Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction

Subadra Kaliyaperumal; Sathasivasubramanian Sankarapandian

doi:10.4103/2278-330X.181645

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HEAD AND NECK CANCER : Original Article

05 (

); 073-079

doi:

10.4103/2278-330X.181645

Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction

Subadra Kaliyaperumal^1,, Sathasivasubramanian Sankarapandian¹

1 Department of Oral Medicine and Radiology, Faculty of Dental Sciences, Sri Ramachandra University, Porur, Chennai, Tamil Nadu

*Corresponding author: Dr. Subadra Kaliyaperumal. subssaai@gmail.com

Published: 2020-12-28

MedIntel Services Pvt Ltd. This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial-License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/.)

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Abstract

Aims: The aim of this study was to quantitatively investigate the hypermethylation of p16 gene in buccal cells and saliva of oral submucous fibrosis (OSMF) patients using real-time quantitative methylation-specific polymerase chain reaction (PCR) and to compare the values of two methods. Subjects and Methods: A total of 120 samples were taken from 60 subjects selected for this study, of which 30 were controls and 30 patients were clinically and histopathologically diagnosed with OSMF. In both groups, two sets of samples were collected, one directly from the buccal cells through cytobrush technique and the other through salivary rinse. We analyzed the samples for the presence of p16 hypermethylation using quantitative real-time PCR. Results: In OSMF, the hypermethylation status of p16 in buccal cells was very high (93.3%) and in salivary samples, it was partially methylated (50%). However, no hypermethylation was found in controls suggesting that significant quantity of p16 hypermethylation was present in buccal cells and saliva in OSMF. Conclusions: This study indicates that buccal cell sampling may be a better method for evaluation than the salivary samples. It signifies that hypermethylation of p16 is an important factor to be considered in epigenetic alterations of normal cells to oral precancer, i.e. OSMF.

Keywords

Acknowledgment

My sincere thanks to Dr. V. Vetrichelvi Ph.D., Assistant Professor, Department of Human Genetics for her immense help, suggestion and expertise and MS.C.D. Mohanapriya Msc., human genetics, application specialist (genomics and molecular biology) for her unhesitating help and support.

My special thanks to Dr. Sankara Aravind Warrier M.D.S., Reader for enthusiastic suggestions, critical judgment and advices and Dr. T. Malarkodi M.D.S. Senior Lecturer, for giving untiring support and offering her guidance, ideas and suggestions through every step of my project.

