Pepsin Increases the Proliferation of Vocal Cord Leukoplakia Epithelial Cells by Inducing Autophagy.

OBJECTIVE: To investigate the role of H+ /K+ ATPase in the proliferation of pepsin-induced vocal cord leukoplakia (VCL) cells. STUDY DESIGN: Translation research. SETTING: Affiliated Hospital of University. METHODS: Immunohistochemistry was used to detect pepsin, H+ /K+ ATPase (ATP4A and ATP4B subunits) in VCL cells with varying degrees of dysplasia. After primary cultures of VCL cells had been established, the effects of acidified pepsin on the proliferation, autophagy, and H+ /K+ -ATPase distribution of VCL cells were investigated. RESULTS: The levels of pepsin, ATP4A, and ATP4B were significantly higher in VCL tissue with moderate-to-severe dysplasia than in normal tissue (p < .05); these levels gradually increased according to dysplasia severity. The expression levels of ATP4A and ATP4B were significantly correlated with the amount of pepsin in VCL cells (p < .01). Acidified pepsin enhanced the levels of proliferation and autophagy in human VCL epithelial cells. The cloning- and autophagy-promoting effects of acidified pepsin on VCL cells were partially reversed by pantoprazole; these effects were completely blocked by the autophagy inhibitor chloroquine. Finally, acidified pepsin promoted the colocalization of H+ /K+ -ATPase and lysosomes in VCL cells; it also mediated lysosome acidification. CONCLUSION: Pepsin and H+ /K+ -ATPase may contribute to the progression of VCL. Specifically, acidified pepsin may regulate lysosome acidification by promoting lysosomal localization of H+ /K+ -ATPase.

Evaluation of P53 gene expression by immunohistochemistry to diagnosis oral precancerous lesions.

Oral Precancerous lesions include leukoplakia, erythroplakia, and mucosa palate changes due to reverse smoking. Assessing the prevalence of these lesions in a cross-sectional study can be effective in the timely prevention and treatment of lesions, in any community. Hence, in the present study, evaluation of P53 gene expression was done by immunohistochemistry method to diagnosis oral precancerous lesions. For this purpose, 111 Chinese patients (54 women and 57 men) were selected for examination. The age range of these patients was 22 to 69 years, and their average age was 32.6 years. All patients were examined by one physician. Oral mucosa was used for immunohistochemical evaluations. All samples taken from patients' mucosa were evaluated by one pathologist under a light microscope. 80 cases of the 111 patients were smokers and 27 were non-smokers. Among the 80 smokers, 56.25% had leukoplakia, 3.75% had erythroplakia, and 40% had mucosa palate changes. Regarding non-smokers, 74.07% had leukoplakia and 25.93% had erythroplakia. None of the non-smokers had mucosa palate changes. In terms of the lesion location, in patients with leukoplakia 89.23%, and patients with erythroplakia 90% of the lesion was located in the cheek mucosa and buccal vestibule. Also, in patients with leukoplakia 9.23%, and patients with erythroplakia 10% of the lesion was located in the lips vestibular mucosa. Only 1.54% of leukoplakia had a lesion in the vermilion border, and none of the erythroplakia patients had a lesion on the vermilion border. 76 patients (68.46%) showed positive expression of the P53 gene. The expression level of the P53 gene did not show a significant relationship with age, and the genders did not have a statistically significant difference in terms of gene expression. The expression level of the P53 gene was 59.8% in leukoplakia, 70% in erythroplakia, and 40% in Mucosa palate changes. The present study showed that the evaluation of P53 gene expression was well able to detect oral precancerous lesions and their severity by increasing their expression rate.

Relationship Between Pepsin Expression and Dysplasia Grade in Patients With Vocal Cord Leukoplakia.

