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Carcinogenesis Advance Access originally published online on August 18, 2006
Carcinogenesis 2007 28(2):465-470; doi:10.1093/carcin/bgl148
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Microsomal glutathione transferase 1 in anticancer drug resistance

Katarina Johansson*, Karin Åhlen, Rosanna Rinaldi1, Karin Sahlander2, Atchasai Siritantikorn3 and Ralf Morgenstern

Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet SE-171 77 Stockholm, Sweden
1 Istituto Scientifico San Raffaele, via Olgettina 58 20132 Milano, Italy
2 Lung and Allergy Research, Division of Physiology, National Institute of Environmental Medicine, Karolinska Institutet Stockholm, Sweden
3 Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University BKK 10330, Thailand

*To whom correspondence should be addressed. Tel: +46 8 5248 7566; Fax: +46 8 343849; Email: katarina.johansson{at}ki.se


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
 References
 
Glutathione transferases (GSTs) are often upregulated in tumors and have been suggested to play an important role in multiple drug resistance in cancer chemotherapy. As a consequence GST-dependent pro-drugs and inhibitors are being developed. Little is known, however, on the potential role of membrane-bound GSTs in drug resistance despite the fact that detoxication of cytostatic drugs and upregulation in tumors has been demonstrated. Therefore, we have studied the involvement of membrane-bound microsomal GST1 (MGST1) in cellular resistance to anticancer drugs. As a tool we have developed a cell system utilizing MCF7 cells stably overexpressing MGST1. Here, we show for the first time that MGST1 can protect cells from several cytostatic drugs, chlorambucil, melphalan and cisplatin in an acute toxicity test (MTT assay) as well as a long-term colony forming efficiency cytotoxicity test. It is of note that these cells do not overexpress multidrug transporters, a prerequisite for protection with certain other GSTs investigated in this system. The cytostatic drugs used comprise both those that are known/predicted to be substrates as well as non-substrates. Thus, the mechanism most probably entails both direct detoxication and downstream protection of the cells from oxidative stress.

Abbreviations: BSA, bovine serum albumin; CDNB, 1-chloro-2,4-dinitrobenzene; CFE, colony forming efficiency; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); FBS, fetal bovine serum; G418, geneticin; GSH, glutathione; GST, glutathione transferase; GPX, glutathione peroxidase; MGST1, microsomal glutathione transferase 1; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; NEM, N-ethylmaleimide; SOD, superoxide dismutase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Concluding remarks
 References
 
Although tremendous advances are made in mechanistic cancer research, it is arguably so that improvement in treatment has had the most significant impact for patients. However, treatment failure due to drug resistance is a serious issue. In this context mechanistic insight into cell biology and drug metabolism are hoped to lead to improved treatment strategies. Tumor cell drug resistance is associated with multiple biochemical changes such as changes of the drug influx and efflux, increased glutathione (GSH) synthesis, activation of enzymes involved in detoxification of the cytotoxic drug [e.g glutathione transferase (GST)] and alterations of genes and proteins involved in the control of cell cycle and apoptosis. Cancer cells can be intrinsically resistant to cytotoxic drugs or resistance can be acquired during chemotherapy. In the latter process recurring tumors become resistant to the parent cytostatic drug used for treatment and, in addition, cross-reactivity to other cytostatics can develop. Several strategies to overcome drug resistance are currently attempted and inhibition of drug metabolism is addressed here.

It was noted early on (1) that inactivation of chemotherapeutic drugs is catalysed by GSTs (2,3). These enzymes catalyze the conjugation of GSH to a variety of electrophilic compounds, including carcinogens, as well as endogenous reactive compounds (4). Indeed GSTs are one of the enzyme systems induced by anti-carcinogens and thus can prevent tumor formation (5). GSTs have also been suggested to play an important role in multiple drug resistance in cancer chemotherapy and there are numerous reports showing that GSTs can protect cancer cells in culture (2,6). These studies have focused on cytosolic GSTs of which there are 17 human forms (7). Little is known, however, on the contribution of membrane-bound microsomal GST1 (MGST1) although the enzyme recently was shown to display activity towards chlorambucil (8) and melphalan (9), and is upregulated in several tumor tissues (10–14). MGST1 is a membrane protein chiefly located in the endoplasmic reticulum and in the outer membrane of mitochondria and is involved in the cellular conjugation of reactive compounds arising during xenobiotic metabolism or oxidative stress (15–17). In addition, the enzyme is a glutathione peroxidase (GPX) (17).

