286 Novel cell death pathways induced by N-(4-hydroxyphenyl)retinamide: therapeutic implications 1 1 Roberta Vene`, Giuseppe Arena, membrane potential (#ym), and ATP depletion. We found Alessandro Poggi,2 Cristina D’Arrigo,3 that 4HPR causes lysosomal membrane permeabilization Michele Mormino,3 Douglas M. Noonan,4 and cytosolic relocation of cathepsin D. Pepstatin A Adriana Albini,5 and Francesca Tosetti1 partially rescued cell viability and reduced DNA fragmen- tation and cytosolic cytochrome c. The antioxidant N- 1Department of Translational Oncology, Molecular Oncology acetylcysteine attenuated cathepsin D relocation into the Laboratory, and 2Immunology Laboratory, National Cancer cytosol, suggesting that lysosomal destabilization is 3 Research Institute (IST); Institute for Macromolecular Studies dependent on elevation of reactive oxygen species and (ISMAC), National Research Council (CNR), Section of Genova, Genoa, Italy; 4Universita`degli Studi dell’Insubria, Varese, Italy; precedes mitochondrial dysfunction. Activation of AKT, and 5IRCCS MultiMedica, Milan, Italy which regulates energy level in the cell, by the retinal survival factor insulin-like growth factor I was impaired and insulin-like growth factor I was ineffective against Abstract ATP and #ym loss in the presence of 4HPR. Lysosomal We previously reported that N-(4-hydroxyphenyl)retina- destabilization, associated with mitochondrial dysfunc- mide (4HPR) inhibits retinoblastoma tumor growth in a tion, was induced by 4HPR also in other cancer cell lines, murine model in vivo and kills Y79 retinoblastoma cells including PC3 prostate adenocarcinoma and the vascular in vitro. In this work, we assayed different cell death– tumor Kaposi sarcoma KS-Imm cells. The novel finding of related parameters, including mitochondrial damage and a lysosome-mediated cell death pathway activated by caspase activation, in Y79 cells exposed to 4HPR. 4HPR 4HPR could have implications at clinical level for the induced cytochrome c release from mitochondria, cas- development of combination chemoprevention and thera- pase-3 activation, and oligonucleosomal DNA fragmenta- py of cancer. [Mol Cancer Ther 2007;6(1):286–98] tion. However, pharmacologic inactivation of caspases by the pan-caspase inhibitor BOC-D-fmk, or specific caspase- Introduction 3 inhibition by Z-DEVD-fmk, was not sufficient to prevent The cancer chemopreventive and therapeutic retinoid N- cell death, as assessed by loss of 3-(4,5-dimethylthiazol-2- (4-hydroxyphenyl)retinamide (4HPR; ref. 1) has extensively yl)-2,5-diphenyltetrazolium bromide reduction, lactate been studied in preclinical models and clinical trials of dehydrogenase release, disruption of mitochondrial trans- cancer prevention due to its favorable toxicity profile in comparison with natural retinoids (2). 4HPR induces cell death in a number of tumor cell types, and different molecular mediators have been identified, suggesting the activation of multiple mechanisms depending on dose and Received 6/12/06; revised 10/4/06; accepted 11/16/06. cell type. In several cellular systems, exposure to 4HPR Grant support: Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Sanita`-Italy USA, Ministero della Sanita`Progetto Finalizzato, results in reactive oxygen species (ROS)–mediated cell the Ministero dell’Istruzione, dell’Universita`e della Ricerca, Progetto death (3, 4). However, ROS-independent mechanisms have Finalizzato and Fondo per gli Investimenti della Ricerca di Base, and the been also reported (5). 4HPR can induce mitochondrial Compagnia di San Paolo. dysfunction and cytochrome c release, leading to caspase- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked dependent apoptosis (6, 7). Other mechanisms include the advertisement in accordance with 18 U.S.C. Section 1734 solely to implication of ceramide/sphingomyelin pathways (8–11), indicate this fact. 12-LOX (8), Bax and/or Bak (7), c-jun NH2-terminal kinase Note: Previous address for A. Albini: Molecular Oncology Laboratory, (12), c-fos (13), GADD153 (14), and caspase-8 (15). Istituto Nazionale per la Ricerca sul Cancro (IST), c/o Centro di Biotecnologie Avanzate, Largo Rosanna Benzi 10, 16132 Genoa, Italy. The identity of the retinoblastoma cell of origin and the The monoclonal antibody H4A3 against human Lamp-1, developed definite molecular events leading to retinal cell transforma- by J.T. August and J.E.K. Hildreth, was obtained from the Developmental tion in humans are still debated (16). Defective cell death– Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Resources and maintained by the regulating pathways in multipotential stem cells of the Department of