Topical tacrolimus with different frequency for exfoliative cheilitis: a pilot study.

BACKGROUND: Tacrolimus is a new type immunosuppressant. The aim of this study was to evaluate the effectiveness of topical tacrolimus 0.1% ointment at 2 different frequencies in treating patients with exfoliative cheilitis. METHODS: A total of 40 patients with exfoliative cheilitis were randomly divided into the QD group receiving topical tacrolimus 0.1% ointment once a day or the QOD group receiving topical tacrolimus 0.1% ointment once-two-day. Patients were also applied wet dressing of saline twice a day. The effectiveness of treatment was defined as the percentage of improvement in signs or symptoms. RESULTS: 37 patients completed the 2-week treatment. And, a full set was analyzed. The effectiveness of topical tacrolimus 0.1% ointment for relief in objective sign and subjective symptom was 50% and 67.5%% in the QD group, respectively. For the QOD group, the effectiveness of sign and symptom relief was 50% and 73.5%. There was no significant difference of effectiveness between application topical tacrolimus once a day and once 2 days. CONCLUSION: Our data suggested that application of topical tacrolimus 0.1% ointment once a day and once 2 days had similar clinical effectiveness for sign and symptom relief in patients with exfoliative cheilitis.

Loss of TACC1 variant25 inducing cell proliferation and suppressing autophagy in head and neck squamous carcinoma.

Transforming acidic coiled-coil containing protein1 (TACC1) is closely related to transcription, translation and centrosome dynamics. Dysregulation of TACC1 is associated with multiple malignancies. Alternative splicing (AS) of TACC1 produces multiple variants, which are of great significance in cancer biology. However, the expression and biological functions of TACC1 variants in head and neck squamous cell carcinoma (HNSCC) remain unclear. In this study, we found for the first time that TACC1 variants exhibited a characteristic expression pattern and that TACC1 variant25 (TACC1v25) was downregulated in HNSCC tissues and cell lines. Overexpression of TACC1v25 in Cal27 and Fadu cells significantly inhibited proliferation and promoted autophagy. Moreover, expression levels of nuclear pERK and p-mTOR were significantly decreased, while the expression of Beclin-1 and the LC3II/LC3I ratio were increased in TACC1v25-overexpressed Cal27 and Fadu cells. After the addition of AKT activator SC79 to TACC1v25-overexpressed Cal27 and Fadu cells, the autophagy levels were remarkably rescued. In conclusion, TACC1v25 inhibits HNSCC progression through the ERK and AKT/mTOR pathways by inhibiting proliferation and increasing autophagy. TACC1v25 might have potential use as a tumour suppressor in HNSCC.

A model for comprehensive oral biological age score with oral and systemic clinical parameters.

BACKGROUND: Biological age reflects the functional status of an individual. The purpose of the study was to develop a model for estimating oral biological age with oral and systemic parameters. METHODS: A total of 248 subjects who had a routine health check were assessed with oral and general clinical examination. Chi-square test was performed to screen oral clinical candidate indicators. General parameters were analyzed by Pearson correlation coefficient and principal component analysis to develop a general biological age score. A final comprehensive model of oral biological age score was established by combining oral and general biological age score. RESULTS: A total of eight oral indicators (mucosal blood blister, mucosal dryness, impacted tooth, missing teeth, residual crowns, dental calculus, gingival hyperemia, and gingival recession) and 10 general clinical indicators (triglyceride, creatinine, blood urea nitrogen, glucose, total cholesterol, mean erythrocyte hemoglobin concentration, mean erythrocyte hemoglobin, uric acid, body weight, and systolic blood pressure) were selected for oral and general biological age score, respectively (r > 0.25, P < 0.05). A model of comprehensive oral biological age score was then formed by principal component analysis: 0.046 triglyceride + 0.010 creatinine + 0.141 blood urea nitrogen + 0.048 glucose + 0.068 total cholesterol + 0.014 mean erythrocyte hemoglobin concentration + 0.082 mean erythrocyte hemoglobin + 0.001 uric acid + 0.020 body weight + 0.005 systolic blood pressure + 0.037 oral biological age score -10.908. The score was increased accordingly with CA. CONCLUSION: Oral biological age can be easily estimated clinically by the model of comprehensive oral biological age score using oral and systemic clinical parameters by general practitioners.

