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© The Author 2006. Published by Oxford University Press.
ARTICLE |
Improvement of the Therapeutic Index of Anticancer Drugs by the Superoxide Dismutase Mimic Mangafodipir
Affiliation of authors: Université Paris-René Descartes, Faculté de Médecine, Assistance Publique-Hopitaux de Paris, Hôpital Cochin, EA 1833, Paris, France
Correspondence to: Jérôme Alexandre, MD, Unité d'Oncologie Médicale, Service de Médecine Interne, Groupe hospitalier Cochin-Saint Vincent de Paul, 27 rue du Faubourg Saint Jacques, 75679 Paris cedex 14, France (e-mail: jerome.alexandre{at}cch.aphp.fr).
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ABSTRACT |
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Background: Anticancer drugs act by increasing intracellular hydrogen peroxide levels. Mangafodipir, a superoxide dismutase (SOD) mimic with catalase and glutathione reductase activities, protects normal cells from apoptosis induced by H2O2. We investigated its and other oxidative stress modulators' effects on anticancer drug activity in vitro and in vivo. Methods: Cell lysis and intracellular reactive oxygen species levels were assessed in vitro in human leukocytes from healthy subjects and in murine CT26 colon cancer cells. Cells were exposed to the chemotherapeutic agents paclitaxel, oxaliplatin, or 5-fluorouracil, either in the presence or absence of mangafodipir and other oxidative stress modulators. Cell viability was evaluated by the methylthiazoletetrazolium assay. The effects of mangafodipir and other oxidative stress modulators on peripheral blood counts and on tumor growth were studied in BALB/c mice that were implanted with CT26 tumors and treated with 20 mg/kg paclitaxel. Survival of BALB/c mice infected with Staphylococcus aureus was also examined by treatment group. Statistical tests were two-sided. Results: In vitro lysis of leukocytes exposed to paclitaxel, oxaliplatin, or 5-fluorouracil in combination with mangafodipir was decreased by 46% (95% confidence interval [CI] = 44% to 48%), 30.5% (95% CI = 29% to 32%), and 15% (95% CI = 10% to 20%), compared with lysis of cells treated with anticancer agent alone. Mangafodipir also statistically significantly enhanced in vitro anticancer drug cytotoxicity toward CT26 cancer cells. In vivo, mangafodipir protected mice against paclitaxel-induced leukopenia. Moreover, the survival rate of mice infected with S. aureus and treated with paclitaxel was higher when mangafodipir was also administered (survival: 3 of 17 versus 14 of 17, P<.001). In addition, mangafodipir amplified the inhibitory effect of paclitaxel on CT26 tumor growth in mice. Conclusions: Mangafodipir decreased hematotoxicity and enhanced cytotoxicity of anticancer agents.
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INTRODUCTION |
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Reactive oxygen species, such as superoxide anions, and hydrogen peroxide (H2O2) are normal byproducts of cellular aerobic metabolism. Superoxide anions (O2–) are secondarily converted to H2O2 by the superoxide dismutases (SODs). H2O2 has a dual role in cellular homeostasis. Low levels of intracellular H2O2 stimulate cell growth (1), whereas high levels of H2O2 lead to cell senescence and apoptosis (2,3). Several anticancer agents, such as 5-fluorouracil (4), platinum (5,6), arsenic trioxide (7), paclitaxel (8), and anthracyclines (9,10) increase levels of reactive oxygen species. Tumor cells have higher levels of reactive oxygen species than normal cells and are therefore more sensitive to the additional oxidative stress generated by anticancer agents (5,6,11).
Cells also contain a variety of free radical scavenging systems that protect them from the effects of drugs that generate oxygen free radicals. Catalase and glutathione peroxidase are enzymes that detoxify H2O2. High levels in tumor cells of reduced glutathione, the cofactor of glutathione peroxidase, have been associated with a multidrug resistance phenotype (12). Recent studies have suggested that compounds that target such protective mechanisms could enhance the activity of anticancer agents and reverse the multidrug resistance phenotype. For example, buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, can increase the cytotoxicity of melphalan by preventing glutathione peroxidase activity and increasing H2O2 levels (13). However, BSO depletes glutathione in both normal and cancer cells, which increases melphalan's hematologic toxicity and prevents any enhancement of the therapeutic index of this anticancer agent (14,15). Conversely, N-acetylcysteine protects normal cells from the cytotoxic effects of anticancer drugs by preventing elevation of intracellular H2O2 through its catalase- and glutathione reductase–like properties (6).
