1 Childrens Hospital, University of Helsinki, 00029 Helsinki; 2 Finnish Institute of Occupational Health, 00250 Helsinki; 4 Department of Internal Medicine, University of Oulu, 90220 Oulu Finland; and 3 Department of Environmental Health, University of Washington, Seattle, Washington 98105-6099
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ABSTRACT |
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The development of drug resistance of
tumors is multifactorial and still poorly understood. Some cytotoxic
drugs generate free radicals, and, therefore, antioxidant enzymes may
contribute to drug resistance. We investigated the levels of manganese
superoxide dismutase (Mn SOD), its inducibility, and its protective
role against tumor necrosis factor- and cytotoxic drugs (cisplatin, epirubicin, methotrexate, and vindesin) in human pleural mesothelioma (M14K) and pulmonary adenocarcinoma (A549) cells. We also studied other
major antioxidant mechanisms in relation to oxidant and drug resistance
of these cells. A549 cells were more resistant than M14K cells toward
both oxidants (hydrogen peroxide and menadione) and all the cytotoxic
drugs tested. M14K cells contained higher basal Mn SOD activity than
A549 cells (28.3 ± 3.4 vs. 1.8 ± 0.3 U/mg protein), and Mn SOD
activity was significantly induced by tumor necrosis factor-
only in
A549 cells (+524%), but the induction did not offer any protection
during subsequent oxidant or drug exposure. Mn SOD was not induced
significantly in either of these cell lines by any of the cytotoxic
drugs (0.007-2 µM, 48 h) tested when assessed by
Northern blotting, Western blotting, or specific activity. A549 cells
contained higher catalase activity than M14K cells (7.6 ± 1.3 vs. 3.6 ± 0.5 nmol
O2 · min
1 · mg
protein
1). They also contained twofold higher levels
of glutathione and higher immunoreactivity of the heavy subunit of
-glutamylcysteine synthetase than M14K cells. Experiments with
inhibitors of
-glutamylcysteine synthetase and catalase supported
our conclusion that mechanisms associated with glutathione contribute
to the drug resistance of these cells.
oxidant; hydrogen peroxide; drug; A549 cells; superoxide dismutase; catalase; glutathione; -glutamylcysteine synthetase
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INTRODUCTION |
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HUMAN MALIGNANT MESOTHELIOMA is an uncommon and fatal tumor associated in most cases with exposure to asbestos fibers (33). One typical feature of mesothelioma is its high resistance to chemotherapeutic agents and to radiation. Lung tumors such as squamous cell carcinoma and adenocarcinoma are often primarily resistant to chemotherapy and/or they develop the resistance rapidly during the first courses of therapy. Because both radiation and several anticancer drugs work by generating free radicals, intracellular antioxidants may partly contribute to the drug resistance of this and other malignant cells.
Manganese superoxide dismutase (Mn SOD) is one of the most important antioxidant enzymes scavenging superoxide radicals in the mitochondria (8, 23, 46, 50). This enzyme has low activity in most cancer cells (37, 38), and it has been suggested that Mn SOD may be a tumor suppressor gene (29). Recent studies have, however, documented high levels of Mn SOD in many malignant tumors such as mesothelioma, glioma, thyroid carcinoma, and colon carcinoma (7, 19, 20, 28, 36). One study also suggested that the survival of colon cancer patients with elevated Mn SOD immunoreactivity in the tumor cells was shorter than that of patients with low Mn SOD immunoreactivity (19). In recent studies by our laboratory, Mn SOD expression was found to be higher in malignant mesothelioma than in healthy human mesothelium (20) and higher in four malignant mesothelioma cell lines than in nonmalignant mesothelial cell cultures (25). It has, however, remained unclear to what extent Mn SOD may contribute to the resistance of human mesothelioma cells against cytotoxic drugs used in the treatment of this disease.