Effect of Suo Quan Wan on the bladder function of aging rats based on the ß-adrenoceptor.

Suo Quan Wan (SQW) has been used to treat lower urinary tract symptoms (LUTS) in elderly patients for hundreds of years in China. ß-adrenoceptors (ß-ARs), particularly ß3-adrenoceptor (ß3-AR), was reported to be important in the bladder dysfunction of the elderly. The present study was conducted to explore the effect of ß-AR, and particularly the ß3-adrenoceptor, in aging rat bladder function in vitro and to test the therapeutic effect of SQW on LUTS in an aging rat model based on the ß3-adrenoceptor. Briefly, the bladder detrusor muscles of young (age, 3 months) and aging (age, 15 months) female rats were separated. A ß-AR non-selective agonist, isoprenaline (ISO), subtype ß3-AR agonist (BRL37344A) and ß3-AR antagonist (SR59230A) were used to define the tension change of detrusor muscles between young and aging rats in vitro. For blank controls, 12 young rats were marked, and 48 aging female rats were randomly divided into four groups as follows: Model, SQW high, SQW middle and SQW low. Following oral administration of SQW for 6 weeks in aging rats, urodynamic and bladder detrusor tests were used to evaluate the therapeutic effect of SQW. The expression of ß3-AR mRNA was investigated using reverse transcription-quantitative polymerase chain reaction. Using ISO and BRL37344A in vitro, maximum relaxation (Emax), intrinsic activity (IA), and log (50% effective concentration) (PD2) were significantly decreased in aging rats compared with that in young rats (P<0.05). Significant changes were also observed in the ß3-AR antagonist experiment, which blocked ISO-induced relaxation, with significant decreases observed in Emax, IA and PD2, and a significant increase observed in PA2 for the aging rats compared with the young controls (P<0.05). SQW was demonstrated to enhance bladder control, storage and contraction ability. Furthermore, SQW was able to increase the sensitivity and expression of ß3-AR in an aging rat. In conclusion, the decrease in ß3-AR sensitivity in aging rats and the expression resulted in bladder detrusor dysfunction. In addition, the therapeutic effect of SQW against LUTS relies on the former's effect on the urethral sphincter, bladder detrusor and ß3-AR.

Absence of high-risk HPV 16 and 18 in Chinese patients with oral squamous cell carcinoma and oral potentially malignant disorders.

BACKGROUND: The critical role of human papillomavirus (HPV) in cancer has been recognized, but the involvement of HPV in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD) is still controversial. The aim of this study was to identify and verify the prevalence of high-risk HPV infection (HPV16 and 18) in Chinese patients with OSCC or OPMD using real-time PCR and DNA sequencing. METHODS: Paired tissue and serum DNA samples were extracted from 40 Chinese patients with OSCC and 59 with OPMD. A SYBR Green-based real-time PCR assay was developed to detect the E6 gene of HPV16 and HPV18. Suspicious positive samples were then sequenced to eliminate false positives. RESULTS: We found that none of the tissue and serum samples of OSCCs and OPMDs were positive for HPV16 E6 or 18 E6, using both real-time PCR and DNA sequencing. Overall, 3 of 198 (1.52 %) and 7 of 198 (3.54 %) samples were false-positive for HPV16 E6 and HPV18 E6, respectively, using real-time PCR. CONCLUSION: The lack of HPV16 and HPV18 detected in this study indicates that high-risk HPV 16 and 18 infections are uncommon in Chinese patients with OSCC and OPMD. Real-time PCR followed by DNA sequencing for HPV DNA detection is an effective strategy to rule out false positives.

[Effect of Zhuangyao Jianshen Wan (ZYJCW) on P2X1 and P2X3 mRNA expressions in rats with diuresis caused by kidney deficiency].

