title: Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer.
abstract: 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.
authors.affiliation: Department of Hematology/Oncology, Martin-Luther-University of Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mueller
authors.forename: Wieland
authors.initials: W
authors.lastname: Voigt
authors.forename: Heike
authors.initials: H
authors.lastname: Simon
authors.forename: Angelika
authors.initials: A
authors.lastname: Fruehauf
authors.forename: Andrej
authors.initials: A
authors.lastname: Bulankin
authors.forename: Axel
authors.initials: A
authors.lastname: Grothey
authors.forename: Hans-Joachim
authors.initials: HJ
authors.lastname: Schmoll
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002945:Cisplatin / Q000494:pharmacology*; D018736:DNA Adducts / Q000096:biosynthesis; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000378:metabolism; D004334:Drug Administration Schedule; D019008:Drug Resistance, Neoplasm; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008297:Male; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D013736:Testicular Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D019014:fas Receptor / Q000096:biosynthesis
chemical_list: D061945:Caspase Inhibitors; D018736:DNA Adducts; D004273:DNA, Neoplasm; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C057270:cisplatin-DNA adduct; D019014:fas Receptor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002945:Cisplatin