Suppression of NF-κB/p65 Inhibits the Proliferation in Oral Squamous Cancer Cells

Naoki Anbo, Kazuhiro Ogi, Yohei Sogabe, Makoto Shimanishi, Takeshi Kaneko, Hironari Dehari, Akihiro Miyazaki, Hiroyoshi Hiratsuka

Department of Oral Surgery, School of Medicine, Sapporo Medical University, Sapporo, Japan.

DOI: 10.4236/jct.2013.44100                

Abstract

Object: Hypoxia occurs when oxygen tension drops below normal limits, and malignant tumors often experience hypoxia, which activates the expression of genes through oxygen-sensitive transcription factors, including the hypoxiainducible factor (HIF) and the nuclear factor-κB (NF-κB). NF-κB pathway represents an attractive therapeutic target in both cancer cells and ischemic cells, which involves immune response. To investigate the expression of NF-κB target genes in oral squamous cancer carcinoma (OSCC) under hypoxia, we performed cDNA plate array analyses of 23 NF-κB-regulated genes in different cell lines. Our aim was to clarify the functions of NF-κB in OSCC under hypoxia. Results: We conducted an NF-κB reporter assay to examine NF-κB activation under hypoxia. This luciferase-based reporter assay showed that hypoxia induced NF-κB activation after 24 h of hypoxia. We also found that vascular endothelial growth factor-C (VEGF-C) of NF-κB-regulated genes was upregulated in both cell lines under hypoxia. We then tested the influence of NF-κB/p65 knockdown on these cells and NF-κB/p65 knockdown inhibited the proliferation of OSCC cells by colony assay. Conclusion: Based on these findings, we postulate that one mechanism by which hypoxic OSCC cells may involve NF-κB-mediated upregulation. Our results also indicate that the knockdown of NF-κB/p65 subunit lead to growth inhibition during hypoxia in OSCC cells. These results suggest that knockdown of NF-κB/p65 subunit could be a potential therapeutic option for patients with OSCC.

