Molecular mechanisms of oral submucous fibrosis and oral cancer: A review

Ashwini Dhopte; Hiroj Siddharth Bagde

doi:10.4103/jpo.jpo_23_22

REVIEW ARTICLE

Year : 2022 | Volume : 2 | Issue : 2 | Page : 75-82

Molecular mechanisms of oral submucous fibrosis and oral cancer: A review

Ashwini Dhopte¹, Hiroj Siddharth Bagde²
¹ Department of Oral Medicine and Radiology, Rama Dental College, Kanpur, Uttar Pradesh, India
² Department of Periodontology, Rama Dental College, Kanpur, Uttar Pradesh, India

Date of Submission	28-Oct-2022
Date of Decision	15-Nov-2022
Date of Acceptance	23-Nov-2022
Date of Web Publication	06-Feb-2023

Correspondence Address:
Dr. Ashwini Dhopte
Department of Oral Medicine and Radiology, Rama Dental College, Kanpur, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_23_22

Abstract

Chewing betel quid (BQ) increases the risk of oral cancer and oral submucous fibrosis (OSMF), a potentially malignant oral premalignant condition (OPMD). BQ constituents including areca nut (AN), trauma by coarse AN fibre, catechin, copper, alkaloids, increased reactive oxygen species, inflammation, and cytotoxicity are hypothesised to be the causative factors. They may stimulate tissue inflammation, fibroblast proliferation and collagen deposition, myofibroblast differentiation and contraction, collagen cross-links, and impede collagenphagocytosis, ultimately leading to the formation of oral squamous cell carcinoma (OSMF) and oral cancer. Through modulation of transforming growth factor1, plasminogen activator inhibitor1, cystatin, lysyl oxidase, tissue inhibitors of metalloproteinases, and matrix metalloproteinases, BQ componentinduced alterations in extracellular matrix turnover facilitate these events. In addition, genetic predisposition plays a role in many disease processes. Understanding the molecular pathways underlying BQinduced OSMF and oral cancer can aid in the future prevention and treatment of disease.This paper provides a comprehensive review of the molecular processes involved in BQ-induced OSMF and oral cancer, as well as future preventative prospects.

Keywords: Areca nut, betel quid, extracellular matrix turnover, genetic susceptibility, oral cancer, submucous fibrosis

How to cite this article:
Dhopte A, Bagde HS. Molecular mechanisms of oral submucous fibrosis and oral cancer: A review. J Precis Oncol 2022;2:75-82

How to cite this URL:
Dhopte A, Bagde HS. Molecular mechanisms of oral submucous fibrosis and oral cancer: A review. J Precis Oncol [serial online] 2022 [cited 2023 Jun 8];2:75-82. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/2/75/369218

Introduction

Oral submucous fibrosis (OSMF) is defined as an insidious chronic disease affecting any part of the oral cavity and sometimes pharynx. Clinically, OSMF presents with burning and pain of the mouth, oral mucosal atrophy with fibrosis of submucosal tissues, mucosal rigidity, and reduction in mouth opening. OSMF is common in India, Sri Lanka, Taiwan, and other Southeast Asian countries, and has started spreading to Europe and North America.^[1] In Taiwan, the prevalence of OSMF increased from 8.3/100,000 people in 1996 to 16.2/100,000 people in 2013. The prevalence of betel quid (BQ) chewing, tobacco smoking, and alcohol drinking habits in patients with oral premalignant lesions is about 82.9%, 95%, and 22.7%, respectively. OSMF may occur at any age but is frequently seen at the age of 21-30 years. The male-to-female ratio is around 11:1,^[2] possibly due to intrinsic differences between genders with/without the BQ chewing habit. Middle-aged chewers are the more commonly involved population in India. BQ components are suggested to be the major etiologic factors for OSMF and oral squamous cell carcinoma (OSCC), due to their content of inflammatory, genotoxic, carcinogenic, and fibrogenic factors such as areca nut (AN), lime, arecoline, catechin, catechol, copper, and reactive oxygen species (ROS).

