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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 33  |  Issue : 3  |  Page : 248-251

Analysis of downregulated salivary proteins in oral submucous fibrosis – A quantitative proteomic pilot study


Department of Oral Medicine and Radiology, Yenepoya Dental College, Mangalore, Karnataka, India

Date of Submission04-Mar-2021
Date of Decision05-Aug-2021
Date of Acceptance06-Aug-2021
Date of Web Publication28-Sep-2021

Correspondence Address:
Dr. Anu Babu
Department of Oral Medicine and Radiology, Yenepoya Dental College, Yenepoya University, Derlakkate, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_59_21

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   Abstract 


Background: Oral submucous fibrosis (OSMF) is a chronic debilitating disease of the oral cavity which is strongly associated with chewing of areca nut. The rate of malignant transformation varies between 3% and 19%. As oral lesions are in direct contact with saliva, the altered composition of saliva has shown promising results in surveillance of malignant transformation. Aim: To do quantitative proteomic profiling of saliva in OSMF and to study the downregulated proteins. Methodology: Quantitative proteomic profiling was carried out using Liquid chromatography with Tandem mass spectroscopy (LC–MS/MS) on saliva obtained from patients with OSMF. Results: A total of 172 proteins were identified in saliva samples. Five proteins were downregulated in the samples (fold change average of disease vs. control ratio ≤0.6). Conclusion: Downregulated proteins could serve as potential biomarkers in the early detection of malignant transformation in OSMF.

Keywords: Biomarker, malignant transformation, mass spectrometry, oral submucous fibrosis, proteomics, quantitative proteomic profiling, saliva


How to cite this article:
Babu A, Chatra L, Shenoy P. Analysis of downregulated salivary proteins in oral submucous fibrosis – A quantitative proteomic pilot study. J Indian Acad Oral Med Radiol 2021;33:248-51

How to cite this URL:
Babu A, Chatra L, Shenoy P. Analysis of downregulated salivary proteins in oral submucous fibrosis – A quantitative proteomic pilot study. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Nov 29];33:248-51. Available from: https://www.jiaomr.in/text.asp?2021/33/3/248/326886




   Introduction Top


Oral cancer is a global public health problem. India accounts for nearly 33% of the global burden of disease (Globocan 2018). Smokeless tobacco and betel quid are identified as the main etiology for oral cancer.[1] The use of smokeless tobacco with or without betel quid is highly prevalent among the South Asian population which has led to the surge in the case numbers in recent times.[2] Oral submucous fibrosis (OSMF) is one such condition seen in patients with chronic betel quid habit.[3] This condition does not regress or revert even after cessation of the habit. Studies suggest that epithelial dysplasia is seen in about 25% of biopsied OSMF cases. The rate of transformation to malignancy varies from 3% to 19%. This establishes an increased risk for the development of oral squamous cell carcinoma; however, the biology underlying this association is yet to be elucidated.[4] It is hypothesized that alkaloids and other substances present in betel quid contribute to malignant transformation by the formation of reactive oxygen species, DNA adducts resulting in DNA strand breakage.[5] Often the clinical and histological appearance of the oral mucosa may not depict the damage at the genetic level.

Saliva is a dynamic body fluid containing an array of analytes which primarily includes proteins, peptides, nucleic acids and enzymes.[6] Saliva directly comes in contact with the oral mucosa and contains exfoliated cells making it a favorable body fluid for the discovery of biomarkers. Due to ease in sample collection and non-invasive collection technique, saliva is preferred over serum and tissue.[7] We attempt to analyze the protein profile of saliva collected from patients diagnosed with OSMF to aid in the discovery of proteins that may serve as potential biomarkers.


   Aim and Objectives Top


This study aims to identify the potential protein biomarkers in saliva samples collected from patients with OSMF using mass spectrometry and studying the downregulated proteins is the scope of this article.


   Materials and Methods Top


Patient samples

The study was conducted in the Department of Oral Medicine and Radiology, in a reputed Dental College, Mangalore. The study was approved by the Institutional Ethical Committee (vide ethical committee protocol number 2015/326) and followed all the recommendations of the Helsinki Declaration (2013). Patients who were clinically and histologically diagnosed with OSMF were included in the case group. Written consent was obtained from the individuals before undergoing the biopsy procedure for histopathological confirmation. Healthy volunteers with pan-chewing habits without lesions were taken as control. After the confirmation, written consent was obtained for including them in the study. Saliva samples were collected from participants of both groups. They were instructed to refrain from food/drink for at least 1 h before collection. The patients were asked to rinse the mouth with water. Saliva was collected by asking the patient to sip 15 ml of sterile saline, swish, and gargle in the mouth for 10 s. Samples were then collected in a 15-ml tube. These samples were stored in an icebox and transported to the research lab (YU-IOB Center for Systems Biology and Molecular Medicine).