References

Kao SY, Chu YW, Chen YW, Chang KW, Liu TY. Detection and screening of oral cancer and pre-cancerous lesions. J Chin Med Assoc 2009;72:227-33.
Nair U, Bartsch H, Nair J. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004;19:251-62.
Angadi PV, Rao SS. Areca nut in pathogenesis of oral submucous fibrosis: Revisited. Oral Maxillofac Surg 2011;15:1-9.
Takeshima M, Saitoh M, Kusano K, Nagayasu H, Kurashige Y, Malsantha M, et al. High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka. J Oral Pathol Med 2008;37:475-9.
Barros SP, Offenbacher S. Epigenetics: Connecting environment and genotype to phenotype and disease. J Dent Res 2009;88:400-8.
Radhakrishnan R, Kabekkodu S, Satyamoorthy K. DNA hypermethylation as an epigenetic mark for oral cancer diagnosis. J Oral Pathol Med 2011;40:665-76.
Viet CT, Jordan RC, Schmidt BL. DNA promoter hypermethylation in saliva for the early diagnosis of oral cancer. J Calif Dent Assoc 2007;35:844-9.
Xing EP, Nie Y, Song Y, Yang GY, Cai YC, Wang LD, et al. Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma. Clin Cancer Res 1999;5:2704-13.
Timmermann S, Hinds PW, Münger K. Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′- deoxycytidine treatment induces a senescence-like state. Oncogene 1998;17:3445-53.
Sartor M, Steingrimsdottir H, Elamin F, Gäken J, Warnakulasuriya S, Partridge M, et al. Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines. Br J Cancer 1999;80:79-86.
Reed AL, Califano J, Cairns P, Westra WH, Jones RM, Koch W, et al. High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res 1996;56:3630-3.
Kresty LA, Mallery SR, Knobloch TJ, Song H, Lloyd M, Casto BC, et al. Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia. Cancer Res 2002;62:5295-300.
Bahar G, Feinmesser R, Shpitzer T, Popovtzer A, Nagler RM. Salivary analysis in oral cancer patients: DNA and protein oxidation, reactive nitrogen species, and antioxidant profile. Cancer 2007;109:54-9.
King IB, Satia-Abouta J, Thornquist MD, Bigler J, Patterson RE, Kristal AR, et al. Buccal cell DNA yield, quality, and collection costs: Comparison of methods for large-scale studies. Cancer Epidemiol Biomarkers Prev 2002;11(10 Pt 1):1130-3.
Hong J, Resnick M, Behar J, Wang LJ, Wands J, DeLellis RA, et al. Acid-induced p16 hypermethylation contributes to development of esophageal adenocarcinoma via activation of NADPH oxidase NOX5-S. Am J Physiol Gastrointest Liver Physiol 2010;299:G697-706.
Bookout AL, Mangelsdorf DJ. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal 2003;1:e012.
Tilakaratne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol 2006;42:561-8.
Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: Implications for therapy. J Dent Res 2008;87:14-32.
Campo-Trapero J, Cano-Sánchez J, Palacios-Sánchez B, Sánchez-Gutierrez JJ, González-Moles MA, Bascones-Martínez A. Update on molecular pathology in oral cancer and precancer. Anticancer Res 2008;28:1197-205.
Liu M, Feng L, Tang X, Guo S. Gene promoter hyper methylation in leukoplakia of the oral mucosa. Pathol Lab Med Int 2010;2:71-7.
Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg 2006;35:101-8.
Righini CA, de Fraipont F, Timsit JF, Faure C, Brambilla E, Reyt E, et al. Tumor-specific methylation in saliva: A promising biomarker for early detection of head and neck cancer recurrence. Clin Cancer Res 2007;13:1179-85.
Mehrotra R, Gupta A, Singh M, Ibrahim R. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 2006;5:11.
Carvalho AL, Jeronimo C, Kim MM, Henrique R, Zhang Z, Hoque MO, et al. Evaluation of promoter hypermethylation detection in body fluids as a screening/diagnosis tool for head and neck squamous cell carcinoma. Clin Cancer Res 2008;14:97-107.
Kulkarni V, Saranath D. Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues. Oral Oncol 2004;40:145-53.
Ruesga MT, Acha-Sagredo A, Rodríguez MJ, Aguirregaviria JI, Videgain J, Rodríguez C, et al. p16(INK4a) promoter hypermethylation in oral scrapings of oral squamous cell carcinoma risk patients. Cancer Lett 2007;250:140-5.
Hall GL, Shaw RJ, Field EA, Rogers SN, Sutton DN, Woolgar JA, et al. p16 Promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 2008;17:2174-9.
Lee KD, Lee HS, Jeon CH. Body fluid biomarkers for early detection of head and neck squamous cell carcinomas. Anticancer Res 2011;31:1161-7.
Rosas SL, Koch W, da Costa Carvalho MG, Wu L, Califano J, Westra W, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res 2001;61:939-42.
Lopez M, Aguirre JM, Cuevas N, Anzola M, Videgain J, Aguirregaviria J, et al. Gene promoter hypermethylation in oral rinses of leukoplakia patients - A diagnostic and/or prognostic tool? Eur J Cancer 2003;39:2306-9.

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[1] Kao SY, Chu YW, Chen YW, Chang KW, Liu TY. Detection and screening of oral cancer and pre-cancerous lesions. J Chin Med Assoc 2009;72:227-33.

[2] Nair U, Bartsch H, Nair J. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: A review of agents and causative mechanisms. Mutagenesis 2004;19:251-62.

[3] Angadi PV, Rao SS. Areca nut in pathogenesis of oral submucous fibrosis: Revisited. Oral Maxillofac Surg 2011;15:1-9.

[4] Takeshima M, Saitoh M, Kusano K, Nagayasu H, Kurashige Y, Malsantha M, et al. High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka. J Oral Pathol Med 2008;37:475-9.

[5] Barros SP, Offenbacher S. Epigenetics: Connecting environment and genotype to phenotype and disease. J Dent Res 2009;88:400-8.

[6] Radhakrishnan R, Kabekkodu S, Satyamoorthy K. DNA hypermethylation as an epigenetic mark for oral cancer diagnosis. J Oral Pathol Med 2011;40:665-76.

[7] Viet CT, Jordan RC, Schmidt BL. DNA promoter hypermethylation in saliva for the early diagnosis of oral cancer. J Calif Dent Assoc 2007;35:844-9.

[8] Xing EP, Nie Y, Song Y, Yang GY, Cai YC, Wang LD, et al. Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma. Clin Cancer Res 1999;5:2704-13.

[9] Timmermann S, Hinds PW, Münger K. Re-expression of endogenous p16ink4a in oral squamous cell carcinoma lines by 5-aza-2′- deoxycytidine treatment induces a senescence-like state. Oncogene 1998;17:3445-53.