OBJECTIVE: To measure pepsin expression in patients with vocal fold leukoplakia and elucidate its clinical significance. STUDY DESIGN: Retrospective analysis of pathologic archive specimens. SETTING: Affiliated university hospital. SUBJECTS AND METHODS: The study included 45 patients with vocal fold leukoplakia and 19 with vocal fold polyps who underwent surgical treatment between December 2013 and July 2016. Masses were detected on both vocal cords in 5 patients with vocal fold leukoplakia and in 1 patient with vocal fold polyps. Immunohistochemistry was used to assess pepsin expression. In addition, the relationship of pepsin expression level with clinical characteristics of vocal fold leukoplakia was assessed. RESULTS: The rate of pepsin expression was high in the polyp group (75%) and the leukoplakia group (68%); however, the difference between groups was not significant (P > .05). Pepsin expression significantly increased according to grade of dysplasia (mild, 57.1%; moderate, 88.9%; severe, 100.0%; P = .034). Similarly, the percentage of lesions that exhibited strongly positive pepsin expression increased with the grade of dysplasia (mild, 37.1%; moderate, 66.7%; severe, 100.0%; P = .005). The leukoplakia recurrence rate was higher in patients with positive pepsin expression than in patients with negative pepsin expression but without a significant difference (P > .05). CONCLUSION: Our study suggests that pepsin was associated with the grade of dysplasia of vocal cord leukoplakia. Further investigation with appropriate control groups and controlling for other risk factors, such as smoking or alcohol consumption, is needed.

Significance of Cytokeratin-1 Single-Nucleotide Polymorphism and Protein Level in Susceptibility to Vocal Leukoplakia and Laryngeal Squamous Cell Carcinoma.

OBJECTIVE: To investigate the association between the cytokeratin (CK)-1 single-nucleotide polymorphism (SNP), the protein level of CK-1 and the risk of vocal leukoplakia and laryngeal squamous cell carcinoma (LSCC). METHODS: In this case-control study, 155 patients with vocal leukoplakia, 323 patients with LSCC, and 266 healthy controls were genotyped for the CK-1 (SNP RS14024) gene using pyrosequencing. The protein expression level of CK-1 was analyzed in vocal leukoplakia, LSCC, and vocal polyp patients by immunohistochemistry (IHC). RESULTS: Of the CK-1 RS14024 polymorphism, the heterozygote AG and homozygote GG genotype exhibited a significantly increased risk of LSCC (AG: OR = 2.16, p = 0.014; GG: OR = 2.15, p = 0.018) compared to normal controls. A higher protein expression level of CK-1 was detected in patients with LSCC compared to vocal leukoplakia and polyps (both p < 0.001), and a significant increasing trend of CK-1 protein expression level from mild-moderate dysplasia to moderate-severe dysplasia in vocal leukoplakia patients was also observed (p = 0.006). CONCLUSIONS: This study demonstrates that the CK-1 SNP and high protein expression levels are associated with vocal leukoplakia and LSCC and promote the transformation from vocal leukoplakia to LSCC in a Chinese Han population.

[The expression characteristics and clinical significance of candidate molecular markers in vocal cord leukoplakia].

Objective: To investigate the expression of marker proteins in vocal cord leukoplakia, and to find markers for the early stage of diagnosis and prognosis of precancerous lesions. Methods: The study included 119 cases, 68 cases of vocal cord leukoplakia (22 cases with epithelial simple hyperplasia, 46 cases with epithelial dysplasia), and 51 cases of vocal cords benign lesions(31 cases of vocal cord polyps, 20 cases of Reinke's edema). The expression of p53, Ki-67, p21, Survivin, p16, p27, PTEN, c-Myc and vascular endothelial growth factor (VEGF) in vocal cords leukoplakia were detected, vocal cord benign lesions (vocal cord polyps and Reinke's edema) acted as controls, comparing the expression differences of different pathological tissue. Data was analyzed by SPSS 22.0 software. Results: The expression of p53, p16, Ki-67, VEGF in vocal cord benign lesions and vocal cords leukoplakia with epithelial simple hyperplasia did not show significant differences. There was a grading increase in the positive expression of p53, Ki-67 in the vocal cord leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions and vocal cords leukoplakia with epithelial simple hyperplasia (p53: χ(2)=13.340, P=0.002, Ki-67: χ(2)=53.386, P=0.000). The expression of p27, PTEN, c-Myc in vocal cord benign lesions and vocal cords leukoplakia with epithelial dysplasia did not show significant differences. There was a grading increase in the positive expression of p21 Survivin in vocal cords leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions (P<0.05). Expression of Survivin in vocal cords leukoplakia with mild-moderate epithelial dysplasia showed a significant increase than those in vocal cord benign lesions (P<0.05). The positive expression grade of p21 showed a rising trend (P=0.073) between the different grades of dysplasia. Conclusion: The positive expression grade of p53, Ki-67, p21 Survivin showed an increase in vocal cords leukoplakia with epithelial dysplasia contrasting to those in vocal cord benign lesions, which might be an implication for evaluating the diagnosis and prognosis of precancerous lesions. Expression of p21 was correlated to the degrees of dysplasia and expression of Survivin showed a significant difference in early stage of epithelial dysplasia contrasting to benign lesions.

