Cancer Research Meeting Calendar EMT and Cancer Progression and Treatment
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Feger, F.
Right arrow Articles by Mossalayi, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feger, F.
Right arrow Articles by Mossalayi, M. D.
[Cancer Research 61, 5289-5294, July 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Role of Iron in Tumor Cell Protection from the Pro-Apoptotic Effect of Nitric Oxide1

Fredéric Feger2, Hélène Ferry-Dumazet2, Maria Mamani Matsuda, Joëlle Bordenave, Maryse Dupouy, Andreas K. Nussler, Michel Arock, Lionel Devevey, Joëlle Nafziger, Jean-Jacques Guillosson, Josy Reiffers and M. Djavad Mossalayi3

Hematology Laboratory, Paris V Faculty of Pharmacy, 75006 Paris, France [F. F., J. B., M. A., L. D., J. N., J-J. G.]; Bone Marrow Transplantation Laboratory, Bordeaux 2 University, 33076 Bordeaux, France [H. F-D., M. M. M., M. D., J. R., M. D. M.]; and Department of Surgery, Berlin University, D-10713 Berlin, Germany [A. K. N.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
F2The host defense against tumor cells is in part based upon the production of nitric oxide (NO) by activated macrophages. However, carcinogenesis may involve mechanisms that protect tumor cells from NO-mediated apoptosis. In the present study, we have assessed the effects of exogenous NO on the proliferation and survival of human liver (AKN-1), lung (A549), skin (HaCat), and pancreatic (Capan-2) tumor cell lines, compared with normal skin-derived epithelial cell cultures. Except to the HaCat cell line, all of the other human epithelioid cells were sensitive to the antiproliferation effect of S-nitroso-N-acetyl-penicillamine or Deta NONOate, whereas tumor cells had low if any response to sodium nitroprusside. Growth inhibition with exogenous NO correlated with increased apoptosis, but was not mediated by cyclic GMP, peroxynitrite generation, or poly(ADP-ribose) polymerase modulation, all of which involved in NO-mediated growth inhibition of normal skin-derived epithelial cell cultures. The simultaneous addition of iron-containing compounds protected tumor cells from NO-mediated growth inhibition and apoptosis. Intracellular iron quantification indicated that, as deferoxamine, exogenous NO significantly decreased intracellular ferric iron levels in tumor cells. Together, the current study reveals that intracellular iron elevation rescues tumor cells from NO-mediated iron depletion and subsequent growth inhibition and apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NO4 is a messenger molecule with complex biological activities including vasodilatation, neurotransmission, immunoregulation, and inflammation (1, 2, 3, 4) . NO is synthesized enzymatically from L-arginine by at least three different NOSs. The endothelial and the neuronal isoforms are constitutively expressed. The third isoform, NOS-II, has to be induced with stimuli that include lipopolysaccharide and cytokines (2) . Once expressed, NOS-II synthesizes large amounts of NO, which can lead to the inhibition of T-cell proliferation, has tumoricidal activity, suppresses the cellular protein synthesis, and leads to oxidative damage and apoptosis (1 , 5) . NO-mediated DNA damage induces apoptotic cell death in tumor cells after the induction of the NOS-II or by the application of an exogenous NO donor (6, 7, 8, 9) . In contrast, some tumor cell lines express or could be induced to express NOS-II without any observed effect on cell proliferation (10 , 11) . This implies that NO is not toxic for these tumor cells or that carcinogenesis enables cells to counteract NO-mediated cytotoxicity. On the other hand, it has been shown that in some instances, high levels of NO synthesis are cytoprotective (11, 12, 13, 14, 15, 16) . This prompted us to examine if tumor cells are still sensitive to the pro-apoptotic effect of NO by applying various exogenous NO-releasing compounds. First, data led us to analyze the role of iron in NO-mediated cytotoxicity.

For few decades, iron has been shown to favor neoplastic cell growth and to display carcinogenic activity, because of its catalytic effect on the formation of hydroxyl radicals, suppression of the activity of host defense cells, and promotion of cancer cell multiplication (17 , 18) . Primary neoplasms develop at body sites of excessive iron deposits (18) . The invaded host attempts to withhold iron from the cancer cells via sequestration of the metal in newly formed ferritin (17) . The host also endeavors to withdraw the metal from cancer cells via macrophage synthesis of NO (19 , 20) . The present study indicates, in various human tumor cell lines, that the pro-tumoral effect of iron may be in part related to its ability to rescue cells from NO-mediated growth inhibition and apoptosis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
NO-releasing chemicals used in this study are SNAP, NOC18 {Deta NONOate; (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazin-1-ium-1,2-diolate; Alexis, Coger S. A., Paris, France}, and SNP (Sigma-Aldrich, Saint Quentin Fallavier, France). NO donors were put in solutions and prepared immediately before use at 5–500 µM. DFX (2–100 µM; Novartis-Pharma, Rueil-Malmaison, France), 3AB (1 mM), bovine SOD (120 IU/ml), catalase (130 IU/ml), Fe3+CN, potassium ferrocyanide, Fe3+Ci (10–200 µM), dibutyrylguanosine cyclic 3'-5' monophosphate (400–800 µM), and camptothecin were purchased from Sigma-Aldrich. ODQ (Alexis) was used to inhibit the NO-sensitive guanylyl cyclase.