[10] Sartor M, Steingrimsdottir H, Elamin F, Gäken J, Warnakulasuriya S, Partridge M, et al. Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines. Br J Cancer 1999;80:79-86.

[11] Reed AL, Califano J, Cairns P, Westra WH, Jones RM, Koch W, et al. High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res 1996;56:3630-3.

[12] Kresty LA, Mallery SR, Knobloch TJ, Song H, Lloyd M, Casto BC, et al. Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia. Cancer Res 2002;62:5295-300.

[13] Bahar G, Feinmesser R, Shpitzer T, Popovtzer A, Nagler RM. Salivary analysis in oral cancer patients: DNA and protein oxidation, reactive nitrogen species, and antioxidant profile. Cancer 2007;109:54-9.

[14] King IB, Satia-Abouta J, Thornquist MD, Bigler J, Patterson RE, Kristal AR, et al. Buccal cell DNA yield, quality, and collection costs: Comparison of methods for large-scale studies. Cancer Epidemiol Biomarkers Prev 2002;11(10 Pt 1):1130-3.

[15] Hong J, Resnick M, Behar J, Wang LJ, Wands J, DeLellis RA, et al. Acid-induced p16 hypermethylation contributes to development of esophageal adenocarcinoma via activation of NADPH oxidase NOX5-S. Am J Physiol Gastrointest Liver Physiol 2010;299:G697-706.

[16] Bookout AL, Mangelsdorf DJ. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl Recept Signal 2003;1:e012.

[17] Tilakaratne WM, Klinikowski MF, Saku T, Peters TJ, Warnakulasuriya S. Oral submucous fibrosis: Review on aetiology and pathogenesis. Oral Oncol 2006;42:561-8.

[18] Choi S, Myers JN. Molecular pathogenesis of oral squamous cell carcinoma: Implications for therapy. J Dent Res 2008;87:14-32.

[19] Campo-Trapero J, Cano-Sánchez J, Palacios-Sánchez B, Sánchez-Gutierrez JJ, González-Moles MA, Bascones-Martínez A. Update on molecular pathology in oral cancer and precancer. Anticancer Res 2008;28:1197-205.

[20] Liu M, Feng L, Tang X, Guo S. Gene promoter hyper methylation in leukoplakia of the oral mucosa. Pathol Lab Med Int 2010;2:71-7.

[21] Shaw R. The epigenetics of oral cancer. Int J Oral Maxillofac Surg 2006;35:101-8.

[22] Righini CA, de Fraipont F, Timsit JF, Faure C, Brambilla E, Reyt E, et al. Tumor-specific methylation in saliva: A promising biomarker for early detection of head and neck cancer recurrence. Clin Cancer Res 2007;13:1179-85.

[23] Mehrotra R, Gupta A, Singh M, Ibrahim R. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions. Mol Cancer 2006;5:11.

[24] Carvalho AL, Jeronimo C, Kim MM, Henrique R, Zhang Z, Hoque MO, et al. Evaluation of promoter hypermethylation detection in body fluids as a screening/diagnosis tool for head and neck squamous cell carcinoma. Clin Cancer Res 2008;14:97-107.

[25] Kulkarni V, Saranath D. Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues. Oral Oncol 2004;40:145-53.

[26] Ruesga MT, Acha-Sagredo A, Rodríguez MJ, Aguirregaviria JI, Videgain J, Rodríguez C, et al. p16(INK4a) promoter hypermethylation in oral scrapings of oral squamous cell carcinoma risk patients. Cancer Lett 2007;250:140-5.

[27] Hall GL, Shaw RJ, Field EA, Rogers SN, Sutton DN, Woolgar JA, et al. p16 Promoter methylation is a potential predictor of malignant transformation in oral epithelial dysplasia. Cancer Epidemiol Biomarkers Prev 2008;17:2174-9.

[28] Lee KD, Lee HS, Jeon CH. Body fluid biomarkers for early detection of head and neck squamous cell carcinomas. Anticancer Res 2011;31:1161-7.

[29] Rosas SL, Koch W, da Costa Carvalho MG, Wu L, Califano J, Westra W, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients. Cancer Res 2001;61:939-42.

[30] Lopez M, Aguirre JM, Cuevas N, Anzola M, Videgain J, Aguirregaviria J, et al. Gene promoter hypermethylation in oral rinses of leukoplakia patients - A diagnostic and/or prognostic tool? Eur J Cancer 2003;39:2306-9.

Evaluation of p16 hypermethylation in oral submucous fibrosis: A quantitative and comparative analysis in buccal cells and saliva using real-time methylation-specific polymerase chain reaction

Abstract

Abstract

Keywords

Acknowledgment

References

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