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During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug-drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.