Dina SABRY, Iman Mahmoud ABOUSHADY, Ghada Abdel Aziz ABDEL-LATİF, Sara El MOSHY

Correlation between Decreased Nuclear Factor Kappa Beta (NFKβ), Kras, and Braf Genes’ Expression and Enhanced Chemosensitivity of Human Squamous Cell Carcinoma Cells to 5-fluorouracil and/or Mesenchymal Stem Cells-derived Microvesicles

OBJECTIVE Oral squamous cell carcinoma (OSCC) comprises about 10% of head and neck cancer. 5-fluorouracil (5- FU) is commonly used for the treatment of OSCC. However, it has many limitations due to its identified side effects. Therefore, this work compared squamous cell carcinoma (SCC) cells’ chemosensitivity to 5-FU, mesenchymal stem cells-derived microvesicles (MVs), and their combination. METHODS Human SCC cell line (SCC152) was subjected to 5-FU or MVs or their combination for 24 h and 48 h. Inverted microscopic evaluation of apoptosis, MTT cell proliferation assay, DNA comet assay, and detection of NFKβ, Kras, and Braf genes’ expression were performed. RESULTS The combination group, compared to 5-FU or MVs treated groups, showed the most apparent apoptotic features. Cell proliferation was significantly decreased, while the tailed DNA% was significantly increased in the combination group versus either the 5-FU or MVs groups. The combination group showed a significant decrease in NFKβ, Kras, and Braf genes’ expression than the 5-FU or MVs treated groups. The correlation between cell proliferation and the studied genes showed a very strong positive linear relationship, while a strong negative linear relationship existed between cell proliferation and tailed DNA%. CONCLUSION The combination of MVs and 5-FU enhanced the genotoxicity and cytotoxicity of the SCC152 cell line compared to using either of them alone. Moreover, downregulation of NFKβ, Kras, and Braf genes’ expression was associated with enhanced apoptotic features, decreased cell proliferation, and enhanced tailed DNA%.

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1. Johnson NW, Jayasekara P, Amarasinghe AH. Squamous cell carcinoma and precursor lesions of the oral cavity: epidemiology and aetiology. Periodontol 2011;57(1):19–37.
2. Patel RS, Clark JR, Dirven R, Wyten R, Gao K, O’Brien CJ. Prognostic factors in the surgical treatment of patients with oral carcinoma. ANZ J Surg 2009;79(1– 2):19–22.
3. Rheeder P, Girdler-Brown B, Abram M, Van Heerden W, Van Zyl AW. Epidemiology of oral squamous cell carcinoma. SADJ 2012;67(10):550–3.
4. Zhang L, Cheung KJ Jr., Lam WL, Cheng X, Poh C, Priddy R, et al. Increased genetic damage in oral leukoplakia from high risk sites: Potential impact on staging and clinical management. Cancer 2001;91(11):2148– 55.
5. Christensen S, Van der Roest B, Besselink N, Janssen R, Boymans S, Martens JW, et al. 5-Fluorouracil treatment induces characteristic T> G mutations in human cancer. Nat Commun 2019;10(1):1–11.
6. Katona C, Kralovánszky J, Rosta A, Pandi E, Fónyad G, Tóth K, et al. Putative role of dihydropyrimidine dehydrogenase in the toxic side effect of 5-fluorouracil in colorectal cancer patients. Oncology 1998;55(5):468– 74.
7. Sommer H, Santi DV. Purification and amino acid analysis of an active site peptide from thymidylate synthetase containing covalently bound 5-fluoro- 2’-deoxyuridylate and methylenetetrahydrofolate. Biochem Biophys Res Commun 1974;57(3):689–95.
8. Pettersen HS, Visnes T, Vågbø CB, Svaasand EK, Doseth B, Slupphaug G, et al. UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation. Nucleic Acids Res 2011;39(19):8430–44.
9. Huehls AM, Huntoon CJ, Joshi PM, Baehr CA, Wagner JM, Wang X, et al. Genomically incorporated 5-fluorouracil that escapes UNG-initiated base excision repair blocks DNA replication and activates homologous recombination. Mol Pharmacol 2016;89(1):53–62.
