Dursun Belda, He Zhibin, Somerset Hilary, Oh Dong-Jin, Faubel Sarah, Edelstein Charles L
University of Colorado Health Sciences Center, Department of Medicine, Denver, CO 80262, USA.
Am J Physiol Renal Physiol. 2006 Sep;291(3):F578-87. doi: 10.1152/ajprenal.00455.2005. Epub 2006 Apr 18.
The role of caspases and calpain in cisplatin-induced endothelial cell death is unknown. Thus we investigated whether caspases and calpain are mediators of cisplatin-induced apoptosis and necrosis in endothelial cells. Cultured pancreatic microvascular endothelial (MS1) cells were exposed to 10 and 50 microM cisplatin. Apoptosis or necrosis was determined by Hoechst 33342 and propidium iodide (PI) nuclear staining. Cells treated with 10 microM cisplatin had normal ATP levels, increased caspase-3-like activity, excluded PI and demonstrated morphological characteristics of apoptosis at 24 h. Cells treated with 50 microM cisplatin had severe ATP depletion, increased caspase-3-like activity, and displayed extensive PI staining indicative of necrosis at 24 h. There was a dose-dependent increase in caspase-2-like activity and Smac/DIABLO protein. Calpain activity increased significantly with 50 microM, but not 10 microM cisplatin at 24 h. With 50 microM cisplatin, ATP levels were significantly reduced starting at 18 h, caspase-2- and caspase-3-like activities were significantly increased starting at 18 h, and LDH release started at 8 h with maximum increase at 18-24 h. Calpain activity was not increased before 24 h. The increase in LDH release and the nuclear PI staining with 50 microM cisplatin at 24 h was reduced by either the pancaspase inhibitor, Q-VD-OPH, or the calpain inhibitor, PD-150606. Calpain inhibitor had no effect on caspase-3-like activity. In conclusion, in cisplatin-treated endothelial cells, caspases, the major mediators of apoptosis, can also cause necrosis. A calpain inhibitor protects against necrosis without affecting caspase-3-like activity suggesting that calpain-mediated necrosis is independent of caspase-3.
半胱天冬酶和钙蛋白酶在顺铂诱导的内皮细胞死亡中的作用尚不清楚。因此，我们研究了半胱天冬酶和钙蛋白酶是否为顺铂诱导内皮细胞凋亡和坏死的介质。将培养的胰腺微血管内皮（MS1）细胞暴露于10和50微摩尔的顺铂中。通过Hoechst 33342和碘化丙啶（PI）核染色来确定凋亡或坏死。用10微摩尔顺铂处理的细胞在24小时时ATP水平正常，半胱天冬酶-3样活性增加，排斥PI并表现出凋亡的形态学特征。用50微摩尔顺铂处理的细胞在24小时时出现严重的ATP耗竭，半胱天冬酶-3样活性增加，并显示出广泛的PI染色，表明有坏死。半胱天冬酶-2样活性和Smac/DIABLO蛋白呈剂量依赖性增加。在24小时时，50微摩尔顺铂可使钙蛋白酶活性显著增加，但10微摩尔顺铂则无此作用。使用50微摩尔顺铂时，ATP水平从18小时开始显著降低，半胱天冬酶-2和半胱天冬酶-3样活性从18小时开始显著增加，乳酸脱氢酶（LDH）释放从8小时开始，在18 - 24小时时增加到最大值。在24小时之前钙蛋白酶活性并未增加。使用泛半胱天冬酶抑制剂Q-VD-OPH或钙蛋白酶抑制剂PD-150606均可减少24小时时50微摩尔顺铂导致的LDH释放增加和细胞核PI染色。钙蛋白酶抑制剂对半胱天冬酶-3样活性无影响。总之，在顺铂处理的内皮细胞中，作为凋亡主要介质的半胱天冬酶也可导致坏死。一种钙蛋白酶抑制剂可预防坏死，而不影响半胱天冬酶-3样活性，这表明钙蛋白酶介导的坏死独立于半胱天冬酶-3。
