Nitric Oxide Contributes to Adriamycin\'s Antitumor Effect

JOURNAL OF SURGICAL RESEARCH ARTICLE NO.

69, 283–287 (1997)

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Nitric Oxide Contributes to Adriamycin’s Antitumor Effect D. Scott Lind, M.D., Maria I. Kontaridis, B.S., Paul D. Edwards, M.D., Michael D. Josephs, M.D., Lyle L. Moldawer, Ph.D., and Edward M. Copeland, III, M.D. Department of Surgery, University of Florida, Gainesville, Florida 32610 Submitted for publication November 25, 1996

Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production. Purpose: To determine if adriamycin induces NO production in breast cancer cells in vitro and whether NO contributes to adriamycin’s antitumor effect in vivo. Methods: Murine breast cancer cells (EMT-6) were incubated with adriamycin (ADRIA, 0, 10, 100, 1000 mM) in the presence or absence of the NO synthase inhibitor aminoguanidine (AG, 1 mM). Twenty-four hours later nitrite accumulation (Greiss reagent) and cell viability (MTT assay) were assessed. Supernatants from adriamycin-stimulated cells were also analyzed at 6, 8, and 24 hr for TNF, IL-1, and IFNg (ELISA). For in vivo experiments, 105 EMT-6 cells were injected into the flank of BALB/c mice (n Å 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks later tumor size was measured and in situ tumor cell apoptosis was determined by fluorescent microscopy and flow cytometry. Results: Adriamycin was cytotoxic to EMT6 cells with 100 mM resulting in nearly 100% killing (P õ 0.01). Adriamycin also stimulated nitrite accumulation with 100 mM producing 6.5 { 0.26 mM nitrite (P õ 0.001). AG blocked adriamycin-stimulated nitrite accumulation (P õ 0.05), but did not inhibit cytotoxicity in vitro. In vivo, adriamycin inhibited tumor size by nearly 400% (P õ 0.001), while AG attenuated adriamycin’s effect on tumor growth (P õ 0.05). There was no difference in the detection of apoptotic tumor cells between the adriamycin and adriamycin and AG groups as determined by immunohistochemistry and flow cytometry. Conclusions: These findings suggest that adriamycin stimulated NO production in EMT-6 cells, but adriamycin’s cytotoxicity in vitro was NO-independent. In vivo, adriamycin inhibited tumorigenesis partially via an NO-dependent, nonapoptotic mechanism. q 1997 Academic Press

INTRODUCTION

Adriamycin is the single most effective drug in the treatment of breast cancer [1]. Recently, several antiPresented at the 30th Annual Meeting of the Association for Academic Surgery, Chicago, Illinois, Nov. 13–16, 1996.

