Expression of hTERT in Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma - an Immunohistochemical Analysis.

Human telomerase reverse transcriptase enzyme, the catalytic subunit of telomerase are seen to be frequently reactivated in cancers including Oral squamous cell carcinoma (OSCC). Increased hTERT expression have been seen in potentially malignant conditions including Oral submucous fibrosis (OSMF). The aim of the study was to evaluate the expression levels in OSMF, OSCC in the background of OSMF and OSCC using immunohistochemistry and also to correlate hTERT expression with clinicopathologic parameters. A total of 50 histopathologically diagnosed cases of 20 OSMF, 20 OSCC wherein 5 were OSCC in the background of OSMF and 10 Normal oral mucosae were retrieved from the departmental archives and subjected to immunohistochemical analysis of hTERT. The expression of hTERT increased from normal, OSMF, to OSCC with statistically significant differences in mean labelling score (LS). We also found a shift in cellular localization of stain where, normal mucosal tissues showed a nuclear stain unlike OSMF, where combined nuclear and cytoplasmic staining as noted. The tumor cells in OSCC showed predominant cytoplasmic staining. There was no correlation between hTERT expression and clinicopathological parameters of OSMF. However, a significant increase of hTERT expression was seen with increasing histological grading of OSCC. These results suggest the role of hTERT in the early event of malignant transformation of OSMF. Telomerase could be used as a potent diagnostic marker to identify high-risk group of OSMF.

Immunohistochemical expression of polo-like kinase 1 in oral squamous cell carcinoma and oral submucous fibrosis.

CONTEXT: Polo-like kinase 1 (PLK1) is a critical molecule in the proliferation of several human cancers. Overexpression of PLK1 has been correlated with cancer cell proliferation and lower overall survival rates. Although PLK1 has been studied in various tumors, information regarding its expression in oral cancer and precancer is limited. Aims: This study is aimed at evaluating the expression of PLK1 in a potentially malignant and malignant disorder of the oral cavity, namely, oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively, using the immunohistochemistry technique. It also intended to evaluate the association of the various histological grades of OSCC with the intensity of PLK1 expression. SUBJECTS AND METHODS: Thirty OSMF, thirty OSCC tissues, and thirty control tissues were obtained, and the expression of PLK1 was detected by immunohistochemistry using rabbit antihuman PLK1 polyclonal antibodies (Abcam Ab47867). The association between staining intensity and histological grade of OSCC was evaluated. STATISTICAL ANALYSIS USED: Using SPSS 20 version, a test for proportions, nonparametric Chi-square/correlation analysis was used to compare differences in proportions of categorical variables of interest between groups. Results: PLK1 was positively expressed in 27 (90%) OSCC tissues. OSMF showed no detectable staining in 27 (90%) tissues and positive staining in 3 (10%) tissues. PLK1 showed no staining (0%) in normal tissues. Statistically significant associations were not found between staining intensity and histological grade of OSCC. CONCLUSIONS: PLK1 could be a promising progression marker for OSCC. Therapeutically, targeting PLK1 may be a new approach to fight oral cancer.

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation.

OBJECTIVES: Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation. MATERIALS: Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2', 7'-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. RESULTS: TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05). CONCLUSIONS: Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation. CLINICAL RELEVANCE: TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

Evaluation of lactate dehydrogenase enzyme activity in saliva and serum of oral submucous fibrosis patients.

BACKGROUND: Of all oral precancerous conditions, Oral Submucous Fibrosis is of greater concern because of its disabling nature and relative greater chances of malignant transformation. This malignant transformation involves glycolytic pathways that can alter lactate dehydrogenase levels. Therefore the aim of this study was to estimate the LDH levels in saliva and serum of subjects with OSMF and to compare them with healthy controls and to correlate the relationship between pathogenesis of OSMF and the LDH enzyme. METHODS: Sixty Subjects were recruited for this study and divided into two groups, 30 subjects with OSMF (Group A) and 30 healthy controls (Group B). Venous blood and unstimulated whole saliva measuring 1 ml was collected from each of these evaluated for LDH levels using the standard kit method. The data obtained were subjected to statistical analysis using the SPSS software version 17. RESULTS: The average salivary LDH value for Group A was 606.83 ± 60.09 U/l and for Group B was 80.73 ± 20.06 U/l. salivary LDH was greater in group A than Group B and this was statistically significant. On comparing the serum and salivary LDH in Group A with the clinical staging of OSMF, the results were not statistically significant. Similarly no statistically significant relationship was found on comparing the serum and salivary LDH in Group A (OSMF subjects) with duration of habit. CONCLUSION: This study provides additional rationale for the role of salivary LDH in the early diagnosis and prognosis of oral submucous fibrosis.

