Melphalan, and Etoposide

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[CANCER RESEARCH56, 3577â€”3582,August1, 19961 Transfection of Glutathione S-Transferase (GST)-n' Antisense Complementary DNA Increases the Sensitivity of a Colon Cancer Cell Line to Adriamycin, Cisplatin, Melphalan, and Etoposide

Noriyoshi Ban, Yasuo Takahashi, Tetsuji Takayama, Toshiro Kura, Tatsuro Katahira, Sumio Sakamaki, and Yoshiro Nlitsu'

Department oflnternal Medicine (Section 4), Sapporo Medical University, South-i, West-16, Chuo-ku, Sapporo 060, Japan

ABSTRACT cancer agents before treatment, which suggests that GST-ir is in volved in intrinsic resistance as well (12). However, these studies The goal ofthis study was to demonstrate that glutathione S-transferase have not shed light directly on the question of whether the expression (GST)-ii' is directly involved in the Intrinsic and acquired resistance of of GST-ir is in fact a cause of drug resistance or merely a reactive cancer cells to anticancer drugs. To this end, GST.i@ antisense eDNA was transfected Into the cultured human colon cancer cell line M7609, which change that accompanies the development of resistance. expresses an Innately high level of GST.ir and shows intrinsic drug Accordingly, various investigators have attempted to obtain direct resistance, and Into an M7609 strain with acquired resistance to Adria proof of the involvement of GST-'rr in anticancer drug resistance by mycin (ADRLe., M7609/ADR cells). The changes in the sensitivity of these the transfection of GST-ii@expression vectors to cultured cancer cells. transfectants to various anticancer drugs were investigated. The intracel For example, Moscow et a!. (13) transfected a GST-ir expression lular concentrations of GST.ir in M7609/anti.1 cells and M7609/anti-2 vector to MCF-7 cells (a cultured human breast cancer cell line) and cells, two clones that were established by transfection of GST-i@antisense showed that the cells acquired resistance to etoposide and ethacrynic eDNA Into M7609 cells, were decreased to approximately half of those acid, but there was no change in the cells' sensitivity to CDDP or detected in the parent cells (M7609) and in the control cells transfected melphalan. Another group, Miyazaki et a!. (14), transfected a GST-ir with vector alone (M7609/pLJ). The sensitivities of the antisense transfec expression vector to Chinese hamster ovarian cells and found that the tents In relation to ADR, cisplatin, melphalan, and etoposide were in cells were resistant to CDDP but not to ADR. Nakagawa et a!. (15) creased â€”3.3-fold, 2.3-fold, 2.2-fold, and 2.1-fold, respectively, compared with those of M7609 and M7609/pLJ. On the other hand, the sensitivities transfected a GST-ir expression vector to NIH3T3 cells that had been of the antisense transfectants to Taxol, vincristine, S-fluorouradll, and transformed with H-ras and found that although the resistance to ADR mitomycin C were not significanfly changed. Similarly, the transfection of had been increased, the cells did not acquire resistance to alkylating antisense cDNA Into M7609/ADR cells resulted in the reduction of intra. agents. In yet another study, Black et al. (16) transfected a GST-ir cellular GST-ir concentration (by about half) and an Increased sensitivity expression vector to Saccharomyces cerevisiae and reported that to ADR (4.4-fold), but no increase In 5-fluorouradil sensitivity. Thus, resistance was acquired to ADR and chlorambucil. However, the GST-@risconsidered to be a multidrug resistance factor that Is responsible results of these experiments involving the transfection of GST-ir gene for both the intrinsic and