The aim of this study was to determine whether MGST1 could be involved in anticancer drug resistance. As a tool we have developed a cell system utilizing MCF7 cells overexpressing MGST1. Here, we show, for the first time, that MGST1 can protect cells from several cytostatic drugs, both those that are predicted to be substrates and a non-substrate where the mechanism most probably entails downstream protection of the cells from oxidative stress.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Concluding remarks
 References
 
Chemicals
All cell culture media and their ingredients were obtained from GIBCO/BRL, Middlothian, UK. Geneticin® (G418) was purchased from GE Healthcare. FuGENETM 6 Transfection Reagent and superoxide dismutase (SOD) were from Roche. Fluoromount-GTM was from Southern Biothechnology Associates, Inc. 1-chloro-2,4-dinitrobenzene (CDNB) and hydrogen peroxide were obtained from Merck. Catalase and xanthine oxidase were from Boehringer Mannheim. Fluorescein-isothiocyanate (FITC)-conjugated swine anti-rabbit IgG and horseradish peroxidase-labeled anti-rabbit IgG as the secondary antibody were purchased from DAKO, A/S, Glostrup Denmark. ECL kit was from Amersham biosciences. Micro BCATM protein reagent assay kit was obtained from Pierce Biotechnology Rockford, USA. Chlorambucil, melphalan, cisplatin and carmustine and 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT), formalin, EDTA, sodium azide, glutathione reductase, GSH and NADPH, cytochrome c, hypoxanthine were all purchased from Sigma-Aldrich, Sweden AB.

Cell line and culture conditions
All cell lines were derived from parental MCF7wt human breast carcinoma cells, which naturally express low endogenous MGST1 (18). MCF7 cells were cultured in DMEM, without sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mM sodium pyruvate at 37°C and 5% CO2 in a humidified environment. Transfected cells were maintained in the above media supplemented with 1 mg/ml G418.

Stable transfection of cells
MCF7 cells were transfected with vectors pCGS (sense) or pCGAS (antisense), respectively (18). The vector contains the powerful promoter–regulatory region of the human cytomegalovirus. Co-transfection was performed for both variants with the vector pcDNA1neo, which introduces resistance to the aminoglycoside G418. Transfection was carried out using FuGENETM 6 Transfection Reagent. The day prior to transfection 4.7 x 105 cells/well were seeded in a 6-well plate to be 50–80% confluent in ~24 h. On the day of transfection FuGENETM 6 was diluted in serum-free media, supplemented with 12.5 mM HEPES, mixed with the plasmids (1.1 µg/0.12 µg) and added to the cells. The relation, (w/w), between the expression vector of interest and the neomycin resistance vector was 9:1. Cells were incubated with the transfection reagent at 37°C for 48 h. Stable transfectants were isolated by selection with 1 mg/ml G418 for ~2 weeks. Several stable clones were selected for further characterization and were maintained in the selection media. It should be pointed out that antisense (AS) transfected MCF7 cells against rat MGST1 appear not to suppress endogenous MGST1. In addition, our results show that AS cells behave as MCF7wt. This demonstrates that G418 does not influence the results and that AS is a good control for the sense vector.

In situ immunofluorescence
Characterization of stably transfected clones was performed using in situ immunofluorescence. An aliquot of 8 x 104 cells/well were seeded in a 4 chamber slide (NUNC Lab-Tek®TC) and incubated at 37°C until almost confluent (24 h). The cells were rinsed twice with 1x PBS and fixed with 50/50 ice-cold methanol/acetone for 7 min. Slides were rinsed with 1x PBS three times, incubated in PBS/1% Triton X-100 for 10 min at room temperature and then for 30 min in PBS/1% Triton X-100/2% bovine serum albumin (BSA) at room temperature. Subsequently, the cells were incubated with the primary antibody, rabbit anti-rat MGST1, at a dilution of 1:400 in PBS/1% Triton X-100/2% BSA at 4°C over night. The following day, cells were washed three times with PBS/1% Triton X-100 for 5 min at room temperature where after incubation with the secondary antibody, FITC-conjugated swine anti-rabbit IgG (diluted 1:30 in PBS/1% triton X-100), was performed for 1 h at room temperature. This was followed by three washes with PBS for 5 min and mounting with Fluoromount-GTM. Expression of the enzyme was determined in a fluorescence microscope.

Harvesting of cells and sonication
Confluent cells in a 75 cm2 bottle were taken for harvest. Cells were washed with 1x PBS and trypsinated followed by a wash in 5 ml 1x PBS and centrifugation for 5 min at 1000 r.p.m. The last step was repeated. After the second centrifugation the cell-pellet was suspended in 500 µl 1x PBS, transferred to eppendorf tubes and kept on ice. Sonication was performed 2 x 20 sec at 6 amps on ice with Soniprep 150 MSE.

