Sarcopenia results in poor survival rates in oral cavity cancer patients.

OBJECTIVE: This study aimed to determine the impact or survival of low skeletal muscle mass (SMM) among patients with oral squamous cell carcinoma (OSCC) undergoing primary surgery. DESIGN: This study was a retrospective cohort study. SETTING: Oral squamous cell carcinoma patients treated at our referral centre from April 2005 to March 2014 were examined. PARTICIPANTS: The cohort comprised 276 patients with OSCC undergoing primary surgery. MAIN OUTCOME MEASURES: Estimated SMM was measured by calculating the cervical skeletal muscle mass from a CT scan of the head and neck. The 5-year overall survival (OS) and disease-specific survival (DSS) were analysed using a multivariable Cox regression model. RESULTS: There were 276 patients with a male-to-female ratio of 12:1. A low SMM (<47.5 cm2 /m2 ) was associated with worse survival. After adjustment for other factors, the result remained robust for OS (hazard ratio [HR] 1.74, 95% confidence interval [CI] 1.14-2.67) and disease-specific survival (HR 1.67, 95% CI 1.04-2.67). In the subgroup analysis, worse OS and DSS were particularly noted in male patients (HR = 1.90, 95% CI 1.22-2.97; HR = 1.91, 95% CI 1.27-3.19) and in those younger than 60 years of age (HR = 1.91, 95% CI 1.14-3.22; HR = 2.12, 95% CI 1.23-3.64) with low SMM. CONCLUSIONS: Low SMM was a significant independent factor that was associated with lower survival in patients who have oral cavity cancers and are undergoing primary surgery. Preoperative CT scans of the head and neck could be utilised to evaluate SMM, predict treatment outcomes and facilitate nutrition management.

Log margin-to-thickness ratio improves disease-specific survival prediction in oral cancer: A single cancer centre database.

OBJECTIVE: We examined whether dynamic margin criteria margin-to-thickness (MTR) ratio has superior predictive value compared with the resection margin or tumour thickness alone in the survival outcome in oral squamous cell carcinoma (OSCC). DESIGN: This is a retrospective cohort study. SETTING: Oral squamous cell carcinoma patients treated in Kaohsiung Veterans General Hospital Cancer Center between January 2006 and December 2013. PARTICIPANTS: A cohort of 302 patients with OSCC who had undergone surgical management. MAIN OUTCOMES MEASURES: Log MTR was calculated for each patient, and survival data were analysed using a multivariable Cox regression model. Discriminative analysis was performed using chi-square, Akaike information criterion (AIC) and Harrell's C tests. RESULTS: After assessing for discriminative ability, the linear trend of log MTR surpassed those of resection margin and tumour thickness in chi-square, AIC and Harrell's C tests for the advanced pathologic T (pT) category. A multivariate Cox proportional hazard regression model revealed that log MTR <33% was associated with less favourable 5-year disease-specific survival (DSS) (P = 0.006) in the entire oral cancer study cohort. Other significant factors included perineural invasion (P = 0.021), pT category, (P = 0.005), pathologic N category (P < 0.001) and differentiation category (P = 0.022). CONCLUSIONS: Log MTR < 33% may be a predictor of less favourable outcome in the DSS of OSCC. Log MTR outperformed both resection margin and tumour thickness alone in terms of discriminative analysis. Our study could help in presurgical planning for high-risk patients and in aiding the decision-making process for adjuvant treatment.

The variant of pri-mir-26a-1 polymorphism is associated with decreased risk of betel quid-related oral premalignant lesions and oral squamous cell carcinoma.

OBJECTIVES: This case-control study evaluated the association of the single nucleotide polymorphism rs7372209 (T>C) in pri-mir-26a-1 with the risk and progression of betel quid (BQ)-related oral premalignant lesions (OPLs) and oral squamous cell carcinoma (OSCC). STUDY DESIGN: In total, 597 BQ chewers were recruited: 196 healthy controls, 241 patients with OPLs, and 160 patients with OSCC. Genotypes were determined using the TaqMan real-time assay. RESULTS: The C/T + T/T genotypes and T allele in pri-mir-26a-1 were correlated with a decreased risk of BQ-related OPLs (P = .038 and .005, respectively), oral leukoplakia (P = .01 and .001, respectively), and advanced-stage OSCC (P = .021 and .004, respectively). The effects of the C/T + T/T genotypes and T allele on the decreased risk of OPLs were potent in the older age group (both Pinteraction < .001), heavy smokers (Pinteraction ≤ .003 and .006, respectively) and alcohol drinkers (Pinteraction ≤ .004 and .001, respectively). Furthermore, among patients with OSCC, the C/T + T/T genotypes and T allele were associated with a decreased risk of advanced pathologic stage (P = .032) and lymph node involvement (P = .017). CONCLUSIONS: BQ chewers carrying the T allele or C/T + T/T genotypes in pri-mir-26a-1 may have a decreased risk of oral leukoplakia, OPLs, and advanced-stage OSCC.