acquired resistance of cancer cells to anticancer are not entirely unambiguous. drugs such as ADR, cisplatin, melphalan, and etoposide. In an attempt to explain these various findings, Tew (17) proposed a number of hypotheses, including: (a) the possibility that the level of INTRODUCTION reduced GSH in the target cells was below that necessary for the activation of the GSHJGST detoxification system; (b) the possibility GST-ir 2 expression is increased in various human cancer tissues, that the level of the cell membrane-bound GSH-conjugate export including gastric cancer, colon cancer, lung cancer, oral cavity cancer, pump was insufficient; (c) the possibility that the level of native GST and uterine cancer; thus it is employed in cancer research as a tumor expression by the cells used was already at a maximum; (d) the marker (1â€”5).There have also been numerous reports showing that the possibility that the transcription efficiency of the transfected GST-'rr expression of GST-ir is elevated in various cultured cells lines pos gene was insufficient or that the turnover was accelerated; and (e) the sessing resistance to anticancer drugs such as ADR (6), melphalan (7), possibility that the importance of the GSH/GST detoxification system CDDP (8), cyclophosphamide (9), and chiorambucil (10) as well as in in the overall anticancer drug resistance mechanism was low. Tew in vivo cancer tissues that have become resistant to therapy after thus suggested that the approach of transferring the GST-ir gene to administration of anticancer agents (11). We have recently estab target cells and thereby causing an increase in GST-ir activity had not lished, from a human colon cancer cell line (M7609), an ADR been adequate. resistant cell line (M7609/ADR) that showed increased expression of In consideration of this background, we designed the present study GST-ir but not of p-glycoprotein, multidrug-resistance associated on the basis of the reverse supposition that if the expression of GST-ir protein (MRP), and topoisomerase ll.@ These observations suggest by cells was inhibited, then the activity of GST-ir should decrease that the expression of GST-ii@is involved in the acquisition of resist without any dependence on the level of GSH in the cells or the ance to anticancer drugs. Furthermore, elevated expression of GST-ir GSH-conjugate export pump. We tested the validity of this approach has been demonstrated even in cancers that show resistance to anti by transfection of GST-ir antisense cDNA to the M7609 human colon cancer cell line and to its ADR-resistant subline, M7609/ADR, and Received 2/5/96; accepted 5124/96. The costs of publication of this article were defrayed in part by the payment of page then testing these cell lines for multidrug sensitivity. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom inquests for reprints should be addressed, at Department of Internal MATERIALS AND METHODS Medicine (Section 4), Sapporo Medical University School of Medicine, South-l, West-l6, Chuo-ku, Sapporo 060, Japan. Phone: 81-I 1-611-2111, extension 3260; Fax: 81-11-612- Cell Linesand Cell CUltUre.Humancoloncancercell lineM7609(18) 7987. was kindly provided by Dr. S. Machida (Hirosaki University, Hirosaki, Japan). 2 Abbreviations used are: GST-ir, glutathione S-transferase ir, ADR, Adriamycin; This cell line was established from a colon cancer patient who had not been CDDP, cisplatin; OSH, glutathione; 5-Ri, 5-fluorouracil; VCR, vincristine; MMC, mit omycin C. treated with anticancer drugs. M7609/ADR, an ADR-resistant cell line, was 3 Unpublished data. established from M7609 cells in our laboratory according to the method of 3577

