Shyun Yeu Liu

Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan.

BACKGROUND Many single nucleotide polymorphisms (SNPs) have been found to be associated with oral cancer but the biological interactions through SNPs are seldom addressed. In this study, we focused on the joint effect for SNP combinations of four DNA repair genes, X-ray repair cross-complementing groups (XRCCs) 1-4, involved in major cancer-related pathways. METHODS Single nucleotide polymorphism genotyping was determined using by polymerase chain reaction-restriction fragment length polymorphism in this study (case = 103, control = 98). Different numbers of combinational SNPs with genotypes called the pseudo-haplotypes from these chromosome-wide genes were used to evaluate their joint effect on oral cancer risk. RESULTS Except for XRCC2 rs2040639-AG, none of these SNPs was found to individually contribute to oral cancer risk. However, for two combined SNPs, the proportion of subjects with oral cancer was significantly higher in the pseudo-haplotype with AG-CC genotypes in rs2040639-rs861539 (XRCC2-XRCC3) compared with those with non-AG-CC genotypes. Similarly, the pseudo-haplotype of rs2040639-rs861539-rs2075685 (XRCC2-XRCC3-XRCC4) and rs2040639-rs861539-rs2075685-rs1799782 (XRCCs 1-4) with specific genotype pattern (AG-CC-TG and CT-AG-CC-TG) among three and four combinational SNPs were significantly associated with oral cancer. After controlling for age, gender, smoking, drinking, and betel nut chewing, the estimated odds ratio of oral cancer were 2.45, 5.03, and 10.10 for two, three and four specific SNP combinations, respectively, comparing these specific pseudo-haplotypes to their corresponding non-pseudo-haplotypes. CONCLUSION We have identified the potential combined XRCCs 1-4 SNPs with genotypes that were associated with oral cancer risk and may have an impact on identification of a high-risk population.

Serine protease inhibitor (SERPIN) B1 promotes oral cancer cell motility and is over-expressed in invasive oral squamous cell carcinoma

Lymph node metastasis is the hallmark of malignant neoplasms in patients of oral cancer, accounting for the poor diagnosis and reduced 5-year survival rate. Here we sought to identify cell motility-associated proteins of oral cancer by proteomic approach. We compared the proteomes of two oral cancer cells, CAL-27 and SAS, with the highest and the lowest migration potential, respectively, amongst six different oral cancer cell lines. Subsequent identification of differentially expressed proteins by LC-MS/MS and Western analysis revealed that SERPINB1 (serine protease inhibitor, clade B, member1) was highly expressed in CAL-27, the high-motility oral cancer cells. Semi-quantitative and real-time PCR further confirmed differential expression of SERPINB1 in these two cell lines at mRNA level. To verify the motility-promoting function of SERPINB1 in oral cancer cells, we showed that endogenous expression of SERPINB1 correlated positively with cell migration. Moreover, ectopic expression of SERPINB1 in oral cancer cells, SAS, Ca9-22, CAL-27 and HSC-3, increased cell migration by 25%, 52%, 90% and 100%, respectively. Finally, we found that the expression of SERPINB1 was significantly higher in 5 of 8 (62.5%) oral cancer tissues compared with the matched adjacent normal tissues. Besides, immunohistochemical results indicated over-expression of SERPINB1 in clinicopathologically invasive oral squamous cell carcinoma (OSCC) but not in normal oral mucosa (p<0.01). Together, our findings have provided a possible biomarker for oral cancer metastasis.

Matrix metalloproteinases (MMP) 1 and MMP10 but not MMP12 are potential oral cancer markers.