Sample size analysis

As this study is in discovery phase of proteins, sample size was usually considered in multiples of 5. Sample size (n) was calculated to be 5 by the formula n =Z 2p( 1-p)/2 with Confidence interval = 95%, level of confidence (Z)= 1.96 where prevalence was 19%, (margin of error )= 4.8. So, depending on the availability of cases, 5 cases and 5 control was decided for the study .

The samples were then centrifuged at 2000 rpm for 5 min and the supernatant was transferred to another tube. Both the cell suspension and saliva were stored separately in a −80°C freezer until further use.

Sample preparation

The saliva samples were centrifuged at 3000 rpm, for 30 min at room temperature, and the supernatant was used for further analysis. Protein estimation was carried out with a bicinchoninic acid assay (BCA assay) kit and about 1 mg of protein was taken for depletion.

Depletion of abundant protein salivary amylase

Salivary amylase was depleted using starch-based affinity chromatography. Around 1.5 g of potato starch was loaded onto a 2.5-ml syringe, whose nozzle was fitted with Whatman® filter paper. The salivary samples were loaded onto the syringe and passed through the starch column. Postdepletion, the protein was again quantified using a BCA assay. To check the depletion, the pre- and post-depleted samples were analyzed using SDS–PAGE gel electrophoresis.

Tryptic digestion of depleted saliva samples

In-solution digestion of proteins was carried out with 5-mm dithiothreitol (DTT), for 20 min at 60°C followed by alkylation with 20-mm iodoacetamide (IAA), for 10 min at room temperature, in dark. Trypsin was added at a ratio of 1:20 (trypsin: sample) and left for overnight incubation at 37°C. Once the digestion was confirmed using the SDS page, the samples were dried in speed vac and stored at −20°C till further processing.

TMT labeling

Labeling of the samples was carried out with tandem mass tag (TMT) mass tags 10-plex kit, Thermo Scientific. The dried peptides were suspended in 100 μl of 50-mm triethylammonium bicarbonate. To each of the mass tags, 41 μl of acetonitrile anhydrous was added. The samples and the tags were incubated at room temperature for 1 h. The labeling reaction was quenched using 5% hydroxylamine. The samples were all pooled by taking 16 μl from each sample and dried using a speed vac.

Fractionation

The fractionation was carried out with SCX (strong cation exchange) stage tips.

LC–MS/MS analysis

The peptides from SCX fractionation were analyzed on Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) connected to Easy-nlc-1200 nanoflow liquid chromatography system (Thermo Scientific). For MS/MS analysis, data were acquired at top speed mode with 3 s cycles and subjected to higher collision energy dissociation with 35% normalized collision energy. MS/MS scans were carried out at a range of 400–1200 m/z using an ion trap mass analyzer. The maximum injection time was 75 ms. Quantitation was done at MS3 mode for MS/MS/MS analysis. Data were acquired at top-speed mode with 3 s cycles and subjected to higher collision energy dissociation with 65% normalized collision energy. MS3 scans were carried out at a range of 100–500 m/z using an Orbitrap mass analyzer with a resolution of 60,000. The maximum injection time was 150 ms.

MS/MS data analysis

Mass spectrometry-derived data were analyzed with Proteome Discoverer software, version 2.1 (Thermo Scientific, Bremen, Germany) with the Sequest and Mascot (version 2.2.0, MatrixScience, London, UK) search algorithms. It was searched against the Human refseq81 protein database. The parameters used were carbamidomethylation of cysteine as static modification, acetylation of protein N-terminus, and oxidation of methionine as dynamic modifications. Precursor mass tolerance error was set to 10 ppm, the fragment mass tolerance as 0.05 Da. The minimum length of the peptide was assigned as six amino acids and two missed tryptic cleavage. The data were searched against the decoy database and an FDR score cutoff of 1% was used for the analysis. Relative protein quantification was carried out using the reporter ions quantifier node of Proteome Discoverer.


   Results Top


A total of 172 proteins were identified using LC–MS/MS. Out of all proteins identified, five were downregulated (fold change average of disease vs. control ratio ≤0.6) [Table 1].
Table 1: List of the downregulated proteins in the saliva samples

Click here to view



   Discussion Top


OSMF is a potentially malignant disorder affecting all the subsites of the oral cavity and sometimes even the pharynx. The present study was targeted toward identifying various proteins that allow the clinicians for risk stratification in patients with OSMF due to its increased rate of malignant transformation. Mass spectrometry has become a method of choice to identify differentially expressed proteins facilitating the discovery of potential biomarkers and drug targets. We employed a TMT-based quantitative proteomic approach to identify differentially expressed proteins in the saliva of patients with OSMF.

