cytochrome-c-t and Sarcoma--Ewing

The Ewing's sarcoma family of tumours (ESFT) are small round cell tumours characterized by the non-random EWS-ETS gene rearrangements. We have previously demonstrated that ESFT are highly sensitive to fenretinide-induced death, effected in part through a reactive oxygen species (ROS)-dependent pathway. Here, we demonstrate for the first time that the sensitivity of ESFT cells to fenretinide-induced cell death is decreased following downregulation of the oncogenic fusion protein EWS-Fli1; siRNA targeting EWS-Fli1 attenuated fenretinide-induced cell death in cell lines expressing EWS-Fli1, but not EWS-ERG. This decrease in cell death was independent of the level of ROS produced following exposure to fenretinide, but was effected through EWS-Fli1-dependent modulation of p38(MAPK) activity. Furthermore, inhibition of p38(MAPK) activity and knockdown of EWS-Fli1 reduced fenretinide-induced mitochondrial permeabilization, cytochrome c release, caspase and PARP cleavage, consistent with the hypothesis that p38(MAPK) is critical for activation of the death cascade by fenretinide in ESFT cells. These data demonstrate that expression of EWS-Fli1 enhances fenretinide-induced cell death in ESFT and that this is effected at least in part through modulation of p38(MAPK) activity.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Cytochromes c; Down-Regulation; Electroporation; Fenretinide; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Membrane Potentials; Mitochondria; Oncogene Proteins, Fusion; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Protein c-fli-1; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Protein EWS; RNA, Small Interfering; Sarcoma, Ewing; Transcription Factors; Tumor Cells, Cultured

When administrated by isolated limb perfusion, tumor necrosis factor alpha (TNFalpha) is an efficient antitumor agent that improves drug penetration and destroys angiogenic vessels. Moreover, the pronounced potentiation of TNFalpha-induced apoptosis by NF-kappaB inhibitors suggest that these compounds could enhance TNFalpha antitumor efficacy through direct induction of tumor cell apoptosis. Therefore, attempts at amplifying signaling pathways that mediate TNFalpha antitumor effects could help to design combination therapies improving its efficiency. We report that nanomolar concentrations of all-trans retinoic acid (ATRA) amplify TNFalpha-induced apoptosis in APL cells expressing a specific repressor of NF-kappaB activation. This effect is abolished by the pan-caspase inhibitor, Z-VAD-fmk and by caspase-8 and -9 inhibitors. Cell death is accompanied by a drop of mitochondrial potential and by poly (ADP-ribose) polymerase (PARP) activation. Using specific PARP-1 inhibitors and siRNAs, we show that PARP-1 is essential for the synergistic apoptotic effect and c-Jun N-terminal kinase 1 (JNK1) activation triggered by the ATRA/TNFalpha combination. JNK1 siRNAs reduce ATRA/TNFalpha-induced apoptosis, mitochondrial release of cytochrome c and caspase-9 activation. Altogether, these results identify a novel mechanism of PARP-1-induced apoptosis, in which JNK1 provides a link between PARP-1 activation and mitochondrial pathway of caspase-9 activation. This study also suggests that inclusion of nanomolar doses of ATRA could be clinically beneficial in amplifying TNFalpha-induced antitumor signals.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cytochromes c; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Humans; Immunoblotting; Leukemia, Promyelocytic, Acute; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 8; NF-kappa B; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Recombinant Proteins; RNA, Small Interfering; Sarcoma, Ewing; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

There is an urgent need for new therapeutic strategies in Ewing's sarcoma family of tumors (ESFT). In this study, we have evaluated the effect of fenretinide [N-(4-hydroxyphenyl)retinamide] in ESFT models.. The effect of fenretinide on viable cell number and apoptosis of ESFT cell lines and spheroids and growth of s.c. ESFT in nu/nu mice was investigated. The role of the stress-activated kinases p38(MAPK) and c-Jun NH(2)-terminal kinase in fenretinide-induced death was investigated by Western blot and inhibitor experiments. Accumulation of reactive oxygen species (ROS) and changes in mitochondrial transmembrane potential were investigated by flow cytometry.. Fenretinide induced cell death in all ESFT cell lines examined in a dose- and time-dependent manner. ESFT cells were more sensitive to fenretinide than the neuroblastoma cell lines examined. Furthermore, fenretinide induced cell death in ESFT spheroids and delayed s.c. ESFT growth in mice. p38(MAPK) was activated within 15 minutes of fenretinide treatment and was dependent on ROS accumulation. Inhibition of p38(MAPK) activity partially rescued fenretinide-mediated cell death in ESFT but not in SH-SY5Y neuroblastoma cells. c-Jun NH(2)-terminal kinase was activated after 4 hours and was dependent on ROS accumulation but not on activation of p38(MAPK). After 8 hours, fenretinide induced mitochondrial depolarization (Deltapsi(m)) and release of cytochrome c into the cytoplasm in a ROS- and p38(MAPK)-dependent manner.. These data show that the high sensitivity of ESFT cells to fenretinide is dependent in part on the rapid and sustained activation of p38(MAPK). The efficacy of fenretinide in preclinical models demands the evaluation of fenretinide as a potential therapeutic agent in ESFT.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fenretinide; Flow Cytometry; Humans; Imidazoles; Intracellular Membranes; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mice; Mice, Nude; Mitochondria; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Reactive Oxygen Species; Sarcoma, Ewing; Xenograft Model Antitumor Assays