Whelan et a!. (19). M7609/ADR shows a resistance to ADR approximately six labeled with 32P, using the random primer method (25) and were used as times greater than that of M7609. M7609/ADR also shows cross-resistance to probes. Hybridization and washings were performed according to the proce CDDP, etoposide, and melphalan but not to 5-FU, VCR, and MMC. In dures described in â€œSouthernBlotting.â€• addition, M7609/ADR shows two times greater expression of GST-ir than the GST-ii Quantitation by ELISA. Afterwashingeach cell preparationtwo parent cell line but shows no increases of p-glycoprotein, MRP, and topoi times in cold PBS (10 mMsodium phosphate buffer containing 0.9% NaCl), somerase II.@ Both of these cell lines were cultured in RPM! 1640 (Life the cells were adjusted to a concentration of 1 X l0@/miinthe same buffer and Technologies, Inc., Grand Island, NY) containing 10% FCS (Flow Laborato were homogenized with a Dounce homogenizer. The lysates were then cen lies, North Ryde, Australia) in tissue-culture flasks; incubation was performed trifuged at 12,000 rpm for 15 mm, and the concentration of GST-ir in each at 37Â°Cinan atmosphere of air containing 5% CO2. supernatant was measured by the sandwich ELISA established in our labora Construction of a GST-i@ Antisense Vector. The plasmid pGpi2 (20), tory as described previously (27, 28). containing GST-ir cDNA, was obtained from the Japanese Cancer Research Sensitivities to Various Anticancer Drugs. ADR, 5-FU, and MMC were Resources Bank (Tokyo, Japan), and the p11 vector (21) was kindly provided purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), whereas by Dr. G. Wu (University of Connecticut, Farmington, Cl'). pGpi2 was etoposide and CDDP were obtained from Nippon Kayaku Co., Ltti (Tokyo, digested with EcoRI, and a 0.7-kb EcoRI-EcoRI fragment containing the Japan), and VCR was purchased from Shionogi Co., Ltd. (Tokyo, Japan). whole coding region for GST-ir was recovered. Both ends of this fragment Melphalan and ethacrynic acid were obtained from Sigma Chemical Co. (St. were then blunted with the Klenow fragment (Takara Shuzo Co., Ltd., Kyoto, Louis, MO), and Taxol was supplied by Bristol-Myers (Tokyo, Japan). The Japan). The pll vector was linearized with BamHI, the blunting of both sensitivities of each cultured cell line to these various anticancer drugs were terminals was similarly performed using the Kienow fragment, and was de determined by the dye-uptake method (29). Briefly, cultured cells were sus phosphorylated with bacterial alkaline phosphatase (Takara Shuzo, Co., Ltd.). pended at a density of 1 X l0@cells/mi in RPMI 1640 containing 10% FCS, Both of these processed fragments were ligated with T4 ligase (Takara Shuzo lOO-pialiquots were dispensed to each well of 96-well culture plates, and the Co., Ltd.), and a clone was selected in which the GST-ir cDNA was inserted plates were incubated for 24 h at 37Â°Cinan atmosphere of air containing 5% @ in the reverse direction. This clone was named pU/antiGST--rr (Fig. 1). In CO2.In some experiments,to obtainpositivecontrols,3.3 ethacrynicacid, consideration of in vivo experiments anticipated for the future, the retroviral a known substrate for GST-ir(30), were added to M7609 or M7609/ADR with vector (Pu) was employed as the expression vector for GST-ii@antisense the anticancer drugs. Otherwise, various anticancer drugs were added without cDNA in the present study. ethacrynic acid to the wells at 10 different concentrations. Then the cells were Gene Transfer. The transfection ofthe pU/antiGST-ir into the M7609 and incubated for another 48 h at 37Â°C.Next, 25 @xlofa 25% glutaraldehyde M7609/ADR cells was performed by the lipofection method (22). Briefly, solution was added to each well to fix the cells, and the plates were then 2.5 X l0@cells were dispersed in a 3.5-cm culture dish and were incubated for washed with water, were dried, were stained with a 0.05% methylene blue solution, and were eluted with 0.33 N HC1. The absorbance at 665 nm was then 24 h. The attached cells were then washed three times with RPM! 1640 (Life measured with an ELISA reader (MS-3096F; SLT-LAB Instruments Co., Technologies, Inc.), followed by the addition of 3 ml of the same culture Salzburg, Austria). For each well on the culture plates, the cell survival rate medium to the dish. Next, 100 @xlof plasmid lipofectin reagent (Life Tech nologies, Inc.) were mixed with 3 ,xgof pU/antiGST-ir and were incubated at was calculated by taking the absorbance value of the control well (to which no anticancer drug had been added) as 100%. room temperature for 15 mm. This mixture was then added to each culture Statistical Analysis. Paired Student's t tests were used to compare GST-ir dish, and the dishes were incubated at 37Â°Cfor 6 h in an atmosphere of air concentrations and IC50 values for each cell type. containing 5% CO2. RPM! 1640 (3 ml) containing 10% FCS was added to each culture dish, and incubation was continued for another 72 h. G4l8 (Life Technologies, Inc.) was added to the culture medium in each dish to a pU SV4O pBR322 concentration of 400 p.g/ml, and the cells were cultured for approximately 2 LTR orI@ Ne&' Orl UTR @ weeks at 37Â°Cin an atmosphere of air containing 5% CO2 The G418-resistant F@ I â€¢ I strains of each cell line were obtained and designated M7609/anti and M7609/ I Ii I ADRJanti, respectively. The M7609/anti cells were further cloned by limiting x B H x dilution, and 2 of the 12 resultant clones, M7609/anti-l and M7609/anti-2, Bani HI were selected for subsequent experiments. pu vector without GST-ir antisense E GST-'r @ cDNA was transfected to M7609 and M7609/ADR cells to obtain each vector kb) control transfectants, M7609/pU and M7609/ADR/pu, respectively.