Increased expression of carbonic anhydrase IX in oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: Cumulative evidence has demonstrated that carbonic anhydrase IX (CAIX) is upregulated in many types of human cancers. We attempted to evaluate plasma levels of CAIX in patients with oral cancer and investigated whether plasma CAIX is correlated with the progression of this disease. METHOD: In total, 191 patients with oral cancer, 30 patients with oral submucous fibrosis and 100 controls were recruited in this study. The plasma samples were collected and the levels of soluble CAIX in plasma were determined by the enzyme-linked immunosorbent assay (ELISA). Furthermore, the normal buccal mucosa fibroblast was challenged by arecoline, the major areca nut alkaloid, to assess the relationship between the levels of CAIX and areca nut chewing in oral cancer patients. RESULTS: Results showed that patients with oral cancer exhibited significantly higher levels of soluble CAIX compared to controls (p<0.001). Plasma levels of CAIX in oral cancer patients were associated with clinical stages after adjusting for age and areca nut chewing (p<0.05). In addition, patients with areca nuts chewing had higher CAIX levels than those who have not chewed areca nuts. Total carbonic anhydrase activity and CAIX mRNA levels were significantly higher in oral submucous fibrosis fibroblasts than in normal buccal mucosa fibroblasts. Moreover, arecoline elevated CAIX expression in a dose-dependent manner in normal buccal mucosa fibroblasts. CONCLUSIONS: Our results suggest that determining plasma levels of CAIX may be used as a non-invasive method for monitoring oral cancer progression and the involvement of areca quid chewing in oral carcinogenesis may be related to a higher expression of CAIX.

EGCG blocks TGFß1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts.

OBJECTIVES: Transforming growth factor ß (TGFß) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) and cyclooxygenase-2 (COX-2) were found to overexpress in OSF. The aim of this study was to investigate the molecular mechanism underlying the TGFß-induced CCN2 expressions in human buccal mucosal fibroblasts (BMFs) to identify the potential targets for drug intervention or chemoprevention of OSF. MATERIALS AND METHODS: TGFß-induced CCN2 expression and its signaling pathways were assessed by Western blot analyses in BMFs. RESULTS: TGFß1 stimulated CCN2 synthesis in BMFs. Pretreatment with c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, and activin receptor-like kinase 5 (ALK5) inhibitor SB431542 significantly reduced TGFß1-induced CCN2 synthesis. Epigallocatechin-3-gallate (EGCG) completely blocked TGFß1-induced CCN2 synthesis by inhibiting the phosphorylation of JNK and p38 MAPK. Prostaglandin E(2) (PGE(2)) inhibited the TGFß1-induced CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs. CONCLUSIONS: The TGFß1-induced CCN2 synthesis in BMFs could be mediated by the ALK5, JNK, and p38 MAPK pathways. EGCG blocks TGFß1-induced CCN2 by suppressing JNK and p38 in BMFs. CLINICAL RELEVANCE: The exceptional signal transduction pathways of TGFß1-induced CCN2 production in BMFs contribute to the resistance of PGE(2) downregulation of CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. EGCG may serve as a useful agent in controlling OSF.

Regulation of protease-activated receptor-1 expression in human buccal fibroblasts stimulated with arecoline.