Cell Culture.
The human pulmonary adenocarcinoma (A549), skin (HaCat), and pancreas carcinoma (Capan-2) cell lines were obtained from the American Type Culture Collection (Manassas, VA). The hepatic carcinoma cell line AKN-1 is described elsewhere (21) . Tumor cells were cultured in RPMI 1640 supplemented with 10% FCS, penicillin/streptomycin, and glutamine (all of these from Life Technologies, Inc., Cergy-Pontoise, France). Normal skin-derived epithelial cell cultures (keratinocytes) were obtained from skin biopsies as described elsewhere (22) . For keratinocytes, we used a special serum-free culture medium supplemented with epidermal growth factor and bovine pituitary extracts (all of these from Life Technologies, Inc.). After expansion, the cells were transferred into plastic flasks and incubated with the serum-free culture medium. For various tests, cells were harvested after trypsin-EDTA treatment of culture surfaces, washed, counted, and suspended before cultures at 37°C in 5% CO2 atmosphere.

Cell Proliferation.
Each cell line was seeded in 6-well culture plates at a density of 30,000 cells/3 ml/well for AKN-1, HaCat, Capan-2, and normal cells or 15,000 cells/3 ml/well for A549 cells, which had a rapid cell division rate compared with the other cells tested. After cell adhesion (4–24 h), the various reagents were added simultaneously as described under "Results." After incubation, cells were harvested by trypsin treatment of plates, washed with culture media, and counted with trypan blue exclusion. The effect of various treatments on the cell nucleus was also assessed after cell centrifugation on slides, fixation with 2% paraformaldehyde, permeabilization with 0.1% Triton X-100, and coloration with DAPI (Vectashield; Vector Laboratories, Burlingame, CA). Cells were analyzed using a fluorescent microscope. To assess cell proliferation, various cell lines were cultured on 96-well plates at 4000 cells/well/50 µl. After adherence (24 h), wells were supplemented with various chemicals in an additional 100 µl/well. [3H]thymidine was added 24 h after cultures, and radioactivity uptake was measured 24 h later. The levels of nitrites were measured in cell supernatants using the colorimetric Greiss method as described previously (22) .

Cell Cycle and Apoptosis.
To analyze the cell cycle, treated cells were washed in PBS containing 1% BSA. Pellets were then treated with 200 µl of lysis solution [calcium- and magnesium-free PBS, 0.5% NP40, 20 µg/ml propidium iodide, 0.2 mg/ml RNase A, and 0.5 mM EDTA (pH 7.2); all of these from Sigma-Aldrich] at room temperature in the dark for 20 min before measurement. DNA analysis was performed using the System II Coulter program (XL Coulter Instrument; Coultronics, Margency, France). To measure apoptosis, externalization of membrane phosphatidylserine was analyzed using annexin-V-FITC and propidium iodide kit (Immunotech, Marseille, France).

Northern Blot Analysis.
Total RNA was extracted using RNA PLUS (Quantum Appligène, Illkirch, France), electrophoresed, and blotted on nylon membrane (Hybond N; Amersham, Saclay, France). For hybridization, human probes for c-myc (610-bp BclI-ClaI fragment of exon III) and p53 (1.8-kb BamHI fragment) were used.

Protein Extraction and Western Blot Analysis.
Whole cell lysates were prepared by pelleting 107 cells, followed by a 5-min incubation in lysis buffer. Proteins were resolved on 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham). Nonspecific interactions were blocked by preincubation of the membranes with PBS-Tween-5% dry milk. Membranes were then incubated with mouse anti-p53 monoclonal antibody and polyclonal goat antihuman actine (both from Santa Cruz Biotechnology, Perray en Yvelines, France). Detection was performed using peroxidase-coupled antibodies and enhanced chemiluminescence detection (Amersham). Ramos lymphoma cell line was used as positive control for p53 expression.