@ Southern Blotting. Genomic DNA was extracted from each cell line as Eco RI ______Blunt end @@ described by Hoggan et aL (23). Each DNA sample (10 p@g)was digested with dephosphor@ KpnI, was subjected to 0.8% agarosegel, and was electrophoresedat 45 V for 6 h. The DNA fragments were then transferred to nitrocellulose filters by the method of Southern (24), followed by baking at 80Â°Cfor2 h. The GST-ir AmpR @4'â€¢'Eâ€”@Ligation cDNA (0.7-kb EcoRI-EcoRI fragment) was labeled with 32Pby the random primer method (25), yielding a probe with a specific activity of 2.0 X 108 cpm/@xg.Thenitrocellulose filters were soaked in a hybridization buffer [50% Blunt end formamide, 4 X SSC, 50 mM HEPES, 10 X Denhardt's solution, and 100 @xg/mlof denatured salmon sperm DNA (pH 7.2)] to which the 32P-labeled pU/anti GST-n probe had been added and then were incubated at 42Â°Cfor18 h. The filters SV4O pBR322 LTR on NeoR on LTR were then washed for 1h in a solution of 2 X SSC and 0.1% X SDS, followed I-I lâ€¢ S â€”@ I I @ by washing for 2 h in a solution of 0.5 X SSC and 0.1% X SDS, and then GST-ir I autoradiography was performed. antlsense H x cDNA Northern Blotting. poly(A)@RNA was extracted from each cell line ac 1 1kb I cording to the method of Badley et a!. (26). Each RNA sample (3.5 @xg)was analyzed by electrophoresis on 1.0% agarose gel containing 2.2 Mformalde Fig. 1. Construction of human GST-ir antisense expression vector. A 0.7-kb EcoPJ hyde. The fractionated RNAs were then transferred to nitrocellulose filters, fragment containing GST-ir cDNA was excised from pGpi-2 and was blunt-ended with a Kienow fragment. Similarly, the pU vector was linearized by BamHI digestion, blunt followed by baking at 80Â°Cfor2 h. The 0.7-kb EcoRI-EcoPJ fragment of ended with a Klenow fragment, and dephosphorylated with a bacterial alkaline phospha human GST-ir and the 0.8-kb PstI-XbaI fragment of human GAPDH (pur tase. These two fragments were then ligated with T4 ligase, and a clone containing the chased from the American Type Culture Collection, Rockville, MD) were 0.7-kb EcoRI-EcoRI fragment in the opposite direction was selected and was named pU/antiGST-ir. LTR, long terminal repeat; SV4Oon, SV4Oorigin containing the SV4O early promoter, Neoâ€•,neomycinresistance gene; pBR322 on, origin of pBR322; Anip'@, 4 Unpublished data. ampicillin resistance gene. 3578

