Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer

• Published in: Cancer research (Chicago, Ill.) Ill.), 2003-01, Vol.63 (2), p.513-521
• Main Authors: MUELLER, Thomas, VOIGT, Wieland, SIMON, Heike, FRUEHAUF, Angelika, BULANKIN, Andrej, GROTHEY, Axel, SCHMOLL, Hans-Joachim
• Format: Article
• Language: English
• Subjects: Adult; Apoptosis - drug effects; Apoptosis - physiology; Biological and medical sciences; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases - metabolism; Cisplatin - pharmacology; DNA Adducts - biosynthesis; DNA Damage; DNA, Neoplasm - metabolism; Drug Administration Schedule; Drug Resistance, Neoplasm; Enzyme Activation - drug effects; fas Receptor - biosynthesis; General aspects (metabolism, cell proliferation, established cell line...); Humans; Male; Medical sciences; Proto-Oncogene Proteins c-bcl-2 - biosynthesis; Testicular Neoplasms - drug therapy; Testicular Neoplasms - enzymology; Testicular Neoplasms - pathology; Tumor cell; Tumor Cells, Cultured; Tumor Suppressor Protein p53 - biosynthesis; Tumors
• ISSN: 0008-5472; 1538-7445

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, ca