Intracellular Iron Quantification.
Intracellular total and Fe2+ levels were measured using a standard analytic procedure as detailed elsewhere (23) . Fe3+ levels were obtained after subtraction of Fe2+ levels from total iron values. Iron levels were shown as nmol/mg protein.

Statistical Analysis.
Statistical analysis was performed using Systat software. For a better comparison of cell growth, results were expressed as a percentage of viable cells compared with the number of cells incubated in medium alone.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumor Cells Differ in their Sensitivity to Exogenous NO.
The effect of exogenous NO on continuous growth of tumor cell lines derived from various tissues was analyzed. SNAP was used as an exogenous NO source (see below). As shown in Fig. 1Citation , proliferation of tumor cells was variably affected by NO addition. Although HaCat cells showed low or no sensitivity to NO, the proliferation of other tumor cells significantly decreased in the presence of exogenous NO (P < 0.001). SNAP breakdown and NO generation did not vary in all of the cell lines, as controlled by the quantification of the levels of nitrites in cell supernatants (data not shown). These experiments indicate that most tumor cells remain sensitive to the antiproliferation effect of NO.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Effect of exogenous NO (SNAP) on the growth of various tumor cell lines. Top, cells were incubated for 3 days and mean percentage values, compared with control, from three different experiments are shown (SE, <19%). Bottom, [3H]thymidine uptake by tumor cells after a 48-h incubation with various concentrations of SNAP. Values are the mean from sextuples (SE, <9%) from each cell line. Each cell line was tested twice.

 
Whether NO-sensitive cells respond to other formulations of exogenous NO was then investigated. In addition to SNAP, which is a rapid (<40-min half-life), relatively pure NO-releasing chemical, we added slower NO-releasing SNP and the very slow (>48-h half-life) NO-releasing donor, Deta NONOate (NOC18). The effect of these molecules on the growth of normal skin-derived epithelial cell (keratinocytes) cultures in response to epidermal growth factor was also quantified. As shown in Fig. 2Citation , the addition of SNAP or NOC18 clearly inhibited the proliferation of epithelial cells, whereas SNP had a significantly low (P < 0.001), if any, effect on the growth of most tumor cell lines. Of interest, NO-releasing molecules differed in their effect on keratinocytes. Compared with tumor cell lines, keratinocytes presented significantly higher sensitivity to SNP (P < 0.02) and less response to SNAP (P < 0,01), but had a similar response to NOC18. These data indicate that, by contrast to SNAP and NOC18, SNP had little or no effect on tumor cell growth. In addition, slow NO release by NOC18 seems to be very toxic for both tumor and normal skin-derived cell cultures and, therefore, was excluded for further analysis.



View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. The ability of various chemical NO donors to inhibit the proliferation of tumor cells or skin-derived epithelial cell cultures (keratinocytes). Cells were incubated for 3 days with various concentrations of NO-releasing compounds. Values are the mean percentage of cell numbers, compared with controls, of three different experiments, each done in duplicate (SE, <16%).

 
Effects of NO on Cell Cycle and Apoptosis in Tumor Cells.
Because SNAP showed a higher effect on tumor cells, we have used this chemical to assay mechanisms of cell growth inhibition and antitumor effects of NO. After a 48-h cell incubation with SNAP (200 µM), a significant dose-dependent increase of apoptotic cells was observed in tumor cells (Fig. 3Citation ; P < 0.003), as quantified by analyzing phosphatidylserine externalization. This was further confirmed by the appearance of a sub-G1 DNA peak without evidence of nuclear cell cycle modification (see below). Apoptotic cells also lost their adhesion capacity, and >95% of annexin-V+ cells were recovered in nonadherent fraction. This is further evidenced by DAPI staining, which showed increased chromatin condensation and nuclear fragmentation in nonadherent cell fraction but not in adherent tumor cells (Fig. 4)Citation treated with SNAP. To mediate growth inhibition, NO was also shown to regulate various gene expression in target cells such as p53 and c-myc (24) . Expression of p53 and c-myc was then quantified in mRNA or protein levels. We did not observe any constant variations in p53 or c-myc expression after incubation with SNAP (data not shown), which led us to conclude that modulation of p53 and c-myc expression may not account for the antitumor effect of NO.



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Dose-dependent induction of apoptosis in tumor cells by NO. Cells were incubated for 2 or 3 days with various concentrations of the NO-releasing compound, SNAP. NO induced a dose-dependent increase in the percentage of annexin-V+ cells after a 48-h incubation. This effect corroborated with the growth inhibitory effect of the same SNAP concentrations in cells incubated 72 h. Representative data in AKN-1 and Capan-2 cells (mean ± SD of two different experiments).