[The role of NF-κB signaling pathway in laryngeal leukoplakia recurrent with laryngeal reflux].

Objective: To study the mechanism of vocal mucosal barrier damage mediated by NF-κB and NF-κB-regulated signaling pathway via probing the expression of inflammatory factors and essential proteins for node of NF-κB signaling pathway. Methods: The patients suffering from vocal leukoplakia accompanied with larygopharyngeal reflux(LPR) were treated with oral administration of proton pump inhibitor(PPI). Mucosal specimens of vocal cord were collected from all patients before PPI treatment. And the mucosal specimens of vocal cord were collected from the patients with suspected recurrence at 8 weeks after PPI treatment. HE staining was used to observe the histopathological changes of the mucosa. ELISA was utilized to detect the levels of inflammatory factors including tumor necrosis factor(TNF)-α, interleukin(IL)-1 and IL-6. Western blot was used to detect the expression of p-p65, p-IKK and p-IκB. Immunofluorescence method was adopted to detect the entrance of p65 to cell nucleus.Data was analyzed by SPSS 23.0 software. Results: In PPI untreated group, the expressions of IL-1ß, TNF-α and IL-6 in the specimens of 8 weeks after operation were not different significantly from those obtained during operation.But in the PPI-treated group, the expressions were down-regulated.The expression of p-p65 in the middle and high grade heterogenous hyperplasia group was higher than that of low level heterogenous hyperplasia group.The difference of p65 and p-p65 expression between 8 weeks after surgery and surgery in PPI-untreated group was statistically insignificant (P>0.05). The difference of p65 expression between PPI-treated group and PPI pre-treatment group was statistically insignificant (P>0.05). The expression of p-p65 in the PPI-treated group was lower than that of the PPI pre-treatment group (P<0.05). The expressions of IL-1ß, TNF-α and IL-6 were positively related with that of NF-κB-p65. Immun of luorescence method revealed the entrance of p65 to cell nucleus in PPI pre-treatment group, which meant that NF-κB was activated. In the PPI-treated group, few activated p65 could be observed in the cell nucleu. Conclusion: The possible mechanism of vocal mucosal barrier damage in vocal leukoplakia accompanied with LPR maybe the vocal mucosal inflammation mediated by NF-κB and NF-κB-regulated signaling pathway activated with refluxed materials.

Biomarkers of Oxidative Stress Associated with the Risk of Potentially Malignant Oral Disorders.

AIM: To investigate the effect of oxidative stress biomarkers on the risk of potentially malignant oral disorders (PMODs). MATERIALS AND METHODS: A total of 208 male adults with PMODs and an equal number of same-age control patients were enrolled. Plasma biomarkers of oxidative stress, measured with 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), were determined using enzyme-linked immunosorbent assay kits. PMODs were diagnosed in accordance with the World Health Organization (WHO) guidelines. RESULTS: A significant association between a high level of 8-ISO and an increased risk of PMODs was identified [odds ratio (OR)=1.71, 95% confidence interval (CI)=1.12-2.63; p=0.013]. This positive association was stronger among patients with PMOD subtype of leukoplakia (OR=1.94, 95% Cl=1.24-3.06; p=0.004). However, no significant association was observed between plasma 8-OHdG levels and overall risk of PMODs or subtypes. CONCLUSION: Our findings suggest that increased plasma 8-ISO levels may indicate the prominence of lipid peroxidation in the development of PMODs, particularly leukoplakia.

Clinicopathological parameters associated with histological background and recurrence after surgical intervention of vocal cord leukoplakia.