SOD enzymes can also affect tumor cell proliferation via their effects on peroxide levels. Indeed, overexpression of SOD in human cancer cell lines increases H2O2 production and reduces tumor growth in the absence of anticancer agents (16,17). We have previously reported that two nonpeptidyl mimics of SOD, copper [II] diisopropylsalicylate (CuDIPS) and manganese [III] tetrakis-(5,10,15,20)-benzoic acid porphyrin (MnTBAP), increase the cytotoxic activity of anticancer drugs by increasing H2O2 levels (6). In addition, mangafodipir, a contrast agent used in magnetic resonance imaging, can protect normal liver cells against reactive oxygen species–induced apoptosis (18). Mangafodipir is a chelate of manganese [II] and of the ligand fodipir, a vitamin B6 derivate. Mangafodipir has SOD-, catalase-, and glutathione reductase–like properties, allowing it to act at multiple steps of the reactive oxygen species cascade.
We hypothesized that the association of those properties in a single molecule such as mangafodipir could be beneficial through its ability to enhance the killing of cancer cells while protecting normal cells from chemotherapeutic toxicity. More specifically, we investigated the ability of mangafodipir to minimize the hematologic toxicity and potentiate the antitumor activity of several commonly used anticancer agents both in vitro and in vivo. The effects of mangafodipir were compared with those of CuDIPS, a Cu/ZnSOD mimic with no catalase or glutathione reductase activity (19); MnTBAP, a MnSOD mimic with catalase activity (20); and N-acetylcysteine, an antioxidant molecule that has both catalase and glutathione-reductase activities but not SOD activity (21), in tumor cell lines, in human leukocytes, and in mice.
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METHODS |
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Chemicals and Cell Lines
All chemicals were from Sigma (Saint Quentin Fallavier, France), except for mangafodipir (Teslascan; Amersham Health, Amersham, U.K.), oxaliplatin (Eloxatine; Sanofi-Aventis, Paris, France), docetaxel (Taxotere; Sanofi-Aventis), and paclitaxel (Taxol; Bristol Myers Squibb, Rueil Malmaison, France), for which commercial solutions were used. CT26 (mouse colon carcinoma, American Type Culture Collection [ATCC] 2638), Hepa1.6 (mouse liver hepatoma, ATCC 1830), and A549 (human lung carcinoma, ATCC CCL-185) cell lines were obtained from ATCC (Manassas, VA). Tumor cells were cultured in Dulbecco's modified Eagle's medium—Glutamax-I (Life Technologies, Cergy Pontoise, France) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies) and antibiotics. All cell lines were routinely tested to ensure that they were free of Mycoplasma infection.
Human Leukocytes
Human leukocytes were obtained from six healthy volunteers and 10 cancer patients. All cancer patients (eight women and two men) were diagnosed with metastatic or locally advanced solid tumors (breast [n = 5], prostate [n = 2], lung [n = 1], stomach [n = 1], and ovary [n = 1]) and were part of a pharmacokinetic study of docetaxel reported elsewhere (22). They had given written inform consent to an additional blood collection for the purpose of the present study. The study was approved by the Ethics Committee of Hopital Cochin, Paris. Ten milliliters of blood were collected before any antitumoral treatment, hemolyzed with 0.15 mM ammonium chloride, and then centrifuged at 1800 rpm (700g) for 5 minutes. The pellet was suspended in RPMI medium–Glutamax-I supplemented with 10% heat-inactivated fetal calf serum and antibiotics (complete medium). Leukocytes (5 x 104 cells/well) were then seeded in 96-well plates (Costar Corning, Inc., Corning, NY) and immediately exposed to anticancer agents and/or to oxidative stress modulators.
In Vitro Cell Viability Assays
Tumor cells (2 x 104 cells/well) or human leukocytes from healthy volunteers (5 x 104 cells/well) were seeded in 96-well plates and incubated for 24 hours in complete medium (DMEM or RPMI, respectively) with the anticancer agent (paclitaxel, oxaliplatin, or 5-fluorouracil) alone or with varying amounts of oxidative stress modulators (mangafodipir, CuDIPS, MnTBAP, or N-acetylcysteine). Tumor cells and leukocytes from healthy volunteers were incubated with 10 and 20 µM paclitaxel, 10 µM and 1 mM oxaliplatin, or 50 µM and 40 mM 5-fluorouracil, respectively. Human leukocytes from cancer patients were incubated with 20, 30, and 40 µM docetaxel alone or with 400 µM mangafodipir. Cell viability was evaluated by the mitochondrion-dependent reduction of methylthiazoletetrazolium (MTT) to formazan. In brief, cells were exposed to 50 µL of MTT (2 mg/mL in phosphate-buffered saline [PBS]) and incubated for 4 hours at 37 °C. MTT reduction, which indicates viable cells, was visualized by addition of 100 µL dimethyl sulfoxide. The absorbance at 550 nm in each well was recorded with an enzyme-linked immunosorbent assay (ELISA) microplate reader (VICTOR2; Perkin Elmer, Paris, France). Results are expressed as percentage of viable cells compared with untreated cells (which have 100% viability).