 


View larger version (79K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Nuclear morphology after 48 h of AKN-1 tumor cell incubation with NO (200 µM SNAP). Nuclear DAPI staining was performed in the following cells: A, adherent cells incubated with medium alone; B, SNAP-treated adherent cells; C, SNAP-treated nonadherent cells.

 
Role of Intracellular Signals after NO Treatment of Tumor Cell Lines.
NO derivatives inhibit cell growth through multiple intracellular targets (1, 2, 3, 4) . Most biological functions of NO are mediated through the induction of intracellular accumulation of cGMP after the activation of guanylyl cyclase (1) . To assay the role of cGMP on tumor cells, we added ODQ (25) , a specific inhibitor of NO-sensitive guanylyl cyclase, to NO-supplemented cultures. ODQ did not affect growth inhibition of tumor cells by SNAP, although it significantly inhibited the antiproliferative effects of SNAP in keratinocytes (Fig. 5)Citation . Accordingly and in contrast to keratinocytes, addition of the cGMP analogue was not toxic for tumor cells (Fig. 5)Citation . NO-mediated cytotoxicity also implies PARP activation and subsequent NAD+ depletion or the generation of the pro-apoptotic peroxynitrites (4) . Inhibition of PARP activation through the use of 3AB had no effect on tumor cells but partially rescued keratinocytes from death (Fig. 6)Citation . The addition of SOD and catalase, which decrease NO reaction with oxygen radicals, did not modify NO effects on tumor cells but increased the growth of keratinocytes (Fig. 6)Citation . Thus, in contrast to normal skin-derived cultures, NO-mediated inhibition of tumor cell growth involves neither the activation of guanylyl cyclase and subsequent generation of cGMP and PARP activation nor the formation of peroxynitrites.



View larger version (76K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Activation of guanylyl cyclase by NO is not involved in the antitumoral effect of exogenous NO. Cells were incubated as shown with various chemicals including dibutyryl-cGMP, SNAP (200 µM), and ODQ (3 µM) during 72 h. Shown are mean percentage values ± SE from two to three experiments performed for each cell line compared with controls. *, P < 0.02; **, P < 0.005.

 


View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Reactive oxygen intermediates and PARP are not involved in the antitumoral effect of exogenous NO. Cells were incubated as shown with various chemicals including SNAP (200 µM), SOD (120 units/ml), catalase (130 units/ml), and/or 3AB (1 mM) during 72 h. Shown are mean percentage values ± SE compared with controls; three experiments were performed for each cell line. *, P < 0.02; **, P < 0.009.

 
Iron Supplementation Rescues Tumor Cells from Death in the Presence of Exogenous NO.
The absence of growth inhibition after SNP addition on tumor cells was intriguing and led us to clarify this response. In addition to NO, SNP contains iron cyanide and sodium cyanide, making it different from other NO-releasing chemicals. We questioned the role of non-NO ferricyanide in this molecule and its effects on tumor cells. As SNP, the addition of Fe3+CN (200 µM) to tumor cells did not affect their growth (Fig. 7A)Citation . In contrast, the addition of ferricyanide to SNAP significantly reversed the antitumor effect of NO in all of the tumor cells tested (Fig. 7A)Citation . The addition of nonferric sodium cyanide (another SNP derivative) had no effect on SNAP-mediated tumor cell inhibition (50–200 µM; data not shown). Interestingly, ferricyanide did not significantly reverse growth inhibition of NO-treated keratinocytes (Fig. 7)Citation . These data may explain the absence of antitumor activity of iron-containing NO donor, SNP. To further confirm the role of iron, we added Fe3+Ci (200 µM) simultaneously with SNAP. Data in Fig. 7BCitation clearly indicate that, similar to Fe3+CN, Fe3+Ci reversed the antiproliferation effect of SNAP. Then, we asked what was the effect of Fe2+ on NO-mediated antitumor effect. As shown in Fig. 7BCitation , Fe2+ significantly reverses the effect of SNAP but remained less potent than the Fe3+ compound in this respect. Surprisingly, dose-response analysis showed that only 2–10 µM Fe3+ salt neutralized 200 µM SNAP (Fig. 7B)Citation , whereas ferrous salt required higher concentrations and remained less efficient in this respect. In addition to increased cell recovery, Fe3+Ci also decreased the sub-G1 DNA peak (Fig. 8A)Citation and significantly rescued tumor cells from NO-induced apoptosis (Fig. 8BCitation ; P < 0.012). Together, these data show the ability of iron, ferric compounds in particular, to protect tumor cells from the antiproliferation effect of NO.