To investigate the urination-reducing effect and mechanism of Zhuangyao Jianshen Wan (ZYJCW). In this study, SI rats were subcutaneously injected with 150 mg · kg(-1) dose of D-galactose to prepare the sub-acute aging model and randomly divided into the model group, the Suoquan Wan group (1.17 g · kg(-1) · d(-1)), and ZYJCW high, medium and low dose groups (2.39, 1.20, 0.60 g · kg(-1) · d(-1)) , with normal rats in the blank group. They were continuously administered with drugs for eight weeks. The metabolic cage method was adopted to measure the 24 h urine volume and 5 h water load urine volume in rats. The automatic biochemistry analyzer was adopted to detect urine concentrations of Na+, Cl-, K+. The ELISA method was used to determine serum aldosterone (ALD) and antidiuretic hormone (ADH). The changes in P2X1 and P2X3 mRNA expressions in bladder tissues of rats were detected by RT-PCR. According to the results, both ZYJCW high and medium dose groups showed significant down-regulations in 24 h urine volume and 5 h water load urine volume in (P <0.05, P <0.01), declines in Na+ and Cl- concentrations in urine (P <0.01), notable rises in plasma ALD and ADH contents (P <0.05, P <0.01) and remarkable down-regulations in the P2X1 and P2X3 mRNA expressions in bladder tissues (P <0.01). The ZYJCW low dose group revealed obvious reductions in Na+ and Cl- concentrations in urine (P <0.01). The results indicated that ZYJCW may show the urination-reducing effect by down-regulating the P2X1 and P2X3 mRNA expressions in bladder tissues of rats with diuresis caused by kidney deficiency.

Decreased expression of GRIM-19 by DNA hypermethylation promotes aerobic glycolysis and cell proliferation in head and neck squamous cell carcinoma.

To identify novel tumor suppressor genes that are down-regulated by promoter hypermethylation in head and neck squamous cell carcinoma (HNSCC), genome-wide methylation profiling was performed using a methylated DNA immunoprecipitation (MeDIP) array in HNSCC and normal mucosa tissue samples. Promoter hypermethylation of the candidate gene, gene associated with retinoid-interferon induced mortality-19 (GRIM-19), was confirmed in HNSCC cell lines. Multivariate regression analysis determined that GRIM-19 hypermethylation was an independent significant factor for HNSCC diagnosis (OR:125.562; P < 0.001). HNSCC patients with lower ratio of GRIM-19/ACTB hypermethylation had increased overall and disease free survival. Furthermore, the optimal cutoff provided 90% sensitivity and 77% specificity of GRIM-19 hypermethylation as a diagnostic marker for HNSCC. Ectopic expression of GRIM-19 in HNSCC cells led to increased oxygen consumption, reduced glycolysis and decreased cell proliferation. HNSCC cells ectopically expressing GRIM-19 displayed increased p53 activity as well as decreased Stat3 and HIF-1α activities. Moreover, GRIM-19 knockdown not only resulted in decreased oxygen consumption and increased aerobic glycolysis but also promoted cell proliferation and tumorigenic capacity in HNSCC cells. Our data indicate that decreased GRIM-19 expression due to promoter hypermethylation may be important in head and neck carcinogenesis by promoting cell proliferation and regulating metabolic activity.

Notch1 mutations are drivers of oral tumorigenesis.

Disruption of NOTCH1 signaling was recently discovered in head and neck cancer. This study aims to evaluate NOTCH1 alterations in the progression of oral squamous cell carcinoma (OSCC) and compare the occurrence of these mutations in Chinese and Caucasian populations. We used a high-throughput PCR-based enrichment technology and next-generation sequencing (NGS) to sequence NOTCH1 in 144 samples collected in China. Forty-nine samples were normal oral mucosa from patients undergoing oral surgery, 45 were oral leukoplakia biopsies, and 50 were chemoradiation-naïve OSCC samples with 22 paired-normal tissues from the adjacent unaffected areas. NOTCH1 mutations were found in 54% of primary OSCC and 60% of premalignant lesions. Importantly, almost 60% of patients with leukoplakia with mutated NOTCH1 carried mutations that were also identified in OSCC, indicating an important role of these clonal events in the progression of early neoplasms. We then compared all known NOTCH1 mutations identified in Chinese patients with OSCC with those reported in Caucasians to date. Although we found obvious overlaps in critical regulatory NOTCH1 domains alterations and identified specific mutations shared by both groups, possible gain-of-function mutations were predominantly seen in Chinese population. Our findings demonstrate that premalignant lesions display NOTCH1 mutations at an early stage and are thus bona fide drivers of OSCC progression. Moreover, our results reveal that NOTCH1 promotes distinct tumorigenic mechanisms in patients from different ethnical populations.