The aim of this study was to investigate the mRNA performance of matrix metalloproteinases (MMP) 1, MMP10 and MMP12 as oral cancer markers. With gingiva as the control, the areas under the receiver- operating characteristic curves (AUCs) of the relative gene expressions for MMP1, MMP10 and MMP12 were 0.715, 0.727 and 0.513, respectively. With the margins or neck platysma muscles as controls, the AUCs of MMP1, MMP10 and MMP12 were 0.746 vs 0.626, 0.712 vs 0.683 and 0.697 vs 0.630, respectively. MMP10 displayed the best sensitivity for oral cancer detection with any controls. MMP1 and MMP10 were suitable markers for cancer detection with gingiva and margin as controls. Using neck tissue as the control, only MMP10 was suitable for cancer detection. With margin and neck controls, there were no significant differences for MMP1, MMP10 and MMP12 in different stages, invasion and locations or different habits. Therefore, MMP1 and MMP10 but not MMP12 are potential oral cancer markers.

Surgical outcomes and prognostic factors of oral cancer associated with betel quid chewing and tobacco smoking in Taiwan

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in geographic regions where betel quid (BQ) chewing is prevalent; OSCC is an extremely malignant neoplasm whose prognostic factors are multiple and complex. The purpose of this study was to assess clinicopathological prognostic factors and treatment outcomes in 698 consecutive OSCC patients who had undergone surgery as the primary treatment in an area with a high prevalence of both betel quid chewing and tobacco smoking. The prognostic factors were predicted using Coxs proportional-hazards regression model, and the survival rate was calculated using Kaplan-Meier analysis. The median followup for all patients was 44 months. The 5-year cumulative overall, disease-specific, and locoregional control survival rates were 61%, 62%, and 46%, respectively. Multivariate analysis showed that the lower level of nodal metastasis, advanced stage, tumor thickness >7 mm, and treatment failures were independent risk factors of overall survival. Furthermore, history of alcohol drinking, lower level of nodal metastasis, advanced stage, poor cell differentiation, and treatment failures were independent predictors of poor disease-specific survival. However, we did not find any significant factor that affected locoregional recurrence. Due to the high frequencies of locoregional recurrence and second primary cancer, our findings emphasize that aggressive surgical excision, adjuvant treatments according to clinicopathological prognostic factors and close surveillance are important to the survival of OSCC patients in an area with a high prevalence of betel quid chewing and tobacco smoking.

Arecoline-mediated inhibition of AMP-activated protein kinase through reactive oxygen species is required for apoptosis induction

Arecoline is the major alkaloid of areca nut (AN) and known to induce reactive oxygen species (ROS) production and apoptosis. The metabolic sensor AMP-activated protein kinase (AMPK), activated by ROS, also regulates apoptosis. This study used several types of cells as the experimental model to analyze the roles of ROS and AMPK in arecoline-induced apoptosis. We found that arecoline dose-dependently increased intracellular ROS level, and two antioxidants, N-acetyl-L-cysteine (NAC) and glutathione, attenuated arecoline-induced apoptotic cell death. Interestingly, arecoline dose- and time-dependently inhibited rather than stimulated AMPK-Thr(172) phosphorylation, and both NAC and glutathione relieved this inhibition. The AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), also restored the phosphorylation level of AMPK-Thr(172) and attenuated apoptotic cell death under arecoline insult. In contrast, the AMPK inhibitor, compound C, and RNA interference of AMPK expression increased the cytotoxicity of arecoline. Collectively, these results suggest that arecoline may inhibit AMPK through intracellular ROS, responsible for the execution of apoptosis.

Autophagy induction by the 30–100 kDa fraction of areca nut in both normal and malignant cells through reactive oxygen species

