nutlin-3a and Testicular-Neoplasms

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2-p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Drug Synergism; Fas Ligand Protein; fas Receptor; Humans; Imidazoles; Immunoprecipitation; Male; Mutation; Piperazines; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-mdm2; RNA Interference; Signal Transduction; Testicular Neoplasms; Tumor Suppressor Protein p53; Up-Regulation

Inadequate response to cisplatin-based chemotherapy is associated with poor prognosis in patients with advanced malignant testicular germ cell tumours (TGCTs), especially of the nonseminomatous type. Novel chemotherapeutic agents have failed so far to significantly improve the outcome of such patients. The majority of these tumours express low levels of p53, and TP53 mutations are rarely observed. Murine double minute 2 (Mdm2) inhibitors enhance apoptosis in tumours harbouring wild-type p53.. We sought to investigate the potential therapeutic value of Mdm2 in TGCT-derived cell lines with the histology of nonseminoma.. The Mdm2 inhibitor nutlin-3 was evaluated alone and in combination with cisplatin in a panel of germ cell tumour (GCT)-derived cell lines (embryonal carcinomas, being the nonseminomatous stem-cell component) with wild-type (NT2 and 2102EP cells) and mutant (NCCIT cells) p53 status.. Biological consequences of Mdm2 inhibition were determined by analysis of the p53 pathway, cell proliferation, and apoptosis.. Nutlin-3 exhibited significant activity (IC50 2.8 μM) in NT2 and 2102EP (wild-type p53) but not in p53-mutant NCCIT cells (<10% inhibition at 10 μM). At concentrations beyond 500 nM, additive effects were seen for the combination of nutlin-3 and cisplatin in NT2 and 2102EP cells but not in NCCIT cells. This correlated with the induction of p53 and its target p21, suggesting an on-target effect of nutlin-3. Moreover, nutlin-3 (5 μM) and cisplatin (0.5 μM) additively induced caspase cleavage and apoptosis in NT2 cells and 2102-EP cells but not in p53-mutant NCCIT cells.. These results provide strong evidence for further development of pharmacologic Mdm2 inhibition for the treatment of patients suffering from high-risk nonseminomatous TGCT with wild-type p53 status.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Embryonal; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Inhibitors; Humans; Imidazoles; Inhibitory Concentration 50; Male; Mutation; Piperazines; Proto-Oncogene Proteins c-mdm2; Testicular Neoplasms; Tumor Suppressor Protein p53