OSMF is an oral potentially malignant disorder (OPMD) carrying risk for malignant transformation. The malignant transformation rate of OSMF is reported to be about 5.7% after 80.9 months of follow-up or 7%-13%. In a total of 1774 cases of OSMF and OSCC in Pakistan, 765 (43.12%) cases were OSMF alone, 472 (26.60%) cases were shown to have OSCC with malignant transformation from OSMF, whereas 537 (30.27%) cases had OSCC without OSMF. A 6.8-year follow-up study also elucidated that alcohol consumption is associated with the malignant transformation of patients with oral precancer. Other factors including BQ chewing habit, smoking habit, environmental heavy metal exposure,^[3] gender, site of lesion, and histological features such as epithelial dysplasia, loss of heterozygosity, aneuploidy of DNA, and human papillomavirus infection are suggested to stimulate the progression and malignant transformation of OSMF.

Betel quid and OSMF - etiology and clinical and histologic features

OSMF is a chronic, progressive, high-risk precancerous disease characterized by juxta-epithelial inflammatory reaction, fibrosis of lamina propria, thin parakeratinized squamous epithelium with atrophic change, and loss of rete peg. Increased dense collagen fiber deposition in the lamina propria occurs through time and, in the end stage, dense hyalinized fibrous tissue occupies the lamina propria and even superficial submucosa, and thus results in varied degrees of mucosal rigidity. OSMF is accompanied by fibroelastic hyperplasia, with/without epithelial hyperplasia/dysplasia over the oral cavity or oropharynx.^[4],[5] Repeated trauma causes inflammation and aggravates the fibrosis while increased collagen fiber deposition, decreased amount of blood vessels, and atrophic change of the epithelium occur. Progression of OSMF, thereby, may lead to loss of tissue mobility, trismus, and limited mouth opening.^[5] However, current treatment strategies are not so effective for attenuation of OSMF. BQ chewing is shown as the major etiologic factor of OSMF. There are about 200-600 million BQ chewers in the world. The ingredients of BQ vary in different countries. In Taiwan and Papua New Guinea, BQ comprised AN, and slaked lime with/without inclusion of Piper betle inflorescence or Piper betle leaf (betel leaf).^[1],[6],[7] However, in India and Sri Lanka, tobacco is popularly added as one major component of BQ.^[1] Among the BQ ingredients, AN components are considered to be the main causative factors in the disease process of OSMF. This is because OSMF is widespread in Taiwan where tobacco is not added into BQ. The roles of lime, betel leaf, and other ingredients in the pathogenesis of OSMF await further clarification. AN contains mainly alkaloids (such as arecoline, arecaidine, guvacoline, and guvacine), catechol, catechin, transition metals (copper and iron), and fibers.^[1],[7] The contributory role of AN components to the pathogenesis of OSMF is closely associated with the induction of ROS production,^[8] chronic mucositis, ulcers caused by mechanical trauma from coarse AN fibers, activation of the coagulation system, cytotoxicity to oral epithelial cells,^[8] stimulation of fibroblast proliferation/contraction, collagen synthesis/deposition, myofibroblast differentiation,^[9],[10] tissue inflammation,^[9] and the inhibition of collagen degradation and phagocytosis. These AN components include AN extract (ANE), areca alkaloids (arecoline, arecaidine, guvacoline, and guvacine), catechin, catechol, and copper. However, only about 1%-2% of BQ chewers develop OSMF, suggesting the presence of some predisposition factors toward mild or severe OSMF in these affected patients.

Genetic susceptibility and expression in tissue/organ fibrosis

A number of studies have found the association of genetic susceptibility with tissue/organ fibrosis such as pulmonary fibrosis, systemic sclerosis, and liver and kidney fibrosis. While exogenous factors such as viral hepatitis and alcohol abuse are the common causative factors of liver fibrosis, genetic predisposition may contribute to the progression of fibrosis, cirrhosis, liver failure, or hepatic carcinoma. Environmental factors are the key etiologic factors of lung fibrosis, but genetic factors in host defense, aging/senescence, and cell-cell adhesion may also increase the risk of pulmonary fibrosis, subsequent disease progression, and poor prognosis. Systemic sclerosis as an autoimmune disease may involve vascular abnormalities, immune alterations, and fibrosis of skin and other internal organs, where tissue inflammation and genetic susceptibility are present.