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Iron protects tumor cells from antitumoral effects of NO. A, cells were incubated in medium alone (none) or with SNAP (200 µM), Fe3+CN (500 µM), and/or Fe3+Ci (200 µM). Shown are mean ± SE of the number of cells after a 72-h incubation; three to five experiments were performed for each cell line. *, P < 0.006; N. S., not significant. B, dose-dependent rescue of tumor cells from NO (SNAP 200 µM)-mediated cell death by exogenous ferrous (Fe2+) or ferric (Fe3+) compounds. Representative data from AKN-1 cell line. Mean ± SD from two distinct experiments.

 


View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Rescue of tumor cells from NO-induced apoptosis after the addition of exogenous iron. Cells were incubated with SNAP (200 µM), Fe3+Ci (200 µM), or both. A, cell cycle distribution of AKN1 cells and the percentage of apoptotic cells after a 72-h incubation. B, the percentage of cells with membrane phosphatidylserine externalization (annexin-V+) after a 48-h incubation. Representative data in Capan-2 cells.

 
Antitumoral Effect of Exogenous NO Correlated with Rapid Intracellular Iron Depletion.
NO-iron interactions led us to investigate the effect of NO on intracellular iron concentrations. Fig. 9ACitation indicates that the addition of SNAP resulted in a rapid decrease in intracellular iron levels, an effect comparable with that observed with the potent iron chelator, DFX. The rapid intracellular iron depletion in the presence of NO donor SNAP corroborates the short half-life of this compound. The addition of Fe3+Ci did not allow iron depletion by NO, because it restored or increased intracellular iron levels (Fig. 9A)Citation . When compared with tumor cell growth, the decrease of intracellular total or ferric iron in the presence of SNAP or DFX always corroborated with lower cell recovery, whereas stabilization of Fe levels protect cells from iron depletion and the growth inhibitory effect of NO (Fig. 9B)Citation . Finally, NO-mediated iron depletion was not amplified by DFX, an observation that enforces the potent effect of NO in this respect.



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Decreased intracellular iron levels after tumor cell incubation with NO. Tumor cells were incubated in the presence of SNAP (200 µM), Fe3+Ci (100 µM), and/or DFX (60 µM). A, similar kinetics of intracellular iron chelation were observed in tumor cells after 2-, 4-, or 24-h incubation in the presence of SNAP or DFX and reversion by the addition of Fe3+Ci. B, direct relationship between the decrease of tumor cell numbers ({blacksquare}) and the intracellular levels of total () or ferric ({square}) iron. Representative data obtained in Capan-2 cell line.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NO, initially recognized as a member of the antitumor effector molecules of macrophages (5) , was subsequently shown to be induced in various human or murine cells including tumor cells without any significant antiproliferation activity (10 , 11 , 14) . This implied that tumor cells acquire mechanism(s) that protect them from the pro-apoptotic effect of NO derivatives. The present work indicates that tumor cells from distinct tissues remained sensitive to the growth inhibitory effect of exogenous NO, dependent upon NO formulation. These data also showed, at least in the three cell lines tested, that cGMP generation after guanylyl cyclase activation by NO was not involved in the antitumor activity of NO. Activation of PARP and consequent NAD+ depletion was implied in a variety of NO-mediated cell cytotoxicity (4 , 26 , 27) . PARP inhibition through 3AB addition did not significantly alter tumor cell growth inhibition by NO. Finally, NO may react with oxygen radicals and generate pro-apoptotic radicals such as peroxynitrites. The addition of both catalase and SOD did not inhibit the antitumor effect of NO, although they partially rescued normal cell growth from NO effects. Together, these data revealed that, compared with normal skin-derived epithelial cell cultures, several NO-mediated pro-apoptotic pathways (1 , 4) were no more functional in tumor cells.

The critical finding of this study is the ability of iron radicals to rescue tumor cells from the antitumor activity of NO. This was revealed by the failure of SNP to display a high or any growth inhibitory effect, together with the capacity of exogenous iron to protect tumor cells from the growth inhibitory effect of NO. In our hands, ferric compounds were more efficient than ferrous ones in inhibiting NO effects. This may be attributable to the higher affinity of ferric ions to NO-containing molecules. These findings may provide an explanation for the well-documented pro-cancer action of iron radicals (17) , because authors (18 , 28) have clearly shown that iron accumulation in many organs is correlated with the process of carcinogenesis. The iron effect is suggested to be attributable to in part interactions between iron and oxidative burst, including NO (28, 29, 30, 31) . Iron may protect cells from oxidative radicals through the induction of antioxidants in these cells (30 , 31) , which then protect cells by neutralizing NO-mediated oxidation (32) .