Human calmodulin-like protein (CALML3/CLP) is a calcium-binding protein found in epithelial cell types of various tissues including skin. CLP closely resembles calmodulin, an intracellular Ca2+ sensor that participates in signaling pathways that regulate many cell activities such as growth, proliferation, movement, and terminal differentiation of keratinocytes. In the present study, CLP showed downregulation by 0.4 folds. Results of the present study are consistent with the study of Brook et al.[8] They studied the expression of CLP in normal oral mucosa and oral squamous cell carcinoma using immunohistochemistry and concluded that CLP expression is seen in normal oral mucosa with downregulation of CLP expression in malignant transformation. They concluded that expression of CLP may serve as a marker for non-malignant tissue, and if downregulated, a predictor of malignant transformation. There are two plausible explanations for downregulation of CLP: first, tumor cells hindering the normal differentiation of epithelial cells; second, due to secondary effect of a tumor suppressor at an earlier stage of cell differentiation manifesting at the terminal differentiation level.[8]

Small Proline-Rich Protein 3 (SPRR3), also called esophagin, is a cornified enveloped structural precursor abundantly expressed in oral and esophageal epithelia. Expression of SPRR3 is strongly associated with terminal differentiation of keratinocytes and is considered as a marker for differentiation of squamous epithelia.[9] SPRR3 expression is strongly related to carcinogenesis since this gene is frequently downregulated in esophageal squamous cell carcinoma and esophageal adenocarcinoma when compared to the adjacent paired mucosa.[10]

In the present study, SPRR3 protein was downregulated by 0.4 folds. So far potential correlation with OSMF or oral cancer has not been studied. Expression of SPRR3-v1 was significantly reduced in dysplastic and early esophageal cancer when compared to healthy subjects.[10] Results of SPRR3 in esophageal squamous cell carcinoma can be extrapolated to our study. It was concluded that esophagin expressed in suprabasal cells induce apoptosis in normal mucosa. This helps in maintaining epithelial homeostasis of normal esophageal epithelium providing an antitumor effect.[9]

Alpha-Amylase 1 Isoform X1 (AMY1B) is an isoform of amylase. These secretory proteins catalyze the first step in the digestion of dietary starch and glycogen by hydrolyzing 1,4-alpha-glucoside bonds. The human genome has several amylase genes that are expressed at high levels in either the salivary gland or pancreas. AMY1B encodes an amylase isoenzyme produced by the salivary gland.[11]

In the present study, AMY1B protein was downregulated by 0.4 folds. Ramya et al.[12] compared the levels of salivary amylase. Results showed a significant decrease in salivary amylase levels – in oral cancer patients, during treatment.

Kallikrein-6 isoform A preproprotein (KLK6) is a member of the kallikrein subfamily of the peptidase S1 family of serine proteases. Literature suggests that various kallikreins are implicated in carcinogenesis. The encoded protein is proteolytically processed to generate the mature protease.[13] In the present study, KLK6 protein was downregulated by 0.3 folds. Schrader et al.[14] studied the expression KLK6 and correlated low levels of KLK6 protein in primary tumors from oropharyngeal and laryngeal squamous cell carcinoma (SCC) patients with poor prognosis and overall survival.

Histones are a highly conserved group of proteins associating DNA within the nucleus. There are four core histones, namely, H2A, H2B, H3, H4, and linker histone H1. Literature suggests epigenetic modifications of histones and their implication in oral squamous cell carcinoma. In the present study, histone H4 was downregulated by 0.4 folds. Our study corroborated with the results of Arif et al.[15] where the levels of H4K16ac were low and H4K8ac level was minimally altered. Further exploration is required to study the correlation between histones and the progression of the disease.

There are many methods used for validating the protein expression namely immunohistochemistry, polymerase chain reaction. Das et.al studied differentially expressed proteins in 100 OSMF samples. Results showed upregulation of HSP 70, calreticulin, lumican, and down regulation of protein.[16] In the above mentioned study, results were validated using immunohistochemistry and reverse transcriptase-polymerase chain reaction.

Limitations and Future Prospects

The limitation of this study is the small sample size. In future studies, validation of these downregulated proteins in a large cohort of clinical samples using other methods should prove useful in the identification of salivary biomarkers for risk stratification and early detection of transformation to squamous cell carcinoma.