Histological examination of biopsy shows usefulness in the diagnosis of vocal cord leukoplakia; however, in considerable amount of cases, the examination cannot provide definitive diagnosis of malignancy from benign conditions such as hyperplasia and dysplasia. The present work therefore was aimed to identify clinicopathological factors and molecular markers predictive of recurrence and malignant transformation of vocal cord leukoplakia.Clinical data of 555 cases of vocal cord leukoplakia enrolled from July 1999 to June 2014 were analyzed. The cohort consisted of keratosis (n = 137), hyperplasia (n = 139), dysplasia (n = 177), and primary (n = 10) and invasive (n = 46) carcinoma. Correlations between patients' backgrounds, clinicopathological factors, molecular markers (p53, p16, Ki67, cytokeratin, and proliferating cell nuclear antigen), and histology backgrounds were examined using by Pearson Chi-squared or Fisher exact test. Reflux symptom index (RSI) and reflux finding score (RFS) before and after treatment were compared using Wilcoxon signed-rank test. Risk factors for disease recurrence were identified using Cox proportional hazards models of multivariate analysis. Time to recurrence was analyzed using log-rank test of Kaplan-Meier method.In the present cohort, alcohol drinking was found associated with GRBAS grade (P = .0258) and the site (P = .0298) of leukoplakia. For the different disease types, chief complaint (P = .0179), GRBAS grade (P = .0101), mucosal wave (P < .0001), and molecular markers p53 (P < .0001) and Ki67 (P < .0001) were identified as correlates. RSI and RFS were significantly lowered by surgical intervention. A single side of leukoplakia was predictive of a lower risk of recurrence (odds ratio, 0.378; 95% confidence interval, 0.197-0.723; P = .0033). Absence of mucosal wave was associated with a shorter time-to-recurrence (P = .0357).The present work identified clinicopathological factors and molecular markers associated with the different histology of vocal cord leukoplakia, and also the prognostic factor for the low risk of recurrence after surgery.

Cortactin is a prognostic marker for oral squamous cell carcinoma and its overexpression is involved in oral carcinogenesis.

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.

[The expression and significance of pepsin in laryngeal carcinoma].

OBJECTIVE: To evaluate the relationship between pepsin induced by laryngopharyngeal reflux and laryngeal carcinoma. METHODS: Patients with vocal cord leukoplakia(n=18) and laryngeal carcinoma(n=21) encountered in Nanfang Hospital between December 2012 and April 2014 were included and sixteen healthy volunteers were recruited as control. Laryngeal biopsy specimens were taken from the patients with laryngeal carcinoma, or vocal cord leukoplakia and control subjects for the immunohistochemical study of pepsin. The correlation between pepsin expression and reflux events of 24 hour multichannel intraluminal impedance-pH monitoring (MII-pH) was analyzed. RESULTS: The patients with laryngeal carcinoma showed the highest expression of pepsin, followed by the patients with vocal cord leukoplakia and control subjects, with significant difference among the three groups (in strong positive expression, the constituent ratio of each group are 0/16、1/18 and 4/21, P<0.01). The presence of pepsin was associated with upright and total laryngopharyngeal acid reflux (P<0.05), including acid reflux episodes, the percentage of times that the pH was below four, the percentages of acid reflux time and average acid removal time. There was a significant correlation between the pepsin level and the esophageal acid reflux parameters (P<0.05) except supine the percentage of time that the pH was below four. CONCLUSIONS: Pepsin expression in laryngeal tissue increases in patients with vocal cord leukoplakia and laryngeal carcinoma, contributing to the development of laryngopharyngeal carcinogenesis.

Characterization of dendritic cells in lip and oral cavity squamous cell carcinoma.

BACKGROUND: There may be differences in the antitumor immunity induced by dendritic cells (DCs) during the development of squamous cell carcinoma (SCC) located in the lip rather than in the oral cavity. The aim of this study was to evaluate the number of immature and mature DCs in SCC and potentially malignant disorders of the oral cavity and lip. METHODS: Immunohistochemistry was used to identify the number (cells/mm(2) ) of immature (CD1a(+) ) or mature (CD83(+) ) DCs in samples of oral cavity SCC (OCSCC) (n = 39), lip SCC (LSCC) (n = 23), leukoplakia (LK) (n = 21), actinic cheilitis (AC) (n = 13), and normal mucosa of the oral cavity (OC control, n = 12) and the lip (lip control, n = 11). RESULTS: The number of CD1a(+) cells tended to be higher in the OC control samples compared with the LK (P = 0.04) and OCSCC (P = 0.21). Unlike, this cell population was lower in the lip control than in AC or LSCC (P < 0.05). The number of CD83(+) cells was increased in the LSCC samples compared with the AC and lip control (P = 0.0001) and in OCSCC compared with both the LK (P = 0.001) and OC control (P = 0.0001) samples. LSCC showed an elevated number of CD1a(+) and CD83(+) cells compared with OCSCC (P = 0.03). The population of mature DCs was lower than the population of immature DCs in all of the tested groups (P < 0.05). CONCLUSION: There were a greater number of both mature and immature DC populations in the LSCC samples than in the OCSCC, which could contribute to establishing a more effective immune antitumor response for this neoplasm.