The present study in tumor cells corroborates recent conclusions drawn in hepatitis patients. In vivo, treatment of hepatitis patients with IFN-{alpha} correlated with an in vivo enhancement of NOS · II expression (33) . Authors (34 , 35) have subsequently observed that patients with high iron levels were less responsive to IFN-{alpha} treatment, compared to those with low in vivo iron concentrations. Accordingly, Kim et al. (36) have recently suggested that non-heme iron content determined whether cytotoxic levels of NO resulted in apoptosis versus necrosis in hepatic cells. Regarding the present study, iron availability in hepatic tissue may therefore rescue virus-infected cells from NO-mediated iron depletion and death. Recently, Amoroso et al. (32) have shown that SNP prevented cell death through the stimulation by iron ions of the activity of Na+-Ca+ exchanger in C6 glioma cells. NO affinity to iron may also lead to intracellular Fe loss, which is by itself cytotoxic for tumor cells. This is likely to be the principal mechanism of NO in the present study because, as observed with DFX (37) , rapid decreases in intracellular iron and ferric levels induced apoptotic cell death after tumor cell incubation with NO. Accordingly, iron supplementation and increased intracellular iron levels may decrease in vivo tumor cell sensitivity to the antitumoral activity of NO. This corroborates multiple studies (17 , 18) indicating that cell iron efflux could assist in prevention and management of cancer. Pharmaceutical methods for depriving tumor cells of iron are being developed in experimental and clinical protocols (38) . Thus, the present study may help to define new therapeutic approaches combining NO and cytoreductive chemotherapy.


   ACKNOWLEDGMENTS
 
We thank Jean-Claude Drapier for his suggestions, Daniel Moynet, and Philippe Vincendeau for kindly reviewing our paper. We also thank Ahmad Faili and Jaqueline Breard for their advice.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Ligue Nationale Contre le Cancer, Comité Sud-Ouest, Conseil Regional d’Aquitaine, and Association de Recherche sur le Cancer. Back

2 Contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Bone Marrow Transplantation Laboratory, CNRS UMR5540, Université de Bordeaux 2; Zone Nord, Bat. 1B; 146, Rue Léo Saignat, 33076 Bordeaux Cedex, France. Phone: 33-5-57-57-11-23; Fax: 33-5-57-57-12-10; E-mail: Djavad.Mossalayi{at}umr5540.u-bordeaux2.fr Back

4 The abbreviations used are: NO, nitric oxide; NOS, NO synthase; SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; DFX, deferoxamine mesylate; 3AB, 3-aminobenzamide; SOD, superoxide dismutase; Fe3+CN, potassium ferricyanide; Fe3+Ci, ferric ammonium citrate; ODQ, oxadiazole quinoxalin one; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; Fe2+, ferrous iron; Fe3+, ferric iron; cGMP, cyclic GMP; PARP, poly(ADP-ribose) polymerase; NOC18, Deta NONOate; (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazin-1-ium-1,2-diolate. Back

