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Article

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

1
National Centre for Cell Science, Savitribai Phule Pune University, Ganeshkhind, Pune 411007, India
2
Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada
3
Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON M5G 1X5, Canada
4
Modern College, Ganeshkhind, Pune 411016, India
5
Rasayani Biologics Pvt Ltd, 48/7, Mhalunge—Nande Road, Mhalunge, Pune 411045, India
*
Author to whom correspondence should be addressed.
Current address: Krefting Research Centre, University of Gothenburg, Box-424, SE-405 30 Gothenburg, Sweden.
These authors contributed equally to this work.
Biomedicines 2018, 6(1), 4; https://doi.org/10.3390/biomedicines6010004
Received: 22 November 2017 / Revised: 15 December 2017 / Accepted: 19 December 2017 / Published: 26 December 2017
(This article belongs to the Special Issue Immuno-Active Cancer Therapeutics)
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. View Full-Text
Keywords: apoptosis; Par-4; NF-κB; IFN-γ; TNF-α; neuroblastoma apoptosis; Par-4; NF-κB; IFN-γ; TNF-α; neuroblastoma
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MDPI and ACS Style

Shelke, G.V.; Jagtap, J.C.; Kim, D.-K.; Shah, R.D.; Das, G.; Shivayogi, M.; Pujari, R.; Shastry, P. TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas. Biomedicines 2018, 6, 4. https://doi.org/10.3390/biomedicines6010004

AMA Style

Shelke GV, Jagtap JC, Kim D-K, Shah RD, Das G, Shivayogi M, Pujari R, Shastry P. TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas. Biomedicines. 2018; 6(1):4. https://doi.org/10.3390/biomedicines6010004

Chicago/Turabian Style

Shelke, Ganesh V.; Jagtap, Jayashree C.; Kim, Dae-Kyum; Shah, Reecha D.; Das, Gowry; Shivayogi, Mruthyunjaya; Pujari, Radha; Shastry, Padma. 2018. "TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas" Biomedicines 6, no. 1: 4. https://doi.org/10.3390/biomedicines6010004

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