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

Both the anticancer agent 2-chloro-2'- deoxy-adenosine ( Cladribine) and its derivative 2-chloro-adenosine induce apoptosis 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 gradual and time-dependent activation of "effector" caspase-3, which preceded the appearance of the nuclear signs of apoptosis, suggesting a temporal correlation between these two events. Moreover, the caspase inhibitor N-benzyloxycarbonylVal- 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 concentrations that selectively inhibit these caspases, neither N-benzyloxycarbonylLeu- Glu-His-Asp-fmk nor N-benzyloxycarbonylIle- 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 mitochondrial 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 carbonyl-Val- Asp-Val-Ala-Asp-fmk significantly reduced both adenosine analogs-induced caspase- 2 activation and the associated cell death. We conclude that adenosine analogs induce the apoptosis of human astrocytoma cells by activating an atypical apoptotic cascade involving caspase- 2 as an initiator caspase, and effector caspase-3. Therefore, these compounds could be effectively used in the pharmacological manipulation of tumors characterized by resistance to cell death via either the mitochondrial or caspase- 8/death receptor pathways.