10. Biancone L, Bruno S, Deregibus MC, Tetta C, Camussi G. Therapeutic potential of mesenchymal stem cell-derived microvesicles. Nephrol Dial Transplant 2012;27(8):3037–42.
11. Ho PJ, Yen ML, Tang BC, Chen CT, Yen BL. H2O2 accumulation mediates differentiation capacity alteration, but not proliferative decline, in senescent human fetal mesenchymal stem cells. ARS 2013;18(15):1895–905.
12. Mimeault M, Batra SK. Recent insights into the molecular mechanisms involved in aging and the malignant transformation of adult stem/progenitor cells and their therapeutic implications. Ageing Res Rev 2009;8(2):94–112.
13. Galipeau J. The mesenchymal stromal cells dilemma does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus- host disease represent a death knell or a bump in the road? Cytotherapy 2013;15(1):2–8.
14. Kimbrel EA, Kouris NA, Yavanian GJ, Chu J, Qin Y, Chan A, et al. Mesenchymal stem cell population derived from human pluripotent stem cells displays potent immunomodulatory and therapeutic properties. Stem Cells Dev 2014;23(14):1611–24.
15. Pokharel D, Wijesinghe P, Oenarto V, Lu JF, Sampson DD, Kennedy BF, et al. Deciphering cell-to-cell communication in acquisition of cancer traits: Extracellular membrane vesicles are regulators of tissue biomechanics. OMICS 2016;20(8):462–9.
16. Khawar MB, Abbasi MH, Siddique Z, Arif A, Sheikh N. An update on novel therapeutic warfronts of extracellular vesicles (EVs) in cancer treatment: Where we are standing right now and where to go in the future. Oxid Med Cell Longev 2019;2019:9702562.
17. Kang T, Jones TM, Naddell C, Bacanamwo M, Calvert JW, Thompson WE, et al. Adipose‐derived stem cells induce angiogenesis via microvesicle transport of miRNA‐31. Stem Cells Transl Med 2016;5(4):440–50.
18. Abdul Rahman SN, Abdul Wahab N, Abd Malek SN. In vitro morphological assessment of apoptosis induced by antiproliferative constituents from the rhizomes of Curcuma zedoaria. Evid Based Complement Alternat Med 2013;2013:257108.
19. Stockert JC, Horobin RW, Colombo LL, Blázquez- Castro A. Tetrazolium salts and formazan products in cell biology: Viability assessment, fluorescence imaging, and labeling perspectives. Acta Histochem 2018;120(3):159–67.
20. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/ comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35(3):206–21.
21. Chan YH. Biostatistics 102: Quantitative data-parametric a non-parametric tests. Singapore Med J 2003;44(8):391–6.
22. Marcazzan S, Varoni EM, Blanco E, Lodi G, Ferrari M. Nanomedicine, an emerging therapeutic strategy for oral cancer therapy. Oral Oncol 2018;76:1–7.
23. Mariño R, Haresaku S, McGrath R, Bailey D, Mccullough M, Musolino R, et al. Oral cancer screening practices of oral health professionals in Australia. BMC Oral Health 2017;17(1):1–9.
24. Moongkarndi P, Kosem N, Kaslungka S, Luanratana O, Pongpan N, Neungton N. Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana (mangosteen) on SKBR3 human breast cancer cell line. J Ethnopharmacol 2004;90(1):161–6.
25. Fonsato V, Collino F, Herrera MB, Cavallari C, Deregibus MC, Cisterna B, et al. Human liver stem cell‐derived microvesicles inhibit hepatoma growth in SCID mice by delivering antitumor microRNAs. Stem Cells 2012;30(9):1985–98.
26. Wu S, Ju GQ, Du T, Zhu YJ, Liu G-H. Microvesicles derived from human umbilical cord Wharton’s jelly mesenchymal stem cells attenuate bladder tumor cell growth in vitro and in vivo. PLoS One 2013;8(4):e61366.
27. Del Fattore A, Luciano R, Saracino R, Battafarano G, Rizzo C, Pascucci L, et al. Differential effects of extracellular vesicles secreted by mesenchymal stem cells from different sources on glioblastoma cells. Expert Opin Biol Ther 2015;15(4):495–504.
28. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: Mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3(5):330–8.