MicroRNA-155 in oral squamous cell carcinoma: Overexpression, localization, and prognostic potential.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignancy with poor prognosis. MicroRNAs (miRNAs) play an important role in cancer, but their role in OSCC is not clarified. METHODS: We performed miRNA microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and fluorescence in situ hybridization (FISH) to examine miRNA expression in OSCC and paired adjacent cancer-free mucosal (ACF) tissues. RESULTS: Thirteen miRNAs, including miRNA-155, were upregulated (>2-fold) in OSCC against ACF. MiRNA-155 was confirmed to have significantly higher expression in OSCC against ACF (paired-samples t test; p = .041) and it was localized in the cancer nest, inflammatory area, and vascular endothelium of OSCC. High expression of miRNA-155 in ACF tissue was an independent prognostic indicator for OSCC survival. CONCLUSION: MiRNA-155 was overexpressed in OSCC and it was located in the cancer nest, inflammatory area, and vascular endothelium of OSCC. High miRNA-155 expression level in ACF may predict poor prognosis in patients with OSCC.

Gastroprotective effect and mechanism of patchouli alcohol against ethanol, indomethacin and stress-induced ulcer in rats.

Pogostemonis Herba is an important Chinese medicine widely used in the treatment of gastrointestinal dysfunction. Patchouli alcohol (PA), a tricyclic sesquiterpene, is the major active constituent of Pogostemonis Herba. This study aimed to investigate the possible anti-ulcerogenic potential of PA and the underlying mechanism against ethanol, indomethacin and water immersion restraint-induced gastric ulcers in rats. Gross and histological gastric lesions, biochemical and immunological parameters were taken into consideration. The gastric mucus content and the antisecretory activity were analyzed through pylorus ligature model in rats. Results indicated that oral administration with PA significantly reduced the ulcer areas induced by ethanol, indomethacin and water immersion restraint. PA pretreatment significantly promoted gastric prostaglandin E2 (PGE2) and non-protein sulfhydryl group (NP-SH) levels, upregulated the cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) mRNA expression, and considerably boosted the gastric blood flow (GBF) and gastric mucus production in comparison with vehicle. In addition, PA modulated the levels of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). The levels of glutathione (GSH), catalase (CAT) and malonaldehyde (MDA) were also restored by PA. However, the gastric secretion parameters (pH, volume of gastric juice and pepsin) did not show any significant alteration. These findings suggest that PA exhibited significant gastroprotective effects against gastric ulceration. The underlying mechanisms might involve the stimulation of COX-mediated PGE2, improvement of antioxidant and anti-inflammatory status, preservation of GBF and NP-SH, as well as boost of gastric mucus production.

Phospholipase C-γ1 expression correlated with cancer progression of potentially malignant oral lesions.

BACKGROUND: Phospholipase C-γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. METHODS: In a retrospective follow-up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant-transformed cases (n = 30). The corresponding post-malignant lesions (OSCCs) were also performed. RESULTS: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan-Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre-malignant OPL and that in post-malignant OSCC was significant (P = 0.004). CONCLUSION: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high-risk OPL into OSCC.

Application of acellular dermal matrix in reconstruction of oral mucosal defects in 36 cases.