Several factors, including nutritional deficiency, vitamin deficiencies, and hypersensitivity to various dietary constituents, may also play a part in the pathogenesis of OSMF. Epidemiological studies strongly indicate that AN is the major etiologic agent that releases alkaloids to promote fibroblastic proliferation and increase collagen formation. However, only a small population of BQ chewers develop the disease, indicating that difference in genetic susceptibility plays a role in this event. About 7%12% of OSMF cases progress into oral malignancy. Additionally, a few individuals developed the disease after only a few contacts with BQ.^[11] Therefore, the relationship between gene and OSMF is still uncertain and awaits clarification.

Betel Quid and Collagen Turnover

Collagen-related genes

The extracellular matrix (ECM) provides a three-dimensional scaffold for cells via connection of cell surface receptors with various ECM components such as collagen, fibronectin, elastin, and nonfibrillar proteins including proteoglycan, hyaluronan, and glycoproteins. Hypoxia and collagen-rich conditions also intensify cancer progression. Impairment of ECM and ECM-cell interaction plays important roles in various diseases such as osteoarthritis, fibrosis, cancer, and genetic diseases. OSMF is a collagen-related disorder, with dense collagen deposition in the oral submucosa as its main characteristic feature.^[12] It has been found that fibroblasts from buccal mucosa exposed to areca alkaloid, due to BQ chewing, may result in the accumulation of collagen. Therefore, it is hypothesized that collagen-related genes might play a role in OSMF pathogenesis. Transforming growth factor-β1 (TGF-β1), lysyl oxidase (LOX), cystatin (CST3), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) proved to be involved in the turnover of ECM, wound healing, tumor invasion, and metastasis.^[13] However, few studies have compared protein expression and polymorphisms of the collagen-related genes situated on different chromosomes between OSMF patients and healthy controls. Some noninvasive markers (serum markers, urinary markers, and image tissue stiffness markers) have been developed for evaluation of organ fibrosis but still with some limitations. Further studies on the development of noninvasive early disease markers of OSMF are crucial for disease prevention and treatment in the future.

Role of collagen 1A1 and collagen 1A2 (COL1A1 and COL1A2)

Collagen in OSMF has proved to be histologically, biochemically, and immunochemically normal but increased in amount. Initially, deposition of type I and type III collagen in OSMF is similar to that of normal oral mucosa. However, type III collagen is gradually replaced by type I collagen as the disease progresses, eventually leading to a collagen I predominant microenvironment. In the early and intermediate stages of OSMF, increased tenascin, perlecan, fibronectin, type III collagen, and elastin are found in the lamina propria of oral mucosa, the submucosal layer, and around the muscle. As OSMF progresses, the components of the extracellular matrix (ECM) diminish and are eventually replaced by type I collagen. Concomitant overexpression of type I collagen and collagen/Hsp47, a stress protein and molecular chaperon for collagen, in OSMF tissues was also observed.^[14]

Interestingly, copper levels in serum/plasma/saliva, buccal tissue, and cytological smear of oral mucosa cells are elevated in OSMF patients compared to control subjects. Salivary copper levels showed a positive association with the histological grade of OSMF. Copper is a cofactor for LOX that is important for collagen and elastin cross-linking/maturation as well as insolubility, and maintaining the rigidity and structural integrity of ECM. Its salivary level increases rapidly after BQ chewing. Copper has been shown to stimulate collagen synthesis, but not proliferation of oral fibroblasts, suggesting the involvement of copper in mediating OSMF. Tetrathiomolybdate, a copper chelator, is thus considered to have a therapeutic effect on fibrotic diseases by repressing LOX expression and possibly also for the prevention/treatment of OSMF and OSCC. Furthermore, polyphenol and catechin fractions of AN may induce collagen cross-linking. In addition, ANE and arecoline were shown to stimulate collagen production in fibroblasts in vitro.