A. B.

@ 28S Fig. 2. Southern and Northern blot analyses of M7609 cells transfected with GST-ir antisense 9.4 â€” cDNA.A, Southern blot analysis. Ten @.&gofcellular DNA were digested with KpnI, separated on 0.8% .@- 4.0kb agarose gel, transferred onto a nitrocellulose filter, 6.6 â€” @ 18 S @@ and probed with a 32P-labeled 0.7-kb EcoRI-EcoRI @::i@_@:: 5.1 kb fragment The 5.1-kb band is from transfected GST-ir antisense cDNA. B, Northern blot analysis. @@ poly(A)@RNAs (3.5 @i.g)wereseparated on 1.2% 0.7kb agarose gel, transferred onto a nitrocellulose filter, and probed with the same probe. The 4.0-kb band is L3 â€” from transfected GST-ir antisense cDNA. A 2.0 â€” GAPDH probe was used as an internal quantitative control for the amounts of RNA on the filter (bot tornpanel). â€”

RESULTS Table 1 GST-ir concentration in M7609 cells, M7609 transfected with GST-ir antisense cDNA, and M7609 treated with ethacrynic acid Southern and Northern Blot Analyses of M7609/anti-1 and concentrationa M7609/anti-2 Cells. Southern blot analysis by KpnI digestion was cells)M76091.4Â±0.2M7609/pUCell lineGST-ir (pmol/106 performed on M7609/pU, M7609/anti-1 cells, and M7609/anti-2 .3 Â±0.2 cells, using GST-ir cDNA (0.7-kb EcoRI-EcoRI fragment) as a probe M76090.2M7609/anti-l0.8 + EAb1 1.4 Â± (Fig. 14). In addition to a band at approximately 11 kb that repre 0.lcM76O9/anti-20.8 Â± sented the cellular GST-i@rgene, a band at 5.1 kb was observed in Â±O.lc M7609/anti-1 and M7609/anti-2 cells. This band was considered to a Each value is the mean Â± SD of three separate experiments. b EA, ethacrynic acid, using a noncytotoxic concentration of ethacrynic acid. represent the transfected antisense vector. C The value was significantly lower than that of M7609/pLJ cells; P < 0.01. To examine the expression of endogenous GST-ir and antisense mRNA, Northern blot analysis was performed using the GST-ir cDNA probe (Fig. 2B) Endogenous GST-'rr mRNA was detectable as a 0.7-kb band for all three cells. However, the amounts of the 0.7-kb significant difference between them. On the other hand, the intracel mRNA in M7609/anti-1 and M7609/anti-2 cells were apparently less lular concentrations of GST-ir in the M7609/anti-l cells and the than that in M7609/pU cells. Moreover, an additional minor band at M7609/anti-2 cells were 0.8 Â±0.1 pmol/l06 cells and 0.8 Â±0.1 4.0 kb was detected in the two transfectants that was considered to pmol/106 cells, respectively. These levels decreased to approximately represent antisense RNA. There have been some reports that antisense half of the GST-i@ concentrations detected in the parent M7609 cells RNA of a transfected gene cannot be detected at times because the and the M7609/pU cells (Table 1). sense-antisense hybrids are rapidly degraded (31, 32). Our results Anticancer Drug Sensitivities of M7609/anti-1 and M7609/ suggest that the expression of antisense mRNA is responsible for anti-2 Cells. Both the M7609/anti-l and the M7609/anti-2 cells lowering the levels of endogenous mRNA (perhaps by hybridization), showed significantly elevated sensitivity to ADR, CDDP, meiphalan, resulting in the degradation of double-stranded RNA. and etoposide in comparison to the M7609/pLJ cells. When M7609 Intracellular GST.i@ Concentration in M7609/anti.1 and cells were incubated in the presence of ethacrynic acid, a known M7609/antl-2 Cells. The intracellular concentrations ofGST-ir in the inhibitor of GST-ir, they showed similar patterns of elevated M7609 cells and the M7609/pLI cells were 1.4 Â±0.2 pmol/106 cells sensitivities to ADR, CDDP, melphalan, and etoposide. Neither and 1.3 Â± 0.2 pmol/106 cells, respectively, with no statistically M7609/anti- 1 nor M7609/anti-2 cells showed statistically signifi

Table 2Comparison of the sensitivities ofM7609, M7609/pJ.J, M7609/anti-1, M7609/anti-2,anticancerdrugsIc@ andethacrynic acid-treated M7609 cclIs to various (@)DugM7609M7609/pLJM7609/anti-1(RRâ€•)M7609/anti-2(RR@')M7609

EAC(RRâ€•)ADR + Â±0.02 Â±0.01 Â±0,02â€• Â±0.01â€• Â±001d CDDP 8.9 Â±0.2 9.0 Â±0.1 4.0 Â±02d (2.3) 3.7 Â±0Y' (2.4) 1.71 Â±0.02'@ (5.2) Melphalan 16.8 Â±0.9 17.2 Â±0.8 7.8 Â±0.9â€• (2.2) 7.9 Â±07d (2.2) 5.8 Â±05d (2.9) Etoposide 2.1 Â±0.1 2.2 Â±0.1 1.0 Â±01d (2.2) 1.1 Â±0.1â€• (2.0) 0.8 Â±0,1â€• (2.6) Taxol 0.087 Â±0.001 0.063 Â±0.002 0.054 Â±0.002 (1.2) 0.072 Â±0.002 (1.0) 0.066 Â±0.004 (1.0) VCR 4.0 Â±0.3 4.0 Â±0.2 4.0 Â±0.3 (1.0) 3.9 Â±0.2 (1.2) 3.8 Â±0.2 (1.0) 5-Ri 73.7 Â±3.2 74.8 Â±4.1 74.2 Â±2.4 (1.0) 73.2 Â±4.1 (1.0) 74.2 Â±3.2 (1.0) MMC1.53 6.3 Â±0.21.40 6.4 Â±0.60.42 6.2 Â±0.4(3.3) (1.0)0.44 6.6 Â±0.7(3.2) (1.0)0.22 6.5 Â±0.3(6.5) (1.0)

a ic@, drug concentration that inhibits cell growth by 50%. Each value is the mean of three independent experiments. b p@ relative resistance, ICse for control cells (M76O9/pll)1IC@ for antisense transfectants or ethacrynic acid-treated M7609 cells.