Keywords

Oral Squamous Cell Carcinoma; Hypoxia; NF-κB

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	J. Sudbo and A. Reith, “The Evolution of Predictive Oncology and Molecular-Based Therapy for Oral Cancer Prevention,” International Journal of Cancer, Vol. 115, No. 3, 2005, pp. 339-345. doi:10.1002/ijc.20896
[2]	R. T. Greenlee, M. B. Hill-Harmon, T. Murray, et al., “Cancer Statistics,” A Cancer Journal for Clinicians, Vol. 5, No. 1, 2001, pp. 15-36.
[3]	M. Shimanishi, K. Ogi, T. Kaneko, et al., “Silencing of GLUT-1 Inhibits Sensitization of Oral Cancer Cells to Cisplatin during Hypoxia,” Journal of Oral Pathology and Medicine, Vol. 42, No. 5, 2013, pp. 382-388. doi:10.1111/jop.12028
[4]	A. L. Harris, “Hypoxia—A Key Regulatory Factor in Tumor Growth,” Nature Reviews Cancer, Vol. 2, No. 1, 2002, pp. 38-47. doi:10.1038/nrc704
[5]	G. L. Semenza, “Targeting Hif-1 Alpha for Cancer Therapy,” Nature Reviews Cancer, Vol. 10, No. 3, 2003, pp. 721-732. doi:10.1038/nrc1187
[6]	J. Yang, L. Zhang, P. J. Erbel, et al., “Functions of the Per/ARNT/Sim Domains of the Hypoxia-Inducible Factor,” The Journal of Biological Chemistry, Vol. 280, No. 43, 2005, pp. 36047-36054. doi:10.1074/jbc.M501755200
[7]	G. L. Semenza, “HIF-1: Mediator of Physiological and Pathophysiological Responses to Hypoxia,” Journal of Applied Physiology, Vol. 88, No. 4, 2000, pp. 1474-1480.
[8]	E. P. Cummins and C. T. Taylor, “Hypoxia-Responsive Transcription Factors,” Pflügers Archiv—European Journal of Physiology, Vol. 450, No. 6, 2005, pp. 363-371.
[9]	K. R. Laderoute, “The Interaction between HIF-1 and AP-1 Transcription Factors in Response to Low Oxygen,” Seminars in Cell & Developmental Biology, Vol. 16, No. 4-5, 2005, pp. 502-513. doi:10.1016/j.semcdb.2005.03.005
[10]	T. Tanaka, H. Nakayama, Y. Yoshitake, et al, “Selective Inhibition of Nuclear Factor-κB by Nuclear Factor-κB Essential Modulator-Binding Domain Peptide Suppresses the Metastasis of Highly Metastatic Oral Squamous Cell Carcinoma,” Japanese Journal of Cancer Research, Vol. 103, No. 3, 2012, pp. 455-463. doi:10.1111/j.1349-7006.2011.02174.x
[11]	A. W. Ken, S. M. Karen, H. Jason, et al., “Hypoxia-Inducible Factor and Nuclear Factor Kappa-B Activation in Blood-Brain Barrier Endothelium under Hypoxic/Reoxygenation Stress,” Journal of Neurochemistry, Vol. 92, No. 1, 2005, pp. 203-214. doi:10.1111/j.1471-4159.2004.02871.x
[12]	S. Y. Nam, Y S. Ko, J. Jung, et al., “A Hypoxia-Dependent Upregulation of Hypoxia-Inducible Factor-1 by Nuclear Factor-κB Promotes Gastric Tumour Growth and Angiogenesis,” British Journal of Cancer, Vol. 104, No. 1, 2011, pp. 166-174. doi:10.1038/sj.bjc.6606020
[13]	A. C. Koong, E. Y. Chen and A. J. Giaccia, “Hypoxia Causes the Activation of Nuclear Factor Kappa B through the Phosphorylation of I Kappa B Alpha on Tyrosine Residues,” Cancer Research, Vol. 54, No. 6, 1994, pp. 1425-1414.
[14]	J. F. Schmedtje Jr, Y. S. Ji, R. N. DuBois, et al., “Hypoxia Induces Cyclooxygenase-2 via the NF-KappaB p65 Transcription Factor in Human Vascular Endothelial Cells,” The Journal of Biological Chemistry, Vol. 272, No. 1, 1997, pp. 601-608. doi:10.1074/jbc.272.1.601
[15]	C. T. Taylor, A. L. Dzus and S. P. Colgan, “Autocrine Regulation of Epithelial Permeability by Hypoxia: Role for Polarized Release of Tumor Necrosis Factor Alpha,” Gastroenterology, Vol. 114, No. 4, 1998, pp. 657-668. doi:10.1016/S0016-5085(98)70579-7
[16]	H. Matsui, Y. Ihara, Y Fujio, et al., “Induction of Interleukin (IL)-6 by Hypoxia Is Mediated by Nuclear Factor (NF)-Kappa B and NF-IL6 in Cardiac Myocytes,” Cardiovascular Research, Vol. 42, No. 1, 1999, pp. 104-112. doi:10.1016/S0008-6363(98)00285-5