This study was aimed to discover the downregulated proteins in saliva samples. Although numerous proteins were downregulated in the present study, their role in the progression of OSMF to oral cancer has not been determined. However, studies on squamous cell carcinomas in other organ systems show the expression of some of these proteins and peptides. Hence, these can be studied as biomarkers in early diagnosis. Validation of these downregulated proteins in a large cohort of clinical samples using other methods should prove useful in the identification of salivary biomarkers for risk stratification and early detection of transformation to squamous cell carcinoma.


   Conclusion Top


The downregulated proteins identified in this study could serve as potential biomarker in early detection of malignant transformation of Oral Premalignant disorders (OMPDS). Although proteomic study using saliva in OPMDS is still in its infancy, it could be an ideal alternative for early detection and screening of oral cancer.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgments

Authors thank the help provided by Yenepoya Research Center to carry out proteomic study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Gupta PC, Warnakulasuriya S. Global epidemiology of areca nut usage. Addict Biol 2002;7:77-83.  Back to cited text no. 1
    
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Rajalalitha P, Vali S. Molecular pathogenesis of oral submucous fibrosis-A collagen metabolic disorder. J Oral Pathol Med 2005;34:321-8.  Back to cited text no. 3
    
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Auluck A, Rosin MP, Zhang L, KN S. Oral submucous fibrosis, a clinically benign but potentially malignant disease: Report of 3 cases and review of the literature. J Can Dent Assoc 2008;74(8):735-40.   Back to cited text no. 4
    
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Merchant AT, Pitiphat W. Total, direct and indirect effects of paan on oral cancer. Cancer Causes Control 2015;26:487-91.  Back to cited text no. 5
    
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Sivadasan P, Gupta MK, Sathe GJ, Balakrishnan L, Palit P, Gowda H, et al. Human salivary proteome — A resource of potential biomarkers for oral cancer. J Prot 2015;127:89-95.  Back to cited text no. 6
    
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8.
Brooks MD, Bennett RD, Strehler EE, Sebo TJ, Eckert SE, Carr AB. Calmodulin-like protein (CLP) expression in oral squamous mucosa and in malignant transformation. J Prosthodont 2009;18:11-6.  Back to cited text no. 8
    
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Zhang Y, Feng Y-B, Shen X-M, Chen B-S, Du X-L, Luo M-L, et al. Exogenous expression of Esophagin/SPRR3 attenuates the tumorigenicity of esophageal squamous cell carcinoma cells via promoting apoptosis. Int J Cancer 2008;122:260-6.  Back to cited text no. 9
    
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de A Simão T, Souza-Santos P, de Oliveira D, Bernardo V, Lima S, Rapozo D, et al. Quantitative evaluation of SPRR3 expression in esophageal squamous cell carcinoma by qPCR and its potential use as a biomarker. Exp Mol Pathol 2011;91:584-9.  Back to cited text no. 10
    
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AMY1C amylase, alpha 1C (salivary) [Homo sapiens (human)]-Gene-NCBI. Ncbi.nlm.nih.gov. 2017. [Last accessed on 2017 Nov 14]. Available from: https://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=detailssearch&Term=278.  Back to cited text no. 11
    
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Ramya AS, Uppala D, Majumdar S, Surekha C, Deepak KGK. Are salivary amylase and pH-Prognostic indicators of cancers? J Oral Biol Craniofac Res 2015;5:81-5.  Back to cited text no. 12
    
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KLK6 kallikrein related peptidase 6 [Homo sapiens (human)]-Gene-NCBI. Ncbi.nlm.nih.gov. 2017. Available from: https://www.ncbi.nlm.nih.gov/gene/5653. [Last accessed on 2017 Nov 14].  Back to cited text no. 13
    
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Schrader CH, Kolb M, Zaoui K, Flechtenmacher C, Grabe N, Weber K-J, et al. Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients. Molecular Cancer. 2015;14:107.  Back to cited text no. 14
    
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Arif M, Vedamurthy BM, Choudhari R, Ostwal YB, Mantelingu K, Kodaganur GS, et al. Nitric oxide-mediated histone hyperacetylation in oral cancer: Target for a water-soluble HAT inhibitor, CTK7A. Chem Biol 2010;17:903-13.  Back to cited text no. 15
    
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Das T, Prodhan C, Patsa S, Ray JG, Chaudhuri K. Identification of over expressed proteins in oral submucous fibrosis by proteomic analysis. J Cell Biochem 2018;119:4361-71.  Back to cited text no. 16
    



 
 
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