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Research Articles: Therapeutics, Targets, and Development

Novel cell death pathways induced by N-(4-hydroxyphenyl)retinamide: therapeutic implications

¹ Department of Translational Oncology, Molecular Oncology Laboratory, and ² Immunology Laboratory, National Cancer Research Institute (IST); ³ Institute for Macromolecular Studies (ISMAC), National Research Council (CNR), Section of Genova, Genoa, Italy; ⁴ Università degli Studi dell'Insubria, Varese, Italy; and ⁵ IRCCS MultiMedica, Milan, Italy

Requests for reprints: Adriana Albini, IRCCS MultiMedica, Polo Scientifico e Tecnologico, Settore Ricerca Oncologica, Via Fantoli 15/16, Milan, Italy. Phone: 02-554061; Fax: 11-39-010573-77231. E-mail: adriana.albini{at}multimedica.it

Abstract

We previously reported that N-(4-hydroxyphenyl)retinamide (4HPR) inhibits retinoblastoma tumor growth in a murine model in vivo and kills Y79 retinoblastoma cells in vitro. In this work, we assayed different cell death–related parameters, including mitochondrial damage and caspase activation, in Y79 cells exposed to 4HPR. 4HPR induced cytochrome c release from mitochondria, caspase-3 activation, and oligonucleosomal DNA fragmentation. However, pharmacologic inactivation of caspases by the pan-caspase inhibitor BOC-D-fmk, or specific caspase-3 inhibition by Z-DEVD-fmk, was not sufficient to prevent cell death, as assessed by loss of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, disruption of mitochondrial transmembrane potential (_m), and ATP depletion. We found that 4HPR causes lysosomal membrane permeabilization and cytosolic relocation of cathepsin D. Pepstatin A partially rescued cell viability and reduced DNA fragmentation and cytosolic cytochrome c. The antioxidant N-acetylcysteine attenuated cathepsin D relocation into the cytosol, suggesting that lysosomal destabilization is dependent on elevation of reactive oxygen species and precedes mitochondrial dysfunction. Activation of AKT, which regulates energy level in the cell, by the retinal survival facto]r insulin-like growth factor I was impaired and insulin-like growth factor I was ineffective against ATP and _m loss in the presence of 4HPR. Lysosomal destabilization, associated with mitochondrial dysfunction, was induced by 4HPR also in other cancer cell lines, including PC3 prostate adenocarcinoma and the vascular tumor Kaposi sarcoma KS-Imm cells. The novel finding of a lysosome-mediated cell death pathway activated by 4HPR could have implications at clinical level for the development of combination chemoprevention and therapy of cancer. [Mol Cancer Ther 2007;6(1):286–98]

Grant support: Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Sanità-Italy USA, Ministero della Sanità Progetto Finalizzato, the Ministero dell'Istruzione, dell'Università e della Ricerca, Progetto Finalizzato and Fondo per gli Investimenti della Ricerca di Base, and the Compagnia di San Paolo.

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.

Note: Previous address for A. Albini: Molecular Oncology Laboratory, Istituto Nazionale per la Ricerca sul Cancro (IST), c/o Centro di Biotecnologie Avanzate, Largo Rosanna Benzi 10, 16132 Genoa, Italy.

The monoclonal antibody H4A3 against human Lamp-1, developed by J.T. August and J.E.K. Hildreth, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Resources and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.

⁶ Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 6/12/06; revised 10/ 4/06; accepted 11/16/06.

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