[17]	A. Zampetaki, S. A. Mitsialis, J. Pfeilschifter, et al., “Hypoxia Induces Macrophage Inflammatory Protein-2 (MIP-2) Gene Expression in Murine Macrophages via NF kappaB: The Prominent Role of p42/p44 and PI3 Kinase Pathways,” Official Publication of the Federation of American Societies for Experimental Biology, Vol. 18, No. 10, 2004, pp. 1090-1092.
[18]	E. P. Cummins, E. Berra, K. M. Comerford, et al., “Prolyl Hydroxylase-1 Negatively Regulates IkB Kinase-Beta Giving Insight into Hypoxia-Induced NFkB Activity,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 48, 2006, pp. 18154-18159. doi:10.1073/pnas.0602235103
[19]	J. Rius, M. Guma, C. Schachtrup, et al., “NF-KappaB Links Innate Immunity to the Hypoxic Response through Transcriptional Regulation of HIF-1alpha,” Nature, Vol. 453, No. 7196, 2008, pp. 807-811. doi:10.1038/nature06905
[20]	R. S. Belaiba, S. Bonello, C. Zahringer, et al., “Hypoxia Up-Regulates Hypoxia-Inducible Factor-1alpha Transcription by Involving Phosphatidylinositol 3-Kinase and Nuclear Factor KappaB in Pulmonary Artery Smooth Muscle Cells,” Molecular Biology of the Cell, Vol. 18, No. 12, 2007, pp. 4691-4697. doi:10.1091/mbc.E07-04-0391
[21]	P. van Uden, N. S. Kenneth, S. Rocha, et al., “Regulation of Hypoxia-Inducible Factor-1alpha by NF-KappaB,” The Biochemical Journal, Vol. 412, No. 3, 2008, pp. 477-484. doi:10.1042/BJ20080476
[22]	S. R. Walmsley, C. Print, N. Farahi, et al., “HypoxiaInduced Neutrophil Survival Is Mediated by HIF-1alphaDependent NF-KappaB Activity,” The Journal of Experimental Medicine, Vol. 201, No. 1, 2005, pp. 105-115. doi:10.1084/jem.20040624
[23]	C. T. Taylor, “Interdependent Roles for Hypoxia Inducible Factor and Nuclear Factor-KappaB in Hypoxic Inflammation,” The Journal of Physiology, Vol. 586, No. 187, 2008, pp. 4055-4059. doi:10.1113/jphysiol.2008.157669
[24]	D. B. Mendonca, G. Mendonca, F. J. Aragao, et al., “NF-κB Suppresses HIF-1α Response by Competing for P300 Binding,” Biochemical and Biophysical Research Communications, Vol. 404, No. 4, 2011, pp. 997-1003. doi:10.1016/j.bbrc.2010.12.098
[25]	C. Huang, Z. Sun, Y. Sun, et al., “Association of Increased Ligand Cyclophilin A and Receptor CD147 with Hypoxia, Angiogenesis, Metastasis and Prognosis of Tongue Squamous Cell Carcinoma,” Histopathology, Vol. 60, No. 5, 2012, pp. 793-803. doi:10.1111/j.1365-2559.2011.04130.x
[26]	H. Kubo, R. Cao, E. Brakenhielm, et al., “Blockade of Vascular Endothelial Growth Factor Receptor-3 Signaling Inhibits Fibroblast Growth Factor-2-Induced Lymphangiogenesis in Mouse Cornea,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 13, 2002, pp. 8868-8873. doi:10.1073/pnas.062040199
[27]	S. E. S. Faustino, D. T. Oliveira, S. Nonogaki, et al., “Expression of Vascular Endothelial Growth Factor-C Does Not Predict Occult Lymph-Node Metastasis in Early Oral Squamous Cell Carcinoma,” International Journal of Oral and Maxillofacial Surgery, Vol. 37, No. 4, 2008, pp. 372-378. doi:10.1016/j.ijom.2007.11.021
[28]	T. Naruse, G. Kawasaki, S. Yanamoto, et al., “Immunohistochemical Study of VEGF Expression in Oral Squamous Cell Carcinomas: Correlation with the mTOR-HIF-1alpha Pathway,” Anticancer Research, Vol. 31, No. 12, 2011, pp. 4429-4437.
[29]	S. Shintani, C. Li, T. Ishikawa, et al., “Expression of Vascular Endothelial Growth Factor A, B, C, and D in Oral Squamous Cell Carcinoma,” Oral Oncology, Vol. 40, No. 1, 2004, pp. 13-20. doi:10.1016/S1368-8375(03)00127-1
[30]	B. S. Siriwardena, Y. Kudo, I. Ogawa, et al., “VEGF-C Is Associated with Lymphatic Status and Invasion in Oral Cancer,” Journal of Clinical Pathology, Vol. 61, No. 1, 2008, pp. 103-108. doi:10.1136/jcp.2007.047662
[31]	E. Sasabe, X. Zhou, D. Li, et al., “ The Involvement of Hypoxia-Inducible Factor-1alpha in the Susceptibility to Gamma-Rays and Chemotherapeutic Drugs of Oral Squamous Cell Carcinoma Cells,” International Journal of Cancer, Vol. 120, No. 2, 2007, pp. 268-277. doi:10.1002/ijc.22294