PURPOSE: Artificial grafts have been investigated for use in the repair of oral mucosal defects. The aim of this retrospective study was to present the outcomes of the use of acellular dermal matrix (ADM) grafts to repair oral mucosal defects. MATERIALS AND METHODS: Data from 36 patients with oral mucosal defects reconstructed with ADM grafts from 2003 through 2009 were reviewed. All patients were followed-up for at least 6 months to observe the graft repair, wound-healing time, contracture, color, infection, pain, immunologic reaction, texture of the graft, and clinical course. Graft success was defined as the ADM graft being replaced by new mucosa-like tissue and the oral mucosal defect being covered with the new mucosa-like tissue. Any evidence of incomplete graft re-epithelialization or graft sloughing was considered a graft failure (complete or incomplete). RESULTS: Of the 36 cases, 34 grafts (94.4%) were successfully replaced with new mucosa-like tissues and only 2 grafts (5.6%) failed. No complaints such as pain, immunologic reaction, or infection were observed during the follow-up. Mild graft contraction occurred in 7 patients with lip or buccal defects, especially at approximately 3 to 5 weeks after the reconstructive surgery. CONCLUSIONS: The ADM grafts for oral mucosal defects were safe and effective. The present data support the clinical application of ADM grafts in reconstructing oral mucosal defects caused by various oral diseases.

Upregulation of miR-31* is negatively associated with recurrent/newly formed oral leukoplakia.

BACKGROUND: Oral leukoplakia (OLK) is a potentially malignant disorder of the oral cavity. However, the underlying mechanism of OLK is still unclear. In this study, we explore possible miRNAs involved in OLK. METHODOLOGY/PRINCIPAL FINDINGS: Using miRNA microarrays, we profiled miRNA expression in OLK and malignantly transformed OLK (mtOLK) tissue samples. The upregulation of miR-31*, miR-142-5p, miR-33a, miR-1259, miR-146b-5p, miR-886-3p, miR-886-5p, miR-519d, and miR-301a along with the downregulation of miR-572, miR-611, miR-602, miR-675, miR-585, miR-623, miR-637, and miR-1184 in mtOLK were new observations. Fluorescence in situ hybridization (FISH) analyses confirmed that miR-31* is highly expressed in mtOLK. There was a significant difference between the FISH score (p<0.05) in patients with or without recurrent/newly formed OLK. Functional analyses demonstrated that a miR-31* inhibitor decreased apoptosis in the Leuk-1, which is an immortalized oral epithelial cell line spontaneously derived from an oral leukoplakia lesion. miR-31* regulated apoptosis, cell proliferation, migration, and invasion in the HOIEC, which is a HPV E6/E7-immortalized oral epithelial cell line. Furthermore, miR-31* modulated the biological functions of apoptosis, cell proliferation, cell cycle, migration, and invasion in the oral squamous cell carcinoma cell line, Cal-27. Using bioinformatic analyses and dual luciferase reporter assays, we determined that the 3' untranslated region of fibroblast growth factor 3 (FGF3) is the target of miR-31*. Expression of FGF3 was downregulated or upregulated in the presence of a miR-31* mimic or inhibitor, respectively. CONCLUSIONS/SIGNIFICANCE: Upregulation of miR-31* is negatively associated with recurrent/newly formed OLK. MiR-31* may exert similar but distinguishable effects on biological function in oral cells with different malignant potential. FGF3 is the target of miR-31*. miR-31* may play an important role during OLK progression through regulating FGF3. MiRNA* strands may also have prominent roles in oral carcinogenesis.

Generic and oral quality of life is affected by oral mucosal diseases.

BACKGROUND: The generic and oral health-related quality of life (QoL) has provided opportunity for investigation of the interrelations among generic health, oral health, and related outcomes. The purpose of this study was to identify the generic and oral QoL in the patients with oral mucosal disease (OMD). METHODS: Five hundred and thirty-eight OMDs were recruited in this study. The instruments applied were Chinese version of the 36-item short form health survey (SF-36) and the short-form of Oral Health Impact Profile (OHIP-14). RESULTS: The mean score of sum OHIP-14 was significantly higher in the patients with OMD (10.81 ± 9.01) compared with those in the healthy subjects (HS) (6.55 ± 6.73) (p < 0.001, Mann-Whitney U test). 56.51% of the OMD patients and 12.94% of the HS reported at least one oral negative impact (p < 0.001, Chi-square test). The overall mean score of SF-36 was significantly lower in the patients with OMD (74.54 ± 12.77) compared with those in the HS (77.97 ± 12.39) (p = 0.021, t-test). CONCLUSIONS: Administration of specific and generic questionnaires of QoL can provide us a detailed picture of the impact of OMDs on patients, and both generic and oral QoL were impaired in the patients with OMD.