Several previous studies (8, 47, 50-52) have emphasized the
importance of SOD in protection against oxidants and hyperoxia. Also,
intraperitoneal injection of tumor necrosis factor (TNF)- and
subsequent induction of Mn SOD have rendered adult rats more resistant
to oxygen toxicity (46). Previous studies (30, 34, 48, 51) have also
shown that induction or transfection of the Mn SOD gene blocks
TNF-
- and/or oxidant-mediated cytotoxicity, at least in
fibroblasts, breast cancer cells, tracheal epithelial cells, and H820
lung adenocarcinoma cells. On the other hand, several studies (12, 15,
16, 24) using transfection of the SOD gene, transgenic animals, or
cells treated with TNF-
to cause Mn SOD induction have indicated
that high levels of SOD convey minimal protection or no protection at
all or even increase susceptibility to oxidant effects. Thus the role
of Mn SOD in the host defense against exogenous oxidants remains
inconclusive. Oxidant and/or drug resistance of tumor cells may be
related to the constitutive level of Mn SOD or its inducibility, to the
species and cell type investigated, or to other antioxidant mechanisms of the cells. The most important of these other mechanisms are the
hydrogen peroxide (H2O2) scavenging enzymes
catalase and glutathione peroxidase as well as other antioxidant
mechanisms related to glutathione (44, 45).
To further evaluate the role of Mn SOD and other
H2O2 scavenging mechanisms in malignant cells,
we investigated oxidant and drug resistance of mesothelioma and lung
adenocarcinoma cell lines with high or low Mn SOD activity.
Mesothelioma cells established from primary tumors contain high
constitutive levels of Mn SOD (20, 25). A549 adenocarcinoma cells were
selected for comparison because they represent well-characterized
malignant lung cells and, in our preliminary experiments, low Mn SOD
activity. Because cytotoxic drugs may lead to drug resistance by
induction of intracellular antioxidant enzymes, the induction of Mn SOD
by TNF- was compared with the effects obtained by various cytotoxic
drugs. In additional studies, the levels of catalase and mechanisms
related to glutathione metabolism were assessed in these cells.
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METHODS |
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Cell cultures. Human M14K mesothelioma cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.03% L-glutamine (all from Life Technologies, Paisley, UK) at 37°C in a 5% CO2 humidified atmosphere (40). Human A549 lung adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and were grown in F-12 nutrient mixture supplemented with 15% FCS (Life Technologies).
Exposures. Subconfluent M14K and A549 cells were treated for
24-48 h with 10 ng/ml of TNF- (Boehringer Ingelheim) or exposed to one of four cytotoxic drugs: epirubicin (Farmorubicin), methotrexate (Trexan), vindesin (Eldisine), or cisplatin (Platinol). Anthracyclines (epirubicin) inhibit RNA transcription and may cause changes in the
cellular redox state; cisplatin inhibits DNA synthesis by mechanisms
closely related to anthracyclines; and vinca alkaloids (vindesin)
inhibit microtubule formation in the mitotic spindle. Methotrexate
inhibits dihydrofolic acid reductase, but a recent study (3) also
showed that methotrexate inhibits
-glutamylcysteine synthetase
(
-GCS) and glutathione reductase, at least in HeLa cells. Drug
concentrations of 0.5, 0.05, and 0.005 µg/ml (0.01-1 µM
epirubicin, 0.02-2 µM cisplatin, 0.01-1 µM methotrexate,
and 0.007-1 µM vindesin) were selected on the basis of previous
studies performed in our laboratory and preliminary
viability tests conducted for this study. The exposures varied between
24 and 48 h. In the oxidant experiments, the cells were exposed to
5-100 µM menadione, which generates superoxide in a redox
cycling reaction intracellularly, or to 0.01-1 mM
H2O2. In selected experiments, the cells were pretreated with 0.2-1 mM buthionine sulfoximine (BSO) for 18 h. BSO inhibits the rate-limiting enzyme
-GCS in glutathione synthesis and causes glutathione depletion. To inhibit catalase, the cells were
pretreated with 30 mM aminotriazole (ATZ) for 60 min and were also
incubated with the same concentration of ATZ for the last 24 h of
exposure (31). Previous and present studies have indicated that the BSO
concentration required for nearly complete glutathione depletion
without toxicity is 0.2 mM for M14K cells (22) and 1 mM for A549 cells
(39), the concentration of ATZ being 30 mM (22). The concentrations of
inhibitors used in this study showed no detectable toxicity. Oxidant
and drug resistance of A549 cells pretreated with TNF-
was
investigated by first incubating the cells with TNF-
(10 ng/ml) for
48 h to induce Mn SOD and then exposing the cells either to 0.05 µg/ml of epirubicin for 48 h or to 50 µM menadione for 16 h, both
exposures causing 50% loss of cell viability in control cells.
Cell viability. After the exposures, the cells were collected by trypsinization and counted with a microscope. Cell survival was also assessed by the XTT method with a commercial kit according to the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany) with a spectrofluorometer capable of reading microtiter plates (1420 Wictor multilabel counter, Wallac, Turku, Finland).
