Caspase activation by anticancer drugs: the caspase storm.
This study measures the time-dependence of cellular caspase activation by anticancer drugs and compares it with that of cellular respiration. Intracellular caspase activation and cellular respiration were measured during continuous exposure of Jurkat, HL-60, and HL-60/MX2 (deficient in topoisomerase-II) cells to dactinomycin, doxorubicin, and the platinum (Pt) compounds cisplatin, carboplatin, and oxaliplatin. Caspase activation was measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC). We show that this substrate rapidly enters cells where it is efficiently cleaved at the aspartate residue by specific caspases, yielding the fluorescent compound 7-amino-4-trifluoromethyl coumarin (AFC). Following cell disruption, released AFC was separated on HPLC and detected by fluorescence. The appearance of AFC in cells was blocked by the pancaspase inhibitor benzyloxycarbonyl-val-ala-asp-fluoromethylketone, thus establishing that intracellular caspases were responsible for the cleavage. Caspase activity was first noted after about 2 h of incubation with doxorubicin or dactinomycin, the production of AFC being linear with time afterward. Caspase activation by doxorubicin was delayed in HL-60/MX2 cells, reflecting the critical role of topoisomerase-II in doxorubicin cytotoxicity. For both drugs, caspase activity increased rapidly between approximately 2 and approximately 6 h, went through a maximum, and decreased after approximately 8 h ("caspase storm"). Cisplatin treatment induced noticeable caspase activity only after approximately 14 h of incubation, and the fluorescent intensity of AFC became linear with time at approximately 16 h. Exposure of the cells to all of the drugs studied led to impaired cellular respiration and decreased cellular ATP, concomitant with caspase activation. Thus, the mitochondria are rapidly targeted by active caspases.
Tao
Zhimin
Z
Department of Chemistry, Syracuse University, 1-014 CST, Syracuse, New York 13244, USA.
Goodisman
Jerry
J
Penefsky
Harvey S
HS
Souid
A-K
AK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D016190:Carboplatin / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D019069:Cell Respiration / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003374:Coumarins / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D009944:Organoplatinum Compounds / Q000494:pharmacology; D000077150:Oxaliplatin; D010101:Oxygen Consumption / Q000187:drug effects; D013379:Substrate Specificity; D013997:Time Factors
D000970:Antineoplastic Agents; D003374:Coumarins; D005456:Fluorescent Dyes; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D003609:Dactinomycin; C055467:7-amino-4-trifluoromethylcoumarin; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D016190:Carboplatin; D020169:Caspases; D002945:Cisplatin