Prognostic Performance of a New Staging Category to Improve Discrimination of Disease-Specific Survival in Nonmetastatic Oral Cancer.

Importance: Inflammatory status is associated with outcome in oral squamous cell carcinoma (OSCC). Combining the preoperative neutrophil to lymphocyte ratio (NLR) and histopathologic features may provide clinicians with more exact information regarding the prognosis of OSCC. Objective: To compare the prognostic performance of the routinely used pathologic TNM staging with a new staging category that incorporates the NLR and histopathologic features. Design, Setting, and Participants: This retrospective cohort study included 396 patients with newly diagnosed OSCC who underwent major surgery at a medical center from January 1, 2006, through December 31, 2013. Follow-up was completed on October 31, 2015, and data analysis was performed from January 1, 2016, through April 30, 2016. Main Outcomes and Measures: The multivariate Cox proportional hazards regression model was used to determine the clinical or pathologic factors associated with 5-year disease-specific survival (DSS), and these factors were assigned integer points to create a new staging category. The monotonicity and discriminatory ability of the pathologic TNM staging and new staging category were evaluated with the linear trend χ2 test, Akaike information criterion, and Harrell C statistic. Results: In total, 396 patients who underwent major surgery with curative intent for OSCC with or without adjuvant therapy were included in this study (mean [SD] age, 53 [11] years; 367 men [92.7%] and 29 women [7.3%]). Perineural invasion (adjusted hazard ratio [aHR], 1.74; 95% CI, 1.23-2.46), high NLR (aHR, 1.60; 95% CI, 1.11-2.30), advanced pT (T3 + T4) classification (aHR, 1.59; 95% CI, 1.13-2.25), and advanced pN (N2) classification (aHR, 3.96; 95% CI, 2.78-5.63) were independent prognostic survival factors. The ß coefficients from the Cox proportional hazards regression model were used to develop an integer-based weighted point system (perineural invasion, score of 1; NLR, score of 1; advanced pT, score of 1; and advanced pN, score of 3). The summations of these risk scores were stratified for the new staging category as follows: new stage I, score of 0; new stage II, score of 1; new stage III, score of 2 or 3; and new stage IV, score of 4 to 6. Compared with the American Joint Committee on Cancer staging category, this new staging category provided better monotonicity with a higher linear trend χ2 value (106 vs 49), better discriminatory ability with smaller Akaike information criterion (1497 vs 1533), and greater Harrell C statistic (0.73 vs 0.69) for 5-year DSS. The results remained robust after adjusting other risk factors. Conclusions and Relevance: In this study, new staging category had better DSS discriminatory ability and could help to identify high-risk patients for intense adjuvant therapy.

Initial Factors Influencing Duration of Hospital Stay in Adult Patients With Peritonsillar Abscess.

OBJECTIVES: To review cases of peritonsillar abscess and investigate the initial clinical factors that may influence the duration of hospitalization. To determine the predictive factors of prolonged hospital stay in adult patients with peritonsillar abscess. METHODS: Subjects were adults hospitalized with peritonsillar abscess. We retrospectively reviewed 377 medical records from 1990 to 2013 in a tertiary medical center in southern Taiwan. The association between clinical characteristics and the length of hospital stay was analyzed with independent t-test, univariate linear regression and multiple linear regression analysis. RESULTS: The mean duration of hospitalization was 6.2±6.0 days. With univariate linear regression, a prolonged hospital stay was associated with several variables, including female gender, older ages, nonsmoking status, diabetes mellitus, hypertension, band forms in white blood cell (WBC) counts, and lower hemoglobin levels. With multiple linear regression analysis, four independent predictors of hospital stay were noted: years of age (P<0.001), history of diabetes mellitus (P<0.001), ratio of band form WBC (P<0.001), and hemoglobin levels (P<0.001). CONCLUSION: In adult patients with peritonsillar abscess, older ages, history of diabetes mellitus, band forms in WBC counts and lower hemoglobin levels were independent predictors of longer hospitalization.