In Vivo Hematologic Toxicity Analysis
BALB/c female mice between 6 and 8 weeks of age (Iffa Credo, L'Arbresles, France) were used in all experiments. Mice received humane care in compliance with institutional guidelines. PBS (1.54 mM potassium phosphate monobasic, 155.17 mM sodium chloride, 2.71 mM sodium phosphate dibasic; Life Technologies) at pH 7.2 was used as the vehicle. Mice were injected intraperitoneally with vehicle alone (group 1); with paclitaxel, mangafodipir, MnTBAP, CuDIPS, or N-acetylcysteine alone (groups 2 to 6); or with paclitaxel and one of the oxidative stress modulators in combination (groups 7 to 10). Five mice were treated in each group. Paclitaxel (20 mg/kg) or vehicle alone was administered on days 0, 2, and 4. This dose of paclitaxel was determined in previous experiments (6) and was used because it was associated with a 50% reduction in tumor volume of CT26 at day 15 without any substantial clinical toxicity. On days 0, 2, 4, and 7, mice were administered 10 mg/kg of mangafodipir, MnTBAP, or CuDIPS, 150 mg/kg of N-acetylcysteine, or vehicle. Ten days after the first injection of paclitaxel or oxidative stress modulator, mice were killed by cervical dislocation. Spleen, bone marrow from femoral bone, and 1 mL of blood were then collected. The total number of hematopoietic cells in bone marrow and leukocytes from spleen and blood were counted using a Malassez cell.
Susceptibility of Mice to Bacterial Infection
In another experiment, BALB/c female mice were injected intraperitoneally with either vehicle alone (group 1), paclitaxel (60 mg/kg) alone on days 0, 3, and 6 (group 2), or paclitaxel (60 mg/kg) in combination with mangafodipir (10 mg/kg) on days 0, 3, and 6 (group 3). A sublethal dose of the pathogen Staphylococcus aureus (200 µL of a stock suspension with an optical density of 600 at 550 nm) was then injected intraperitoneally on day 9. The survival rate of mice was evaluated 48 hours following S. aureus injection. A total of 17 mice were treated in each group in three independent experiments.
In Vivo Antitumor Activity
Each BALB/c female mouse was injected subcutaneously in the back of the neck with 2 x 106 of CT26 cells. When the tumor reached a size of 200–500 mm3, mice were grouped in 10 groups so that the sizes of the tumors were not statistically significantly different by group. Mice were injected intraperitoneally with vehicle alone (group 1); with paclitaxel, mangafodipir, MnTBAP, CuDIPS, or N-acetylcysteine alone (groups 2 to 6); or with paclitaxel and one of these oxidative stress modulators in combination (groups 7 to 10). Paclitaxel (20 mg/kg) or vehicle was administered on days 0, 2, and 4. Then 10 mg/kg mangafodipir, MnTBAP, or CuDIPS or 150 mg/kg N-acetylcysteine or vehicle were administered three times a week for 1 month. Ten mice were treated in each group. Tumor size was measured with a Vernier calliper every 3 days at the same time that the oxidative stress modulator was administered. Tumor volume (TV) was calculated as follows: TV (mm3) = (L x W2)/2, where L is the longest and W the shortest radius of the tumor. Results are expressed as the mean of tumor volume within each group. Tumor volumes were compared across treatment groups after each tumor size measurement, i.e., every 3 days.
Intracellular Levels of Superoxide Anions, Hydrogen Peroxide, and Reduced Glutathione
Tumor cells (2 x 104/well) or human leukocytes from one healthy volunteer (5 x 104/well) were seeded in 96-well plates and incubated for 48 hours in complete medium with anticancer agents (oxaliplatin, paclitaxel, or 5-fluorouracil) alone or in combination with 400 µM mangafodipir, 100 µM CuDIPS, 100 µM MnTBAP, or 400 µM N-acetylcysteine. Tumor cells and leukocytes were incubated with 10 and 20 µM paclitaxel, 10 µM and 1 mM oxaliplatin, or 50 µM and 40 mM 5-fluorouracil, respectively. Levels of intracellular superoxide anion (O2–) and H2O2 were assessed spectrofluorometrically (VICTOR2) by oxidation of dihydroethidium (DHE) (Molecular Probes, Leiden, The Netherlands) and of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes), respectively. Cells were washed once with PBS and then incubated with either 250 µM DHE or 200 µM H2DCFDA in PBS for 1 hour at 37 °C. Excitation and emission wavelengths used were 500 and 605 nm for DHE and 490 and 535 nm for H2DCFDA, respectively. The number of adherent cells was evaluated by the crystal violet assay. In brief, cells were stained in 0.5% crystal violet and 30% ethanol in PBS for 30 minutes at room temperature. After two washes in PBS, the stain was dissolved in 50% ethanol, and absorbance was measured at 560 nm on an ELISA multiwell reader. The levels of O2– or H2O2 were calculated in each sample as follows: reactive oxygen species rate (arbitrary units/min/106 cells) = (fluorescence intensity [arbitrary units] at T60 minutes – fluorescence intensity [arbitrary units] at T0)/60 minutes/number of adherent cells as measured by the crystal violet assay. Levels of intracellular reduced glutathione were assessed spectrofluorometrically by monochlorobimane staining (23). Briefly, tumor cells (2 x 104/well) or human leukocytes (5 x 104/well) were seeded in 96-well plates and incubated for 48 hours in complete medium with anticancer agent alone or in combination with an oxidative stress modulator at the concentrations described above. Cells were washed once with PBS and then incubated with 50 µM monochlorobimane diluted in PBS. The fluorescence intensity was measured after 15 minutes at 37 °C. Excitation and emission wavelengths were 380 and 485 nm, respectively. The intracellular glutathione level was expressed as arbitrary units of fluorescence intensity/number of adherent cells as measured by the crystal violet assay.