Expression of NF-κB and IL-6 in oral precancerous and cancerous lesions: An immunohistochemical study.

BACKGROUND: The purpose of this study was to evaluate the immunohistochemical expression of NF-κB and IL-6 in oral premalignant and malignant lesions and to investigate their possible correlation with the presence of subepithelial inflammation. MATERIAL AND METHODS: Thirty two oral premalignant lesions, clinically compatible with leukoplakia or erythroplakia, were investigated. Microscopically, 11 of them showed hyperkeratosis and acanthosis (epithelial hyperplasia) and 21 showed dysplasia of varying degrees. Nine cases of OSCC and four control cases of normal oral mucosa were also included in the study. Immunohistochemical staining with NF-κB (p65) and IL-6 was performed. IL-6 and nuclear NF-κB staining were assessed as positive or negative. For cytoplasmic localization of NF-κB, a total score combining intensity and percentage of positive epithelial cells was additionally calculated. The presence of inflammation was also recorded. RESULTS: Intensity and total scores for NF-κΒ cytoplasmic immunostaining showed a statistically significant gradual increase from normal mucosa to OSCC (p=0.012 and p=0.026 respectively). Non-statistically significant increased NF-κΒ nuclear localization was detected in dysplasias and OSCCs. Positive statistical correlation was detected between the presence of inflammation and IL-6 expression (p=0.015). No correlation between NF-κΒ and IL-6 was detected. CONCLUSIONS: NF-κΒ is activated in the early stages of oral carcinogenesis. IL-6 may have an NF-κΒ-independent role, possibly through regulation of the inflammatory response.

[Clinical and pathological characteristics of vocal fold leukoplakia and influential factors of recurrence and malignancy].

Objective:To investigate the clinical and pathological characteristics of vocal fold leukoplakia and influential factors of recurrence and malignancy. Method:The study included 555 cases of vocal fold leukoplakia. All patients received surgical treatment. The relationship between the clinical and pathological features, and influential factors of recurrence and malignancy were analyzed. Result:The pathological types included squamous epithelial simple hyperplasia in 58.0%, mild hyperplasia in 15.5%, moderate hyperplasia in 10.1%, severe hyperplasia and carcinoma in situ in 8.1%, infiltrative carcinoma in 8.3%. The different pathological types varied among different ages significantly(P<0.01). The tobacco and alcohol abuse had no relationship with pathological types(P>0.05). The more severe the pathology were, the more decreasing of the mucosal waves would be(P<0.01). The pathologic severity had an apparent correlation with p53 positivity and the level of p53 and Ki67 positivity. The p53 positive proportion may increase with the increasing pathological severity(P<0.01), so were the p53 and Ki67 positive levels(P<0.01).Among 232 cases of postoperation follow-ups, 83 cases(35.8%) relapsed while 8 cases(3.4%) malignancy. Bilateral lesions had a higher rate of relapse and malignancy than unilateral lesions(95%CIOR 0.378 ［0.197-0.723］，P<0.01). The relapse and malignancy had a relationship with the pathologic severity. The incidence of disease recurrence and malignancy were positively correlated with pathological severity(P<0.05). p16 positive patients relapsed for more times(P<0.01) and with an uprising tendency of relapse and malignancy（P>0.05）. Conclusion:Pathological types of vocal fold leukoplakia are different. The older men, decreasing of mucosal wave of vocal folds and bilateral lesions were very important factors to predict disease severity. p53 and Ki67 positive rating and pathological severity were positively correlated. The bilateral lesions, pathological severity and p16 positivity had a tendency to increasing relapse and malignancy.

¹H NMR-derived serum metabolomics of leukoplakia and squamous cell carcinoma.