Genetic variants in microRNA-146a (C>G) and microRNA-1269b (G>C) are associated with the decreased risk of oral premalignant lesions, oral cancer, and pharyngeal cancer.

OBJECTIVE: To investigate the relationships between two single-nucleotide polymorphisms at miR-146a C>G (rs2910164) and miR-1269b G>C (rs7210937) and the risk of developing oral premalignant lesions (OPLs), oral squamous cell carcinoma (OSCC), pharyngeal SCC (PSCC), and oral and pharyngeal SCC (OPSCC). DESIGN: Genotyping of miR-146a C>G and miR-1269b G>C was performed in two case-control studies using the TaqMan assay. A total of 197 healthy control subjects, 241 OPLs patients, and 188 OPSCC patients who habitually chewed betel quid (BQ) were recruited into one case-control study. Additionally, 668 cancer-free control subjects and 658 OPSCC patients were recruited into the other case-control study. RESULTS: The G/G genotype at miR-146a C>G was associated with the decreased risk of OSCC [adjusted odds ratio (AOR)=0.66, P=0.040], PSCC (AOR=0.42, P=0.013), and OPSCC (AOR=0.63, P=0.020). Additionally, the C allelic type and C/C genotype at miR-1269b G>C decreased the risk of BQ-related oral leukoplakia (C vs. G: AOR=0.68, P=0.012;C/C vs. G/G: AOR=0.43, P=0.009), BQ-related OPLs (C vs. G: AOR=0.69, P=0.008;C/C vs. G/G: AOR=0.44, P=0.005), and BQ-related OPSCC (C vs. G: AOR=0.65, P=0.003;C/C vs. G/G: AOR=0.47, P=0.011). In OPSCC patients, the G/G genotype of miR-146a was correlated with well-differentiated cells (P=0.041), and the G/C and C/C genotypes of miR-1269b were correlated with the absence of lymph node involvement (P=0.031), especially in OSCC patients (P=0.038 and P=0.007, respectively). CONCLUSION: The genetic variants of miR-146a and miR-1269b are biomarkers against the development of OPLs and OPSCC.

Aberrant DNA hypomethylation of miR-196b contributes to migration and invasion of oral cancer.

MicroRNAs (miRs) are a class of small endogenous non-coding RNAs of ~21-24 nucleotides in length. Previous studies have indicated that miR-196b has either an oncogenic or tumor-suppressive function in various types of cancer. However, the biological role of miR-196b in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, the expression levels of miR-196b were examined in oral cancer tissues and corresponding adjacent normal tissues from 69 OSCC patients using stem-loop reverse transcription-quantitative polymerase chain reaction. The results indicated that miR-196b was significantly overexpressed in OSCC tissues compared with the corresponding adjacent normal tissue samples (64 of 69, 92.7%, P<0.001). Analysis of the methylation status of the miR-196b gene indicated more frequent hypomethylation of the CpG islands located upstream of the miR-196b gene in the OSCC tissues than in the adjacent normal tissues (32 of 69, 46.3%), and the methylation status of miR-196b correlated inversely with its expression levels. Furthermore, the unmethylated status of the miR-196b promoter correlated with poor disease-specific survival in OSCC patients (P=0.035). Functional analysis revealed that ectopic miR-196b expression promoted oral cancer cell migration and invasion abilities, and that silencing of miR-196b could abrogate in vitro migration and invasion of oral cancer cells. Collectively, the present findings indicate that the epigenetic regulation of miR-196b expression plays a crucial role in modulating cell migration and invasion during OSCC progression, and thus may serve as a potential prognosis marker or therapeutic target for OSCC.

Transnasal endoscopic orbital decompression: 15-year clinical experience in Southern Taiwan.

