Expression of p53, Ki-67, sox-2, oct-4, and NANOG in the tongue and trachea of Swiss mice exposed to narghile smoke
DOI:
https://doi.org/10.1590/1678-7765-2025-0839Keywords:
Narghile, Carcinogenesis, Tongue, TracheaAbstract
Introduction: Narghile (waterpipe) tobacco use has increased worldwide, partly due to limited awareness of its harmful effects. Despite the widespread misconception that the water filters toxic substances, the chemical composition of narghile smoke is comparable to that of cigarette smoke. However, its effects on airway tissues remain poorly understood. Objective: This study aimed to evaluate the histological and immunohistochemical profiles of the tongue and trachea in Swiss mice exposed to narghile smoke. Methodology: Sixty animals were divided into six groups: a control group (0 days) and experimental groups exposed daily to narghile smoke for seven, 15, 30, 60, and 90 days. After euthanasia, tongue and tracheal tissues were collected for histological (hematoxylin and eosin staining) and immunohistochemical analyses of NANOG, OCT-4, SOX-2 (neoplastic stem cell markers), Ki-67, and p53 in epithelial cells. Results: Histological evaluation revealed cytological and architectural alterations in the tongue, suggestive of early dysplastic changes. In the trachea, early metaplastic modifications were observed, including cilia loss, epithelial hyperplasia, and mild keratinization. These changes increased with longer exposure duration. Immunohistochemical analysis showed greater expression of NANOG, OCT-4, SOX-2, and p53, peaking at 30 days, whereas Ki-67 expression peaked at 90 days. Elevated p53 levels suggest alterations in cell cycle regulation and apoptosis. Conclusion: Narghile smoke induced histological and molecular changes in the tongue and tracheal epithelium of mice, indicating a potential carcinogenic effect. Further in vitro, in vivo, and clinical investigations are warranted to validate these biomarkers and better elucidate the biological effects of narghile smoke exposure.
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1Knishkowy B, Amitai Y. Water-pipe (narghile) smoking: an emerging health risk behavior. Pediatrics. 2005;116(1):e113–9. doi: 10.1542/peds.2004-2173
» https://doi.org/10.1542/peds.2004-2173
2Alanazi NH, Lee JW, Dos Santos H, Job JS, Bahjri K. The use of planned behavior theory in predicting cigarette smoking among waterpipe smokers. Tob Induc Dis. 2017;15:29. doi: 10.1186/s12971-017-0133-z
» https://doi.org/10.1186/s12971-017-0133-z
3Shihadeh A, Saleh R. Polycyclic aromatic hydrocarbons, carbon monoxide, tar, and nicotine in the mainstream smoke aerosol of the narghile water pipe. Food Chem Toxicol. 2005;43(5):655–61. doi: 10.1016/j.fct.2004.12.013
» https://doi.org/10.1016/j.fct.2004.12.013
4Gissi DB, Gabusi A, Servidio D, Cervellati F, Montebugnoli L. Predictive role of p53 protein as a single marker or associated with Ki-67 antigen in oral leukoplakia: a retrospective longitudinal study. Open Dent J. 2015;9:41–5. doi: 10.2174/1874210601509010041
» https://doi.org/10.2174/1874210601509010041
5Folescu R, Levai CM, Grigoraş ML, Arghirescu TS, Talpoş IC, Gîndac CM, et al. Expression and significance of Ki-67 in lung cancer. Rom J Morphol Embryol. 2018;59(1):227–33.