Statistical Analysis
All values from the in vitro experiments, except for those using leukocytes from cancer patients, are the mean values from three independent experiments, each carried out in triplicate. Data obtained using leukocytes from cancer patients are the mean values from the 10 cancer patients from one experiment carried out in triplicate. Error bars in the figures represent 95% confidence intervals (CIs) and are shown when larger than the symbol. Differences between treated and untreated groups were analyzed by the chi-square test for categorical data and by the Student's t test for comparison of means. P values less than .05 were considered statistically significant.
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RESULTS |
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In Vitro Effect of Mangafodipir and Other Oxidative Stress Modulators on Human Leukocytes Treated With Anticancer Agents
We first investigated the effect of oxidative stress modulators on anticancer drug–induced cytotoxicity of normal leukocytes. Leukocytes from six healthy volunteers were exposed in vitro to the alkylating agent oxaliplatin, the taxane paclitaxel, or the antimetabolite 5-fluorouracil at concentrations approaching IC50 values for 24 hours in the presence or absence of mangafodipir. In one representative healthy volunteer (Fig. 1, A–C), concomitant exposure to increasing concentrations of mangafodipir statistically significantly decreased the cytotoxicity of these three anticancer agents in a dose-dependent fashion compared with any of the anticancer agents alone. For example, a concentration of 400 µM mangafodipir led to an absolute increase in cell survival of 46% (95% CI = 44% to 48%), 30.5% (95% CI = 29% to 32%), and 15% (95% CI = 10% to 20%) after 24 hours of exposure to 20 µM paclitaxel, 1 mM oxaliplatin, or 40 mM 5-fluorouracil, respectively, compared with survival of cells treated with each drug alone. N-Acetylcysteine displayed a similar protective effect against the cytotoxicity of paclitaxel, oxaliplatin, and 5-fluorouracil toward human normal leukocytes. By contrast, CuDIPS and MnTBAP statistically significantly enhanced the cytotoxicity of paclitaxel, oxaliplatin, and 5-fluorouracil against leukocytes, although the effects were more pronounced with CuDIPS than with MnTBAP (Fig. 1, A–C). Thus, concomitant treatment with 100 µM CuDIPS or MnTBAP decreased cell survival by 44% (95% CI = 43% to 45%) and 3.6% (95% CI = 2.9% to 4.3%) after exposure to paclitaxel for 24 hours, respectively, compared with survival of cells treated with paclitaxel alone. We also assessed whether mangafodipir was effective in protecting leukocytes from the toxic effects of another taxane, docetaxel. Mangafodipir protected the leukocytes from the 10 cancer patients against the toxicity of docetaxel. Simultaneous incubation for 24 hours with 20 µM docetaxel and 400 µM mangafodipir increased the viability rate by 19.5% (95% CI = 16% to 23%) compared with the rate of cells incubated with docetaxel alone.
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In Vivo Effect of Mangafodipir and Other Oxidative Stress Modulators on Hematologic Toxicity of Paclitaxel and Susceptibility of Mice to Bacterial Infection
We next investigated the effect of mangafodipir and three other oxidative stress modulators in combination with paclitaxel in BALB/c mice. After 10 days of treatment with paclitaxel alone (group 2), a statistically significant decrease in the absolute number of peripheral lymphocytes, neutrophils, and monocytes was observed in blood drawn from treated mice compared with blood drawn from control mice (group 1) (Table 1). This decrease was accompanied by a decrease in total bone marrow cell numbers. Administering mangafodipir (group 7) or N-acetylcysteine (group 10) in combination with paclitaxel abrogated the hematologic toxicity of paclitaxel. Neither mangafodipir nor N-acetylcysteine seemed to enhance hematopoiesis by itself. Indeed, blood and bone marrow cell counts in mice treated with mangafodipir (group 3) or N-acetylcysteine (group 6) alone were similar to those in untreated mice. By contrast to mangafodipir, the two other SOD mimics, CuDIPS (group 8) and MnTBAP (group 9), did not alter the hematologic toxicity of paclitaxel.