BACKGROUND/PURPOSE: To present the results and evaluate the efficacy of endoscopic transnasal orbital decompression for dysthyroid orbitopathy. METHODS: Retrospective chart review of patients who underwent endoscopic transnasal orbital decompression from 1996 to 2010 in one institution. We included 42 orbits of 25 patients. Preoperative and postoperative examinations included visual acuity, Hertel exophthalmometry, tonometry, exposure keratitis, and diplopia. The measurements of outcome depend on proptosis reduction, intraocular pressure reduction, and visual acuity improvement of 42 orbits of 25 patients. RESULTS: There were no surgical complications for the 42 orbital decompressions except one patient experienced cerebrospinal fluid leak during the operation. Mean proptosis reduction in all orbits was 1.93 ± 0.25 (mean ± standard deviation, p < 0.01) after 1 month postoperatively and 2.07 ± 0.29 (p < 0.01) after 3 months postoperatively. An average reduction of intraocular pressure was 4.40 ± 0.72 (p < 0.01) and 4.38 ± 0.80 (p < 0.01) respectively after 1 and 3 months postoperatively. Visual acuity increased from a preoperative average of 0.45 ± 0.34 to 0.66 ± 0.36 and 0.70 ± 0.35 after 1 and 3 months postoperatively. In addition, postoperative relief of exposure keratitis is also noted. CONCLUSION: The transnasal orbital decompression procedure has statistically significant improvements in proptosis, intraocular pressure, and visual acuity. The procedure has obvious benefit in relieving exposure keratitis. Furthermore, there are favorable cosmetic results and rare complications.

Effect of the pesticide, deltamethrin, on Ca2+ signaling and apoptosis in OC2 human oral cancer cells.

Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Although deltamethrin has been shown to induce cytosolic free Ca(2+) concentration ([Ca(2+)]i) rises and apoptosis in different cancer cells, there is no information concerning the effects of deltamethrin on oral cancer. This study explored the effects of deltamethrin on [Ca(2+)]i and viability in OC2 human oral cancer cells. Deltamethrin, at concentrations of 5-10 µM, increased [Ca(2+)]i in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Deltamethrin-induced [Ca(2+)]i rise was not inhibited by econazole, SK&F96365, phorbol 12-myristate 13 acetate (PMA) or GF109203X, but was inhibited by nifedipine. In Ca(2+)-free medium, 10-µM deltamethrin pretreatment inhibited the [Ca(2+)]i rise induced by the endoplasmic reticulum Ca(2+) pump inhibitor, 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with BHQ inhibited deltamethrin-induced [Ca(2+)]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with phospholipase C (PLC) inhibitor U73122 did not suppress deltamethrin-induced Ca(2+) release. At concentrations between 20 and 100 µM, deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl. Deltamethrin-induced cell death was not caused by a preceding [Ca(2+)]i rise. Annexin V/propidium iodide staining data suggest that deltamethrin (40-60 µM) induced apoptosis in a concentration-dependent manner. To conclude, in OC2 cells, deltamethrin evoked a [Ca(2+)]i rise by inducing PLC-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry by nifedipine-sensitive Ca(2+) channels. Further, deltamethrin induced Ca(2+)-independent cell death might involve apoptosis.

M-3M3FBS-induced Ca² ⁺ movement and apoptosis in HA59T human hepatoma cells.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca² ⁺ concentrations ([Ca² ⁺ ]i ) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca² ⁺ ]i levels in suspended cells by using fura-2 as a Ca² ⁺ -sensitive fluorescent dye. M-3M3FBS at concentrations of 10- 50 µM increased [Ca² ⁺ ]i in a concentration-dependent fashion. The Ca² ⁺ signal was reduced partly by removing extracellular Ca² ⁺ . M-3M3FBS-induced Ca² ⁺ influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca² ⁺ -free medium, 50 µM m-3M3FBS pretreatment inhibited the [Ca² ⁺ ]i rise induced by the endoplasmic reticulum Ca² ⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca² ⁺ ]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca² ⁺ ]i rise. At concentrations between 10 and 40 µM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca² ⁺ with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. M-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca² ⁺ ]i rise by inducing phospholipase C-independent Ca² ⁺ release from the endoplasmic reticulum and Ca² ⁺ entry via protein kinase C-sensitive store-operated Ca² ⁺ channels. M-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.

Effect of the environmental pollutant bisphenol A dimethacylate (BAD) on Ca2+ movement and viability in OC2 human oral cancer cells.