neoplastic agents have been shown to stimulate nitric oxide (NO) production [2–4]. NO may influence several aspects of tumor biology including modulation of cell growth [5], apoptosis [6], differentiation [7], metastatic capability [8], and tumor-induced immunosuppression [9]. The purpose of this study was to determine if adriamycin stimulates NO production in a murine breast cancer cell line and, if so, whether cytokines mediate this response. In addition, we investigated whether adriamycin-stimulated NO contributes to adriamycin’s antitumor effect in vivo by inducing tumor cell apoptosis. METHODS Reagents. All chemicals and solvents were of the highest commercially available grade from Sigma Chemical Co. (St. Louis, MO). Cell culture. The EMT-6 murine breast cancer cell line was kindly provided by Dr. G. Lopez-Berenstein (M.D. Anderson Hospital and Tumor Institute, Houston, TX). The cells were maintained by serial passage in RPMI media with 5% fetal bovine serum supplemented with antibiotic/antimycotics. Cells were grown in 75-cm2 flasks in a CO2-free, humidified, 377C atmosphere. Only low passaged cells (six to eight passages) were used for all experiments. Nitric oxide production. Determination of NO levels was made using a variation of the method described by Greene et al. which measures accumulation of the stable NO end product, nitrite [10]. Briefly, 100 ml culture supernatant was deproteinized with 100 ml of 35% sulfosalicylic acid and centrifuged for 15 min at 5000g. One hundred microliters was removed and added to an equal volume of the Griess reagent [prepared fresh by mixing equal volumes of 1.0% sulfanilamide (1 mg/100 ml) stored in aqueous 2.5% H3PO4 (2.94 ml/ 100 ml) and 0.1% naphthalene diamine in H2O (100 mg/100 ml)] in triplicate on a 96-well microtiter plate. Absorbance was determined after chromophore development at 607C followed by 07C (10 min at each temperature), using an automated 96-well plate reader at an optical density of 540 nm. Results were compared to a standard curve generated with sodium nitrite. For serum measurements, serum nitrate was first reduced to nitrite by nitrate reductase (0.1 unit/105 cells, Sigma Chemical Co.). In addition, flavin adenine dinucleotide (FAD, 50 mM per well) and B-nicotinamide adenine dinucleotide phosphate (NADPH, 500 mM per well) were added to each well, followed by 30 min incubation at 377C. Following this, 10 ml of 500 mM pyruvate and 10 ml/250 ml H2O lactic acid dehydrogenase (LDH) were added to each well, followed by 30 min incubation at 377C. Finally, 100 ml of Griess reagent was added to each well and nitrite/nitrate was measured as described above. MTT assay. Cellular viability was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [11]. Briefly, 5 1 105 EMT-6 cells were seeded onto 24-well culture plates and incubated in a humidified, CO2-free, 377C atmo0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Effect of adriamycin on viable cell number and nitrite accumulation in EMT-6 cells in vitro. Data represent means { SEM. *P õ 0.001 and **P õ 0.01 versus no adriamycin.

sphere for 48 hr. Following this, the cells were incubated with 100 ml MTT solution (5 mg/ml) for 4 hr at 377C for the development of formazan crystals. The crystals were then solubilized with 1.0 ml MTT solution and 750 ml 2-propanol. The optical density was then determined at 570 nm with subtraction of 690-nm background absorbance. Cell count was determined by comparison to a standard curve employing known concentrations of cells. Detection of apoptosis. Measurement of apoptosis was determined using a commercially available immunoperoxidase assay (Apoptag, Oncor, Inc., Gaithersburg, MD). Briefly, tumors were removed, minced into small pieces, and incubated with Oncor protein digesting enzyme (20 mg/ml) for 15 min at room temperature. Cells were then washed four times in phosphate-buffered saline (PBS) and resuspended at 1–2 1 105 in 0.5 ml of PBS and fixed in 1% paraformaldehyde for 15 min on ice. Cells were then centrifuged (1000 rpm 1 5 min) and resuspended in 70% ice-cold ethanol and kept at 0207C until staining for immunohistochemistry or flow cytometry. Following this, 3*-end DNA fragments were visualized using the Apoptag kit. Briefly, digoxigenin-nucleotide (dUTP and dATP) residues were catalytically added to nucleosome-sized DNA fragments by terminal deoxynucleotidyltransferase (TdT). The 3*-end fragments were then visualized with a fluorescein-conjugated anti-digoxigenin antibody. Nuclear counterstaining was performed with 4*-6-diaminido-2-phenylindole (DAPI). Cells were then analyzed by fluorescent microscopy. The percentage of cells with morphological appearance of apoptosis (i.e., smaller size, condensed cytoplasm and chromatin, and nuclear fragmentation) was determined. Cells were also analyzed by flow cytometry using a FACSCAN flow cytometer. Animal experiments. For in vivo experiments, 105 EMT-6 cells were injected into the flank of BALB/c mice (n Å 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/ kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks following tumor cell implantation, tumor size (mean diameter) was measured, mice were then sacrificed, and serum was collected and nitrite/nitrate measured. Statistical analysis. Data were analyzed with Student’s t test and analysis of variance (ANOVA) using SigmaStat software (Jandel Scientific, San Rafael, CA). Statistical significance was predetermined as P õ 0.05.