BACKGROUND: The purpose of this study was to compare the major thrombin receptor protease-activated receptor-1 (PAR-1) expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanisms that may lead to induce PAR-1 expression. METHODS: Thirty OSF and 10 normal buccal mucosa specimens were examined by immunohistochemistry. Buccal mucosal fibroblasts (BMFs) were challenged with arecoline by using Western blot analysis. N-acetyl-L-cysteine (NAC), LY294002, herbimycin A, NS-398, and PD98059 were added to find the possible regulatory mechanisms. RESULTS: PAR-1 expression was significantly higher in OSF specimens (p < .05). Arecoline was found to elevate PAR-1 expression in a dose-dependent and time-dependent manner (p < .05). The addition of NAC, LY294002, herbimycin A, NS398, and PD98059 markedly inhibited the arecoline-induced PAR-1 expression (p < .05). CONCLUSION: PAR-1 expression is significantly upregulated in areca quid chewing-associated OSF. Arecoline-induced PAR-1 expression was downregulated by NAC, LY294002, herbimycin A, NS398, and PD98059.

Estimation of superoxide dismutase and glutathione peroxidase in oral submucous fibrosis, oral leukoplakia and oral cancer--a comparative study.

Present study was undertaken to estimate and compare erythrocyte superoxide dismutase (E-SOD) and Glutathione peroxidase (GPx) levels in oral submucous fibrosis, oral leukoplakia and oral cancer patients and age/sex matched healthy subjects, 25 in each group. Statistically significant (P<0.001) decrease in E-SOD and GPx levels were observed in OSF, oral leukoplakia and oral cancer groups as compared to the control group. Oral leukoplakia group showed lower levels in comparison with OSF (P>0.05). Oral cancer group had the lowest levels amongst the study groups. Imbalance in antioxidant enzyme status may be considered as one of the factors responsible for the pathogenesis of cancer and may serve as a potential biomarker and therapeutic target to reduce the malignant transformation in oral premalignant lesions/conditions.

Association of CYP1A1, GSTM1, and GSTT1 gene polymorphism with risk of oral submucous fibrosis in a section of North Indian population.

Genetic alterations in the genes expressing drug metabolizing enzymes can make an individual susceptible to various cancers. This study detects the polymorphisms at CYP1A1, GSTM1, and GSTT1 genes in a section of North Indian population and determines the susceptibility to oral submucous fibrosis (OSF). In this case-control study one hundred and two OSF patients were genotyped to detect the GSTM1, GSTT1, CYP1A1 polymorphism. Two hundred healthy controls were also included. Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The frequency of GSTM1 and GSTT1 genotype was higher in OSF patients, as compared to controls. A trend risk analysis showed 7.6 fold increase in risk, when both the genes were absent. The frequency of CYP1A1 (m1) and CYP1A1 (m2) genotypes was higher in controls. No polymorphic alleles were detected in the m4 site. CYP1A1 (m1) wild genotype in the absence of GSTM1 null genotype, falls under the highest risk group (OR 3.74). Our findings suggest that CYP1A1 (m1) genotype and (m2) genotype singly acts as a protective factor but in the absence of GSTM1 and/or GSTT1 gene significantly alters risk towards OSF.

Increased secretion of tissue inhibitors of metalloproteinases 1 and 2 (TIMPs -1 and -2) in fibroblasts are early indicators of oral sub-mucous fibrosis and ageing.

Oral submucous fibrosis (OSMF) is associated with paan chewing, altered collagen metabolism, inflammation and the upregulation of numerous cytokines. OSMF fibroblasts accumulate senescent cells at an increased rate because of increased reactive oxygen species production and DNA double-strand breaks (DDBs), generated intrinsically by damaged mitochondria. This results in a reduced replicative lifespan. However, it is still unclear which other changes are intrinsic to the fibroblasts and associated with OSMF rather than the paan chewing habit or the OSMF environment. Both the oral epithelium and the mesenchyme have elevated levels of TGF-ß(1) in OSMF in vivo. However, in cultured fibroblasts, secreted levels of TGF-ß(1,) other cytokines and the matrix metalloproteinases 1 and 2 showed no association with OSMF. In contrast, the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, were increased in 10/11 OSMF fibroblast cultures relative to normal and non-diseased paan user controls. OSMF fibroblast collagen levels were normal. TIMP levels correlated with replicative lifespan of the cultures but not with the presence of senescent cells, as senescent cell depletion in OSMF fibroblast cultures did not result in a reduction in either TIMP-1 or TIMP-2. However, the introduction of unrepairable DDBs into normal oral fibroblasts by ionizing radiation increased TIMP-1 and TIMP-2 secretion by two-fold and seven-fold, respectively, within 5 days, replicating early senescence and the elevation seen in OSMF cultures. Therefore, increased fibroblast TIMP-1/2 levels could be early disease-specific markers of OSMF onset, DDBs and ageing and may have clinical significance, as OSMF can be reversed in its early stages.