C EA, ethacrynic acid, using a noncytotoxic concentration of ethacrynic acid in combination with each drug. @ d value was significantly lower than that of M7609/pLJ cells; P < 0.01. 3579

INCREASEOF DRUG SENSITIVITYBY G5T-ir ANTISENSEcDNA

A. B. kb

231- @ [email protected] 28 S 9.4 â€” @ . 4.0kb Fig. 3. Southern and Northern blot analyses of M7609/ADR cells transfected with GST-ir anti @ 6.6 â€” .â€” 5.1 kb @ sense cDNA. A, Southern blot analysis was per 18 S formed as described in Fig. 2. The 5.1-kb band is from transfected GST-ir antisense cDNA. B, North em blot analysis was performed as described in Fig. 2. The 4.0-kb band is from transfected GST-ir an tisense cDNA. A GAPDH probe was used as an 2.3 â€” internal quantitative control for the amounts of 2.0 RNA on the filter (bottom panel).

cant changes in their sensitivity to Taxol, VCR, 5-FU, or MMC determine whether GST-ir is directly involved in the drug resistance (Table 2). of cancer cells. Antisense methods can be broadly divided into anti Southern and Northern Blot Analyses of M7609/ADR/anti sense oligoDNA methods and antisense RNA methods. However, Cells. Southern blot analysis by KpnI digestion was performed on with the antisense oligoDNA methods, the resultant inhibitory effect M7609/ADR/anti, M7609/ADRJpLJ, and M7609/ADR cells, using is transient and unstable, and it is impossible to obtain permanent GST-ii' cDNA (0.7-kb EcoRI-EcoRI fragment) as a probe (Fig. 3A). transfectants. In contrast, the antisense RNA method is considered to Besides an 11-kb band representing the cellular GST-ir gene, a 5.1-kb be useful in the analysis of specific gene functions (33). For these band was observed in the M7609/ADR/anti cells. This band was reasons, the antisense RNA method was used for the present study. considered to represent the transfected antisense vector. In Northern Permanent transfectants were established and were studied to detect blot analysis, endogenous GST-'rr mRNA was detectable as a 0.7-kb changes in their sensitivities to various anticancer agents. The degree band in all three cell lines (Fig. 3B). However, the amount of endog of inhibition of gene expression by the antisense RNA method de enous mRNA in M7609/ADRIanti was apparently decreased corn pends on many factors, including the levels of expression of the target pared to that in the M7609/ADR/pU and M7609/ADR cells. In gene, the amount of antisense RNA transcribed, the stereochemical addition to the band at 0.7 kb, a minor band at 4.0 kb was detected in configuration of the antisense RNA, and so on. In this study, we M7609/ADR/anti cells that was considered to represent the antisense transfected the GST-ir antisense cDNA to human colon cancer cell mRNA. These results suggest that the expression of antisense mRNA line M7609 and its ADR-resistant subline, M7609/ADR, and found is responsible for lowering the levels of endogenous mRNA. that the intracellular concentration of GST-ir in these two cell lines Intracellular GST-ir Concentration in M7609/ADR/anti Cells. was reduced to approximately half. As a result of this decreased The intracellular concentrations of GST-ir in M7609/ADR and GST-ir concentration, the sensitivities of these cell lines to ADR, M7609/ADR/pLJ cells were 2.5 Â±0. 1 prnol/l06 cells and 2.3 Â±0.2 CDDP, melphalan, and etoposide were increased. Accordingly, it is pmol/106 cells, respectively. The difference between these values was suggested that the increased expression of GST-ir is not merely an not statistically significant. In contrast, the intracellular concentration incidental phenomenon occurring in parallel to the acquisition of of GST-ii' in antisense transfectant M7609/ADR/anti was 0.8 Â±0.1 resistance by cancer cells, but a cause of the resistance to anticancer pmol/l06 cells, which is approximately half of the GST-'rr concentra drugs in cancer cells. On the other hand, the sensitivities to Taxol, tions detected in M7609/ADR cells and M7609/ADR/pLJ cells (Table 3). Anticancer Drug Sensitivities of M7609/ADR/anti Cells. There Table 3 Comparison of the sensitivities of M7609/ADR,M7609/ADR/pU, were no statistically significant differences in the IC50 values of ADR M7609/ADR/anri, and ethacrynic acid-treated M7609/ADR cells and 5-FU for M7609/ADR and M7609/ADR/pLJ. Sensitivity to ADR IC@ () GST-ir concentration of M7609/ADRIanti and that of M7609/ADR incubated in the pres (@@)M7609/ADRCell line (pmol/l06 cells) ADR (RRC) 5}@ ence of ethacrynic acid was elevated by approximately 4.4- and 1.2M7609/ADR/pLJ 2.5 Â±0.1 9.0 Â±0.6 76.5 Â± 6. 1-fold compared with that of M7609/ADR/pL! or M7609/ADR 2.3 Â±0.2 7.0 Â±0.9 81.3 Â±3.2 cells, respectively. Both cell types showed no elevation in their M7609/ADR + EAb 2.5 Â±0.1 1.5 Â±0.1â€• (6.1) 78.2 Â±2.1 (1.0) M76O9/ADR/anti(1.0)a 1.2 Â±01d 1.6 Â±0.2â€•(44) 77.6 Â±5.8 sensitivities to 5-FU (Table 3). ofthreeIC5@, drug concentration that inhibits cell growth by 50%. Each value is the mean independent experiments. DISCUSSION b EA, ethacrynic acid, using noncytotoxic concentrations of ethacryrnc acid in corn bination5-FU.C with ADR or RR, relative resistance, ICan for control cells (M7609/ADR/PU)11C50 for transfec In this study, an antisense RNA method to selectively inhibit the tant or IC@ for ethacrynic acid-treated M7609/ADR cells. expression of GST-i@ in human colon cancer cell lines was used to d The value was significantly lower than that of M7609/ADR cells; P < 0.01. 3580