6Yang XH, Ding L, Fu Y, Chen S, Zhang L, Zhang XX, et al. p53-positive expression in dysplastic surgical margins is a predictor of tumor recurrence in patients with early oral squamous cell carcinoma. Cancer Manag Res. 2019;11:1465–72. doi: 10.2147/CMAR.S192500
» https://doi.org/10.2147/CMAR.S192500
7Yu HH, Featherston T, Tan ST, Chibnall AM, Brasch HD, Davis PF, et al. Characterization of cancer stem cells in moderately differentiated buccal mucosal squamous cell carcinoma. Front Surg. 2016;3:46. doi: 10.3389/fsurg.2016.00046
» https://doi.org/10.3389/fsurg.2016.00046
8Curtarelli RB, Gonçalves JM, dos Santos LG, Savi MG, Nör JE, Mezzomo LA, et al. Expression of cancer stem cell biomarkers in human head and neck carcinomas: a systematic review. Stem Cell Rev Rep. 2018;14(6):769–84. doi: 10.1007/s12015-018-9839-4
» https://doi.org/10.1007/s12015-018-9839-4
9Rizzino A. Sox2 and Oct-3/4: a versatile pair of master regulators that orchestrate the self-renewal and pluripotency of embryonic stem cells. Wiley Interdiscip Rev Syst Biol Med. 2009;1(2):228–36. doi: 10.1002/wsbm.12
» https://doi.org/10.1002/wsbm.12
10Falougy M, Taubitz C, Ragab M, Patil A, Jensen J, Hoppe S, et al. Prognostic value of SOX2 and NANOG expression in recurrent oral squamous cell carcinoma. Cancers (Basel). 2025;17(7):1181. doi: 10.3390/cancers17071181
» https://doi.org/10.3390/cancers17071181
11Peitzsch C, Nathansen J, Schniewind SI, Schwarz F, Dubrovska A. Cancer stem cells in head and neck squamous cell carcinoma: identification, characterization and clinical implications. Cancers (Basel). 2019;11(5):616. doi: 10.3390/cancers11050616
» https://doi.org/10.3390/cancers11050616
12Khabour OF, Alzoubi KH, Bani-Ahmad M, Dodin A, Eissenberg T, Shihadeh A. Acute exposure to waterpipe tobacco smoke induces changes in oxidative and inflammatory markers in mouse lung. Inhal Toxicol. 2012;24(10):667–75. doi: 10.3109/08958378.2012.710918
» https://doi.org/10.3109/08958378.2012.710918
13Shraideh ZA, Najjar HN. Histological changes in tissues of trachea and lung alveoli of albino rats exposed to the smoke of two types of narghile tobacco products. JJBS. 2011;4(4):219–24.
14Shihadeh A, Azar S, Antonios C, Haddad A. Towards a topographical model of narghile water-pipe café smoking: a pilot study in a high socioeconomic status neighborhood of Beirut, Lebanon. Pharmacol Biochem Behav. 2004;79(1):75–82. doi: 10.1016/j.pbb.2004.06.005
» https://doi.org/10.1016/j.pbb.2004.06.005
15Flausino CS, Hoffmeister GF, Pilati PV, Modolo F, Pilati SF. A new adapted machine to simulate narghile smoke. J Cytol Tissue Biol. 2020;7:029. doi: 10.24966/CTB-9107/100029
» https://doi.org/10.24966/CTB-9107/100029
16Nemmar A, Al Hemeiri A, Al Hammadi N, Yuvaraju P, Beegam S, Yasin J, et al. Early pulmonary events of nose-only water pipe (shisha) smoking exposure in mice. Physiol Rep. 2015;3(3):e12258. doi: 10.14814/phy2.12258
» https://doi.org/10.14814/phy2.12258
17Bentur L, Hellou E, Goldbart A, Pillar G, Monovich E, Salameh M, et al. Laboratory and clinical acute effects of active and passive indoor group water-pipe (narghile) smoking. Chest. 2014;145(4):803–9. doi: 10.1378/chest.13-0960
» https://doi.org/10.1378/chest.13-0960
18Embaló B, Parize HN, Rivero ER. Evaluation of cell proliferation in cystic lesions associated with impacted third molars. Microsc Res Tech. 2018;81(11):1241–5. doi: 10.1002/jemt.23128
» https://doi.org/10.1002/jemt.23128