FIG. 2. Effect of the NO synthase inhibitor, aminoguanidine (AG), on adriamycin-stimulated nitrite accumulation in vitro. *P õ 0.01 versus no AG. Aminoguanidine had no effect on adriamycin cytotoxicity (data not shown).

adriamycin stimulated nitrite accumulation in a dosedependent fashion with 100 mM adriamycin producing 6.5 { 0.26 mM nitrite at 24 hr (P õ 0.001, Fig. 1). The NOS inhibitor aminoguanidine significantly blocked adriamycin-stimulated nitrite accumulation (P õ 0.05, Fig. 2), but had no effect on cell viability at 24 hr (data not shown). None of the cytokines (TNF, IL-1, and IFNg) were detectable in the supernatants of adriamycin-treated cells (data not shown). In Vivo Experiments Figure 3 demonstrates that mice receiving adriamycin had tumors that were nearly 400% smaller than tumors in control mice (P õ 0.001). The addition of aminoguanidine attenuated adriamycin’s antitumor effect. Tumors in mice receiving adriamycin and aminoguanidine were nearly twice as large as tumors in mice receiving adriamycin alone (P õ 0.05). Aminoguanidine alone had no effect on tumor size. Mice receiving adriamycin had elevated serum nitrite/nitrate levels (P õ 0.05), while aminoguanidine blocked this effect (P

RESULTS

In Vitro Experiments Adriamycin was cytotoxic in a dose-dependent fashion with 100 mM adriamycin producing nearly 100% killing of EMT-6 cells (P õ 0.01, Fig. 1). In addition,

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FIG. 3. Effect of aminoguanidine (AG) on adriamycin’s antitumor effect in vivo and serum nitrite/nitrate accumulation. *P õ 0.001 versus saline. **P õ 0.05 versus adriamycin alone.

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FIG. 4. (A) Immunohistochemistry of tumors from mice receiving adriamycin. Cells demonstrate characteristic apoptotic morphology including membrane blebbing and fluorescent staining of apoptotic nuclear bodies. There was no difference in the percentage of cells with morphological appearance of apoptosis between tumors from mice treated with adriamycin and mice that received adriamycin and aminoguanidine (data not shown). (B) DNA histograms of tumor cells from adriamycin-treated mice. There is a shift in the mean fluorescent intensity of cells from adriamycin-treated mice consistent with staining of apoptotic cells. There was no difference in the percentage of positive staining cells between tumors from mice treated with adriamycin and mice treated with adriamycin and aminoguanidine (data not shown).

õ 0.05). Adriamycin-treated tumor cells demonstrated characteristic apoptotic morphology including membrane blebbing and fluorescent staining of apoptotic nuclear bodies (Fig. 4A), while DNA histograms of tu-

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mor cells from adriamycin-treated mice revealed a shift in the mean fluorescent intensity consistent with staining of apoptotic cells (Fig. 4B). There was no difference in the percentage of cells with morphological appear-

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ance of apoptosis or the percentage of positive staining cells between tumors from mice treated with adriamycin and mice receiving adriamycin and aminoguanidine (data not shown). DISCUSSION

