TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-gamma) and TumorNecrosis factor-alpha (TNF-alpha) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-kappa B/p65), Akmouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-gamma and TNF-alpha individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flowcytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-gamma, TNF-alpha, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-gamma and TNF-alpha induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-gamma and TNF-alpha together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-gamma, and TNF-alpha have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins-Bcl-2 and Akt and NF-kappa B/p65. Furthermore, the apoptotic effect induced by IFN-gamma-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-gamma and TNF-alpha can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.