19Scotti FM, Mitt VC, Vieira DS, Biz MT, Castro RG, Modolo F. Expression of stem cell markers Nanog and Nestin in lip squamous cell carcinoma and actinic cheilitis. Oral Dis. 2018;24(7):1209–16. doi: 10.1111/odi.12891
» https://doi.org/10.1111/odi.12891
20Liu A, Yu X, Liu S. Pluripotency transcription factors and cancer stem cells: small genes make a big difference. Chin J Cancer. 2013;32(9):483–7. doi: 10.5732/cjc.012.10282
» https://doi.org/10.5732/cjc.012.10282
21Fu TY, Hsieh IC, Cheng JT, Tsai MH, Hou YY, Lee JH, et al. Association of OCT4, SOX2, and NANOG expression with oral squamous cell carcinoma progression. J Oral Pathol Med. 2016;45(2):89–95. doi: 10.1111/jop.12335
» https://doi.org/10.1111/jop.12335
22Tanaka T, Komai Y, Tokuyama Y, Yanai H, Ohe S, Okazaki K, et al. Identification of stem cells that maintain and regenerate lingual keratinized epithelial cells. Nat Cell Biol. 2013;15(5):511–8. doi: 10.1038/ncb2719
» https://doi.org/10.1038/ncb2719
23Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH, et al. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin Cancer Res. 2008;14(13):4085–95. doi: 10.1158/1078-0432.CCR-07-4404
» https://doi.org/10.1158/1078-0432.CCR-07-4404
24Islam F, Qiao B, Smith RA, Gopalan V, Lam AK. Cancer stem cell: fundamental experimental pathological concepts and updates. Exp Mol Pathol. 2015;98(2):184–91. doi: 10.1016/j.yexmp.2015.02.002
» https://doi.org/10.1016/j.yexmp.2015.02.002
25Bianco BC, Scotti FM, Vieira DS, Biz MT, Castro RG, Modolo F. Immunohistochemical expression of matrix metalloproteinase-1, matrix metalloproteinase-2 and matrix metalloproteinase-9, myofibroblasts and Ki-67 in actinic cheilitis and lip squamous cell carcinoma. Int J Exp Pathol. 2015;96(5):311–8. doi: 10.1111/iep.12140
» https://doi.org/10.1111/iep.12140
26Zaid K, Azar-Maalouf E, Barakat C, Chantiri M. p53 overexpression in oral mucosa in relation to shisha smoking in Syria and Lebanon. Asian Pac J Cancer Prev. 2018;19(7):1879–82. doi: 10.22034/APJCP.2018.19.7.1879
» https://doi.org/10.22034/APJCP.2018.19.7.1879
27Yadav P, Malik R, Balani S, Nigam RK, Jain P, Tandon P. Expression of p16, Ki-67 and p53 markers in dysplastic and malignant lesions of the oral cavity and oropharynx. J Oral Maxillofac Pathol. 2019;23(2):224–30. doi: 10.4103/jomfp.JOMFP_299_18
» https://doi.org/10.4103/jomfp.JOMFP_299_18
28Mirza AM, Gysin S, Malek N, Nakayama K, Roberts JM, McMahon M. Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT. Mol Cell Biol. 2004;24(24):10868–81. doi: 10.1128/MCB.24.24.10868-10881.2004
» https://doi.org/10.1128/MCB.24.24.10868-10881.2004
29Lee HJ, Kang YH, Lee JS, Byun JH, Kim UK, Jang SJ, et al. Positive expression of NANOG, mutant p53, and CD44 is directly associated with clinicopathological features and poor prognosis of oral squamous cell carcinoma. BMC Oral Health. 2015;15:153. doi: 10.1186/s12903-015-0120-9
» https://doi.org/10.1186/s12903-015-0120-9
30Kim BR, Van de Laar E, Cabanero M, Tarumi S, Hasenoeder S, Wang D, et al. SOX2 and PI3K cooperate to induce and stabilize a squamous-committed stem cell injury state during lung squamous cell carcinoma pathogenesis. PLoS Biol. 2016;14(11):e1002581. doi: 10.1371/journal.pbio.1002581
» https://doi.org/10.1371/journal.pbio.1002581
31van den Berg RM, van Tinteren H, van Zandwijk N, Visser C, Pasic A, Kooi C, et al. The influence of fluticasone inhalation on markers of carcinogenesis in bronchial epithelium. Am J Respir Crit Care Med. 2007;175(10):1061–5. doi: 10.1164/rccm.200612-1770OC
» https://doi.org/10.1164/rccm.200612-1770OC
