Ching Yu Yen

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.

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.

The expression and prognostic significance of hepatoma-derived growth factor in oral cancer

Hepatoma-derived growth factor (HDGF) participates in oncogenic progression and represents a prognostic factor in several types of cancer. This study aimed to elucidate the role of HDGF during oral carcinogenesis. HDGF expression and the tumorigenic behaviors in human oral cell lines were investigated by immunoblotting, invasion and colony formation assays. Recombinant adenovirus vectors were employed to modulate the HDGF level in oral cancer cells. Immunohistochemical analysis using tissue microarray (TMA) consisting of surgically resected samples from 95 oral cancer patients was performed to delineate the correlation between HDGF expression and clinic-pathological parameters. HDGF expression was higher in malignant oral cancer cells than benign ones. Adenovirus-mediated HDGF overexpression and knockdown demonstrated the cellular HDGF level regulated the tumorigenic behaviors of oral cancer cells. Immunohistochemical analysis revealed increased HDGF expression in the nucleus and cytoplasm in oral cancer tissues. The nuclear HDGF expression was significantly correlated with tumor stage (P=0.004) and grade (P=0.013) while the cytoplasmic HDGF expression was associated with tumor necrosis (P=0.002). Kaplan-Meier analysis revealed that patients with high nuclear HDGF expression had significantly worse 5-year disease-specific survival (P=0.0069), metastasis-free survival (P=0.0168), and local recurrence-free survival (P=0.0047). Multivariate analysis indicated that the nuclear HDGF labeling index was an independent prognostic factor for disease-specific and local recurrence-free survival. HDGF overexpression contributes to the oncogenic processes in oral cancer cells and constitutes a novel prognostic factor for survival outcome of oral cancer patients.

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.

Evaluating the performance of fibronectin 1 (FN1), integrin α4β1 (ITGA4), syndecan-2 (SDC2), and glycoprotein CD44 as the potential biomarkers of oral squamous cell carcinoma (OSCC)

Objectives: Some extracellular matrix genes as prognostic biomarkers for oral squamous cell carcinoma (OSCC) were evaluated. Methods: We investigated gene expression of fibronectin 1 (FN1), integrin α4β1 (ITGA4), syndecan-2 (SDC2), and glycoprotein CD44 in matched OSCC/margin tissues. Results: Areas under receiver-operating characteristic curves (AUCs) of relative mRNA expression of FN1, ITGA4, SDC2, and CD44 were 0.700, 0.677, 0.513, and 0.549, respectively. For tongue/mouth floor and edentulous ridge, AUC for FN1 and ITGA4 were 0.827 and 0.725 and sensitivities/specificities were 80%/84% and 88%/52%, respectively. Conclusion: FN1 and ITGA4 are potential OSCC biomarkers for tongue/mouth floor and edentulous ridge.

DNA methylation, histone acetylation and methylation of epigenetic modifications as a therapeutic approach for cancers.

Epigenetic modifications play important roles in regulating carcinogenesis, and specific epigenetic modifications have emerged as potential tumor markers. Herein, we summarize several types of epigenetic modifications, explore the role played by epigenetic modifications in gene regulation, and describe the patterns of epigenetic modifications in cancers. Since epigenetic modifications have been reported to regulate the Warburg effect in cancers, the roles of epigenetic modifications in sugar metabolism are discussed. In addition, oxidative stress may play an important role in carcinogenesis, and the role of oxidative stress and epigenetic modification in carcinogenesis is addressed. We also discuss the role of epigenetic modifications as therapeutic targets. Finally, the synergistic effects of the combined treatment of epigenetic regulator and anticancer drugs for cancer therapy are described.

Tenuifolide B from Cinnamomum tenuifolium Stem Selectively Inhibits Proliferation of Oral Cancer Cells via Apoptosis, ROS Generation, Mitochondrial Depolarization, and DNA Damage

The development of drugs that selectively kill oral cancer cells but are less harmful to normal cells still provide several challenges. In this study, the antioral cancer effects of tenuifolide B (TFB), extracted from the stem of the plant Cinnamomum tenuifolium are evaluated in terms of their effects on cancer cell viability, cell cycle analysis, apoptosis, oxidative stress, and DNA damage. Cell viability of oral cancer cells (Ca9-22 and CAL 27) was found to be significantly inhibited by TFB in a dose-responsive manner in terms of ATP assay, yielding IC50 = 4.67 and 7.05 μM (24 h), but are less lethal to normal oral cells (HGF-1). Dose-responsive increases in subG1 populations as well as the intensities of flow cytometry-based annexin V/propidium iodide (PI) analysis and pancaspase activity suggested that apoptosis was inducible by TFB in these two types of oral cancer cells. Pretreatment with the apoptosis inhibitor (Z-VAD-FMK) reduced the annexin V intensity of these two TFB-treated oral cancer cells, suggesting that TFB induced apoptosis-mediated cell death to oral cancer cells. Cleaved-poly (ADP-ribose) polymerase (PARP) and cleaved-caspases 3, 8, and 9 were upregulated in these two TFB-treated oral cancer cells over time but less harmful for normal oral HGF-1 cells. Dose-responsive and time-dependent increases in reactive oxygen species (ROS) and decreases in mitochondrial membrane potential (MitoMP) in these two TFB-treated oral cancer cells suggest that TFB may generate oxidative stress as measured by flow cytometry. N-acetylcysteine (NAC) pretreatment reduced the TFB-induced ROS generation and further validated that ROS was relevant to TFB-induced cell death. Both flow cytometry and Western blotting demonstrated that the DNA double strand marker γH2AX dose-responsively increased in TFB-treated Ca9-22 cells and time-dependently increased in two TFB-treated oral cancer cells. Taken together, we infer that TFB can selectively inhibit cell proliferation of oral cancer cells through apoptosis, ROS generation, mitochondrial membrane depolarization, and DNA damage.

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.

Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.