VCR, MMC, and 5-FU were not changed, indicating that GST-i@is human melanoma cell lines showing resistance to ADR and reported not related to the resistance of these anticancer drugs. Previously, we that GST-ir was more strongly correlated than p-glycoprotein in that found that an ADR-resistant strain (M7609/ADR) expressing high resistance in cell lines showing comparatively lower (less than 10- amounts of GST-ir showed resistance not only to ADR but also to fold) resistance to ADR. This finding is in agreement with our results, CDDP, etoposide, and melphalan, indicating that the expression of obtained from the characterization of M7609/ADR cells and the GST-ii@is related to the resistance of these anticancer drugs.5 The present experiment of GST-ir antisense transfection. That is, although results of the present study are therefore in agreement with the the activity of GST-ii@as a resistance factor may not be as effective as resistance properties of the M7609/ADR cells and are consistent with that of p-glycoprotein, it is surmised that GST-'ir is primarily involved the previous reports concerning GST-'rr. in intrinsic drug resistance and in the early stage of acquired resist To date, there have been very few reports concerning whether or ance. Accordingly, we speculate that, in clinical terms, GST-ir may be not GST-ir is involved in the intrinsic resistance of cancer cells to a more important resistance factor than p-glycoprotein in the early drugs. Using the ELISA system for GST-i@ established in our labo stage of chemotherapy of cancer. ratory, we have demonstrated that the level of GST-ir was elevated not only in the malignant tissues but also in the plasma of patients ACKNOWLEDGMENTS with digestive tract cancers, which are generally considered to be intrinsically resistant to anticancer agents (27). Thus, we suggested We thank Dr. I. Listowsky for helpful discussion and Dr. L. W. Stiver for the possibility that the expression of GST-ir is involved in the intrinsic the correction of English in the manuscript. drug resistance of cancer cells. Using the cultured cell line M7609 cells as target cells, we confirmed that GST-ir is one of the resistance REFERENCES factors responsible for intrinsic resistance to anticancer drugs. It was 1. Niitsu, Y., Takahashi, Y., and Saito, T. Serum GST-ir as a tumor marker for also demonstrated that GST-ir is directly involved in acquired resist gastrointestinal malignancies. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. Transfection of Glutathione S-Transferase (GST)-π Antisense Complementary DNA Increases the Sensitivity of a Colon Cancer Cell Line to Adriamycin, Cisplatin, Melphalan, and Etoposide

Noriyoshi Ban, Yasuo Takahashi, Tetsuji Takayama, et al.

Cancer Res 1996;56:3577-3582.

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