Effect of methylxanthines (coffee/tea consumers) on oral precancer and oral cancer patients with smoking and smokeless tobacco habits.

AIM: To study, whether the consumption of regular tea/coffee (methylxanthines) increases the risk of oral cancer in patients with smoking and smokeless tobacco habits. MATERIALS AND METHODS: This study was conducted on a total of 90 oral cancer and precancerous patients, from western Maharashtra (India) males in the age group of 20 to 45 years who were with smoking and smokeless tobacco habits; also regular tea/coffee consumers were subjected to biochemical parameters such as aspartate transaminase (AST) and alanine transaminase (ALT) from saliva and serum of patients with oral precancer (submucous fibrosis, leukoplakia) and oral cancer patients and compared with 90-age and sex-matched controls. Individuals consent was taken to measure their biochemical parameters, by using Hafkenscheid method in whole saliva and serum. Statistical analysis of variance (ANOVA) with Tukey's correction for multiple group comparisons was performed using Student t-test. RESULTS: Results show, that a statistically significant increase in value (p < 0.05) in ALT, AST in both saliva and serum was observed in precancerous and oral cancer patients among the study group as compared to the control group. CONCLUSION: In the present study, there was increase in the levels of ALT, AST enzymes in both saliva and serum levels in the study group as compared to the control group which was statistically significant (p < 0.05) suggesting that long-term exposure of methylxanthines results in impairment of salivary gland antioxidant system which may affect the anticarcinogenic action of saliva. CLINICAL SIGNIFICANCE: Oral fluids may be utilized effectively to study the variations in the biochemical constituents of saliva of leukoplakia, submucous fibrosis and oral cancer patients.

Association of XRCC1, XRCC3, and NAT2 polymorphisms with the risk of oral submucous fibrosis among eastern Indian population.

BACKGROUND: Arecanut and smokeless tobacco usage is a major cause for oral submucous fibrosis (OSF) and its subsequent development to oral squamous cell carcinoma in South-east Asian population. Polymorphisms at N-acetyltransferase 2 locus, coding for an enzyme catalyzing acetylation of aromatic amines, might cause DNA adduct formation because of improper acetylation of these polyaromatic hydrocarbons. DNA repair enzymes remove these adduct to prevent malignancy. METHODS: In this hospital-based study, 100 controls and 88 OSF patients were genotyped at four polymorphic sites on NAT2 481 (C > T; silent), 590 (G > A; Arg197 > Gln), 803 (A > G; Lys268 > Arg), 857 (G > A; Gly286 > Glu) and two on XRCC1 18067 (C > T Arg 194 > Trp), 28152 (G > A Arg 399 > Gln), and one of XRCC3 26304 (C > T Thr 241 > Met) loci by PCR-RFLP to determine the risk of the disease. RESULTS: Heterozygous XRCC3 codon 241 [OR 2.07 (1.05-4.06)], homozygous variant of NAT C481T [OR 2.81 (1.09-7.21)], and both heterozygous and homozygous variants of NAT codon 268 and 286 [OR 2.31 (1.20-4.45) and 4.98 (1.87-13.14), and 6.12 (2.75-13.62) and 2.65 (1.04-6.72)] individually influenced susceptibility to OSF in the population. CONCLUSION: Gene-gene interaction analysis by multifactor dimensionality reduction (MDR) revealed that XRCC3 Thr 241 Met had the largest univariate effect followed by XRCC3 Thr 241 Met - NAT2 A857G in men that presents a highly synergistic interaction as one of the potential combinations of single nucleotide polymorphisms (SNPs) to increase the risk of OSF in men if exposed to arecanut or smokeless tobacco usage. These observations can speculate the impact of the studied SNPs on the etiology of OSF.

Immunohistochemical detection of human telomerase reverse transcriptase in oral cancer and pre-cancer.

