Simultaneous Treatment with P53 Overexpression and Interferon γ Exerts a Dramatic Increase in Apoptosis Induction of U87 Cells
AbstractBackground: Gliomas possess low immunogenicity, which is an inevitable hinder in front of cancer immunotherapy. Different interferons (IFNs) may proceed apoptosis instead in p53-dependent or independent pathways. P53 induces the anti-inflammatory programmed cell death in cancer cells; on the other hand, IFN gamma (IFNγ) is a modulatory/pro-inflammatory cytokine. There are contradictory reports of whether this cytokine can possess an anti- or pro-cancerous impact on tumors. Hence, we aimed to investigate the possible cooperative apoptotic effect of the P53 and IFNγ over expressions on the U87 glioblastoma cell line. Materials and Methods: The P53 expressing vector was amplified by Escherichia coli BL21. This vector was confirmed by the aid of sequencing. At the next step, U87 cells were transfected using lipofectamine. Cells were treated with P53 vector and/or IFNγ. The type of cellular death investigated by flow cytometry and the expression level of cleaved caspase-3 protein was also precisely demonstrated by western blotting. Results: Sequencing results revealed that inserted P53 was identical with human P53. Western blot results revealed that both IFNγ and P53 overexpression could up-regulate cleaved caspase-3 protein expression in this cell line. Interestingly, flow cytometry data determined that concurrent treatment with P53 exogenous overexpression and IFNγ induces about 70% apoptosis in U87; more than the sum of cell death occurs after IFNγ or P53 overexpression alone (~18%+21%=39%). Conclusion: The present study results showed that p53-overexpression and IFNγ could ultimately induce up-regulation of the caspase-3 and ultimately significant apoptosis increasing in the U87 cell line. Although IFNγ is believed to be a pro-inflammatory cytokine and P53 is an anti-inflammatory agent, our results demonstrated that they could act synergistically to induce apoptosis in U87 cells. [GMJ.2021;10:e2270]
Ansari M, Nasrolahi H, Kani AA, Mohammadianpanah M, Ahmadloo N, Omidvari S, et al. Pediatric glioblastoma multiforme: A single-institution experience. Indian J Med Paediatr Oncol. 2012;33(3):155.
Hosseini MM, Karimi A, Behroozaghdam M, Javidi MA, Ghiasvand S, Bereimipour A, Aryan H, Nassiri F, Jangholi E. Cytotoxic and apoptogenic effects of cyanidin-3-glucoside on the glioblastoma cell line. World neurosurgery. 2017;108:94-100.
Wang G, Wang JJ, Fu XL, Guang R, To SS. Advances in the targeting of HIF-1α and future therapeutic strategies for glioblastoma multiforme. Oncol Rep. 2017;37(2):657-70.
Chen J, Xu T. Recent therapeutic advances and insights of recurrent glioblastoma multiforme. Front Biosci (Landmark Ed). 2013;18:676-84.
Robins HI, Chang S, Butowski N, Mehta M. Therapeutic advances for glioblastoma multiforme: current status and future prospects. Curr Oncol Rep. 2007;9(1):66-70.
Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon‐γ: an overview of signals, mechanisms and functions. J Leukoc Biol. 2004;75(2):163-89.
Kelderman S, Schumacher TN, Haanen JB. Acquired and intrinsic resistance in cancer immunotherapy. Mol Oncol. 2014;8(6):1132-9.
Zaidi MR, Merlino G. The two faces of interferon-γ in cancer. Clin Cancer Res. 2011;17(19):6118-24.
Rearden R, Sah A, Doff B, Kobayashi T, McKee SJ, Leggatt GR, et al. Control of B‐cell lymphoma by therapeutic vaccination and acquisition of immune resistance is independent of direct tumour IFN‐gamma signalling. Immunol Cell Biol. 2016;94(6):554-62.
Mattarollo SR, West AC, Steegh K, Duret H, Paget C, Martin B, et al. NKT cell adjuvant-based tumor vaccine for treatment of myc oncogene-driven mouse B-cell lymphoma. Blood, The Journal of the American Society of Hematology. 2012;120(15):3019-29.
John LB, Darcy PK. The double-edged sword of IFN-[gamma]-dependent immune-based therapies. Immunol Cell Biol. 2016;94(6):527.
Newton HB. Molecular neuro-oncology and the development of targeted therapeutic strategies for brain tumors. Part 3: brain tumor invasiveness. Expert Rev Anticancer Ther. 2004;4(5):803-21.
Weller M, Wick W, Aldape K, Brada M, Berger M, Pfister SM, et al. Glioma. Nat Rev Dis Primers. 2015;1(1):1-8., 2015.
Guan X, Hasan MN, Begum G, Kohanbash G, Carney KE, Pigott VM, et al. Blockade of Na/H exchanger stimulates glioma tumor immunogenicity and enhances combinatorial TMZ and anti-PD-1 therapy. Cell Death Dis. 2018;9(10):1-6.
Qian J, Wang C, Wang B, Yang J, Wang Y, Luo F, et al. The IFN-γ/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. J neuroinflammation. 2018;15(1):1-3.
Gul-e-Saba Chaudhry RJ, Zafar MN, Mohammad H, Muhammad TS. Vitex rotundifolia fractions induced apoptosis in human breast cancer T-47D cell line via activation of extrinsic and intrinsic pathway. Asian Pac J Cancer Prev. 2019;20(12):3555.
