[CANCER RESEARCH 61, 5289 –5294, July 1, 2001] Role of Iron in Tumor Cell Protection from the Pro-Apoptotic Effect of Nitric Oxide 1 Frede ´ric Feger, 2 He ´le `ne Ferry-Dumazet, 2 Maria Mamani Matsuda, Joe ¨lle Bordenave, Maryse Dupouy, Andreas K. Nussler, Michel Arock, Lionel Devevey, Joe ¨lle Nafziger, Jean-Jacques Guillosson, Josy Reiffers, and M. Djavad Mossalayi 3 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 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 nitro- prusside. 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 pro- tected tumor cells from NO-mediated growth inhibition and apoptosis. Intracellular iron quantification indicated that, as deferoxamine, exoge- nous 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 NO 4 is a messenger molecule with complex biological activities including vasodilatation, neurotransmission, immunoregulation, and inflammation (1– 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 cyto- kines (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 oxida- tive 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 –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 (11–16). 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 multipli- cation (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 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 M. DFX (2–100 M; Novartis- Pharma, Rueil-Malmaison, France), 3AB (1 mM), bovine SOD (120 IU/ml), catalase (130 IU/ml), Fe 3+ CN, potassium ferrocyanide, Fe 3+ Ci (10 –200 M), dibutyrylguanosine cyclic 3'-5' monophosphate (400 – 800 M), and campto- thecin were purchased from Sigma-Aldrich. ODQ (Alexis) was used to inhibit the NO-sensitive guanylyl cyclase. Cell Culture. The human pulmonary adenocarcinoma (A549), skin (Ha- Cat), 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 gluta- mine (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% CO 2 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 centrifuga- tion on slides, fixation with 2% paraformaldehyde, permeabilization with 0.1% Received 6/28/00; accepted 4/27/01. 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, Comite ´ Sud-Ouest, Conseil Regional d’Aquitaine, and Association de Recherche sur le Cancer. 2 Contributed equally to this work. 3 To whom requests for reprints should be addressed, at Bone Marrow Transplantation Laboratory, CNRS UMR5540, Universite ´ de Bordeaux 2; Zone Nord, Bat. 1B; 146, Rue Le ´o Saignat, 33076 Bordeaux Cedex, France. Phone: 33-5-57-57-11-23; Fax: 33-5-57- 57-12-10; E-mail: Djavad.Mossalayi@umr5540.u-bordeaux2.fr. 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; Fe 3+ CN, potassium ferricyanide; Fe 3+ Ci, ferric ammonium citrate; ODQ, oxadiazole quinoxalin one; DAPI, 4',6- diamidino-2-phenylindole dihydrochloride; Fe 2+ , ferrous iron; Fe 3+ , 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. 5289 Research. on January 13, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from