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Because bacterial infection is the main complication of neutropenia, the susceptibility of mice to S. aureus was tested after administration of paclitaxel alone (group 2) or in combination with mangafodipir (group 3) (Table 2). No deaths were observed in untreated mice inoculated with a sublethal dose of S. aureus. By contrast, 14 of 17 mice died within 48 hours after inoculation with the same dose of S. aureus following treatment with paclitaxel at a dose that induced neutropenia. However, only three of 17 mice inoculated with the bacteria died when mangafodipir had been administered in combination with paclitaxel (P<.001).
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Effect of Mangafodipir on Cytotoxicity of Anticancer Agents Against CT26 Cancer Cells In Vitro
Mangafodipir, as well as CuDIPS and MnTBAP, statistically significantly increased the cytotoxicity of oxaliplatin (Fig. 1, D), paclitaxel (Fig. 1, E), and 5-fluorouracil (Fig. 1, F) toward mouse CT26 colon cancer cells in a dose-dependent fashion. Conversely, N-acetylcysteine decreased the cytotoxicity of oxaliplatin, 5-fluorouracil, and paclitaxel toward CT26 cancer cells (Fig. 1, D–F). Similar results were observed using murine hepatoma Hepa1.6 and human lung cancer A549 cell lines (data not shown).
Effect of Mangafodipir and Other Oxidative Stress Modulators in Mice Implanted with CT26 Tumor Cells and Treated With Paclitaxel
To determine whether oxidative stress modulators inhibit tumor growth, we next treated mice bearing CT26 tumors with either paclitaxel, mangafodipir, MnTBAP, CuDIPS, or N-acetylcysteine alone or with paclitaxel in combination with one of these oxidative stress modulators (Fig. 2). Mice treated with paclitaxel alone (group 2) developed smaller tumors than untreated mice (group 1), but the difference was statistically significant only at day 27 and not before or after (–37%, 95% CI = –7% to –67%, P = .02 versus untreated mice at day 27). Mangafodipir inhibited tumor growth when administered alone (group 3) to a similar extent as paclitaxel (Fig. 2, A) (–34%, 95% CI = –2% to –66% at day 27, P = .05 versus untreated mice). Mice treated simultaneously with mangafodipir and paclitaxel (group 7) had statistically significantly smaller tumors than untreated mice at days 24, 27, and 30 (–49%, 95% CI = –7% to –91%, P = .04 at day 27). The concentrations of mangafodipir and paclitaxel slightly amplified the antitumor effect seen with paclitaxel alone, but the difference was not statistically significant. CuDIPS (group 4) and MnTBAP (group 5) had only minimal antitumoral effects when administered alone and did not amplify the antitumoral effect of paclitaxel (Fig. 2, B and C). By contrast, mice treated with N-acetylcysteine alone (group 6) developed larger tumors than untreated mice, but the difference was not statistically significant (Fig. 2, D). Adding N-acetylcysteine to paclitaxel (group 10) abrogated the antitumor effect of paclitaxel (Fig. 2, D).
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Effect of Mangafodipir and Other Oxidative Stress Modulators on Intracellular H2O2 and O2– Generation in Normal and Cancer Cells Exposed to Anticancer Agents In Vitro
We next investigated the effects of mangafodipir and other oxidative stress modulators on levels of reactive oxygen species in cancer cell lines and normal leukocytes. Basal H2O2 and O2– levels were lower in normal human leukocytes than in CT26 cancer cells (Table 3) (P<.001). Exposure to oxaliplatin, paclitaxel, or 5-fluorouracil for 48 hours statistically significantly increased intracellular levels of O2– and H2O2 in both types of cells. (All comparisons between treated and untreated cells reached P<.001, with the exception of 5-fluorouracil, for which P = .005 and P = .03 for H2O2 and O2– levels, respectively). However, reactive oxygen species levels remained lower in normal leukocytes than in cancer cells after exposure to these agents (Table 3). Exposure to these anticancer agents for 48 hours also statistically significantly decreased the intracellular reduced glutathione pool by 30% to 40% in both cancer and normal cells compared with untreated cells (both P<.005). Similar results were observed with Hepa1.6 cells (data not shown).
