A Key Role for Caspase-2 and Caspase-3 in the Apoptosis Induced by 2-Chloro-2'-deoxy-adenosine (Cladribine) and 2- Chloro-adenosine in Human Astrocytoma Cells STEFANIA CERUTI, ELENA BELTRAMI, PAOLA MATARRESE, ALESSIA MAZZOLA, FLAMINIO CATTABENI, WALTER MALORNI, and MARIA P. ABBRACCHIO Department of Pharmacological Sciences, School of Pharmacy (S.C., E.B., A.M., F.C., M.P.A.) and Center of Excellence for Neurodegenerative Diseases (S.C., F.C., M.P.A.), University of Milan, Milan, Italy; and Department of Ultrastructures, Istituto Superiore di Sanita ` , Rome, Italy (P.M., W.M.) Received November 25, 2002; accepted March 14, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Both the anticancer agent 2-chloro-2'-deoxy-adenosine (Cladribine) and its derivative 2-chloro-adenosine induce apo- ptosis of human astrocytoma cells (J Neurosci Res 60:388 – 400, 2000). In this study, we have analyzed the involvement of caspases in these effects. Both compounds produced a grad- ual and time-dependent activation of “effector” caspase-3, which preceded the appearance of the nuclear signs of apo- ptosis, suggesting a temporal correlation between these two events. Moreover, the caspase inhibitor N-benzyloxycarbonyl- Val-Ala-DL-Asp-fluoromethylketone (fmk) suppressed both caspase-3 activation and apoptosis induction. “Initiator” caspase-9 and caspase-8 were only marginally activated at later times in the apoptotic process. Accordingly, at concen- trations that selectively inhibit these caspases, neither N-ben- zyloxycarbonyl-Leu-Glu-His-Asp-fmk nor N-benzyloxycar- bonyl-Ile-Glu-Thr-Asp-fmk could prevent adenosine analog- induced cell death. To definitively rule out a role for the caspase-9/cytochrome c-dependent mitochondrial pathway of cell death, neither adenosine analog had any effect on mito- chondrial membrane potential, which was instead markedly reduced by other apoptotic stimuli (e.g., deoxyribose, NaCN, and betulinic acid). Consistently, although the latter triggered translocation of mitochondrial cytochrome c to the cytoplasm, no cytosolic accumulation of cytochrome c was detected with adenosine analogs. Conversely, 1 to 7 h after addition of either adenosine analog (i.e., before the appearance of caspase-3 activation), caspase-2 activity was surprisingly and markedly increased. The selective caspase-2 inhibitor N-benzyloxy car- bonyl-Val-Asp-Val-Ala-Asp-fmk significantly reduced both adenosine analogs-induced caspase-2 activation and the as- sociated cell death. We conclude that adenosine analogs in- duce the apoptosis of human astrocytoma cells by activating an atypical apoptotic cascade involving caspase-2 as an initi- ator caspase, and effector caspase-3. Therefore, these com- pounds could be effectively used in the pharmacological ma- nipulation of tumors characterized by resistance to cell death via either the mitochondrial or caspase-8/death receptor path- ways. Apoptosis is a cell death process characterized by cell shrinkage, chromatin condensation, membrane blebbing, and fragmentation of the cell into “apoptotic bodies”, which are recognized and eliminated by phagocytes (Allen et al., 1997). Multicellular organisms eliminate abnormal, damaged or unwanted cells through apoptosis. Besides genetic mutations promoting unrestrained cellular proliferation, the transfor- mation of a normal to a neoplastic cell (and then to more malignant phenotypes) is linked to the acquisition of genetic This work was supported by the Ministero dell’Universita ` e della Ricerca Scientifica e Tecnologica (MURST), Cofinanziamento di Ricerche di Interesse Nazionale 1999 and 2001 on “Recettori purinergici e neuroprotezione” (to F.C.), by the University of Milan, grant F.I.R.S.T., by the Italian National Research Council (CNR) Contributo di Ricerca 99.02360.CT04 (to M.P.A.), and by a grant of the Ministero della Sanita ` , Italy (to W.M.). Part of the present data has been presented at the following meetings: International Symposium “Purine 2000” (Madrid, Spain; published as an ab- stract in Drug Dev Res 50:90, 2000); “XXX Congress of the Italian Pharmaco- logical Society” (Genova, Italy; published as an abstract in Pharmacol Res 43:129, 2001); “3rd Meeting of the Federation of the European Pharmacolog- ical Societies (EPHAR)” (Lyon, France; published as an abstract in Fundam Clin Pharmacol 15:147, 2001). S.C. and E.B. contributed equally to this work. ABBREVIATIONS: 2-CdA, 2-chloro-2'-deoxy-adenosine; m, mitochondrial transmembrane potential; 2-CA, 2-chloro-adenosine; ANOVA, analysis of variance; BetA, betulinic acid; DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-paranitroaniline; dRib, 2-deoxyribose; Eto, etoposide; FBS, fetal bovine serum; IETD-pNA, N-acetyl-Ile-Glu-Thr-Asp-paranitroaniline; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide; LEHD-pNA, N- acetyl-Leu-Glu-His-Asp-paranitroaniline; NaCN, sodium cyanide; PI, propidium iodide; TBS, Tris-buffered saline; zDEVD-fmk, N-benzyloxycarbonyl- Asp-Glu-Val-Asp-fluoromethylketone; zIETD-fmk, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone; zLEHD-fmk, N-benzyloxycarbonyl-Leu- Glu-His-Asp-fluoromethylketone; zVAD-fmk, N-benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone; zVDVAD-fmk, N-benzyloxy carbonyl-Val-Asp- Val-Ala-Asp-fluoromethylketone; zVDVAD-pNA, N-benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-paranitroaniline. 0026-895X/03/6306-1437–1447$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 6 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2303/1068999 Mol Pharmacol 63:1437–1447, 2003 Printed in U.S.A. 1437 at ASPET Journals on August 27, 2017 molpharm.aspetjournals.org Downloaded from