32Lu Y, Futtner C, Rock JR, Xu X, Whitworth W, Hogan BL, et al. Evidence that SOX2 overexpression is oncogenic in the lung. PLoS One. 2010;5(6):e11022. doi: 10.1371/journal.pone.0011022
» https://doi.org/10.1371/journal.pone.0011022
33Ling TY, Kuo MD, Li CL, Yu AL, Huang YH, Wu TJ, et al. Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus infection in vitro. Proc Natl Acad Sci U S A. 2006;103(25):9530–5. doi: 10.1073/pnas.0510232103
» https://doi.org/10.1073/pnas.0510232103
34Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hsieh WJ, et al. Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer Res. 2010;70(24):10433–44. doi: 10.1158/0008-5472.CAN-10-2638
» https://doi.org/10.1158/0008-5472.CAN-10-2638
35Boers JE, ten Velde GP, Thunnissen FB. p53 in squamous metaplasia: a marker for risk of respiratory tract carcinoma. Am J Respir Crit Care Med. 1996;153(1):411–6. doi: 10.1164/ajrccm.153.1.8542151
» https://doi.org/10.1164/ajrccm.153.1.8542151
36Breuer RH, Snijders PJ, Sutedja TG, van der Linden H, Risse EK, Meijer CJ, et al. Suprabasal p53 immunostaining in premalignant endobronchial lesions in combination with histology is associated with bronchial cancer. Lung Cancer. 2003;40(2):165–72. doi: 10.1016/S0169-5002(03)00029-1
» https://doi.org/10.1016/S0169-5002(03)00029-1
37Kitamura H, Kameda Y, Nakamura N, Nakatani Y, Inayama Y, Iida M, et al. Proliferative potential and p53 overexpression in precursor and early stage lesions of bronchioloalveolar lung carcinoma. Am J Pathol. 1995;146(4):876–87.
38Magnani KL, Cataneo DC, Domingues MA, Hasimoto EN, Evaristo TC, Cataneo AJ. Respiratory immunohistochemical study in rats exposed to cigarette smoke and alcohol. Acta Cir Bras. 2015;30(3):178–85. doi: 10.1590/S0102-865020150030000003
» https://doi.org/10.1590/S0102-865020150030000003
39Lee JJ, Liu D, Lee JS, Kurie JM, Khuri FR, Ibarguen H, et al. Long-term impact of smoking on lung epithelial proliferation in current and former smokers. J Natl Cancer Inst. 2001;93(14):1081–8. doi: 10.1093/jnci/93.14.1081
» https://doi.org/10.1093/jnci/93.14.1081
40Cavarga I, Kocan P, Boor A, Belak J, Zak V, Kluchova Z, et al. Immunohistochemical markers of proliferation and vascularisation in preneoplastic bronchial lesions and invasive non-small cell lung cancer. Neoplasma. 2009;56(5):414–21. doi: 10.4149/neo_2009_05_414
» https://doi.org/10.4149/neo_2009_05_414
41Ishikuro A, Dosaka-Akita H, Kato M, Fujino M, Isobe H, Miyamoto H, et al. Ki-67 labeling indices in non-small cell lung cancer: comparison between image cytometry and flow cytometry. Cytometry. 1997;30(4):186–91. doi: 10.1002/(SICI)1097-0320(19970815)30:4<186::AID-CYTO5>3.0.CO;2-I
» https://doi.org/10.1002/(SICI)1097-0320(19970815)30:4<186::AID-CYTO5>3.0.CO;2-I
42Sousa V, Espírito Santo J, Silva M, Cabral T, Alarcão AM, Gomes A, et al. EGFR/erB-1, HER2/erB-2, CK7, LP34, Ki67 and p53 expression in preneoplastic lesions of bronchial epithelium: an immunohistochemical and genetic study. Virchows Arch. 2011;458(5):571–81. doi: 10.1007/s00428-011-1062-5
» https://doi.org/10.1007/s00428-011-1062-5
43Abdallah A, Awny S, Metwally IH, Eid AM, Ayman R, Abdelaal R, et al. Tongue cancer in pregnancy: challenges and outcomes. A case series and review of the literature. Oral Maxillofac Surg. 2025;30(1):11. doi: 10.1007/s10006-025-01494-5
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Copyright (c) 2026 Sarah Freygang Mendes Pilati, Filipe Ivan Daniel, Paulo Vinicius Fontanella Pilati, Mariana Hornung Marins, Aline Cristina Batista Rodrigues, Filipe Modolo
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