The environmental pollutant bisphenol A dimethacylate (BAD) has been used as a dental composite. The effect of BAD on cytosolic Ca(2+) concentrations ([Ca(2+)]i) and viability in OC2 human oral cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)]i. BAD induced [Ca(2+)]i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca(2+). BAD-evoked Ca(2+) entry was suppressed by nifedipine, econazole, and SK&F96365. In Ca(2+)-free medium, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished BAD-induced [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 did not alter BAD-induced [Ca(2+)]i rise. At 10-30µM, BAD inhibited cell viability, which was not reversed by chelating cytosolic Ca(2+). BAD (20-30µM) also induced apoptosis. Collectively, in OC2 cells, BAD induced a [Ca(2+)]i rise by evoking phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. BAD also caused apoptosis.

Effect of sertraline on [Ca2+](i) and viability of human MG63 osteosarcoma cells.

The antidepressant, sertraline, has been shown to have diverse in vitro effects. This study examined whether sertraline altered [Ca(2+)](i) in MG63 human osteosarcoma cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. At 50-200 µM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent manner. Ca(2+) response was decreased by removing extracellular Ca(2+), suggesting that Ca(2+) entry and release contributed to the [Ca(2+)](i) signal. Sertraline-induced Ca(2+) entry was inhibited by nifedipine, La(3+), Gd(3+), and SK&F96365. When extracellular Ca(2+) was removed, pretreatment with the endoplasmic reticulum (ER) Ca(2+) pump inhibitor, thapsigargin, or 2,5-di-tert-butylhydroquinone (BHQ) abolished the sertraline-evoked [Ca(2+)](i) rise. Incubation with sertraline also abolished the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished the sertraline-induced [Ca(2+)](i) rise. At 20-30 µM, overnight treatment with sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data demonstrate that sertraline (30 µM) evoked apoptosis. Sertraline (20 and 30 µM) also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, sertraline evoked a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the ER and Ca(2+) entry by L-type Ca(2+) channels and store-operated Ca(2+) channels. Sertraline induced cell death that may involve apoptosis by mitochondrial pathways.

The mechanism of carvacrol-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in OC2 human oral cancer cells.

Carvacrol is one of the main substances of essential oil which triggers intracellular Ca(2+) mobilization and causes cytotoxicity in diverse cell models. However, the mechanism of carvacrol-induced Ca(2+) movement and cytotoxicity is not fully understood. This study examined the effect of carvacrol on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)), cell viability and apoptosis in OC2 human oral cancer cells. Carvacrol induced a [Ca(2+)](i) rise and the signal was reduced by removal of extracellular Ca(2+). Carvacrol-induced Ca(2+) entry was not altered by store-operated Ca(2+) channel inhibitors and protein kinase C (PKC) activator, but was inhibited by a PKC inhibitor. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited carvacrol-induced [Ca(2+)](i) rise. Conversely, incubation with carvacrol inhibited TG or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol-induced [Ca(2+)](i) rise. Carvacrol decreased cell viability, which was not reversed when cytosolic Ca(2+) was chelated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Carvacrol-induced apoptosis and activation of reactive oxygen species (ROS) and caspase-3. Together, carvacrol induced a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-sensitive, non store-operated Ca(2+) channels. Carvacrol-induced ROS- and caspase-3-associated apoptosis.

Mechanism of [Ca2+]i rise induced by angiotensin 1-7 in MDCK renal tubular cells.

The effect of angiotensin 1-7 (Ang 1-7) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in MDCK renal tubular cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Ang 1-7 at concentrations of 10-50 µM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Ang 1-7 evoked store operated Ca(2+) entry that was inhibited by La(3+) and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin prevented Ang 1-7 from releasing more Ca(2+). Inhibition of phospholipase C with U73122 abolished Ang 1-7-induced [Ca(2+)](i) rise. Ang 1-7-induced [Ca(2+)](i) rise was abolished by the angiotensin type 1 receptor antagonist losartan, but was not affected by the angiotensin type 2 receptor antagonist PD 123,319. In sum, in MDCK cells, Ang 1-7 stimulated angiotensin type 1 receptors leading to a [Ca(2+)](i) rise that was composed of phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A2-sensitive store-operated Ca(2+) channels.

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 µM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 µM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 µM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.

NAT2 slow acetylation haplotypes are associated with the increased risk of betel quid-related oral and pharyngeal squamous cell carcinoma.