Northern blot analysis. The cells were scraped into 4 M
guanidine thiocyanate buffer and immediately frozen at 70°C.
Total RNA was isolated with the acid guanidium method (6). Denatured RNA samples were electrophoresed on 1% agarose gels containing 0.36 M
formaldehyde. After ethidium bromide staining and ultraviolet examination to confirm loading homogeneity, the RNA was transferred onto Hybond-N nylon filters (Amersham) and cross-linked to the filters
by ultraviolet illumination. The filters were prehybridized at
58.5°C in a buffer containing 50% deionized formamide, 5×
saline-sodium citrate, 50 mM sodium phosphate (pH 6.5), 5×
Denhardt's reagent, and 100 µg/ml of herring sperm DNA. The
full-length cDNA of Mn SOD, kindly provided by Dr. Y.-S. Ho (Wayne
State University, Detroit, MI), was cloned into pSP65 vector and
transcribed into a 32P-labeled riboprobe. Purified probe
was added to the prehybridization solution and hybridized overnight at
58.5°C with shaking. The filters were then washed in 2×
saline-sodium citrate and exposed to Kodak (Rochester, NY) X-OMAT AR
photographic film at
80°C. After autoradiography, the same
filter was hybridized with a
-actin control probe transcribed from
the p-TRI-
-actin plasmid (Ambion, Austin, TX).
Western blot analysis. The cells were mixed with the
electrophoresis sample buffer and boiled; 50 µg of cell protein (5) were applied to 12% sodium dodecyl sulfate-polyacrylamide gels (27).
The gels were electrophoresed for 1.5 h (90 V) at room temperature and
transferred onto Hybond ECL (Amersham, Arlington Heights, IL)
nitrocellulose membranes in a Mini-PROTEAN II cell (Bio-Rad). The
blotted membranes were incubated with rabbit antibody to recombinant
human Mn SOD (1:10,000; a gift from Dr. J. D. Crapo, Department of
Medicine, National Jewish Medical and Research Center, Denver,
CO) or with rabbit antisera raised against the peptide ETLQEKGERTNPNHPTL for the -GCS heavy subunit (1:4,000) followed by
treatment with a secondary antibody (1:30,000) conjugated to horseradish peroxidase (Amersham). The reactivity was detected by an
enhanced chemiluminescence system (Amersham).
-Actin expression of
the cells was detected with a monoclonal anti-actin antibody (1:2,500)
followed by sheep anti-mouse antibody conjugated to horseradish
peroxidase (1:3,000; Amersham). The amount of immunoreactive protein
was assessed with the 300A computing densitometer and ImageQuant
software version 3.0 Fast Scan (Molecular Dynamics, Sunnyvale, CA).
Cell protein was measured with the method of Bradford (5) (Bio-Rad,
Hercules, CA).
Mn SOD activity. Total SOD was measured spectrophotometrically with the method of McCord and Fridovich (32). Mn SOD activity was distinguished from Cu/Zn SOD by its resistance to 1 mM potassium cyanide. The activity is expressed as units per milligram of protein.
Catalase activity. Catalase was assayed by measuring oxygen production in cells exposed to H2O2 with a Clark-type oxygen electrode (22). Enzyme activity is expressed as nanomoles of oxygen produced per minute per milligram of protein.
Glutathione S-transferase activity. The activity was measured spectrophotometrically with 1 mM 1-chloro-2,4-dinitrobenzene and 1 mM glutathione (13). Enzyme activity is expressed as units per milligram of protein.
Glutathione. Total glutathione content was determined spectrophotometrically after the reduction of 5,5'-dithiobis-(2-nitrobenzoic) acid by NADPH in the presence of glutathione reductase (4). Glutathione content is expressed as nanomoles per milligram of protein.
Statistical analysis. Results are means ± SE; two groups were compared with two-tailed Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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The basal level of Mn SOD was higher in M14K cells (28.3 ± 5.8 U/mg
protein) than in A549 cells (1.8 ± 0.6 U/mg). TNF- treatment for
48 h enhanced the protein levels of Mn SOD in both cell types (Fig.