The genotypes of type I collagen show an association with the highest risk of OSMF. Type I collagen is processed from type I procollagen, a triple-stranded, rope-like molecule combining two COL1A1 gene-encoded alpha1 chains and one COL1A2 gene-encoded alpha2 chain. Comparing the frequency distribution of CC, CT, and TT on the COL1A1 gene in chromosome 17q, the high OSMF risk allele seems to be CC in the low-exposure group, while TT in the high-exposure group. Comparing the frequency distributions of AA, AB, and BB on the COL1A2 gene on chromosome 7q revealed that the high OSMF risk allele appears to be AA in the low-exposure group, while BB is present in the high-exposure group.^{[14],[15],[16]}

Betel Quid and Matrix Metalloproteinases

MMPs are a family of neutral proteases that degrade ECM produced by a variety of cells. Currently, 28 human MMPs have been identified, and these enzymes are classified according to their substrate specificity and structural similarities.^[15] The major subgroups are collagenases (MMP-1), gelatinases (MMP-2 and MMP-9), stromelysin (MMP-3), and membrane-bound MMPs. One major question is whether inhibition of fibrogenesis and induction of fibrinolysis may resolve tissue fibrosis and thus relieve OSMF. In addition to regulating ECM proteolysis, MMPs may also process a number of biologically active proteins such as cytokine, chemokines, cell-surface proteins, TGF-β1, and other inflammation-related molecules,^[16] which contribute to tissue fibrosis. Moreover, MMP expression is involved in tumor invasion and metastasis of OSCC.

Various types of MMPs are expressed and activated in patients with OSMF as well as head-and-neck squamous cell carcinoma (HNSCC). Gene polymorphisms are suspected to influence the gene transcription and expression level in OPMD and malignant lesions.

A number of studies aimed to explore the association of protein expression and single-nucleotide polymorphisms (SNPs) of MMP-1, MMP-2 (−1306 C/T), MMP-3 (−1171 5A > 6A), and MMP-9 (−1562 C/T) promoter in OSMF and HNSCC. Cases will be discussed below.

Collagenase-1 (COLase, matrix metalloproteinase-1)

Collagenase-1, also called MMP-1 or interstitial collagenase, is one of the major subgroups of the MMP family and the principal human enzyme that cleaves collagens, including types I, II, III, VII, and X. It is shown to be important for photoaging and photocarcinogenesis. It is produced by a wide variety of cells such as stromal fibroblasts, macrophages, endothelial cells, epithelial cells, and tumor cells.^[16],[17] In addition, it is secreted as a latent precursor known as procollagenase and can be activated by many pathways. MMP-1 activity has been found to be lower in OSMF than in normal oral mucosa, varying the collagen metabolism of the patients. However, there is no statistically significant difference between different histological grades of OSMF. On the contrary, an elevated expression of MMP-1 in OSMF (n = 30) is found in comparison with normal mucosa (n = 10), but no difference is noted between different histological grades of OSMF.

The MMP-1 promoter region (−1607 1G/2G) has been found to affect its transcriptional activity and may contribute to carcinogenesis and metastasis of cancers. Chaudhary et al. (2011) found that SNPs in the MMP-1 promoter region (−1607 1G/2G) are associated with the susceptibility of BQ chewers to OSMF and HNSCC in India. Habitual BQ chewing and alcohol consumption may enhance the expression of the 2G allele of MMP-1 genes in OSMF and HNSCC patients. Similarly, the 2G genotype of the MMP-1 promoter is observed in higher frequency in both OSCC and OSMF patients, compared to controls. However, there is also a study, indicating that the MMP-1 promoter region (−1607 1G/2G) polymorphism increases the risk of OSCC, but not OSMF.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 (gelatinase-A and gelatinase-B)

Both MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) are Zn2⁺-dependent endopeptidases with similar structures. However, their physiological distributions show some differences. MMP-2 is expressed by a wide variety of cell types in normal conditions, while MMP-9 is expressed in only a few cell types including trophoblasts, osteoclasts, leukocytes, dendritic cells, and their precursors.^[18] MMP-2 primarily degrades proteins in the ECM and basement membrane as its primary function. It also degrades type I, IV, V, VII, and X collagens, laminin, elastin, fibronectin, and proteoglycans. The MMP-2 gene is located on chromosome 16q. Price et al. reported that C to T substitution at

