BackgroundFOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.MethodsThe effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.ResultsWe report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.ConclusionThe combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-224) contains supplementary material, which is available to authorized users.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3??g/mL (CI95: 25.9?28.9??g/mL) corresponding to 9.6??M (CI95: 9.1?10.1??M) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR. The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5???0.2 units (calculated average MW 2853.5) for the active fraction and 11.4???0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6???1.0?% active; 80.7???2.7?% inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7?, 4?6?) and single linked B-type units. This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

HighlightsDiscovery of a novel XIAP-inhibitory natural compound from Morus root bark (Sanggenon G).Sanggenon G binds specific to the BIR3 domain of XIAP in a low μM range.Sanggenon G interferes with XIAP-BIR3-substrate binding in living cells.Sanggenon G acts as chemosensitizer in tumor cell lines with high XIAP expression.