Abstract
The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) 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-κB/p65), Ak mouse 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-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), 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-γ, TNF-α, 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-γ and TNF-α 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-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α 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-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-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-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process.
Highlights
Neuroblastoma (NB) is the most common extracranial solid tumor in children and accounts for 8 to 10% of pediatric tumors [1]
Our results demonstrate that IFN-γ and TNF-α up-regulate pro-apoptotic apoptotic protein-prostate apoptosis response-4 (Par-4) with a concomitant decrease in the pro-survivaland protein-Bcl-2, protein-Par-4 with a concomitant decrease in the expression of expression pro-survivalofprotein-Bcl-2, activation and activation of
We show that IFN-γ and TNF-α together down-regulated suppressor of cytokine signaling-3 (SOCS3)
Summary
Neuroblastoma (NB) is the most common extracranial solid tumor in children and accounts for 8 to 10% of pediatric tumors [1]. Neuroblastoma is characterized by high rate of spontaneous regression suggesting activation of an apoptotic/differentiation program; advanced stages are associated with poor prognosis and resistance to chemotherapy [2]. Despite advancements in conventional therapies and the development of a recent line of treatments that include blood stem cell transplantation, differentiation therapy with retinoic acid, and passive immunotherapy with anti-disialoganglioside (GD2) antibodies, there is no significant improvement in the survival rate [3]. Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine with multifaceted functions. In vitro and in vivo studies have demonstrated strong anti-tumor activity of TNF-α in different types of cancers [4,5,6]. Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine belonging to the type II class of interferons. The biological activity of IFN-γ as an anticancer agent is attributed to inhibition of proliferation, induction, and modulation of gene expression mediated by activation of Janus
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