Rachakhom W, Khaw-On P, Pompimon W, Banjerdpongchai R. Dihydrochalcone derivative induces breast cancer cell apoptosis via intrinsic, extrinsic, and ER stress pathways but abolishes EGFR/MAPK pathway. Biomed Res Int. 2019;2019.
Lu Z, Zhou H, Zhang S, Dai W, Zhang Y, Hong L, et al. Activation of reactive oxygen species-mediated mitogen-activated protein kinases pathway regulates both extrinsic and intrinsic apoptosis induced by arctigenin in Hep G2. J Pharm Pharmacol. 2020;72(1):29-43.
Engels IH, Stepczynska A, Stroh C, Lauber K, Berg C, Schwenzer R, et al. Caspase-8/FLICE functions as an executioner caspase in anti-cancer drug-induced apoptosis. Oncogene. 2000;19(40):4563-73.
Tekautz T, Teitz T, Lahti JM, Kidd VJ. Proapoptotic Gene Silencing Via Methylation in Human Tumors. InDeath Receptors in Cancer Therapy 2005 (pp. 207-229). Humana Press.
Reed JC, Tomaselli KJ. Drug discovery opportunities from apoptosis research. Curr Opin Biotechnol. 2000;11(6):586-92.
Tekautz TM, Zhu K, Grenet J, Kaushal D, Kidd VJ, Lahti JM. Evaluation of IFN-γ effects on apoptosis and gene expression in neuroblastoma-preclinical studies. Biochim Biophys Acta. 2006;1763(10):1000-10.
Afshar G, Jelluma N, Yang X, Basila D, Arvold ND, Karlsson A, et al. Radiation-induced caspase-8 mediates p53-independent apoptosis in glioma cells. Cancer Res. 2006;66(8):4223-32.
Cerrato JA, Yung WA, Liu TJ. Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis. Neuro-oncology. 2001 Apr 1;3(2):113-22.
Li L, Chen SN, Laghari ZA, Huang B, Huo HJ, Li N, et al. Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi. Dev Comp Immunol. 2019;97:98-112.
Dos Santos RS, Marroqui L, Velayos T, Olazagoitia-Garmendia A, Jauregi-Miguel A, Castellanos-Rubio A, et al. DEXI, a candidate gene for type 1 diabetes, modulates rat and human pancreatic beta cell inflammation via regulation of the type I IFN/STAT signalling pathway. Diabetologia. 2019;62(3):459-72.
Chen S, Wu Z, Zhang J, Wang M, Jia R, Zhu D, et al. Duck stimulator of interferon genes plays an important role in host anti-duck plague virus infection through an IFN-dependent signalling pathway. Cytokine. 2018;102:191-9.
Fleming SB. Viral inhibition of the IFN-induced JAK/STAT signalling pathway: development of live attenuated vaccines by mutation of viral-encoded IFN-antagonists. Vaccines. 2016;4(3):23.
Park GB, Hur DY, Kim YS, Lee HK, Yang JW, Kim D. TLR 3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea. J Cell Mol Med. 2015 May;19(5):1042-54.
Chow YL, Lee KH, Vidyadaran S, Lajis NH, Akhtar MN, Israf DA, et al. Cardamonin from Alpinia rafflesiana inhibits inflammatory responses in IFN-γ/LPS-stimulated BV2 microglia via NF-κB signalling pathway. Int Immunopharmacol. 2012;12(4):657-65.
Bambard ND, Mathew SO, Mathew PA. LLT1‐mediated Activation of IFN‐γ Production in Human Natural Killer Cells Involves ERK Signalling Pathway. Scand J Immunol. 2010;71(3):210-9.
Wei B, Baker S, Wieckiewicz J, Wood KJ. IFN‐γ triggered STAT1‐PKB/AKT signalling pathway influences the function of alloantigen reactive regulatory T cells. Am J Transplant. 2010 Jan;10(1):69-80.
Liu LD, Dong CH, Shi HJ, Zhao HL, Wang LC, Ma SH, et al. A novel type II membrane receptor up‐regulated by IFN‐α in fibroblasts functions in cell proliferation through the JAK‐STAT signalling pathway. Cell prolif. 2006;39(2):93-103.
Imaizumi T, Kumagai M, Taima K, Fujita T, Yoshida H, Satoh K. Involvement of retinoic acid-inducible gene-I in the IFN-γ/STAT1 signalling pathway in BEAS-2B cells. Eur Respir J. 2005;25(6):1077-83.
Ruiz-Ruiz C, Muñoz-Pinedo C, López-Rivas A. Interferon-γ treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program. Cancer Res. 2000;60(20):5673-80.
Westphal D, Kluck RM, Dewson G. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ. 2014;21(2):196-205.
Luna-Vargas MP, Chipuk JE. Physiological and pharmacological control of BAK, BAX, and beyond. Trends Cell Biol. 2016;26(12):906-17.
Peña‐Blanco A, García‐Sáez AJ. Bax, Bak and beyond-mitochondrial performance in apoptosis. FEBS J. 2018;285(3):416-31.
Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, et al. Interferon-γ modulates a p53-independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem. 1997;272(26):16351-7.
Castro F, Cardoso AP, Gonçalves RM, Serre K, Oliveira MJ. Interferon-gamma at the crossroads of tumor immune surveillance or evasion. Front Immunol. 2018;9:847.
Copyright (c) 2021 Galen Medical Journal
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).