1306 in the promoter region of MMP-2, disrupts the Sp1-binding site and results in reduction of its transcriptional activity. On the contrary, the − 1306C allele may enhance its transcription level. Therefore, individuals who carry the CC genotype express higher MMP-2 protein than those who carry the TT or CT genotype. Lin et al. (2004) assessed the association of MMP-2 genotype with the risk of OSMF and OSCC by comparing 58 OSMF cases, 121 OSCC cases, and 147 controls. Their data suggested no significant association between subjects carrying the CC genotype and the development of OSMF, but the results showed that subjects carrying the CC genotype had a two-fold increased risk for developing OSCC when compared with the CT or TT genotypes.

MMP-9 proteolytically degrades ECM components such as decorin, elastin, fibrillin, laminin, gelatin, and type IV, V, XI, and XVI collagen. Moreover, it activates growth factors such as proTGF-β and proTNF-α. Expressions of MMP-2, MMP-9, TIMP-1, and TIMP-2 in OSMF are found to be higher than in healthy oral mucosa. Increased levels of various MMPs in serum have been shown to be valuable biomarkers in liver cirrhosis and other fibrotic diseases. Arecoline may stimulate TIMP-1 expression but inhibits MMP-2 and MMP-9 expression of buccal mucosal fibroblasts. Interestingly, oral keratinocytes and SAS tongue cancer cells express MMP-9, and ANE stimulates MMP-9 but suppresses TIMP-1 and TIMP-2 secretion via differential signaling pathways. Increased expressions of MMP-2, MMP-3, MMP-9, MMP-10, and MMP-13 immediately after acute liver injury prior to fibrogenesis have been reported and contribute to hepatocyte necrosis, suggesting that MMPs participate in the acute phase of hepatic injury and the activation of tissue fibrosis. The concomitant synthesis and degradation of ECM is a hallmark of the dynamic nature of liver cirrhosis.^[19] These results indicate that expression of MMPs can vary in different stages of OSMF and OSCC. The stimulation of MMPs by BQ components also contributes to inflammation, epithelial cell necrosis, and oral mucosa atrophy and activates fibrogenesis in OSMF tissues. The MMP-9 gene is located on chromosome 20q. The C to T transition at −1562 in the promoter region of MMP-9 leads to differential transcription and is associated with increased susceptibility to various diseases.

In 2011, matrix metalloproteinase-3 (stromelysin-1)

MMP-3 is able to degrade components in the basal membrane, and collagen types II, V, IX, and X. It can also induce the activation of other MMPs such as MMP-1 and MMP-9. The MMP-3 gene is located near chromosome 11q, and the insertion or deletion of a single adenosine at position-1171 in the promoter region of MMP-3 gene could result in different transcriptional activities.

In MMP-3-1171 5A >6A, the insertion or deletion of a single adenosine could alter the transcription level of the MMP-3 gene. For MMP-3, the frequency of the 5A genotype in the MMP-3 promoter region was higher in the OSMF group than in the control group and had a greater than two-fold risk for developing OSMF compared to controls. However, the 5A/5A carrier alleles showed an association only in patients <45 years of age. Further studies with larger sample sizes are warranted.

Betel Quid and Transforming Growth Factor-β

The synthesis of collagens is influenced by a wide variety of mediators, including growth factors, hormones, and cytokines. Among these mediators, TGF-β1 is a prominent one, which controls the proliferation, differentiation, and other functions in many cell types. It also stimulates the production of collagens through regulation of intramembrane proteolysis and activation of CREB3 L1.^[20]