Phosphorylated p120-catenin expression has predictive value for oral cancer progression.

AIMS: Recent studies have shown that phosphorylation of p120-catenin (p120) promotes progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to evaluate the usefulness of phosphorylated p120-catenin (pp120) as a biomarker for predicting clinical behaviour in the carcinogenesis of potentially malignant oral lesions. METHODS: In a retrospective follow-up study, the expression pattern of pp120 protein was determined using immunohistochemistry in samples from 68 patients with potentially malignant oral lesions, including patients with untransformed lesions (n=38) and patients with malignant transformed lesions (n=30). Analysis of corresponding post-malignant lesions (OSCCs) was also performed. RESULTS: There was high expression of pp120 in 35 of 68 (51.5%) of general potentially malignant oral lesions and 23 of 30 (76.7%) of OSCCs compared with expression in normal oral mucosa. Kaplan-Meier analysis revealed that patients with potentially malignant oral lesions expressing high levels of membranous pp120 had a significantly higher incidence of OSCC than those expressing low expressing pp120 (p=0.002; log-rank test). Cox regression analysis revealed that this pp120 expression pattern was significantly associated with a 3.43-fold increase in the risk of malignant progression (p=0.007). In addition, there was a significant correlation between high levels of membranous expression of pp120 in pre-malignant lesions and cytoplasmic expression in post-malignant lesions (p=0.028). CONCLUSIONS: The data indicated that a high level of membranous expression of pp120 in potentially malignant oral lesions is an early event during oral carcinogenesis, and that the mislocalisation of expression of pp120 from the cell membrane to the cytoplasm is associated with oral cancer progression. pp120 may serve as a useful marker for the identification of a high risk of potentially malignant oral lesions progressing to OSCC.

DAPK promoter hypermethylation in tissues and body fluids of oral precancer patients.

Death-associated protein kinase (DAPK) has been suggested as a tumor suppressor gene. A high frequency of DAPK promoter hypermethylation has been noted in head and neck cancers and other solid tumors, and it has been used as a tumor marker in molecular detection strategies. Our aim was to examine DAPK promoter hypermethylation in tissue, blood, and salivary rinse samples of oral precancer patients (OPs) and to explore the potential role in oral carcinogenesis. DAPK hypermethylation was analyzed in 77 OPs and 32 oral squamous cell carcinomas (OSCCs) by real-time quantitative methylation-specific PCR (QMSP). We compared the hypermethylation expression between two groups and analyzed the associations with clinicopathologic parameters. The promoter hypermethylation frequency of DAPK in tissue (46.9%) and blood (52.2%) of OSCCs was significantly higher than those in OPs (19.5%, P = 0.004; 22.4%, P = 0.007, respectively). DAPK promoter hypermethylation expression in blood was correlated with its expression in tissue (r = 0.49, P < 0.000). The OP patients who smoked more than 20 years were found 40.0% tissue DAPK hypermethylation in contrast with 10.7% tissue DAPK hypermethylation in the patients whose smoking duration ≦20 years (P = 0.010). Our results suggest that DAPK hypermethylation is an early event in oral carcinogenesis and blood DAPK hypermethylation might be a potential minimal invasive biomarker for OSCC early detection.

Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance.

PURPOSE: Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP). EXPERIMENTAL DESIGN: Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1. RESULTS: We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8-80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2-6.5; P = 0.016). CONCLUSIONS: We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.

Podoplanin and ABCG2: malignant transformation risk markers for oral lichen planus.