29. Sax JK, Fei P, Murphy ME, Bernhard E, Korsmeyer SJ, El-Deiry WS. BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol 2002;4(11):842–9.
30. Saran R, Tiwari RK, Reddy PP, Ahuja YR. Risk assessment of oral cancer in patients with pre-cancerous states of the oral cavity using micronucleus test and challenge assay. Oral Oncol 2008;44(4):354–60.
31. McKenna DJ, McKeown SR, McKelvey-Martin VJ. Potential use of the comet assay in the clinical management of cancer. Mutagenesis 2008;23(3):183–90.
32. Mozaffarieh M, Konieczka K, Hauenstein D, Schoetzau A, Flammer J. Half a pack of cigarettes a day more than doubles DNA breaks in circulating leukocytes. Tob Induc Dis 2010;8(1):1–4.
33. Lee TL, Yang XP, Yan B, Friedman J, Duggal P, Bagain L, et al. A novel nuclear factor-κB gene signature is differentially expressed in head and neck squamous cell carcinomas in association with TP53 status. Clin Cancer Res 2007;13(19):5680–91.
34. Yan M, Xu Q, Zhang P, Zhou XJ, Zhang ZY, Chen WT. Correlation of NF-κB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma. BMC Cancer 2010;10(1):1–13.
35. Anbo N, Ogi K, Sogabe Y, Shimanishi M, Kaneko T, Dehari H, et al. Suppression of NF-κB/p65 Inhibits the proliferation in oral squamous cancer cells. J Cancer Ther 2013;2013:32578.
36. Liu F, Bardhan K, Yang D, Thangaraju M, Ganapathy V, Waller JL, et al. NF-κB directly regulates Fas transcription to modulate Fas-mediated apoptosis and tumor suppression. J Biol Chem 2012;287(30):25530–40.
37. Dajee M, Lazarov M, Zhang JY, Cai T, Green CL, Russell AJ, et al. NF-κB blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature 2003;421(6923):639–43.
38. Shi Z, Hu Z, Chen D, Huang J, Fan J, Zhou S, et al. MicroRNA 200a mediates nasopharyngeal carcinoma cell proliferation through the activation of nuclear factor κB. Mol Med Report 2016;13(2):1732–8.
39. Anderson JA, Irish JC, McLachlin CM, Ngan BY. H-ras oncogene mutation and human papillomavirus infection in oral carcinomas. AOHNS 1994;120(7):755–60.
40. Das N, Majumder J, DasGupta U. Ras gene mutations in oral cancer in Eastern India. Oral Oncol 2000;36(1):76–80.
41. Weber A, Langhanki L, Sommerer F, Markwarth A, Wittekind C, Tannapfel A. Mutations of the BRAF gene in squamous cell carcinoma of the head and neck. Oncogene 2003;22(30):4757–9.
42. Caulin C, Nguyen T, Longley MA, Zhou Z, Wang XJ, Roop DR. Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity. Cancer Res 2004;64(15):5054–8.
43. Howell R, Wong F, Fenwick R. A transforming Kirsten ras oncogene in an oral squamous carcinoma. J Oral Pathol Med 1990;19(7):301–5.
44. Duran A, Linares JF, Galvez AS, Wikenheiser K, Flores JM, Diaz-Meco MT, et al. The signaling adaptor p62 is an important NF-κB mediator in tumorigenesis. Cancer Cell 2008;13(4):343–54.
45. Chenette E. Cancer: A Ras and NF-kappaB pas de deux. Nat Rev Drug Discov 2009;8(12):932.
46. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature 2009;462(7269):108–12.
47. Mercer KE, Pritchard CA. Raf proteins and cancer: BRaf is identified as a mutational target. Biochim Biophys Acta 2003;1653(1):25–40.
48. Khosravi-Far R, White MA, Westwick JK, Solski PA, Chrzanowska-Wodnicka M, Van Aelst L, et al. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol Cell Biol 1996;16(7):3923–33.
49. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417(6892):949–54.
50. Bissada E, Abboud O, Abou Chacra Z, Guertin L, Weng X, Nguyen-Tan PF, et al. Prevalence of K-RAS codons 12 and 13 mutations in locally advanced head and neck squamous cell carcinoma and impact on clinical outcomes. Int J Otolaryngol 2013;2013:848021.