Received 6/28/00. Accepted 4/27/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hobbs A. J., Ignarro L. J. Nitric oxide-cyclic GMP signal transduction system. Methods Enzymol., 269: 134-148, 1996.[Medline]
  2. Moncada S., Higgs E. A. Molecular mechanisms and therapeutic strategies related to nitric oxide. FASEB J., 9: 1319-1330, 1996.[Abstract]
  3. MacMicking J., Xie Q. W., Nathan C. F. Nitric oxide and macrophage function. Annu. Rev. Immunol., 15: 323-350, 1997.[Medline]
  4. Weinberg J. B. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol. Med., 4: 557-591, 1998.[Medline]
  5. Stuehr D. J., Nathan C. F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med., 169: 1543-1555, 1989.[Abstract/Free Full Text]
  6. Jaiswal M., LaRusso N. F., Burgart L. J., Gores G. J. Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholangiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Res., 60: 184-190, 2000.[Abstract/Free Full Text]
  7. Geng Y. J., Hellstrand K., Wennmalm A., Hansson G. K. Apoptotic death of human leukemic cells induced by vascular cells expressing nitric oxide synthase in response to {gamma}-interferon and tumor necrosis factor-{alpha}. Cancer Res., 56: 866-874, 1996.[Abstract/Free Full Text]
  8. Ouaaz F., Sola B., Issaly F., Kolb J. P., Davi F., Mentz F., Arock M., Paul-Eugene N., Korner M., Dugas B., Debré P., Mossalayi M. D. Growth arrest and terminal differentiation of leukemic myelomonocytic cells induced through ligation of surface CD23 antigen. Blood, 84: 3095-3104, 1994.[Abstract/Free Full Text]
  9. Bruns C. J., Shinohara H., Harbison M. T., Davis D. W., Nelkin G., Killion J. J., McConkey D. J., Dong Z., Fidler I. J. Therapy of human pancreatic carcinoma implants by irinotecan and the oral immunomodulator JBT 3002 is associated with enhanced expression of inducible nitric oxide synthase in tumor-infiltrating macrophages. Cancer Res., 60: 2-7, 2000.[Abstract/Free Full Text]
  10. Doi K., Akaike T., Horie H., Noguchi Y., Fujii S., Beppu T., Ogawa M., Maeda H. Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth. Cancer (Phila.), 77: 1598-1604, 1996.[Medline]
  11. Mannick J. B., Asano K., Izumi K., Kieff E., Stamler J. S. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell, 79: 1137-1146, 1994.[Medline]
  12. Gorbunov N. V., Yalowich J. C., Gaddam A., Thampatty P., Ritov V. B., Kisin E. R., Elsayed N. M., Kagan V. E. Nitric oxide prevents oxidative damage produced by tert-butyl hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron. Electron paramagnetic resonance evidence. J. Biol. Chem., 272: 12328-12341, 1997.[Abstract/Free Full Text]
  13. De Nadai C., Sestili P., Cantoni O., Lievremont J. P., Sciorati C., Barsacchi R., Moncada S., Meldolesi J., Clementi E. Nitric oxide inhibits tumor necrosis factor-{alpha}-induced apoptosis by reducing the generation of ceramide. Proc. Natl. Acad. Sci. USA, 97: 5480-5485, 2000.[Abstract/Free Full Text]
  14. Zhao H., Dugas N., Mathiot C., Delmer A., Dugas B., Sigaux F., Kolb J. P. B-cell chronic lymphocytic leukemia cells express a functional inducible nitric oxide synthase displaying anti-apoptotic activity. Blood, 92: 1031-1043, 1998.[Abstract/Free Full Text]
  15. Li J., Bombeck C. A., Yang S., Kim Y. M., Billiar T. R. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J. Biol. Chem., 274: 17325-17333, 1999.[Abstract/Free Full Text]
  16. Melino G., Bernassola F., Catani M. V., Rossi A., Corazzari M., Sabatini S., Vilbois F., Green D. R. Nitric oxide inhibits apoptosis via AP-1-dependent CD95L transactivation. Cancer Res., 60: 2377-2383, 2000.[Abstract/Free Full Text]
  17. Toyokuni S. Iron-induced carcinogenesis: the role of redox regulation. Free. Radic. Biol. Med., 20: 553-566, 1996.[Medline]
  18. Weinberg E. D. Iron, asbestos, and carcinogenicity. Lancet, 1: 1399-1400, 1989.
  19. Drapier J. C., Pellat C., Henry Y. Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells co-cultured with activated macrophages. J. Biol. Chem., 266: 10162-10167, 1991.[Abstract/Free Full Text]
  20. Bastian N. R., Yim C. Y., Hibbs J. B., Jr., Samlowski W. E. Induction of iron-derived EPR signals in murine cancers by nitric oxide. Evidence for multiple intracellular targets. J. Biol. Chem., 269: 5127-5131, 1994.[Abstract/Free Full Text]
  21. Nussler A. K., Vergani G., Gollin S. M., Dorko K., Morris S. M., Jr., Demetris A. J., Nomoto M., Beger H. G., Strom S. C. Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver. In Vitro Cell. Dev. Biol. Anim., 35: 190-197, 1999.[Medline]
  22. Bécherel P. A., Mossalayi M. D., Dugas B., Ouaaz F., Paul-Eugène N., Le Goff L., Kilchherr E., Guillosson J. J., Debré P., Arock M. Involvement of the cAMP and nitric oxide pathway in the IgE-dependent activation of normal human keratinocytes. J. Clin. Investig., 93: 2275-2279, 1994.