The activation of TGF-β/Smad2 and Smad4 signaling has been found in keratinocytes and myofibroblasts in OSMF tissues.^[10] Overexpression of both TGF-β1 and TGF-β2 was reported in OSMF tissues, with a higher expression of TGF-β1 than TGF-β2. TGF-β1 is expressed mainly in epithelial cells, perivascular cells, infiltrated inflammatory cells, fibroblasts, and muscle cells. The signal of TGF-β2 was mainly localized in the submucosal area with minimal involvement of the epithelium. The mRNA level of TGF-β1 was higher in the early and middle stages of OSMF tissues than that in healthy counterparts in patients of Hunan, China. An increased expression of CD105, a TGF-β1 receptor, was associated with hypoxia-induced neoangiogenic activity in OSMF, and this feature was linked to transformation from normal oral mucosa to mild and severe epithelial dysplasia. Its expression was also associated with differentiation status, TNM stage, metastasis, and 3-year survival rate of OSCC patients with OSMF.

Secretion of TGF-β1 in cultured fibroblasts harvested from the aforementioned specimens also showed no marked difference, but recent real-time PCR and immunohistochemical staining studies found the markedly elevated expression of TGF-β1, p-Smad2, connective tissue growth factor (CTGF), and MMP-3 and decreased expression of bone morphogenetic protein-7 in OSMF. They also showed the possible stimulatory effect of areca components (ANE, arecoline, and others) on epithelial-mesenchymal transition and the expression of TGF-β1, p-Smad2, smooth muscle actin, CTGF, and LIM domain kinase 1 in epithelial cells. Local injection of ANE and pan masala extract to buccal mucosa of Sprague–Dawley rats on alternate days for 48 weeks induced OSMF-like changes with a concomitant elevated expression of TGF-β1, suggesting the contribution of AN and pan masala components. Polyphenol, tannin, catechin, and areca alkaloids of AN components are able to stimulate TGF-β2 and p-Smad in keratinocytes, but not gingival fibroblast. We also found that ANE stimulates TGF-β1 and Smad2 signaling in oral keratinocytes and SAS oral cancer cells, implicating the involvement of AN in the pathogenesis of OSMF. Accordingly, smad2 overexpression is reported in OSMF tissues relative to healthy tissues. Curcumin may attenuate the increased expression of TGF-β1, inducible nitric oxide synthase, and p53 in OSMF. Unexpected expression of Smad 7, an inhibitor of TGF-β, is elevated in OSMF and OSCC tissues relative to normal tissues, and has been suggested as a promoter and diagnostic marker. Interestingly, for the treatment of OSMF, glabridin as an isoflavone is shown to attenuate the arecoline-induced TGF-β1, p-smad2, collagen, and fibroblast contraction,^[21] implicating its potential use for the prevention and treatment of OSMF. However, initial attempts for systemic inhibition of TGF-β1 are disappointing because of an increase in generalized tissue/organ inflammation.^[22]

The association of genotypes of TGF-β1 with the risk of OSMF has been studied. Comparing the frequencies of TT, TC, and CC alleles on the TGF-β1 gene on chromosome 19q, high OSMF risk seems to be associated with CC alleles in both low- and high-exposure groups.

Betel quid and lysyl oxidase

LOX, also known as protein-lysine 6-oxidase, is a copper-dependent extracellular enzyme that functions in crosslinking of collagens and elastin through posttranslational oxidative deamination of peptidyl lysine residues in their precursors. This modification stabilizes the collagen fibrillar array,^[23],[24] and contributes to ECM stiffness and mechanical property. LOX and LOX-like proteins are involved in atherosclerosis, tissue fibrosis, tumorigenesis, and metastasis through changes in protein expression and regulation of signal transduction.^[25] LOX overexpression may affect the tumor microenvironment and tumor desmoplasia (fibrosis) and also stimulate anchorage-independent growth of OSCC cells.

LOX mRNA expression in blood cells from OSMF (n = 127) and control patients (n = 127) has been analyzed. LOX expression in blood cells from patients of OSMF can be similar (n = 89), lower (n = 11), or higher (n = 27) than age- and sex-matched controls, suggesting that changes of LOX in circulating blood cells of OSMF are not evident. The activity of LOX is found to be elevated in fibroblasts cultured from OSMF patients relative to fibroblasts cultured from normal oral mucosa. An epidemiological study showed elevated LOX expression in OSCC tissues relative to adjacent oral mucosa. Similarly, overexpression of LOX was also found in OSMF tissues.^[26] This can be partly explained by stimulation of LOX expression in oral keratinocytes by ANE. The presence of copper in BQ is found to stimulate LOX expression of fibroblasts, and thus increases collagen cross-linking and resistance to degradation.