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Simultaneous treatment of CT26 cells and human leukocytes with mangafodipir, CuDIPS, or MnTBAP decreased O2– accumulation induced by all three anticancer agents by 20% to 30% in cancer cells (P<.01 compared with cells treated with anticancer agents alone). N-Acetylcysteine did not alter the O2– level in cells treated with anticancer agents.
In CT26 cancer cells, CuDIPS, MnTBAP, and mangafodipir increased H2O2 accumulation (Fig. 3, A–C) and glutathione depletion (Fig. 4, A–C) induced by oxaliplatin, paclitaxel, and 5-fluorouracil. Similar results were obtained when the same set of experiments were conducted in Hepa1.6 cancer cells (data not shown). By contrast, in normal human leukocytes treated with paclitaxel, concomitant exposure to mangafodipir led to a 33% decrease in H2O2 accumulation (95% CI = 29% to 37%, P<.001) compared with normal human leukocytes treated with paclitaxel alone, whereas concomitant exposure to CuDIPS or MnTBAP led to 28% (95% CI = 27% to 29%, P<.001) and 34% (95% CI = 29% to 39%, P<.001) increases in H2O2 accumulation, respectively (Fig. 3, E–G).
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P<.01, ***P<.001 by Student's t test when cells treated with both an anticancer agent plus one of the oxidative stress modulator were compared with cells not incubated with an oxidative stress modulator.Glutathione is an essential cofactor in H2O2 catabolism. Exposure to mangafodipir increased glutathione levels by 45% (95% CI = 40% to 50%, P<.001) in leukocytes also exposed to paclitaxel, whereas CuDIPS and MnTABP decreased glutathione levels by 15% (95% CI = 10.5% to 19.5%, P<.001) and 26.5% (95% CI = 15% to 38%, P<.001), respectively, when compared with leukocytes exposed to paclitaxel alone (Fig. 4, E–G). Similar effects of mangafodipir were observed in cells treated with oxaliplatin or 5-fluorouracil (Fig. 4, G). Simultaneous treatment with N-acetylcysteine statistically significantly lowered H2O2 accumulation and glutathione depletion induced by all three anticancer agents, both in cancer and normal cells compared with cells treated with anticancer agents alone (Fig. 3, D and H, and Fig. 4, D and H).
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DISCUSSION |
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We report what is, to our knowledge, the first evidence that mangafodipir, a molecule that is currently used as a contrast agent for magnetic resonance imaging, has properties that may be useful in cancer therapy. We observed that mangafodipir protected human peripheral blood leukocytes drawn from healthy volunteers and cancer patients from the toxicity of several different anticancer agents that have distinct mechanisms of action. We also observed that mangafodipir protected BALB/c mice treated with paclitaxel from developing leukopenia infections and also amplified the antitumoral effect of paclitaxel toward implanted tumors.
To investigate whether the SOD-, catalase-, and glutathione reductase–like activities of mangafodipir were responsible for its protective action, three other oxidative stress modulators—N-acetylcysteine, CuDIPS, and MnTBAP—were also studied to determine whether they protected leukocytes against toxicity from anticancer agents. In our in vitro experiments, N-acetylcysteine, like mangafodipir, protected normal leukocytes from the cytotoxic effects of paclitaxel, oxaliplatin, and 5-fluorouracil. By contrast, CuDIPS and MnTBAP enhanced the toxicity of these drugs in leukocytes. The absence of a protective effect of the latter two compounds may be due to their lack of glutathione reductase activity, which reduces their capacity to detoxify H2O2. By contrast, the protective effect of mangafodipir and N-acetylcysteine could result from the presence of glutathione reductase activity, which limits depletion of glutathione and in turn enables detoxification of H2O2.
Similar observations were made in vivo in mice treated with the hematotoxic anticancer agent paclitaxel. Simultaneous administration of mangafodipir, but not of CuDIPS or MnTBAP, protected mice from paclitaxel-induced hematologic and bone marrow toxicities at day 10. Moreover, coadministration of mangafodipir and paclitaxel dramatically improved the survival rate of mice infected with S. aureus. N-Acetylcysteine also had protective effects similar to those of mangafodipir, in agreement with previous data showing that N-acetylcysteine can abrogate the hematologic toxicity of alkylators in mice (24). However, the use of N-acetylcysteine in association with anticancer drugs has been limited by its capacity to accelerate tumor growth, which neutralizes the antitumoral activity of the drugs (6,8).
Other nonpeptidyl SOD mimics have also been shown to decrease the toxicity of anticancer agents or ionizing radiation in normal cells (25–27). For example, M40403, a pure MnSOD mimic, is able to block interleukin-2–induced hypotension in mice, presumably by scavenging extracellular interleukin-2–induced O2–, which inactivates circulating catecholamines (25,26). Moreover, in a randomized trial, orgotein, a Cu/ZnSOD mimic that does not cross cell membranes, abrogated the late but not the acute side effects of pelvic irradiation (27). This protective effect of orgotein was attributed to the inhibition of the acute-phase inflammatory events mediated by radiation-induced O2–. The two pure SOD mimics explored in these studies prevented specific side effects related to extracellular O2– accumulation, whereas in this study, mangafodipir was observed to prevent hematologic toxicity related to intracellular H2O2 accumulation.