1), whereas none of the cytotoxic drugs at
two concentrations had any effect (data not shown). The specific
activity of Mn SOD increased significantly with TNF-
only in A549
cells (+524%; Fig. 2), suggesting that at
least part of the immunoreactive protein in M14K cells may be
enzymatically inactive. The specific activity of Mn SOD was not changed
significantly by any of the drugs (Table 1). All the cytotoxic drugs with the
exception of methotrexate (0.05 and 0.005 µg/ml) caused a modest
increase in the mRNA level of the 4-kb transcript of Mn SOD in A549
cells (177% for cisplatin, 183% for epirubicin, and 164% for
vindesin); this change was, however, small compared with the effect of
TNF-
, which was the only agent causing an increase also in the 1-kb
transcript (Fig. 3).
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A549 cells were more resistant than M14K cells against menadione and to
epirubicin as shown in Fig. 4. Cisplatin at
the highest concentration (2 µM) for 48 h reduced the viability in
A549 cells by 16%, whereas the estimated concentration causing 50%
loss of M14K cells was 1.7 µM. The concentrations of methotrexate
causing 50% cytotoxicity in 48 h were 1 µM in A549 cells and 0.01 µM in M14K cells, and those of vindesin were 0.07 and 0.007 µM,
respectively. To investigate the hypothesis that Mn SOD induction may
increase oxidant and drug resistance, TNF--pretreated A549 cells
were exposed either to menadione for 16 h or to epirubicin for 48 h at
concentrations, leading to ~50% loss of control cells. Mn SOD induction did not protect these cells against either exposure (Fig.
5).
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A549 cells were also more resistant than M14K cells against exogenous
H2O2 (Fig.
6). This greater oxidant
resistance is in line with the finding that both catalase and
glutathione concentrations were higher in A549 cells than in M14K
cells. Catalase activity was 7.6 ± 1.3 nmol
O2 · min1 · mg
protein
1 in A549 cells and 3.6 ± 0.5 nmol
O2 · min
1 · mg
protein
1 in M14K cells (P < 0.05; n = 4 experiments). Total glutathione concentrations are shown in Fig.
7A. The heavy subunit
of
-GCS, the rate-limiting enzyme in glutathione synthesis, was
prominently expressed in A549 cells but not in M14K cells (Fig.
7B), supporting the importance of
-GCS in regulating
cellular glutathione levels. Total glutathione S-transferase
(GST) activity was 56 ± 4.8 U/mg protein in A549 cells and 74 ± 6.3 U/mg in M14K cells when analyzed in three separate experiments in
duplicate. Thus the level of total GST seems not to be responsible for
the higher resistance of A549 cells.
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Kinnula et al. (22) previously found that total depletion of glutathione with BSO increased the toxicity and decreased the high-energy nucleotide pool in human mesothelioma M14K cells during exposure to H2O2 and epirubicin, whereas catalase inhibition by ATZ enhanced only the toxicity caused by H2O2. The present study confirmed this finding with A549 cells because both BSO and ATZ pretreatments made A549 cells more vulnerable to the oxidant injury caused by H2O2 and ATZ pretreatment had no effect on the toxicity caused by epirubicin (data not shown). These additional results suggest that glutathione-related mechanisms may be more important than catalase in the drug resistance of malignant lung cells.
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DISCUSSION |
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The present findings with human mesothelioma cells and adenocarcinoma
A549 cells suggest that glutathione-associated mechanisms play an
important role in the resistance of these cells to exogenous oxidants
and cytotoxic drugs in vitro. Mn SOD was induced by TNF- but not
significantly by cytotoxic drugs, but neither the basal level of Mn SOD
nor its inducibility explained the resistance of these cells to
exogenous oxidant or drug exposures.
Recent findings (12, 15, 16) with transgenic animals containing high Mn
SOD activities, with cells carrying Mn SOD-transfected cDNA, and with
cells pretreated with TNF- to cause Mn SOD induction have shown
controversial results. Transfection of one single gene may result in a
disturbance of the antioxidant-oxidant balance of the cell, with
consequent changes, e.g., the levels of superoxide and/or
H2O2 (12). The effects of TNF-
are complex,
and besides Mn SOD, other enzymes are simultaneously induced, including
-GCS (41). On the other hand, enzymes also associated with the
initiation of the apoptotic cascade may be activated by TNF-
(53).