LOX is encoded by the LOX gene on chromosome 5q, and defects in this gene have been linked with predisposition to thoracic aortic aneurysms and OSMF. The frequencies of three genotypic variants (AA, AG, and GG) of LOX genes in patients of OSMF and controls were evaluated. The high OSMF risk allele seems to be AA in the low-exposure group, while GG is more prevalent in the high-exposure group. The differences of Arg158Gln SNPs of the LOX genotype between elder BQ chewers (control, 216 patients, without OSMF) and OSMF patients (83 patients with OSMF) through PCR-RFLP and direct sequencing were found.^[27]

Betel Quid and Cystatin C (CST3)

In addition to LOX, cystatin C (CST3) is another molecule responsible for the prevention of ECM degradation. The terminal regions of each collagen molecule are composed of terminal peptides, which function in cross-linking and enhance the strength of collagen fibers. These areas are resistant to attacks by collagenases but are susceptible to other serine and cysteine proteinases. These groups of enzymes belong to the cystatin superfamily, namely the type 1 cystatins (stefins A, B), type 2 cystatins, and the kininogens. Cystatin C is one of the type 2 cystatins, a class of cysteine proteinase inhibitors found in a variety of human body fluids and secretions. The major functions of this enzyme are thought to provide the protection and stabilization of the collagen fibrils.

Cystatin was shown to play crucial roles in fibrosis and carcinogenesis of various organs such as lung, kidney, and liver. Cystatin C expression is significantly higher in OSMF tissues from patients than in normal oral mucosa. It is mainly expressed by fibroblasts, endothelial cells, and inflammatory cells. Fibroblasts from OSMF were shown to have higher cystatin expression than normal fibroblasts. In addition, arecoline was found to promote cystatin C mRNA and protein expression in a dose-dependent manner.^[28] An elevated cystatin M was shown to promote metastasis of OSCC by blocking cathepsin B activity and rescue tumor cells from TNF-α-induced apoptosis. Salivary cystatin B level was also found to be a valuable prognostic marker for OSCC patients. More studies are needed to clarify the role of various cystatins in OSMF and OSCC.

Cystatin C is encoded by the CST3 gene on chromosome 20p. A mutation in this gene has been associated with amyloid angiopathy by reducing the expression of cystatin C. The frequency distribution of AA, AB, and BB (with A as the normal allele and B as the mutated allele) on the CST3 gene in patients of OSMF and healthy counterparts has been probed. The high OSMF risk allele seems to be AA in both low- and high-exposure groups. However, special attention should be paid to the reduced levels of CST3 when OSMF transforms into malignancy. Further research is required to identify the individual mechanisms operating at various stages and progression to carcinogenesis.

Betel Quid and Plasminogen Activator Inhibitor-1

PAI-1 regulates ECM homeostasis and wound healing by suppression of urokinase plasminogen activator/tissue plasminogen activator (tPA)-mediated conversion of plasminogen to plasmin that activates MMPs and fibrinolysis.^[29] A number of reports using fibrosis models of internal organs (liver, lung, and kidney) have found that PAI-1 deficiency or inhibition of PAI-1 activity attenuates organ fibrosis. TGF-β may stimulate PAI-1 expression via ROS and smad-dependent (ALK5/smad2/3) and smad-independent (Src/EGFR/MEK/ERK) pathways.^[30] Interestingly, PAI-1 and tPA secretion is increased in fibroblasts derived from OSMF when compared to normal buccal fibroblasts. The ratio of PAI-1/tPA is also increased in OSMF fibroblasts. Arecoline was shown to stimulate PAI and tPA secretion and also increase PAI-1/tPA ratio in buccal mucosal fibroblasts. In addition, hypoxia-inducible factor-1α overexpression in fibroblasts, epithelial cells, and inflammatory cells was found in OSMF tissues relative to healthy tissues. Hypoxia enhanced the arecoline-induced PAI-1 and ECM production by buccal mucosal fibroblasts,^[30] leading to clinical OSMF. Furthermore, PAI-1 expression was elevated in OSCC tissues relative to normal tissues, but PAI-1 showed little association with the survival rate of OSCC patients.