Microsomal and cytosolic fraction preparation
To measure the GST, catalase and SOD activity in cells, sonicated cells (1–2 x 106 cells/ml) were centrifuged at 10 000 g for 10 min at 4°C. A 1:1 mixture 0.8 M CaCl2/0.5 M MgCl2 (10 µl) was added to the supernatant (1 ml) and incubated for 10 min. After centrifugation at 1500 g for 10 min, the supernatant (cytosol) was saved and put on ice. The pellet was resuspended in 1 ml of 0.15 M Tris–HCl pH 8.0, 1 mM EDTA and 20 µl of the CaCl2/MgCl2 mixture was added. After centrifugation at 1500 g for 10 min the pellet (microsomal fraction) was resuspended in 100 µl 0.25 M sucrose, 1 mM EDTA, pH 7.5, and kept on ice.

5,5'-Dithiobis-(2-nitrobenzoic acid) thiol assay
To estimate the amount of free thiols in the cells these were mixed with TCA (to a final concentration of 5% TCA) followed by centrifugation, 5 min at 1000 r.p.m. The supernatant was saved and the amount of GSH was measured spectrophotometrically at 412 nm ({varepsilon} = 13 600 M–1 cm–1), as the amount of product, 2-nitro-5-thiobenzoate anion (TNB2–), formed in the reaction between GSH and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (19). An aliquot of 43 µl sodium phosphate buffer, 2 M pH 7.5 and 43 µl cell suspension (TCA-precipitated) was mixed in a microcuvette and the absorbance recorded, 15 µl 10 mM DTNB was added and the resulting absorbance measured. The background absorbance of 15 µl 10 mM DTNB was subtracted by measuring the absorbance after a second addition. The content of GSH was calculated after subtraction of the blank (buffer and cell suspension) and the absorbance of DTNB itself. The results are presented as nmol GSH/mg protein.

GST activity
GST activity in microsomal and cytosolic fractions were measured using GSH and CDNB as substrates essentially according to the method of Habig (20). The activity was determined in 0.1 M potassium phosphate, pH 6.5, containing 0.1% Triton X-100 with 5 mM GSH and 0.1 mM CDNB at 30°C. The rate of product formation was monitored by measuring the change in absorbance at 340 nm ({varepsilon} = 9600 M–1 cm–1) using a single-beam Philips PU8700 UV/visible spectrophotometer (Philips Scientific and Analytical Equipment, Cambridge, UK). Enzyme activities were calculated after correction for the non-enzymatic reaction.

GPX activity
The activity of GPX was determined as described (15). The cytosolic fraction, collected as described above, was added to a buffer, containing 50 mM potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM sodium azide, 1 mM GSH and 0.2 mM NADPH, glutathione reductase (1:100, 100–300 U/mg). The reaction was started by the addition of 0.25 mM H2O2 and estimated spectrophotometrically by recording the NADPH oxidation at 340 nm in the coupled assay system at 30°C.

Catalase activity
The catalase activity measurement was based on method of Aebi (21). The reaction was started by the addition of the cytosolic fraction to PBS, containing 10 mM H2O2. The activity was measured as the rate of decomposition of H2O2 by monitoring the absorbance decrease at 240 nm ({varepsilon} = 43.6 M–1 cm–1) at 30°C.

SOD activity
SOD activity, was determined by the method of McCord and Fridovich (22) at 30°C. Superoxide was generated at a constant rate by adding 0.1 U xanthine oxidase to a buffer containing PBS, cytochrome c (40 µM), hypoxanthine (100 µM) and catalase (20 µg/ml final). The reduction of cytochrome c at 550 nm and inhibition by added SOD activity in the cytosolic fractions were estimated from calibration curves with commercial SOD (500 U/mg lyophilizate).

Colony forming efficiency
The assay used to measure cellular ability to form colonies was as previously described by Sundqvist et al. (23), with slight modifications. Cells were seeded at a density of 50 cells/cm2 in a 60 mm petri dish. After 24 h, anticancer drugs were added at different concentrations and incubated for 3 h, and then media was changed to DMEM (see supplements in Cell line and culture conditions) without anticancer drugs. After 7 days, cells were washed with 1x PBS, fixed with 10% formalin and stained with aqueous crystal violet. A colony was defined as at least 16 cells and was counted in a light microscope. Each experiment was repeated at least three times.