Biological Sciences, University of Iowa, Iowa City, developing retina lacking the tumor suppressor pRB-1 have IA 52242. been indicated as a cause of retinoblastoma insurgence in Requests for reprints: Adriana Albini, IRCCS MultiMedica, Polo Scientifico humans (17). Current treatment of intraocular retinoblasto- e Tecnologico, Settore Ricerca Oncologica, Via Fantoli 15/16, Milan, Italy. Phone: 02-554061; Fax: 11-39-010573-77231. ma includes a three-drug regimen of high-dose chemother- E-mail: [email protected] apy, in addition to radiotherapy and intensive focal therapy. Copyright C 2007 American Association for Cancer Research. Because of concerns with the systemic toxicity of conven- doi:10.1158/1535-7163.MCT-06-0346 tional chemotherapy agents and the related risk of MolCancerTher2007;6(1).January2007 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 287 developing extraocular tumors, the identification of novel concentrations indicated in figure legends. The following molecular targets for the development of more effective and reagents were used at the indicated final concentration: less toxic therapeutic interventions is of great importance to BOC-D-fmk at 10 to 40 Amol/L (stock solution 4 mmol/L in control retinoblastoma progression. Recently, 4HPR has DMSO); Z-DEVD-fmk and Z-LEHD-fmk at 1 to 50 Amol/L been shown to induce cell death in brain tumor cells, (stock solution 5 mmol/L in DMSO); bafilomycin A1 at including glioblastoma and primary meningioma cells (18), 0.25 to 0.5 Amol/L (stock solution 10 mmol/L in DMSO); and has been tested as a chemotherapy drug in gliomas (19). pepstatin A at 200 Amol/L (stock solution 25 mmol/L in We previously reported that inhibition of retinoblastoma DMSO); DL-buthionine-[S,R]-sulfoximine at 150 Amol/L tumor growth by 4HPR in a murine model in vivo is (stock solution 100 mmol/L in water); N-acetylcysteine at associated with an increase in the intracellular levels of 10 mmol/L (stock solution 1 mol/L in culture medium, pH ROS leading to a necrosis-like form of death (20). 7.4); and desferrioxamine mesylate at 200 Amol/L (stock In this work, we show that 4HPR activates caspase- solution 50 mmol/L in water). dependent and caspase-independent cell death pathways CellViability and CytotoxicityAssays through lysosomal membrane permeabilization. We ob- Y79 cell viability was determined by the 3-(4,5-dime- served induction of lysosomal damage also in other cancer thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell lines, including PC3 prostate adenocarcinoma and assay as previously described (20). Cytotoxicity was Kaposi sarcoma KS-Imm cells. Recently, the activation of measured as cell membrane lysis and release of lactate mechanisms of cell death alternative to classic apoptosis by dehydrogenase (LDH) into the culture medium. Y79 cells in cancer therapeutics has been highlighted as a promising suspension were seeded at 1 Â 104 to 1.5 Â 104 per well in tool to overcome drug resistance following conventional poly-D-lysine–coated 96-well microtiter dishes in 100 ALof chemotherapy (21). Because 4HPR as a single agent gave complete medium. The medium was replaced after encouraging results in children affected by neuroblastoma, 24 h with 100 AL of defined N1 medium per well. 4HPR a pediatric tumor of neuroectodermal origin (22), the data dissolved in ethanol (0.1% final ethanol concentration) was shown here could represent an experimental background to added to the cells and incubated for 4 or 24 h at 37jC. The plan novel drug combination strategies for the treatment of LDH assay was carried out with the colorimetric CytoTox pediatric neurogenic tumors. 96 assay kit (Promega, Milan, Italy) following the instruc- tions of the manufacturers. The data obtained were expressed as percent LDH release relative to total LDH in Materials and Methods culture. Absorbance at 570 nm (MTT) or 490 nm (LDH) was Chemicals determined with an automatic microtiter plate reader 4HPR was kindly provided by Dr. James A. Crowell (Molecular Devices Corp., Sunnyvale, CA). (Division of Cancer Prevention, National Cancer Institute, Detection of Apoptosis and Necrosis by Flow Cyto- Bethesda) and Dr. Gregg Bullard (McKessonBio, Rockville, metry, Histone-Associated DNA Fragments, and Cyto- MD). The following reagents were used: the pan-caspase chrome c Release inhibitor Boc-Asp(OMe)-CH2F (BOC-D-fmk), the caspase-3 Fluorescence-activated cell sorting (FACS) analysis was inhibitor Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2F done using an Annexin V-FITC/propidium iodide apopto- (Z-DEVD-fmk), and the caspase-9 inhibitor Z-Leu- sis detection kit (Oncogene Research, Boston, MA) based