Adriamycin is the single most effective chemotherapeutic agent in the treatment of breast cancer [1]. Adriamycin’s tumoricidal activity may involve several mechanisms including the generation of reactive oxygen intermediates, DNA fragmentation, and programmed cell death (apoptosis) [12]. Recently, several antineoplastic agents have been demonstrated to induce NO production [2–4]. NO is a multifunctional messenger molecule derived from the amino acid L-arginine in a reaction catalyzed by NO synthase (NOS) [13]. NO may influence several aspects of tumor biology including cell growth [5], apoptosis [6], differentiation [7], metastatic capability [8], and tumor-induced immunosuppression [9]. The purpose of this study was to determine if adriamycin stimulates NO production in a murine breast cancer cell line in vitro and whether cytokines mediate this response. In addition, we investigated whether adriamycin-stimulated NO contributes to adriamycin’s antitumor effect in vivo by inducing tumor cell apoptosis. Although adriamycin was cytotoxic to EMT-6 cells, it also induced nitrite accumulation. Nitrite accumulation was not simply a manifestation of cell death, however, because we have previously treated EMT-6 cells with another antineoplastic agent, taxol, that was cytotoxic to EMT-6 cells but failed to induce nitrite accumulation. Adriamycin-treated cells may be induced to accumulate nitrite prior to cell death. The NOS inhibitor aminoguanidine blocked nitrite accumulation, but had no effect on adriamycin cytotoxicity, suggesting that adriamycin’s killing in vitro was NO-independent. It is possible that the amount of NO generated by EMT-6 cells following exposure to adriamycin was not sufficient to be cytotoxic. We have previously demonstrated that inflammatory mediators stimulate NO production in EMT-6 cells and this results in an autocrine antiproliferative effect [14]. The amount of nitrite accumulated in these experiments was two- to fivefold that produced by adriamycin. It is also possible, however, that by only examining cell number at a single time point (i.e., 24 hr), any effect adriamycin-stimulated NO production might have on cell proliferation was missed. We have also previously demonstrated that endotoxin stimulates transmembrane arginine transport and NO production in porcine pulmonary artery endothelial cells through an autocrine mechanism involving IL-1 and TNF [15]. Tumor cells have been demonstrated to produce cytokines [16] and adriamycin has been shown to stimulate cytokine secretion [17]. Therefore, we hypothesized that adriamycin might stimulate nitrite accumulation indirectly through the production of cytokines. However, IL-1, TNF, and IFNg were not detectable in the supernatants of adriamycin-treated

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cells, making an indirect mechanism involving cytokines unlikely. Adriamycin may directly stimulate NOS activity. Recently, Joshi et al. demonstrated that NOS activity was increased in brain extracts after treatment with daunorubicin [18]. Adriamycin may also act through other means to accumulate nitrite. We have demonstrated that NOS inducers also stimulate transmembrane arginine transport [15]. Therefore, it is possible that adriamycin stimulates transmembrane arginine transport to provide increased substrate. Alternatively, constitutive expression of mRNA for inducible NO has been reported [19], so adriamycin could increase NO production posttranscriptionally through the stabilization of iNOS mRNA. In vivo adriamycin inhibited tumor growth, while aminoguanidine attenuated adriamycin’s antitumor efficacy. Aminoguanidine also blocked serum nitrite/nitrate production, suggesting that NO was partially responsible for adriamycin’s antitumor effect in vivo. While it is possible that aminoguanidine affected intracellular adriamycin concentration independent of its NO-inhibiting capabilities, the failure of aminoguanidine to have any effect on adriamycin’s cytotoxicity in vitro makes this an unlikely mechanism for aminoguanidine’s attenuation of adriamycin’s antitumor efficacy in vivo. The source of NO in vivo remains undefined. It is possible that adriamycin directly or indirectly stimulated NO production from other NO-producing cell types which, in turn, acted on tumor cells. Manthey et al. have recently demonstrated that the anticancer drug taxol activates macrophages to kill tumor cells in an NO-dependent fashion [20]. The antitumor efficacy of adriamycin correlates with its ability to induce apoptosis [21]. Several studies have implicated NO in the induction of apoptosis [6, 22–24]. Although adriamycin induced apoptosis, AG had no effect on programmed cell death, suggesting it was an NO-independent event. NO may affect cell proliferation via nonapoptotic mechanisms such as the inactivation of iron/sulfur-containing enzymes responsible for mitochondrial respiration [25] or the inhibition of ribonucleotide reductase [26]. NO also enhances cellular oxidative injury [27] and may affect adriamycin metabolizing enzymes or cellular drug efflux pump mechanisms. Preliminary data from our laboratory suggest that NO may inhibit p-glycoprotein expression. In summary, we have demonstrated that adriamycin stimulates nitrite accumulation in a murine breast cancer cell line through a mechanism that does not involve IL-1, TNF, or IFNg secretion. Adriamycin’s cytotoxicity in vitro was NO-independent; however, in vivo adriamycin inhibited tumor growth partially via an NO-dependent, nonapoptotic mechanism. Further experiments are required to determine if NO’s contribution to adriamycin’s antitumor effect is a consistent finding in other NO-producing tumor cells. If so, adriamycinstimulated NO production could represent a mechanism to enhance the efficacy of this antineoplastic agent.

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