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

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.

Keywords: IFN-γ; NF-κB; Par-4; TNF-α; apoptosis; neuroblastoma.

Figure 1

Dose-dependent effect of Interferon-gamma (IFN-γ) and Tumor necrosis factor-alpha (TNF-α) alone or in combination treatment on neuroblastoma cell lines. SK-N-MC cells were treated for 48 h with increasing concentrations of (A) TNF-α; and (B) IFN-γ alone and in combination and the viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of combination treatment was tested on other Neuroblastoma cell lines; SH-SY-5Y and SK-N-SH (C). Cells were treated with TNF-α (20 ng/mL) and IFN-γ (10 ng/mL) alone and in combination for 48 h and the viable cells were quantified by MTT assay. The data represented is the mean ± standard error of mean (n = 5). * p < 0.05; ** p < 0.005. −: untreated; +: treated.

Figure 2

Combination of IFN-γ (interferon gamma) and TNF-α (tumor necrosis factor-alpha) enhances apoptosis in SK-N-MC cells. Cells were treated with IFN-γ (10 ng/mL) and TNF-α 20 ng/mL) alone and in combination for 48 h, and analyzed for apoptosis: (A) hypodiploid population by flow-cytometry; (B) DNA fragmentation; (C) Poly (ADP-ribose)polymerase (PARP) cleavage by immunoblotting; and (D) Casapase-8 activity assay. The figures are representative of 2 similar experiments.

Figure 3

Combination of IFN-γ and TNF-α up-regulates the expression of Prostate apoptosis response-4 (Par-4). SK-N-MC cells were treated with IFN-γ (10ng/mL) and TNF-α (20 ng/mL) alone and in combination and analyzed for the expression of Par-4 by: (A) RT-PCR; (B) immunoblotting. Actin was used as loading control in both; (C) Immunofluorescence shows merged images with Par-4 (red) and nucleus (blue); Bar = 20 μm and (D) Immunoblotting represents level of Par-4 in cytoplasmic, and nuclear fraction of SK-N-MC cells at 48 h with actin and lamin-a/c as loading control, respectively.

Figure 4

Down-regulation of anti-apoptotic proteins in SK-N-MC cells treated with the combination of IFN-γ and TNF-α. (A) SK-N-MC cells were treated with IFN-γ (10 ng/mL) and TNF-α (20 ng/mL) alone and in combination for 24 h and 48 h. Lysates were subjected to SDS-PAGE, followed by western blot analysis with the antibodies indicated; (B) SK-N-MC cells grown on glass cover slips were exposed to IFN-γ (10 ng/mL) and TNF-α (20 ng/mL) and in combination for 48 h, and analyzed for the localization NF-κB/p65 by immunofluorescence (Bar = 20 μm); and (C) immunoblotting analysis. Bcl-2: B-cell lymphoma-2; Ak mouse strain thymoma: Akt.

Figure 5

Activation of Signal Transducer and Activator of Transcription-1 (STAT1) on phosphorylation by IFN-γ alone and in combination with TNF-α. (A) Activation of phospho-STAT1 (Try-701) in IFN-γ and in combination with TNF-α by immunoblotting; (B) Decrease in total PARP in cells treated with STAT1 inhibitor (Stattic) at 2.5 µg/mL; and (C) Expression of suppressor of cytokine signaling-3 (SOCS3) on combination treatment for 48 h determined by immunoblotting. −: untreated; +: treated.

Figure 6

Correlative gene-expression profile of IFN-γ and TNF-α in relation with Par-4 of human neuroblastoma datasets. Three datasets from neuroblastoma patients were obtained from gene expression omnibus (GEO) database (A) We evaluated the individual correlation between Par-4 with IFN-γ or TNF-α and significance value described for one dataset; (B) positive (upper panel; also upper panel in (C)); and negative (lower panel; also lower panel in (C)) synergies were determined in the datasets by determining the correlation between differentially regulated Par-4 with IFN-γ and TNF-α together. GSE: GEO accession number; *: detail analysis presented in (B,C).

Figure 7

Expression levels of IFN-γ, TNF-α, and PAR-4 in neuroblastoma patients and their survival. Normalized gene expression of IFN-γ, TNF-α, and PAR-4 were analyzed in patients with their survival outcome. Dunn’s Multiples comparison was used to determine significant differences between groups (**** p < 0.0001).