PURPOSE: Telomerase is a specialized ribonucleoprotein complex that stabilizes telomeres by adding "TAG" repeats to the end of chromosomes. The catalytic subunit of telomerase is human telomerase reverse transcriptase (hTERT), whose expression is the critical determinant of telomerase activity. Telomeres and telomerases play an important role in the longevity of cell and are known to conform "immortalization" on neoplastic cells. Although there exists a lot of information on telomerase in oral cancer, very little is known about their expression in leukoplakia and oral submucous fibrosis (OSF). This study addresses this lacuna. MATERIALS AND METHODS: In this preliminary study, immunohistochemistry (IHC) was used to detect the expression of hTERT protein in oral squamous cell carcinoma (OSCC) (n=30), leukoplakia (n=15), OSF (n=15) and normal oral mucosa (n=10). The cellular localization of immunostain, intensity of stain, mean nuclear labeling index (LI) and mean nuclear labeling score (LS) of hTERT protein were studied. A total number of 1000 cells were counted in each slide. All the data were analyzed using SPSS software version 10.0.2. The cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity and LI were compared across the groups using Pearson's χ2 test. The mean LI and LS for OSF, leukoplakia, OSCC and normal were compared using analysis of variance (ANOVA). A P-value <0.05 was considered to be statistically significant. RESULTS: The mean nuclear LI increased from OSF (22.46±4.53), through normal (28.3±12.3) to OSCC (47.56±21.30) (P=0.002) and from normal (28.3±12.3), through leukoplakia (44.06±14.6), to OSCC (47.56±21.30) (P=0.00). The mean nuclear labeling score was observed to increase from OSF (37.8±15), through normal (64.9±30.7), to OSCC samples (106.9±29.77) (P=0.00) and from normal (64.9±30.7), through leukoplakia (85.6±25.1) to OSCC samples (106.9±29.77) (P=0.00). CONCLUSION: There was increased expression of hTERT protein in OSCC and leukoplakia samples when compared to normal oral mucosa. The cellular localization, LI and LS in OSF were significantly different from OSCC and leukoplakia.

Matrix metalloproteinase-1 expression in oral submucous fibrosis: an immunohistochemical study.

CONTEXT: Oral submucous fibrosis (OSF) is a form of pathological fibrosis affecting the oral mucosa. There is compelling evidence to implicate the habitual chewing of areca nut with the development of OSF. Because collagens are the major structural components of connective tissues, including oral submucosa, the composition of collagen within each tissue needs to be precisely regulated to maintain tissue integrity. Arecoline stimulates fibroblasts to increase the production of collagen by 150%. AIM: As the role of collagenase is implicated in cleaving the collagen under physical conditions, this study was carried out to evaluate the role of collagenase-1 (matrix metalloproteinase [MMP]-1) in a pathologic condition like OSF. SETTINGS AND DESIGN: A total of 40 patients were included in the study, comprising of 30 OSF as Group 1 and 10 normal buccal mucosa tissue as Group 2. MATERIALS AND METHODS: Both the groups were stained for MMP-1 by the immunohistochemical method using the streptavidin HRP-biotin labeling technique. MMP-1 expression intensity in the epithelium and connective tissue was decreased in Group 1 when compared to Group 2. STATISTICAL ANALYSIS USED: Chi-square test of association was used to determine the difference in the expression of MMP-1 between OSF and normal buccal mucosa and among different histological gradings of OSF. RESULTS: The results were statistically significant. However, there was no statistically significant difference between the expression of MMP-1 among different histological grades of OSF in Group 1.

Expression of BUBR1 in human oral potentially malignant disorders and squamous cell carcinoma.

