Tao Zhimin, Goodisman Jerry, Penefsky Harvey S, Souid A-K
Department of Chemistry, Syracuse University, 1-014 CST, Syracuse, New York 13244, USA.
Mol Pharm. 2007 Jul-Aug;4(4):583-95. doi: 10.1021/mp070002r. Epub 2007 Apr 17.
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.
本研究测定了抗癌药物诱导细胞半胱天冬酶激活的时间依赖性，并将其与细胞呼吸的时间依赖性进行比较。在Jurkat细胞、HL-60细胞和HL-60/MX2（拓扑异构酶II缺陷）细胞持续暴露于放线菌素、阿霉素以及铂（Pt）化合物顺铂、卡铂和奥沙利铂的过程中，对细胞内半胱天冬酶激活和细胞呼吸进行了测定。使用荧光化合物N-乙酰-asp- glu-val-asp-7-氨基-4-三氟甲基香豆素（Ac-DEVD-AFC）测定半胱天冬酶激活情况。我们发现该底物能迅速进入细胞，在细胞内被特定的半胱天冬酶在天冬氨酸残基处有效切割，产生荧光化合物7-氨基-4-三氟甲基香豆素（AFC）。细胞裂解后，释放的AFC通过高效液相色谱（HPLC）分离并通过荧光检测。全半胱天冬酶抑制剂苄氧羰基-val-ala-asp-氟甲基酮可阻断细胞内AFC的出现，从而确定细胞内半胱天冬酶是切割反应的原因。在用阿霉素或放线菌素孵育约2小时后首次观察到半胱天冬酶活性，之后AFC的产生与时间呈线性关系。阿霉素诱导的HL-60/MX2细胞半胱天冬酶激活延迟，这反映了拓扑异构酶II在阿霉素细胞毒性中的关键作用。对于这两种药物，半胱天冬酶活性在大约2至大约6小时之间迅速增加，达到最大值，在大约8小时后下降（“半胱天冬酶风暴”）。顺铂处理仅在孵育约14小时后诱导出明显的半胱天冬酶活性，并且AFC的荧光强度在大约16小时时与时间呈线性关系。将细胞暴露于所有研究的药物都会导致细胞呼吸受损和细胞ATP减少，同时伴有半胱天冬酶激活。因此，线粒体迅速成为活性半胱天冬酶的作用靶点。