Areca nut (AN) is an addictive carcinogen used by about 200-600 million people worldwide. Some AN components are shown to induce apoptosis; however, we previously demonstrated that AN extract (ANE) and the 30-100kDa fraction of ANE (ANE 30-100K) induced autophagy-like responses, such as swollen cell morphology, empty cytoplasm, acidic vesicles, and LC3-II accumulation, in an oral cancer cell line, OECM-1. To further assess the responses of other cell types to ANE 30-100K, we used both normal and malignant cells as the targets of ANE 30-100K and found that normal oral fibroblasts (CMT415), peripheral blood lymphocytes (PBLs), Jurkat leukemia T cells, and esophageal carcinoma cells (CE81T/VGH) exhibited similar responses after ANE 30-100K challenge. ANE 30-100K drastically increased acidic vesicle-containing PBLs isolated from two independent donors (from 0.1% to 92.1% and 2.9% to 64.2%). Furthermore, both ANE- and ANE 30-100K-induced LC3-II accumulation in CMT415 and CE81T/VGH was further increased in the presence of the lysosomal protease inhibitors (pepstatin A, E64d, and leupeptin). On the other hand, ANE 30-100K also increased the level of intracellular reactive oxygen species (ROS), and the ROS scavengers, N-acetylcysteine (NAC) and Tiron, inhibited ANE 30-100K-induced cell death and LC3-II accumulation. Collectively, these results suggest the existence of an autophagy-inducing AN ingredient (AIAI) in ANE 30-100K, which renders ANE as an autophagic flux inducer through ROS in both normal and malignant cells.

Upregulation of matrix metalloproteinase-1 (MMP-1) expression in oral carcinomas of betel quid (BQ) users: Roles of BQ ingredients in the acceleration of tumour cell motility through MMP-1

Matrix metalloproteinases (MMPs) are commonly expressed in carcinomas including oral squamous cell carcinomas (OSCCs). On the other hand, some evidences suggested that ingredients of betel quid (BQ) inhibit the activity and/or expression of some MMPs thought to be the pathogenesis of oral submucous fibrosis. This study was to analyse whether MMP-1 expression is inhibited in OSCC specimens from BQ users and in cell lines survived from the challenge of BQ ingredients. We found that MMP-1 mRNA was expressed in all the tested 27 OSCC. Levels of MMP-1 mRNA and protein were significantly elevated in the tested five OSCC specimens than in their adjacent tissues (P<0.001 and 0.05, respectively). Esophageal carcinoma (CE81T/VGH) and OSCC (OECM-1) cell lines survived from the cytotoxic BQ extract (BQE) and arecoline selection process were found to express higher MMP-1 mRNA and protein levels, or to exhibit a significant acceleration of two-dimensional (2D) motility than their non-selected parental cells. The enhanced motility was further demonstrated to be specifically and significantly inhibited by the MMP-1 neutralizing antibody and/or by the transfection of an MMP-1 specific antisense oligodeoxynucleotide. These results suggest that in some carcinomas of the upper aerodigestive tract, BQ usage may upregulate MMP-1 expression in the survived tumour cells, and increase their mobility in an MMP-1-dependent manner.

Arecoline and the 30–100 kDa fraction of areca nut extract differentially regulate mTOR and respectively induce apoptosis and autophagy: a pilot study

Areca nut (AN) is recognized as a human carcinogen; however, few studies of the cytotoxic effects of AN ingredients on cells have been reported. In Taiwan, AN, lime and inflorescence of Piper betle are the common components of betel quid (BQ). We recently noticed that extract of AN (ANE), but not those of lime and inflorescence of Piper betle, induces rounding cell morphology and nuclear shrinkage in different types of carcinoma cells. In this study, the rounding cell activity was first traced to the partially purified >or=10 kDa fraction (ANE >or= 10 K) and subsequently to the 30-100 kDa fraction (ANE 30-100 K). ANE and ANE >or=10 K stimulated nuclear shrinkage (P < 0.001 in both cases) and the clearance of the cytoplasm. ANE, ANE >or= 10 K, and ANE 30-100 K induced the cleavage of LC3-I (P < 0.05, 0.01, and 0.05, respectively) and the emergence of autophagic vacuoles (AVs) and acidic vesicles. On the other hand, arecoline (Are, the major alkaloid of AN) triggered caspase-3 activation, peri-nuclear chromatin condensation, and micronucleation. Meanwhile, ANE 30-100 K, but not Are, inhibited the phosphorylation of the mammalian target of rapamycin (mTOR)-Ser(2448). In conclusion, this study demonstrates that different AN ingredients exerting differential impact on mTOR-Ser(2448) phosphorylation are capable of triggering apoptosis and autophagy.

Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity

BACKGROUND We previously demonstrated the autophagy-inducing activity in the crude extract of areca nut (ANE) and its 30-100 kDa fraction (ANE 30-100 K). This study aimed to analyze whether chronic ANE and ANE 30-100 K stimulations lead to higher stress resistance and autophagic activity in oral cells, and whether the resulting autophagic status in stimulated cells correlates with stress resistance. MATERIALS AND METHODS Malignant cells from the mouth oral epidermoid carcinoma Meng-1 (OECM-1) and blood (Jurkat T) origins were stimulated with non-cytotoxic ANE and ANE 30-100 K for 3 months. Sensitivity to anticancer drugs of and autophagy status in stimulated cells, analyzed respectively by XTT assay and calculating microtubule-associated protein 1 light chain 3-II LC3-II/β-actin ratios from Western blot, were compared to non-treated cells. Autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), were used to assess whether autophagy inhibition interferes the altered chemoresistance. RESULTS Areca nut extract-stimulated (ANE-s) and ANE 30-100 K-stimulated (30-100 K-s) OECM-1 and Jurkat T cells generally exhibited higher cisplatin and 5-fluorouracil (5-FU) resistances, compared to non-stimulated cells. Most stimulated cells expressed significantly higher levels of LC3-II and Atg4B proteins. Interestingly, these cells also showed stronger tolerances against hypoxia environment and expressed higher LC3-II levels under glucose-deprived and hypoxia conditions. Finally, both 3-MA and CQ alleviated, albeit to different degrees, the increased chemoresistance in ANE-s and/or 30-100 K-s cells. CONCLUSIONS Chronic stimulations of ANE or ANE 30-100 K may increase tolerance of oral cancer and leukemia T cells to anticancer drugs, as well as to glucose deprivation and hypoxia conditions, and cause an elevation of autophagy activity responsible for increased drug resistance.

Outcome of treatment with total main tumor resection and supraomohyoid neck dissection in oral squamous cell carcinoma.

BACKGROUND/PURPOSE Supraomohyoid neck dissection (SOHND) is commonly used to treat oral squamous cell carcinoma (OSCC) patients with clinical N0 or selected N1 status. The purpose of this study was to evaluate the clinical outcome of OSCC patients treated with SOHND. METHODS This retrospective study reviewed the clinical outcome of 257 patients (247 men, 10 women) with N0, N1 and N2a OSCC treated with wide excision of the main tumor and SOHND between 1992 and 1999. All patients were followed up for at least 5 years. Survival distributions were analyzed using Kaplan-Meier curves. N status was compared using chi2 and log rank tests. RESULTS The neck failure rate was 20% for clinically false negative cases, 6.1% for clinically true negative cases, 21.8% for clinically false positive cases, and 40% for clinically true positive cases. The 3- and 5-year overall neck disease-free survival rates were 79.8% and 77.6%, respectively. The 3- and 5-year neck disease-free survival rates were 86.7% and 84.2% for pathologic N0 cases, 56.9% and 56.9% for pathologic N1 cases, and 27.5% and 27.5% for pathologic N2 cases, respectively. Log rank test showed that the p value for difference in survival at 3-5 years was 0.064 for pathologic N0 vs. N1 cases, < 0.0001 for pathologic N0 vs. N2 cases, and 0.008 for pathologic N1 vs. N2 cases. CONCLUSION This study showed that SOHND is effective for pathologic N0 OSCC, relatively effective for pathologic N1, and less effective for pathologic N2a. These findings also support that when SOHND is used to treat N2a OSCC, postoperative radiotherapy or radical neck dissection may be needed to improve the neck disease-free survival rate.