MTT assay
This cell proliferation assay was used as a quantitative colorimetric method for measurements of cellular cytotoxicity as previously described by Mosman (24) with slight modifications. Sense transfected cells, AS transfected cells and MCF7wt cells were run in parallel. Briefly, cells were seeded at a density of 1 x 104 cells/well in a 96-well plate, after 24 h culturing the media was changed to DMEM without phenol red and serum containing anticancer drugs at different concentrations. Four replicates were used for each concentration. After 24 h the media was changed to media supplemented with 0.5 mg/ml MTT and incubated 4 h. Formazan crystals where dissolved in DMSO for 5 min and read spectrophotometrically (Vmax Kinetic Microplate Reader, Molecular Devices) at 590 nm with a reference at 650 nm. The MTT assay was performed 3–7 times for each anticancer drug.

Western blot
Protein levels of MGST1 in the cells were estimated by western blotting. Cells were lysed in 1% SDS, 1% Triton X-100 in dH2O. Twenty micrograms of protein from cell lysate was run on SDS–polyacrylamide gel electrophoresis in a 15% gel, transferred to a nitrocellulose membrane and immunologically stained using polyclonal rabbit IgG against rat MGST1 as the primary antibody, and with horseradish peroxidase-labeled anti-rabbit IgG as the secondary antibody. Blots where developed by enhanced chemiluminescence using an ECL kit. Protein determination was performed with the Micro BCATM protein assay kit in a 96-well plate.

Statistical analysis
Statistical analysis was performed with Student's t-test.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Concluding remarks
 References
 
Expression of MGST1 in cultured MCF7 cells
Several independent stable transfectants of MCF7 cells were screened for the presence of MGST1 by in situ immunofluorescence (data not shown) and western blot (Figure 1). Clones that displayed comparatively high expression of the enzyme were selected for further characterization (0.2–0.5 µg/mg total protein as determined by western blots). The clone displaying the highest expression was used for most studies described here. It should be noted that these cells express physiologically relevant levels of MGST1 (~10-fold less than rat hepatocytes), a level encountered in many tissues (25,26). As endogenous MGST1 is present in almost all cell types examined (18) it was important to ascertain that our transfected enzyme makes a significant contribution to overall GST activity in microsomes isolated from MCF7 cells. Indeed a low activity could be measured in untransfected MCF7 and AS cells that most likely stems from low amounts of endogenous MGST1 and cytosolic GSTs which are known to adhere to membranes (27). The CDNB activity in microsomes from MGST1 sense transfected cells was significantly higher than controls and could be activated by N-ethylmaleimide (NEM) (Table 1). Activation by NEM is a hallmark of MGST1 catalysis (28) that was not observed in control and AS transfected cells indicative of low levels of endogenous MGST1. In conclusion, we have developed a system to selectively investigate the cellular function of MGST1.


Figure 1
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  		Fig. 1 Western blot analysis of MGST1 expression in selected clones of sense and AS transfected MCF7 cells. Microsomes were prepared; SDS–polyacrylamide gel electrophoresis (15%), transfer to nitrocellulose and immunodetection were performed as described under methods. Purified enzyme was used as standard. Clones are indicated by numbers and AS transfected by AS.

 


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  		Table 1 GST activity in the microsomal fraction of antisense and sense MGST1 transfected MCF 7 cells

 
Although MCF7 cells contain low amounts of endogenous GSTs, these and other detoxification systems, especially those protecting from oxidative stress, could be altered upon stable expression of MGST1. Therefore, we measured the activities of key enzymes that protect from oxidative stress as well as GSH levels. As is evident from Table 2 neither cytosolic GST, GPX, SOD, catalase nor GSH levels were altered in cells that overexpress MGST1. The levels of GPX correlate well with values in the literature (29). It is therefore reasonable to assume that the potential role of MGST1 involvement in anticancer drug resistance is studied in a specific fashion.


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  		Table 2 Biochemical characterization of antisense and sense transfected cells

 
Two independent tests were used to assess cell toxicity, the acute MTT cell viability test (mitochondrial function) and the colony formation efficiency test that measures cellular capacity to divide. In both these tests MGST1 stable transfectants were protected from chlorambucil and melphalan (Figure 2A–D). Most likely this protection results from direct conjugation of the drugs since these substances function as substrates for MGST1 (8,9). In addition, protection from oxidative stress that could be caused by these compounds is also possible (vide infra).


Figure 2
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  		Fig. 2 Cellular toxicity of chlorambucil (A and B), melphalan (C and D), carmustine (E and F) and cisplatin (G and H). MGST1 sense- (filled square, line) and AS-transfected MCF 7 cells (triangle, dashed line) as well as MCF 7wt (triangle, dotted line). Cells were exposed to the doses indicated and viability assayed by the MTT assay (A, C, E and G), exposure time 24 h (n = 4). CFE assay (B, D, F and H), exposure time 3 h (n = 2). For further details see Materials and methods. Experiments were performed at least three times with comparable results. The results are expressed as mean values ± SD. Significance levels are *P < 0.05, **P < 0.01 and ***P < 0.001.