BACKGROUND: NAT2, the most important phase II metabolic enzyme for betel quid (BQ), might modify the risk of BQ-related oral and pharyngeal squamous cell carcinoma (OPSCC) in Taiwan. STUDY DESIGN: PCR-RFLP and TaqMan assay were conducted for genotyping of NAT2 in 172 OPSCC cases and 170 healthy controls who habitually chewed BQ. RESULTS: The genotypic and allelic type of T341C and C481T in NAT2 are associated with the risk of OPSCC. There were linear trends between increased risk of OPSCC and slowness of NAT2 acetylation haplotypes (P = .017), especially for young subjects (P < .001), light BQ chewers (P = .005), light smokers (P = .023), and alcohol drinkers (P = .001). The interactions on risk of OPSCC were found for NAT2 acetylation haplotypes with status of age, BQ chewing, and alcohol drinking. CONCLUSIONS: The NAT2 acetylation haplotypes might be genetic markers for risk of BQ-related OPSCC.

Investigation of carvedilol-evoked Ca²+ movement and death in human oral cancer cells.

The effect of carvedilol on cytosolic free Ca²âº concentrations ([Ca²âº](i)) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²âº](i) levels in suspended OC2 cells by using fura-2 as a Ca²âº-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²âº](i) in a concentration-dependent fashion. The Ca²âº signal was decreased by 50% by removing extracellular Ca²âº. Carvedilol-induced Ca²âº entry was not affected by the store-operated Ca²âº channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin did not change carvedilol-induced [Ca²âº](i) rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²âº release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²âº](i) release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²âº](i) rise. Carvedilol at 5-50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²âº was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²âº](i) rise by causing phospholipase C-independent Ca²âº release from mitochondria and non-endoplasmic reticulum stores, and Ca²âº influx via protein kinase C-regulated channels. Carvedilol (up to 50 µM) induced cell death in a Ca²âº-independent manner that involved apoptosis.

Interaction between tumour necrosis factor-α gene polymorphisms and substance use on risk of betel quid-related oral and pharyngeal squamous cell carcinoma in Taiwan.

OBJECTIVE: Betel quid (BQ) components induce the secretion of tumour necrosis factor-alpha (TNF-α) in oral keratinocytes, which promotes oral mucosal inflammation and oral cancer. This study was carried out to evaluate the association of TNFA genetic variants (-308G>A and -238G>A) with the risk and prognosis of BQ-related oral and pharyngeal squamous cell carcinoma (OPSCC). DESIGN: A total of 403 subjects (205 cancer cases and 198 healthy controls) who habitually chewed BQ were recruited. The genotypes were determined by TaqMan real-time assays. RESULTS: G allele and G/G genotype at TNFA -308 were associated with a 1.95-fold (95%CI: 1.16-3.28, p(corrected)=0.024) and 2.28-fold (95%CI: 1.30-4.00, p(corrected)=0.008) increased risk of cancer as compared to those with A allele or A/A+A/G genotypes, respectively. In addition, G allele (p=0.080) and G/G genotype (p=0.076) at TNFA -238 were associated with a borderline but statistically significant increased risk of OPSCC. The combined G/G+G/G genotype at both loci had a 2.37-fold increased risk of OPSCC as compared to those with other combined genotypes (95%CI: 1.41-4.00, p=0.001). Interactions between combined genotypes and smoking status were also found to contribute to risk of BQ-related OPSCC. There was no association of TNFA genotypes with clinicopathologic findings or the survival of OPSCC patients. CONCLUSIONS: BQ-chewers who carry the G allele or G/G genotype in TNFA -308 may have an increased risk of OPSCC. The intensity of cigarette smoking modulates the effect of the combined TNFA genotypes on risk of BQ-related OPSCC.

Effect of methoxychlor on Ca2+ handling and viability in OC2 human oral cancer cells.

The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in human oral cancer cells (OC2) by using the Ca(2+)-sensitive fluorescent dye fura-2. Methoxychlor at 5-20 µM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by 70% by removing extracellular Ca(2+). Methoxychlor-induced Ca(2+) entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished methoxychlor-induced [Ca(2+)](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor-induced [Ca(2+)](i) rise. At 5-20 µM, methoxychlor killed cells in a concentration-dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that methoxychlor (10 and 20 µM) evoked apoptosis in a concentration-dependent manner. Together, in human OC2, methoxychlor induced a [Ca(2+)](i) rise probably by inducing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive channels. Methoxychlor induced cell death that may involve apoptosis.