BACKGROUND: Oral cancer (OC) is the sixth commonest cancer worldwide with alarming mortality. If identified at an early stage, the survival rate would be improved. METHODS: We appraised the feasibility of using (1)H nuclear magnetic resonance ((1)H NMR) based metabolomics in the identification of signature metabolites in serum from patients suffering with oral leukoplakia (OLK, n=100), oral squamous cell carcinoma (OSCC, n=100), and healthy control (HC, n=75). (1)H NMR derived data were processed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to reveal discriminating metabolites among these groups. Receiver operating characteristic (ROC) curve evaluation was also executed. RESULTS: NMR-derived serum metabolomics reveals eight differentially expressed biomarkers. Among them four biomarkers (glutamine, propionate, acetone, and choline) were able to accurately (ROC; 0.97) segregate 93.5% of OC cases equated to HC with substantial sensitivity and specificity. Similarly, four biomarkers (glutamine, acetone, acetate, and choline) were able to precisely (ROC; 0.96) discriminate, 92.4% of OLK cases from OSCC with considerable sensitivity and specificity. (1)H NMR-based metabolic fingerprint obtained for oral cancer is remarkable, even for OLK stage. CONCLUSION: There is a systemic metabolic response to initial stage of cancer, which carries immense possibility for early appraisal.

Increased VEGFR2 and MMP9 protein levels are associated with epithelial dysplasia grading.

The present study aimed to compare levels of VEGFR2 and MMP-9 among control, epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC) groups. We analyzed 48 patients with oral leukoplakia (OL), 20 patients with OSCC and 21 patients without OL and OSCC. Immunohistochemistry of VEGFR2 and MMP9 were performed and compared among groups. Analysis of tissue immunolocalization of VEGFR2 and MMP-9 assumed non-parametrical distribution and comparison between groups was performed using the Mann-Whitney and Kruskal-Wallis statistical tests. VEGFR2 and MMP9 immunoexpression appeared to correlate with the degree of dysplasia and was observed to increase in lesions with more severe dysplasia as compared to those with lower degrees of dysplasia. Immunoreactivity of MMP-9 was lower in the OL samples compared to the OSCC samples (p = 0.004). We observed no difference in VEGFR2 protein levels between OL and OSCC samples. A positive correlation was found between VEGFR2 and MMP-9 in OL samples (r = +0.452, p = 0.001), however, no correlation was found in OSCC samples (r = -0.042, p = 0.861). In conclusion, the results of the current study suggest that expression of MMP9 and VEGFR2 is associated with ED grading and MMP9 levels are increased in OSCC.

Expression and significance of p53 and mdm2 in patients with leukoplakia cancer.

OBJECTIVE: To study the relationship of the expressions of p53 and mdm2 in leukoplakia cancer. METHODS: RT-PCR was used to detect the mRNA of p53, mdm2 in patients with leukoplakia cancer. The frequencies of p53, mdm2 in peripheral blood were detected by flow cytometric analysis. RESULTS: The expression of p53mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were 7.7%, 27.3%,33.3%, 56.8%, respectively. The frequencies of p53 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were (0.3±0.1)%, (1.6±0.9)%, (1.9±1.1)%, (3.4±1.8)%. The expression of mdm2 mRNA in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were 0.0%, 6.8%, 11.1%, 37.8%, respectively. The frequencies of mdm2 in normal oral mucosa, simple oral leukoplakia, no-simple oral leukoplakia and leukoplakia cancer were (0.1±0.1)%, (0.8±0.6)%, (1.2±0.8)%, (1.2±0.8)%. There was a positively correlation between p53 mRNA and mdm2 mRNA. CONCLUSIONS: The positive rate of p53 and mdm2 cells in the peripheral blood increases in patients with leukoplakia cancer tissue and has positive correlation with the severity of leukoplakia cancer.

Association of cytokeratin 17 expression with differentiation in oral squamous cell carcinoma.

PURPOSE: The aim of this study was to confirm the expression profile of cytokeratin (CK)17 in comparison with that of CK13 in oral squamous cell carcinoma (OSCC) and leukoplakia and to clarify an association of CK17 with the OSCC differentiation. MATERIALS: The expression of CK17 and CK13 was immunohistochemically examined in 105 patients with OSCC and 108 patients with leukoplakia. A correlation of CK expression with clinicopathological variables was carried out. The over-expression levels of CK17 mRNA were analyzed by real-time RT-PCR in 5 OSCC cell lines (HSC-2, HSC-3, SAS, SQUU-A, SQUU-B). RESULTS: CK17 and CK13 were detected in 101 (96.2 %) and three (2.9 %) of the 105 OSCCs, respectively. CK17 was significantly expressed in well-differentiated OSCC compared to moderately/poorly differentiated OSCC (p < 0.01). As detected in 19 of the 34 dysplastic leukoplakias (55.9 %) and 36 of the 74 hyperplastic leukoplakias (48.6 %), CK17 was significantly expressed in dysplastic leukoplakias (p < 0.01). As detected in 11 of the 34 dysplastic (32.4 %) and 52 of the 74 hyperplastic leukoplakias (70.3 %), CK13 was significantly expressed in hyperplastic leukoplakias (p < 0.01). The relative expression of CK17 mRNA in HSC-2 was significantly higher than in HSC-3 and SAS (p < 0.05). Moreover, the relative expression of CK17 mRNA in SQUU-A was significantly higher than in SQUU-B (p < 0.05). CONCLUSION: CK17 expression could be associated with the differentiation and the malignancy of OSCC. A combination pattern of CK17/CK13 might be a suitable marker of malignant transformation.