Little is known about the role of PAI-1 polymorphism in the pathogenesis of OSMF and OSCC. The presence of PAI-1 promoter polymorphism − 675 4G/5G and high plasma PAI-1 level were reported to increase the risk of keloid in Chinese Han population, as well as the risk of liver cirrhosis, hepatocarcinoma, and pulmonary fibrosis. Recently, PAI-1 −675 4G/5G genotypes were found to be strongly associated with overall stage and early stage of OSCC relative to control subjects in the European population.^[31] More studies are needed to clarify the role of PAI-1 polymorphism in the prediction of risk and survival of OSMF and OSCC patients.

Betel Quid and TIMPs

There are four TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) that show differential inhibitory effects on MMPs, a disintegrin and metalloproteinases (ADAMs), thus preventing ECM proteolysis and leading to accumulation of ECM/tissue fibrosis.^[32] Immunohistochemical staining and enzyme zymography analysis found simultaneous increases of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 expression in OSMF tissues relative to normal tissues.

Arecoline and safrole (a component of the Piper betle inflorescence) are shown to stimulate TIMP-1 mRNA and protein expression of buccal fibroblasts.^[33],[34] Fibroblasts from OSMF tissues secreted more TIMP-1 than fibroblasts from adjacent healthy tissues,^[35],[36] suggesting their involvement in the pathogenesis of OSMF. On the contrary, fibroblasts from early-stage OSMF showed similar levels in secretion of TGF-β1, MMP-1, MMP-2, interleukin-6 (IL-6), IL-8, and MMP-3 compared to fibroblasts from healthy tissues, but increased TIMP-1 and TIMP-2 secretion of OSMF fibroblasts was noticed.^[37]

Conclusions

OSMF is an OPMD with a potential for malignant transformation. The pathophysiology of OSMF is very complex. The predisposing and risk factors of OSMF and malignant transformation in BQ chewers may vary depending on exposure periods, amount of BQ consumption, with/without other oral habits (tobacco, alcohol), and genetic susceptibility (nucleotide polymorphism of collagen, MMPs, TIMPs, TGF-β1, CST3, and LOX, etc.). BQ components including trauma by coarse fiber in AN, catechin, copper, alkaloids, ROS, inflammation, genotoxicity, and cytotoxicity are shown as the major contributing factors. These toxic components may stimulate inflammation in the lamina propria of the buccal mucosa, proliferation of fibroblasts and collagen deposition, myofibroblast differentiation and fibrotic contracture, and cross-linking of collagen, and may inhibit collagen phagocytosis. The net effect of these events leads to OSMF and oral cancer, but more studies are necessary to fully unravel the underlying molecular mechanisms. BQ components are found to induce ECM deposition via upregulation of TGF-β1, PAI-1, cystatin, LOX, and TIMPs. However, only a small population of BQ chewers develop these diseases, suggesting that genetic background plays a role in the development of OSMF. The high-risk alleles and genotypes of collagen, MMPs, TGF-β1, and LOX found in OSMF patients with high frequency may change the transcriptional activity and the functions of corresponding proteins, and increase the risk of OSMF. Treatment of OSMF includes preventing mucosal damage, reducing oxidative stress, and controlling inflammation by stopping BQ chewing, smoking, and drinking alcohol. Surgical management, natural products, low-power laser irradiation, enzymes, corticosteroid, vasodilator, and antioxidants have been used for the treatment of OSMF but their efficacy is limited. New therapies, such as targeting therapy toward TGF-β signaling, PAI-1, cystatin, or LOX, as well as antioxidative and anti-inflammatory therapy, are urgently needed. It is, therefore, important to further clarify the molecular mechanisms of BQ-induced OSMF and oral cancer in order for future prevention and treatment of BQ-chewing-related diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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