One major concern of the finding that mangafodipir prevents hematologic toxicity induced by anticancer agents is the possibility that mangafodipir could abrogate the toxicity of anticancer agents in cancer cells, as it does in normal cells. However, under the same in vitro experimental conditions as above, mangafodipir enhanced the antitumoral activity of the three agents tested. In vivo, mangafodipir abrogated the growth of implanted tumors when administered alone and enhanced the antitumoral effects of paclitaxel in combination. Therefore, mangafodipir may enhance the therapeutic index of anticancer agents by both protecting normal cells and increasing the antitumoral activity of these agents.
The opposing effects of mangafodipir in normal and cancer cells may be related to differences in the redox status of these cells. Indeed, we have confirmed here that the basal concentration of reactive oxygen species and particularly of H2O2 is higher in cancer cells than in normal cells (6). The higher oxidative stress in tumor cells seems to be related to a higher rate of production of superoxide anions by the respiratory chain (6,28) and cytoplasmic NADPH oxidase (29). Furthermore, tumor cells express lower levels of catalase, glutathione peroxidase, reductase, and their respective cofactors than normal cells (6,10,30,31). Because anticancer agents induce the generation of H2O2 in both normal and cancer cells (5,6), the threshold of toxicity is reached more easily in cancer cells than in normal cells (6). This difference leads to preferential inhibition of cell proliferation and increasing cell death in cancer cells.
A limitation of our study is that the antitumoral effect of mangafodipir was studied only in established tumor cell lines. Further studies are warranted to evaluate its activity against primary human tumors. Moreover, relatively high doses of mangafodipir were used in mice compared with those used in the imagery diagnostic setting. Associations between pharmacokinetic and pharmacodynamic parameters need to be evaluated in animals and in humans.
In conclusion, our results support investigation of the use of mangafodipir in cancer patients, because mangafodipir may enhance the therapeutic index of anticancer agents by both protecting normal cells and increasing the antitumoral activity of these agents. The safety of mangafodipir administered as a contrast agent in magnetic resonance imaging has already been demonstrated (32). Clinical studies are planned to evaluate the safety of repeated administrations of mangafodipir in human subjects and their ability to reproduce the biologic and clinical activities observed in mice.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
(1) Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 1995;270:296–9.
(2) Polyak K, Xia Y, Zweier JL, Kinzler KW, Vogelstein B. A model for p53-induced apoptosis. Nature 1997;389:300–5.[CrossRef][Medline]
(3) Bernard D, Gosselin K, Monte D, Vercamer C, Bouali F, Pourtier A, et al. Involvement of Rel/nuclear factor-
B transcription factors in keratinocyte senescence. Cancer Res 2004;64:472–81.
(4) Hwang PM, Bunz F, Yu J, Rago C, Chan TA, Murphy MP, et al. Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat Med 2001;7:1111–7.[CrossRef][Web of Science][Medline]
(5) Benhar M, Dalyot I, Engelberg D, Levitzki A. Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress. Mol Cell Biol 2001;21:6913–26.
(6) Laurent A, Nicco C, Chéreau C, Goulvestre C, Alexandre J, Alves A, et al. Controlling tumor growth by modulating endogenous production of reactive oxygen species. Cancer Res 2005;65:948–56.
(7) Jing Y, Dai J, Chalmers-Redman RM, Tatton WG, Waxman S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 1999;94:2102–11.
(8) Alexandre J, Batteux F, Nicco C, Chereau C, Laurent A, Weill B, et al. The accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo. Int J Cancer. In press.
(9) Hussain SP, Amstad P, He P, Robles A, Lupold S, Kaneko I, et al. p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis. Cancer Res 2004;64:2350–6.
(10) Hirpara JL, Clement MV, Pervaiz S. Intracellular acidification triggered by mitochondrial-derived hydrogen peroxide is an effector mechanism for drug-induced apoptosis in tumor cells. J Biol Chem 2001;276:514–21.
(11) Pelicano H, Carney D, Huang P. ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 2004;7:97–110.[CrossRef][Web of Science][Medline]
(12) Hamaguchi K, Godwin AK, Yakushiji M, O'Dwyer PJ, Ozols RF, Hamilton TC. Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines. Cancer Res 1993;53:5225–32.
(13) Skapek SX, Colvin OM, Griffith OW, Elion GB, Bigner DD, Friedman HS. Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in a human medulloblastoma xenograft in athymic mice. Cancer Res 1988;48:2764–7.