  23. Strähic J., Schweda E. Quantitative analysis Jander G. Blasius E. eds. . Einführung In Das Anorganisch-Chemische Praktikum, : 377-379, S. Hirzel Verlag Stuttgart, Leipzig 1995.
  24. Calmels S., Hainaut P., Ohshima H. Nitric oxide induces conformational and functional modifications of wild-type p53 tumor suppressor protein. Cancer Res., 57: 3365-3369, 1997.[Abstract/Free Full Text]
  25. Moro M. A., Russel R. J., Cellek S., Lizasoain I., Su Y., Darley-Usmar V. M., Radomski M. W., Moncada S. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc. Natl. Acad. Sci. USA, 93: 1480-1485, 1996.[Abstract/Free Full Text]
  26. Pieper A. A., Verma A., Zhang J., Snyder S. H. Poly (ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol. Sci., 20: 171-181, 1999.[Medline]
  27. Mossalayi M. D., Becherel P. A., Debre P. Critical role of nitric oxide during the apoptosis of peripheral blood leukocytes from patients with AIDS. Mol. Med., 5: 812-819, 1999.[Medline]
  28. Goldstein S. R., Yang G. Y., Chen X., Curtis S. K., Yang C. S. Studies of iron deposits, inducible nitric oxide synthase and nitrotyrosine in a rat model for esophageal adenocarcinoma. Carcinogenesis (Lond.), 19: 1445-1449, 1998.[Abstract/Free Full Text]
  29. Park S. H., Aust A. E. Participation of iron and nitric oxide in the mutagenicity of asbestos in hgprt-, gpt+ Chinese hamster V79 cells. Cancer Res., 58: 1144-1148, 1998.[Abstract/Free Full Text]
  30. Wiegant F. A., Malyshev I. Y., Kleschyov A. L., van Faassen E., Vanin A. F. Dinitrosyl iron complexes with thiol-containing ligands and S-nitroso-D-L-penicillamine as inductors of heat shock protein synthesis in H35 hepatoma cells. FEBS Lett., 455: 179-182, 1999.[Medline]
  31. Nikitovic D., Holmgren A. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J. Biol. Chem., 271: 19180-19185, 1996.[Abstract/Free Full Text]
  32. Amoroso S., Tortiglione A., Secondo A., Catalano A., Montagnani S., Di Renzo G., Annunziato L. Sodium nitroprusside prevents chemical hypoxia-induced cell death through iron ions stimulating the activity of the Na+-Ca2+ exchanger in C6 glioma cells. J. Neurochem., 74: 1505-1513, 2000.[Medline]
  33. Sharara A. I., Perkins D. J., Misukonis M. A., Chan S. U., Dominitz J. A., Weinberg J. B. Interferon (IFN)-{alpha} activation of human blood mononuclear cells in vitro and in vivo for nitric oxide synthase (NOS) type 2 mRNA and protein expression: possible relationship of induced NOS2 to the anti-hepatitis C effects of IFN-{alpha} in vivo. J. Exp. Med., 186: 1495-1502, 1997.[Abstract/Free Full Text]
  34. Barton A. L., Banner B. F., Cable E. E., Bonkovsky H. L. Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy. Am. J. Clin. Pathol., 103: 419-424, 1995.[Medline]
  35. Akiyoshi F., Sata M., Uchimura Y., Suzuki H., Tanikawa K. Hepatic iron stainings in chronic hepatitis C patients with low HCV RNA levels: a predictive marker for IFN therapy. Am. J. Gastroenterol., 92: 1463-1466, 1997.[Medline]
  36. Kim Y. M., Chung H. T., Simmons R. L., Billiar T. R. Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition. J. Biol. Chem., 275: 10954-10961, 2000.[Abstract/Free Full Text]
  37. Fan L., Iyer J., Zhu S., Frick K. K., Wada R. K., Eskenazi A. E., Berg P. E., Ikegaki N., Kennett R. H., Frantz C. N. Inhibition of N-myc expression and induction of apoptosis by iron chelation in human neuroblastoma cells. Cancer Res., 61: 1073-1079, 2001.[Abstract/Free Full Text]
  38. Weinberg E. D. Development of clinical methods of iron deprivation for suppression of neoplastic and infectious diseases. Cancer Investig., 17: 507-513, 1999.[Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
M. D. Mossalayi, I. Vouldoukis, M. Mamani-Matsuda, T. Kauss, J. Guillon, J. Maugein, D. Moynet, J. Rambert, V. Desplat, D. Mazier, et al.
CD23 Mediates Antimycobacterial Activity of Human Macrophages
Infect. Immun., December 1, 2009; 77(12): 5537 - 5542.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. Wang, C. Fillebeen, G. Chen, B. Andriopoulos, and K. Pantopoulos
Sodium Nitroprusside Promotes IRP2 Degradation via an Increase in Intracellular Iron and in the Absence of S Nitrosylation at C178.
Mol. Cell. Biol., March 1, 2006; 26(5): 1948 - 1954.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J.-H. Yang, W.-D. Le, S. F. Basinger, S. M. Wu, and C.-y. Yang
Mechanisms of Apoptosis in Human Retinal Pigment Epithelium Induced by TNF-{alpha} in Conditions of Heavy Metal Ion Deficiency
Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 1039 - 1046.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Feger, F.
Right arrow Articles by Mossalayi, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feger, F.
Right arrow Articles by Mossalayi, M. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online