X., Jaiswal, A. K., Lyttle. M. H., and Tew. K. D. Modulation valuable discussions; Dr. Kenneth Tew for providing a GST-a polyclonal of detoxification gene expression in human colon HT29 cells by glutathione 5- antibody; Dr. Alan Townsend for providing purified human GST Al and A2 transferase inhibitors. Mol. Pharmacol., 48: 639-647, 1995. 26. O'Dwyer, P. J., LaCreta. F. 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Peter O. Beaumont, Malcolm J. Moore, Kamran Ahmad, et al.

Cancer Res 1998;58:947-955.

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