(14) Bailey HH, Mulcahy RT, Tutsch KD, Arzoomanian RZ, Alberti D, Tombes MB, et al. Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol 1994;12:194–205.[Abstract]
(15) Bailey HH, Ripple G, Tutsch KD, Arzoomanian RZ, Alberti D, Feierabend C, et al. Phase I study of continuous-infusion L-S,R-buthionine sulfoximine with intravenous melphalan. J Natl Cancer Inst 1997;89:1789–96.
(16) Cullen JJ, Weydert C, Hinkhouse MM, Ritchie J, Domann FE, Spitz D, et al. The role of manganese superoxide dismutase in the growth of pancreatic adenocarcinoma. Cancer Res 2003;63:1297–303.
(17) Zhang Y, Zhao W, Zhang HJ, Domann FE, Oberley LW. Overexpression of copper zinc superoxide dismutase suppresses human glioma cell growth. Cancer Res 2002;62:1205–12.
(18) Bedda S, Laurent A, Conti F, Chereau C, Tran A, Tran-Van Nhieu J, et al. Mangafodipir prevents liver injury induced by acetaminophen in the mouse. J Hepatol 2003;39:765–72.[CrossRef][Web of Science][Medline]
(19) Leuthauser SW, Oberley LW, Oberley TD, Sorenson JR, Ramakrishna K. Antitumor effect of a copper coordination compound with superoxide dismutase-like activity. J Natl Cancer Inst 1981;66:1077–81.
(20) Patel M, Day BJ. Metalloporphyrin class of therapeutic catalytic antioxidants. Trends Pharmacol Sci 1999;20:359–64.[CrossRef][Medline]
(21) Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 1989;6:593–7.[CrossRef][Web of Science][Medline]
(22) Alexandre J, Rey E, Dieras V, Grabar S, Tran A, Montheil V, et al. Prospective study of predictive factors of docetaxel (DCX)-induced febrile neutropenia (FN): relevance of in vivo cytochrome 3A (CYP3A) phenotyping. Proc Am Soc Clin Oncol 2005; abstr 2046.
(23) Rice GC, Bump EA, Shrieve DC, Lee W, Kovacs M. Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. Cancer Res 1986;46:6105–10.
(24) Neuwelt EA, Pagel MA, Hasler BP, Deloughery TG, Muldoon LL. Therapeutic efficacy of aortic administration of N-acetylcysteine as a chemoprotectant against bone marrow toxicity after intracarotid administration of alkylators, with or without glutathione depletion in a rat model. Cancer Res 2001;61:7868–74.
(25) Samlowski WE, Petersen R, Cuzzocrea S, Macarthur H, Burton D, McGregor JR, et al. Nonpeptidyl mimic of superoxide dismutase, M40403, inhibits dose-limiting hypotension associated with interleukin-2 and increases its antitumor effects. Nat Med 2003;9:750–5.[CrossRef][Web of Science][Medline]
(26) Macarthur H, Westfall TC, Riley DP, Misko TP, Salvemini D. Inactivation of catecholamines by superoxide gives new insights on the pathogenesis of septic shock. Proc Natl Acad Sci U S A 2000;97:9753–8.
(27) Esco R, Valencia J, Coronel P, Carceller JA, Gimeno M, Bascon N. Efficacy of orgotein in prevention of late side effects of pelvic irradiation: a randomized study. Int J Radiat Oncol Biol Phys 2004;60:1211–9.[CrossRef][Web of Science][Medline]
(28) Carew JS, Zhou Y, Albitar M, Carew JD, Keating MJ, Huang P. Mitochondrial DNA mutations in primary leukemia cells after chemotherapy: clinical significance and therapeutic implications. Leukemia 2003;17:1437–47.[CrossRef][Web of Science][Medline]
(29) Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, et al. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 1997;275:1649–52.
(30) Jaruga P, Zastawny TH, Skokowski J, Dizdaroglu M, Olinski R. Oxidative DNA base damage and antioxidant enzyme activities in human lung cancer. FEBS Lett 1994;341:59–64.[CrossRef][Web of Science][Medline]
(31) Li S, Yan T, Yang JQ, Oberley TD, Oberley LW. The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res 2000;60:3927–39.
(32) Federle MP, Chezmar JL, Rubin DL, Weinreb JC, Freeny PC, Semelka RC, et al. Safety and efficacy of mangafodipir trisodium injection for hepatic MRI in adults; results of the US multicenter phase III clinical trials. J Magn Reson Imaging 2000;12:186–97.[CrossRef][Web of Science][Medline]
Manuscript received June 15, 2005; revised November 30, 2005; accepted December 29, 2005.
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J Natl Cancer Inst 2006 98: 223-225.
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