Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer.
Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer.
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发表时间：
2003-01
期刊：
影响因子：
11.2
通讯作者：
T. Mueller;W. Voigt;Heike Simon;Angelika Fruehauf;A. Bulankin;A. Grothey;H. Schmoll
中科院分区：
文献类型：
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作者：
T. Mueller;W. Voigt;Heike Simon;Angelika Fruehauf;A. Bulankin;A. Grothey;H. Schmoll
睾丸生殖细胞癌是极少数对以顺铂为基础的化疗高度敏感并可以治愈的癌症之一，即使在晚期也是如此。然而，在少数情况下，对顺铂产生耐药性，患者随后死于进展性疾病。这种抗性的分子基础仍有待确定。本研究以顺铂敏感的人睾丸生殖细胞癌细胞系(2102EP和H12.1)和顺铂耐药的人睾丸生殖细胞癌细胞系(1411HP)为研究对象，重点研究了caspase-2、caspase-3、caspase-7、caspase-8和caspase-9在顺铂作用下诱导细胞凋亡的分子机制。与IC(90)S的敏感细胞株2102EP和H12.1相比，1411HP细胞对顺铂的耐药率是2102EP和H12.1的3.3倍，且与治疗方案无关(2或24小时孵育)。通过Apo 2.7检测、DNA梯形扫描、活性caspase-3的免疫组织化学和末端脱氧核苷酸转移酶介导的缺口末端标记试验，证实顺铂耐药与体外和衍生裸鼠移植瘤中的细胞凋亡显著减少有关。在等摩尔剂量顺铂处理后，用ELISA法评估顺铂耐药细胞和敏感细胞之间的DNA总平台数没有差异。在对早期和晚期细胞的单独分析中，顺铂诱导的细胞凋亡的启动似乎更多地是由caspase-9而不是caspase-8介导的。耐药的1411HP细胞在IC(90)剂量顺铂诱导细胞凋亡的过程中未能激活caspase-9。有趣的是，在敏感的H12.1中，抑制caspase-9几乎完全阻止了细胞的凋亡，并诱导了与1411HP相同程度的顺铂耐药，因此只有在3.3倍的顺铂剂量下才能诱导细胞凋亡。此外，在caspase-9阻断的细胞中，凋亡的启动不依赖于caspase-9，同时伴随着caspase-2和caspase-3的激活，这是耐药1411HP细胞的固有特征。Caspase-9激活失败和顺铂耐药与P53、Bcl2家族蛋白、Fas受体和Fas配体的表达无关。总之，caspase-9通路的激活失败导致顺铂诱导睾丸癌细胞凋亡的细胞阈值更高。然而，这一更高的阈值可以通过更高的顺铂剂量来克服，可以想象的是，通过使用另一种不依赖于caspase-9的凋亡途径。这支持了目前对化疗难治性生殖细胞肿瘤患者进行大剂量化疗的临床策略。然而，进一步明确并最终针对阻止caspase-9激活的确切分子机制可能会导致更具选择性的治疗方法，以克服生殖细胞癌对顺铂的耐药性。
Testicular germ cell cancer is one of the very few cancers that are highly sensitive to and curable by cisplatin-based chemotherapy even in an advanced stage. However, in a few cases resistance to cisplatin occurs and patients subsequently die from progressive disease. The molecular basis for this resistance remains to be determined. Using two cisplatin-sensitive (2102EP and H12.1) and one cisplatin-resistant human testicular germ cell cancer cell line (1411HP), we investigated molecular mechanisms in the induction of apoptosis after cisplatin-treatment focusing on the cleavage and activation of caspase-2, caspase-3, caspase-7, caspase-8, and caspase-9. The cell line 1411HP showed a 3.3-fold cisplatin resistance when compared with the sensitive cell lines 2102EP and H12.1 by IC(90)s, which was treatment schedule independent (2- or 24-h incubation). Cisplatin resistance was associated with substantially decreased apoptosis in vitro and in derived nude mice xenografts as determined by Apo 2.7 detection, DNA-laddering, immunohistochemistry of active caspase-3, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Total DNA platination as assessed by ELISA after cisplatin treatment in equimolar doses did not differ between cisplatin-resistant or -sensitive cells. In separate analysis of cells of early and late apoptotic stages, initiation of cisplatin-induced apoptosis appeared to be rather mediated by caspase-9 than by caspase-8. Resistant 1411HP cells failed to activate caspase-9 during the induction of apoptosis after cisplatin treatment at the IC(90) dose. Interestingly, inhibition of caspase-9 in sensitive H12.1 almost completely blocked apoptosis and induced cisplatin resistance to the same extent as in 1411HP so that apoptosis could only be induced by 3.3-fold higher cisplatin doses. Furthermore, in caspase-9 blocked cells, initiation of apoptosis occurred in a caspase-9 independent manner accompanied by activation of caspase-2 and caspase-3, which are intrinsic characteristics of resistant 1411HP cells. Failure of caspase-9 activation and cisplatin resistance was independent of the expression of p53, Bcl-2 family proteins, Fas receptor, and Fas ligand. In conclusion, failure of activation of the caspase-9 pathway induces a higher cellular threshold for cisplatin-mediated induction of apoptosis in testicular cancer cells. However, this higher threshold can be overcome by higher cisplatin doses, conceivably by using an alternate, caspase-9-independent apoptotic pathway. This supports the current clinical strategy of high-dose chemotherapy in patients with chemorefractory germ cell tumors. However, additional defining and eventually targeting the exact molecular mechanism blocking caspase-9 activation might lead to more selective therapeutic approaches to overcome cisplatin resistance in germ cell cancer.