Denmeade S R, Lin X S, Tombal B, Isaacs J T
Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Prostate. 1999 Jun 1;39(4):269-79. doi: 10.1002/(sici)1097-0045(19990601)39:4<269::aid-pros7>3.0.co;2-f.
Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells.
Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure.
During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU.
Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.
半胱天冬酶是一类半胱氨酸蛋白酶家族，其特点是能够在天冬氨酸残基后进行特异性切割。半胱天冬酶家族的不同成员（如半胱天冬酶8和9）被认为是信号传导阶段的关键启动因子，而其他成员（如半胱天冬酶3、6和7）则与细胞凋亡的效应或执行阶段有关。毒胡萝卜素（TG）能够诱导前列腺癌细胞发生不依赖细胞增殖的凋亡。本研究旨在确定半胱天冬酶抑制是否能预防TG或5-氟脱氧尿苷（5-FrdU）诱导的前列腺癌细胞凋亡。
通过蛋白质印迹分析视网膜母细胞瘤（Rb）蛋白的切割情况来评估半胱天冬酶活性，Rb蛋白是TG诱导前列腺癌细胞死亡过程中的半胱天冬酶底物。此外，还在TG处理细胞的裂解物中检测了半胱天冬酶特异性荧光肽底物的水解情况。在用TG和5-FrdU处理大鼠AT3和人TSU-Pr1前列腺癌细胞系时，在有或没有肽半胱天冬酶抑制剂的情况下进行克隆形成存活试验。对转染了编码具有半胱天冬酶抑制活性病毒蛋白的crmA基因的AT3.1细胞，在暴露于TG和5-FrdU后也进行了克隆形成存活检测。
在TG处理期间，Rb首先去磷酸化，然后被蛋白水解切割成100 kDa和40 kDa的形式，这表明有半胱天冬酶活性。在TG或5-FrdU处理24小时后，观察到半胱天冬酶底物Z-DEVD-AFC的II类（即半胱天冬酶3、7和10）水解增加了6 - 8倍。表达crmA（即半胱天冬酶1、4和8的抑制剂）的AT3细胞不能免受TG或5-FrdU诱导的凋亡。半胱天冬酶抑制剂Z-DEVD-fmk（即半胱天冬酶3、7和10的抑制剂）和Z-VAD-fmk（即一种通用的半胱天冬酶抑制剂）也不能保护TSU和AT3细胞免受TG或5-FrdU诱导的凋亡。
半胱天冬酶激活可能在凋亡级联反应的下游效应阶段起作用；然而，在本研究中，半胱天冬酶抑制并不能预防由具有不同细胞毒性机制的两种药物TG或5-FrdU诱导的凋亡信号传导阶段。这些结果表明，最近描述的内源性半胱天冬酶抑制剂对半胱天冬酶的抑制不应导致对TG产生耐药性。目前正在开发一种针对TG对转移性前列腺癌细胞独特细胞毒性的策略。