 
In comparison with chlorambucil and melphalan, MGST1-dependent protection from carmustine was less evident in the MTT test (Figure 2E). In the colony forming efficiency (CFE) test no significant protection of the MGST1 overexpressing cell line could be seen (Figure 2F). Carmustine is an alkylator (however, not bifunctional) but also includes a nitroso group as a toxicophore. It is conceivable that a hypothetical conjugate can still cause toxicity via this group thus making protection less efficient. This cytostatic drug may therefore be better suited to overcome drug resistance to pure alkylators when GSTs are involved.

Cisplatin is used to treat a variety of cancers, including tumors of the head, neck, lung, lymphoma, testicular, ovarian and genitourinary tract (30). It has been shown that cytosolic GSTs are involved in the resistance against cisplatin (31), whereas MGST1 has not been studied in this context. Cisplatin is different from the alkylators described above since the mechanism of GST protection probably does not involve direct detoxication of the compound but rather protection from its deleterious effects (among which are oxidative stress) (32–34). As MGST1 has been shown to protect membranes from lipid peroxidation (which is increased by cisplatin) and cells from hydrogen peroxide (35) we hypothesized that the enzyme should be protective. Indeed MGST1 expressing cells are markedly protected from cisplatin toxicity both in the MTT and CFE assay (Figure 2G and H). The protective effect of MGST1 most likely results from a general protection from lipid peroxidation. However, cisplatin can also induce apoptosis (36) via caspase-2 that leads to pore formation in outer mitochondrial membranes and also disrupts the interaction of cytochrome c and anionic phospholipids, especially cardiolipin (37). If MGST1 protects specific phospholipids from oxidation it could act as an anti-apoptotic agent, thus preventing cisplatin toxicity by an alternate mechanism. Certainly, the high concentration of MGST1 in mitochondria (26) is consistent with this notion.

Apart from the above cytostatic drugs we have also investigated busulfan, cytarabine and doxorubicin. No protection was evident in neither the MTT assay nor the CFE assay (data not shown).

In several studies it has been shown that MGST1 is overexpressed in malignant compared with non-malignant tissues notably brain-, colorectal-, lung- and prostate cancer cells (10–14). Our findings thus take on added relevance since cisplatin and carmustine are prominent cytostatics used to treat prostate and brain cancer, respectively. Melphalan and chlorambucil are used in chemotherapy against ovarian cancers that have high endogenous MGST1 expression also in normal tissues (26). These compounds are also used for treatment of various blood cancers where MGST1 expression has not been studied. It is of interest to note that MGST1 has been proposed as a good marker to detect colorectal and lung tumors at an early stage (12,13). Taken together, further studies defining the content of MGST1 in tumors and possible relation to drug resistance appear founded.

According to Paumi et al. (4) MCF7 cells contain very low levels of multidrug resistance protein 1 (MRP1). Thus, when MCF7 cells, overexpressing GSTs, are treated with melphalan, chlorambucil and CDNB (38) toxic conjugates are not efficiently excreted from the cell and protection from these compounds occurred only, or was much more efficient, when MRP1 was co-expressed/upregulated. The effect of MRP1 overexpression needs to be examined also in our system but it adds the complication of parent drug efflux. Since here we do observe protection from chlorambucil and melphalan which should form conjugates it appears that the MGST1 levels in our experimental system do not result in a build-up of toxic conjugate levels or that the additional protection from downstream toxicity (oxidative stress) is comparatively significant.


   Concluding remarks
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
References
 
We have demonstrated that physiologically relevant expression of MGST1 in mammary tumor cells (MCF7) results in resistance against chlorambucil, melphalan and cisplatin. This is, to our knowledge, the first demonstration that MGST1 could be involved in anticancer drug resistance. Our findings are significant since it has been shown that MGST1 is upregulated in many tumors. The cellular system developed here is well suited to study the role of MGST1 in resistance against anticancer drugs and we aim to develop and study the efficiency of different inhibitors of MGST1 that can counteract resistance.


   Acknowledgments
 
This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council, the Swedish National Board for Laboratory Animals, Astra-Zeneca, the Blanceflor Boncompagni Ludovisi nee Bildt foundation and funds from Karolinska Institutet.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
 References
 

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Received May 31, 2006; revised August 9, 2006; accepted August 11, 2006.


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