The induction of Mn SOD may also be mediated by factors other than TNF-
(10, 49), and these mechanisms may be even more complicated in
cancer cells. It has been suggested that the level of Mn SOD is
consistently low in tumor cells and also that Mn SOD is not induced in
malignant cells (38, 51). However, Warner et al. (48) showed that H820
human pulmonary adenocarcinoma cells treated with TNF-
(50 ng/ml, 48 h) had elevated Mn SOD activities and were more resistant to paraquat
than nontreated cells that contained low Mn SOD activity. Another study
(17) with insulinoma cells showed that transfection of Mn SOD prevented
cytokine-induced toxicity. In addition, myeloid leukemic cells, which
are sensitive to the cytotoxic effects of TNF-
, showed low Mn SOD
activities and significant upregulation of Mn SOD with TNF-
, whereas
resistant leukemic cells had higher levels of Mn SOD and no induction
by TNF-
(26). The present study suggests that Mn SOD can be induced in malignant lung cells and also that this change is more prominent in
those cells that originally showed low Mn SOD activity. The basal or
induced level of Mn SOD did not predict the development of oxidant or
drug resistance in these cells, and, if anything, TNF-
pretreatment
potentiated oxidant and drug sensitivity.
Few studies have investigated Mn SOD induction by cytotoxic drugs.
Akashi et al. (1) have shown that the anticancer drug OK-432 leads to
elevation of Mn SOD in human granulocytes. Das and White (11) showed
that anticancer drugs such as paclitaxel, vinca alkaloids (vinblastine
and vincristine), and anthracyclines (daunomycin and doxorubicin) may
cause activation of nuclear factor-B, and recently, Das et al. (9)
also reported that mRNA of Mn SOD was upregulated by cytotoxic drugs
through a protein kinase-dependent mechanism. In their study, the doses
of the drugs were higher and the exposure times shorter (4-8 h)
than in the present study, which may explain the more marked increase
in the mRNA levels of Mn SOD in their experiments. They did not
measure immunoreactive protein or Mn SOD activity or effects of Mn SOD
induction on the oxidant or drug resistance. The results of our and
other recent studies (1, 9, 11) cannot rule out the
possibility that Mn SOD activity may also be induced by cytotoxic drugs
in vivo.
The role of Mn SOD in relation to other antioxidant enzymes and oxidant resistance of cancer cells has not been earlier investigated with the exception of recent studies by our laboratory (20, 22) in which human M38K mesothelioma cells were found to contain higher specific activities of Mn SOD, catalase, and GST and a higher glutathione content than M14K mesothelioma cells. However, these findings did not support the importance of Mn SOD in the oxidant resistance of these cells.
Very little is known about catalase in malignant cells or about its role in drug resistance, but a recent study by our laboratory (22) suggests that catalase is not important in the drug resistance of human mesothelioma cells. The present study indicated that even though catalase was higher in A549 cells than in M14K cells, inhibition of catalase with ATZ did not enhance drug-related toxicity. ATZ does not lead to complete inhibition of catalase (22, 31), and, therefore, the final role of catalase in malignant cells remains unclear.
Previous studies (18, 35, 42, 44, 45) have suggested that the
resistance to cytotoxic drugs is dependent on glutathione, GST, and/or
glutathione-dependent multidrug resistance-associated glycoprotein in
numerous malignant cells. The present study showed that A549
adenocarcinoma cells contained a higher level of glutathione and
-GCS and a lower level of Mn SOD than M14K mesothelioma cells. Furthermore, inhibition of
-GCS with BSO enhanced the oxidant sensitivity of these cells significantly. A recent report (43) has
shown that the
-GCS light subunit, which exerts regulatory control
over
-GCS enzyme activity, maps within a critically deleted region
of human malignant mesothelioma. On the basis of our laboratory's (22)
and other studies (2, 14, 21), we conclude that glutathione metabolism
is important in the resistance of lung adenocarcinoma and human
malignant mesothelioma cells against oxidants and oxidant-generating
cytotoxic drugs.
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ACKNOWLEDGEMENTS |
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We thank Prof. Y.-S. Ho (Wayne State University, Detroit, MI) for providing the cDNA of manganese superoxide dismutase (Mn SOD) and Prof. J. D. Crapo (National Jewish Medical and Research Center, Denver, CO) for providing the antibody to Mn SOD. The skillful technical assistance of Raija Sirviö is acknowledged.
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FOOTNOTES |
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This study was supported in part by the Universities of Oulu and Helsinki, the Finnish Antituberculosis Association Foundation, and the Sigrid Juselius Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: V. Kinnula, Univ. of Oulu, Dept. of Internal Medicine, Kajaanintie 50A, 90220 Oulu, Finland (E-mail: vuokko.kinnula{at}oulu.fi).
Received 27 May 1999; accepted in final form 22 December 1999.
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