Increased expression of the PRL-3 gene in human oral squamous cell carcinoma and dysplasia tissues.

Phosphatase of regenerating liver (PRL) belongs to a class of the protein tyrosine phosphatase family, which is known so far to consist of 3 members, PRL-1, PRL-2, and PRL-3. The aim of this study was to uncover the role of PRL genes in development of oral malignancy. We analyzed expression levels of the 3 PRL genes in 50 human oral squamous cell carcinomas (OSCCs), 11 dysplasia and 12 normal mucosa tissues by a real-time RT-PCR method. PRL-3 but not PRL-1 or PRL-2 expressions were significantly higher in OSCC and dysplasia than in normal mucosa tissues. Additionally, PRL-3 expressions were significantly higher in OSCC tissues harboring dominant-negative p53 or recessive p53 mutation than in those harboring wild-type p53. These results suggest that PRL-3 plays a role in oral cancer development and can be useful as a marker of pre-malignant and malignant lesion of oral mucosa.

Estrogen and cytochrome P450 1B1 contribute to both early- and late-stage head and neck carcinogenesis.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the United States. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP) 1B1, examine the effect of estrogen on gene expression, and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation, and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER) ß were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3- to 3.6-fold relative to vehicle-treated controls (P = 0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, whereas supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERß (91.9%), CYP1B1 (99.4%), and 17ß-estradiol (88.4%). CYP1B1 and ERß were elevated in HNSCCs relative to normal epithelium (P = 0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification of new targets for chemopreventive intervention.

Human oral cancers have altered expression of Bcl-2 family members and increased expression of the anti-apoptotic splice variant of Mcl-1.

Expression of Bcl-2 family proteins in tumours can modulate apoptosis, influencing tumour behaviour and treatment. To investigate their role in oral tumourigenesis, nine Bcl-2 family transcripts were examined in three oral cell lines and 25 oral tumours, using ribonuclease protection assay. Since Mcl-1 mRNA was elevated in these samples, Mcl-1 splice variants were assessed by RT-PCR and Mcl-1 protein was studied in normal, premalignant and malignant oral tissues and cell lines, by immunohistochemistry and/or immunoblotting. The cell lines exhibited significantly higher levels of 7/9 Bcl-2 family transcripts as compared to those in normal tongue, and significantly higher (p=0.030, p=0.004) anti-apoptotic versus pro-apoptotic transcripts. Elevated Mcl-1 mRNA was observed in 11/25 (44%) tumours as compared to normal tissues with a five- to ten-fold higher expression of full-length anti-apoptotic Mcl-1 transcript versus the pro-apoptotic short isoform. Strong cytoplasmic Mcl-1 immunoreactivity was detected predominantly in differentiated epithelia in 27/33 (82%) oral tumours, 18/20 (90%) leukoplakia, 25/30 (83%) submucous fibrosis and 3/3 oral cell lines, with weak staining in 8/15 (53%) normal mucosa samples. Mcl-1 positivity in malignant and premalignant tissues was comparable but significantly higher (p<0.01) than that in normal mucosa. The expression of bcl-2 family genes, including Mcl-1 in tumours, did not correlate significantly with clinicopathological parameters. This is the first report delineating the in vivo expression patterns of Mcl-1 protein and Mcl-1 transcripts in oral cancers and premalignant lesions. The observed imbalance between expression of anti-apoptotic and pro-apoptotic Bcl-2 family genes may promote survival in the oral cell lines. Since the majority of oral tumours associated with tobacco-chewing evolve from premalignant lesions, the sustained expression of full-length anti-apoptotic Mcl-1 protein in these tissues suggests an important role for Mcl-1, early in oral cancer pathogenesis in protecting cells from apoptosis via neutralization of pro-apoptotic members and could be a potential therapeutic target for oral cancers.