OBJECTIVE: BUBR1 is one of the key components of the spindle assembly checkpoint (SAC) machinery and is activated in response to kinetochore tension. Defects in the SAC contribute to an increased rate of aneuploidization during tumorigenesis. The aim of the present study was to examine the immunohistochemical expression of BUBR1 protein for human oral squamous cell carcinogenesis. STUDY DESIGN: A total of 120 samples of squamous cell carcinoma (SCC, n = 43) and 5 types of potentially malignant disorders (PMDs: oral epithelial dysplasia, n = 11; hyperkeratosis/epithelial hyperplasia, n = 20; lichen planus, n = 16; submucous fibrosis, n = 19; and verrucous hyperplasia, n = 11) of human oral mucosa (1991-2001) from our institution were retrieved and immunohistochemical staining were performed. Normal oral mucosa (n = 9) and fibrous hyperplasia (n = 9) from patients without the aforementioned oral habits were also included in the study. RESULTS: BUBR1 staining was detected at the basal and suprabasal layers in 75 (97.4%) of 77 samples of PMD and 43 (100%) of 43 samples of SCC of oral mucosa but was absent in all samples of normal oral mucosa (n = 9) and fibrous hyperplasia (n = 9). BUBR1 expression of various types of PMD and SCC of oral mucosa was significantly overexpressed as compared respectively with normal mucosa (P < .001) and fibrous hyperplasia (P < .001). Moreover, the expression of oral SCC was significantly higher as compared respectively with the 5 types of oral PMD; on the other hand, BUBR1 expression of verrucous hyperplasia was significantly higher than that of the other 4 types of PMD of oral mucosa (P < .001). CONCLUSION: Our results may interpret that BUBR1 protein is suggested to be one of the contributing factors involved in the pathogenesis of oral SCC. These also hypothesize that BUBR1 protein is a putative biomarker for human oral squamous cell carcinogenesis.

Augmented heme oxygenase-1 expression in areca quid chewing-associated oral submucous fibrosis.

OBJECTIVES: Heme oxygenase-1 (HO-1) is known as a stress-inducible protein and functions as an antioxidant enzyme. HO-1 is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare HO-1 expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce HO-1 expression. METHODS: Twenty OSF specimens and 10 normal buccal mucosa were examined by immunohistochemistry. The mRNA levels of HO-1 from fibroblasts cultured from OSF and normal buccal mucosa fibroblasts (BMFs) were evaluated by reverse transcription polymerase chain reaction. The effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanism that may lead to induce HO-1 expression. RESULTS: Heme oxygenase-1 expression was significantly higher in OSF specimens (P < 0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher HO-1 mRNA expression than BMFs (P < 0.05). Arecoline was also found to elevate HO-1 mRNA and protein expression in a dose-dependent manner (P < 0.05). CONCLUSIONS: Taken together, the data presented here demonstrated that HO-1 expression is significantly upregulated in OSF from areca quid chewers, and arecoline may be responsible for the enhanced HO-1 expression in vivo.

Transglutaminase-2 regulation by arecoline in gingival fibroblasts.

Transglutaminase-2 (TGM-2) stabilizes extracellular matrix (ECM) proteins by cross-linking and has been implicated in several fibrotic disorders. Arecoline present in betel quid has been proposed as one of the causative factors for oral submucous fibrosis (OSMF). Hence, we hypothesize that arecoline may regulate TGM-2 and may have a role in the pathogenesis of OSMF. The expression of TGM-2 was studied in OSMF tissues by real-time RT-PCR analysis, and significant overexpression was observed in most OSMF tissues (P=0.0112) compared with normal tissues. Arecoline induced TGM-2 mRNA and protein expression as well as TGM-2 activity in human gingival fibroblast cells. The addition of methocramine hemihydrate (M-2 muscarinic acetylcholine receptor selective antagonist) or 8'-bromo-cAMP abolished arecoline-mediated TGM-2 induction, suggesting a role for M-2 muscarinic acid receptor and a repressor role for cAMP. Our study provides evidence for TGM-2 overexpression in OSMF and its regulation by arecoline in oral fibroblasts.

Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers.

OBJECTIVE: Areca use is the major cause for oral squamous cell carcinoma and oral submucous fibrosis (OSF) in South Asians. Lysyl oxidase (LOX) is a copper-activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The presence of a G to A polymorphism at nucleotide 473 caused a non-conservative Arg158Gln change in the LOX amino acid sequence. OSF is a precancerous lesions characterized by the accumulation of collagen in oral submucosa. The aim of this study was to investigate the relationship between LOX Arg158Gln polymorphism and the risk of OSF. METHOD: PCR-restriction fragment length polymorphisms and direct sequencing was utilized to compare LOX polymorphic allelotype in male areca-chewing controls (n = 216) and OSF (n = 83) patients. RESULTS: There was a borderline of statistically significant difference in Arg158Gln genotype lying between control and OSF patients. However, the G/A+A/A of LOX Arg158Gln in OSF patients older than 50 year was statistically significantly higher than controls older than 50 year (odd's ratio: 4.48; 95% CI = 1.58-12.67). CONCLUSION: The elder OSF patients were increased in LOX Arg158Gln. Our findings may suggest a potential application in risk population selection using LOX polymorphism for preventive intervention of OSF genesis in a subset of areca chewers.

Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis.

OBJECTIVE: We tested the hypothesis that inducible nitric oxide synthase (iNOS) modulates angiogenesis in human models and this information could be extrapolated in elucidating the pathophysiology of oral submucous fibrosis (OSF). A hypothesis which looks inadequate, but is deep rooted in literature is the epithelial alteration ("atrophy") seen in OSF and the events that lead to its causation. This aspect was tried to be addressed and an alternative pathogenetic pathway for the disease is proposed. MATERIALS AND METHODS: This immunohistochemical study sought to investigate the expression of iNOS in OSF samples (n=30) a using monospecific antibody (SC- 2050, Santa Cruz Biotechnology, Inc) to the protein and also to correlate it with different grades of epithelial dysplasia associated with the disease. Twenty (20) healthy adults acted as controls. RESULTS: iNOS staining was not demonstrated in normal oral epithelium. In oral epithelial dysplasia, staining was seen in all cases (100%) in the basal layers of the epithelium and in 30% of cases it extended into the parabasal compartments as well. iNOS staining was uniformly positive in moderate dysplasia with an increase in intensity and distribution noted as the severity of dysplasia progressed. There were highly significant differences in overall positivity for iNOS in epithelium between cases and controls (Mann-Whitney U=11.000, Wilcoxon W=221.00, P=0.000). Significant comparisons were made of mild Vs moderate dysplasia (Mann-Whitney U=48.000, P=0.014) CONCLUSIONS: This study supports our earlier morphological assessment (image analysis) of the nature of vascularity in OSF mucosa. The significant vasodilation noticed in these cases argues against the concept of ischemic atrophy of the epithelium. This observation of vascularity and iNOS expression helped to explain the vasodilation noticed (sinusoids) in this disease; NO being a net vasodilator. The mechanism of activation of iNOS in dysplasia is difficult to explain. The role of contingent paracrine-activating factors on keratinocytes and macrophages is discussed.

Functional -1562 C-to-T polymorphism in matrix metalloproteinase-9 (MMP-9) promoter is associated with the risk for oral squamous cell carcinoma in younger male areca users.

BACKGROUND: Circulating matrix metalloproteinase-9 (MMP-9) is a prognostic factor for gastric cancer and vascular diseases, and has been associated with head and neck cancers. The -1562 C-to-T polymorphism in MMP-9 promoter (abbreviated MMP-9 -1562 C>T polymorphism) leads to differential transcription, and is associated with increased susceptibility to neoplastic and vascular diseases. Thus, our aim was to determine whether a functional MMP-9 polymorphism might also influence the risk or affect the progression of areca-associated oral cancers. METHODS: Genomic DNAs were obtained from peripheral blood cells of male subjects with areca-associated oral squamous cell carcinoma (OSCC) (n = 192), oral submucosal fibrosis (OSF) (n = 73), and non-diseased areca users (n = 191). The PCR-based restriction fragment length polymorphism analysis was performed for MMP-9 genotyping. RESULTS: MMP-9 -1562 C>T polymorphism was not associated with the risk of OSCC or OSF. However, when subjects were stratified by the median age, an association with the risk of OSCC was found in younger patients (P = 0.029). The T allele frequency was significantly higher in the subset of older patients with buccal mucosa OSCC than older patients with OSCC in counterpart locations. The joint MMP-9 -1562 C>T and MMP-3 -1171 5A>6A functional polymorphisms were not associated with OSCC risk or patient survival. CONCLUSION: Aberrant MMP-9 expression is closely related to tumor invasiveness and the prognosis of head and neck cancers. However, functional MMP-9 -1562 C>T polymorphism is associated with OSCC risk only in younger areca chewers. The impact of aging or areca-related effect on this functional polymorphism should be elucidated.