BACKGROUND: Oral lichen planus (OLP) is a potentially malignant disorder associated with an increased risk for oral cancer. The purpose of this study was to determine protein expression of podoplanin and ATP-binding cassette, G2 subfamily (ABCG2) in patients with OLP and evaluate their use as biomarkers for OLP malignant transformation risk. METHODS: Podoplanin and ABCG2 expressions were determined in samples from 110 patients with untransformed OLP and 9 patients with malignant transformed OLP (mean follow-up of 5.1 years). We compared podoplanin expression, ABCG2 expression, and clinicopathologic parameters between the two groups. RESULTS: Podoplanin expression was observed in 48 of 110 (43.6%) cases of untransformed OLP and in 8 of 9 (88.9%) cases of transformed OLP. ABCG2 expression was found in 23 of 110 (20.9%) cases of untransformed OLP and in 6 of 9 (66.7%) cases of transformed OLP. Multivariate regression analysis revealed that podoplanin or ABCG2 expression was associated with 17.13-fold [95% confidence interval (95% CI), 1.71-171.22; P = 0.016] or 6.04-fold (95% CI, 1.20-30.36; P = 0.029) increased risk of malignant transformation, respectively. The risk of OLP malignant transformation was considerably higher with coexpression of podoplanin and ABCG2 than without coexpression of podoplanin and ABCG2 (odds ratio, 25.24; 95% CI, 4.48-142.27; P < 0.001). CONCLUSIONS: The expressions of podoplanin and ABCG2 in OLP were significantly associated with malignant transformation risk. IMPACT: Our data suggested that podoplanin and ABCG2 may be used as biomarkers for risk assessment of oral malignant transformation in patients with OLP.

Cellular transformation by cigarette smoke extract involves alteration of glycolysis and mitochondrial function in esophageal epithelial cells.

Cigarette-smoking increases the risk of developing various types of human cancers including esophageal cancers. To test the effects of chronic cigarette smoke exposure directly on esophageal epithelium, cellular resistance to mainstream extract (MSE), or sidestream smoke extract (SSE) was developed in chronically exposed nonmalignant Het-1A cells. Anchorage-independent growth, in vitro invasion capacity and proliferation of the resistant cells increased compared with the unexposed, sensitive cells. An epithelial marker E-cadherin was down-regulated and mesenchymal markers N-cadherin and vimentin were up-regulated in the resistant cells. Het-1A cells resistant to MSE or SSE consumed more glucose, and produced more lactate than the sensitive cells. The increased anchorage-independent cell growth of the resistant cells was suppressed by a glycolysis inhibitor, 2-deoxy-D-glucose, indicating that these cells are highly dependent on the glycolytic pathway for survival. Decreased mitochondrial membrane potential and ATP production in the resistant cells indicate the presence of mitochondrial dysfunction induced by chronic exposure of cigarette smoke extract. Increased expression of nuclear genes in the glycolytic pathway and decreased levels of mitochondrial genes in the resistant cells support the notion that cigarette smoking significantly contributes to the transformation of nonmalignant esophageal epithelial cells into a tumorigenic phenotype.

Chronic cigarette smoke extract treatment selects for apoptotic dysfunction and mitochondrial mutations in minimally transformed oral keratinocytes.

Cigarette smoke demonstrates a carcinogenic effect through chronic exposure, not acute exposures. However, current cell line models study only the acute effects of cigarette smoke. Using a cell line model, we compared the effects of acute versus chronic cigarette smoke extract (CSE) on mitochondria in minimally transformed oral keratinocytes (OKF6). OKF6 cells were treated with varying concentrations of CSE for 6 months. Cells were analyzed monthly by flow cytometry for mitochondrial membrane potential (MMP), cytochrome c release, caspase 3 activation and viability after CSE exposure. At each time point, the same assays were performed after 24 hr of valinomycin (MMP-depolarizing agent) treatment. The mitochondrial DNA of chronically CSE-treated cells was sequenced. After 6 months of CSE treatment, the cells were increasingly resistant to CSE-mediated and valinomycin-induced cell death. In addition, chronic CSE treatment caused chronic depolarization of MMP, cytochrome c release and caspase activation. Cells grown in the presence of only CSE vapor also exhibited the same resistance and chronic baseline apoptotic activation. Mitochondrial DNA sequencing found that chronic CSE-treated cells had more amino acid-changing mitochondrial mutations than acutely treated cells. CSE treatment of normal cells select for apoptotic dysfunction as well as mitochondrial mutations. These findings suggest that chronic tobacco exposure induces carcinogenesis via selection of apoptosis resistance and mitochondrial mutation in addition to previously known genotoxic effects that were found by acute treatments. Chronic models of tobacco exposure on upper aerodigestive epithelia may be more insightful than models of acute exposure in studying head and neck carcinogenesis.