Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation.

OBJECTIVES: Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation. MATERIALS: Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2', 7'-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. RESULTS: TGM-2 expression was significantly higher in OSF specimens than normal specimens (p < 0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p < 0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p < 0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p < 0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p < 0.05). CONCLUSIONS: Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation. CLINICAL RELEVANCE: TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

BPDE-like DNA adduct level in oral tissue may act as a risk biomarker of oral cancer.

OBJECTIVE: Most reports have shown that PAH-related DNA adducts are positively correlated with the smoking status of oral cancer patients. However, these reports did not focus on a specific carcinogen in cigarette smoke. The purpose of this study was to elucidate the role of the BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)-DNA adduct in the development of oral cancer in Taiwanese patients. DESIGN: We enrolled 158 oral cancer patients and 64 non-cancer controls to investigate whether there were differences in susceptibility to cigarette smoke exposure in the formation of DNA adducts between cancer patients and controls. Immunohistochemistry and ELISA (enzyme-linked immunosorbent assay) were used to evaluate BPDE-DNA adduct levels in this study. RESULTS: Our data showed that the BPDE-DNA adduct levels were positively correlated with gender, smoking status, betel nut chewing and alcohol consumption. The difference in DNA adduct levels could be explained by genetic polymorphisms of glutathione S-transferase M1 (GSTM1), but not by cytochrome P-4501A1 (CYP1A1). Patients with high DNA adduct levels (≧34.03 adducts/10(8) nucleotides) had an approximately 9.936-fold risk of oral cancer compared with those with low DNA adduct levels (<34.03 adducts/10(8) nucleotides) (p<0.001). CONCLUSIONS: We suggest that genetic background and carcinogen exposure may increase the risk of developing oral cancer.

A preliminary study of the use of bioimpedance in the screening of squamous tongue cancer.

Oral cancers are the 11th most common malignancy reported worldwide, accounting for 3% of all newly diagnosed cancer cases, and one with high mortality ratios among all malignancies. The objective of this study was to study the electrical properties of cancerous tongue tissue (CTT) and normal tongue tissue (NTT). Five tongue cancer patients participated in this study. A disposable probe incorporating four silver electrodes was used to measure the electrical properties of CTT and the surrounding NTT of patients. Measurements were performed at six frequencies: 20 Hz; 50 kHz; 1.3 MHz; 2.5 MHz; 3.7 MHz; and 5 MHz, with the amplitude of the applied voltage limited to 200mV. Four measurement parameters of impedance (Z), phase angle (theta), real part of impedance (R), and imaginary part of impedance (X) of tongue tissue were assessed to see if there was any significant difference in the values obtained in CTT and surrounding NTT. The intraclass correlation coefficient showed that all measurements were reliable. A significant difference (P < 0.05 for the four measurement parameters) was found at 50kHz between CTT and surrounding NTT. It was also found that Z and R of CTT were generally smaller than that of surrounding NTT. In conclusion, bioimpedance at a particular frequency is a potentially promising technique for tongue cancer screening.

The use of bioimpedance in the detection/screening of tongue cancer.

Oral cancers are the 11th most common malignancy reported worldwide, accounting for 3% of all newly diagnosed cancer cases, and one with high mortality ratios among all malignancies. The objectives of this study were therefore to study the electrical properties of cancerous tongue tissue and normal tongue tissue, as well as to investigate a new approach for low-cost, noninvasive, and real-time screening of oral cancer. Twelve tongue cancer patients and twelve healthy subjects participated in this study. A disposable probe with four silver electrodes was used to measure the electrical properties of patient's and healthy subject's tongue tissues at six different frequencies, which were 20Hz, 50kHz, 1.3MHz, 2.5MHz, 3.7MHz and 5MHz. The amplitude of the applied voltage was limited to 200mV. Four measurement parameters of impedance, phase angle, real part of impedance, and imaginary part of impedance of tongue were assessed to see if significant difference in values obtained in patient's and healthy subject's tongue tissues existed. Intraclass correlation coefficient showed that all measurements had good reliability and validity (ICC>0.95 for all measurements). Significant differences were found at 20Hz (p<0.05-0.001 for the four measurement parameters) and 50kHz (p<0.001 for the four measurement parameters) between patient's and healthy subject's tongue tissues. In conclusion, bioimpedance at a particular frequency is a potentially promising technique for tongue cancer screening.

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by interleukin-1alpha in human pulp cells.

Tissue type plasminigen activator (t-PA) is one of the important proteolysis factors in the pathogenesis of pulpal inflammation. However, the mechanisms and signal transduction pathways involved in the production of t-PA in human pulp cells are not fully understood. The purpose of this study was to investigate the t-PA activity in human pulp cells stimulated with various pharmacological agents. IL-1alpha was used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search possible signal transduction pathways, p38 inhibitor SB203580, MEK inhibitor U0126, and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with IL-1alpha during 2 day cultured period (p < 0.05). From the results of casein zymography and ELISA, SB203580, U0126, and LY294002 significantly reduced the IL-1alpha-stimulated t-PA production, respectively (p < 0.05). Our findings demonstrated that IL-1alpha enhance t-PA production in human pulp cells, and the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of t-PA. SB203580, U0126, and LY294002 suppress t-PA activity and may also have important implication for pharmacological intervention.

The upregulation of matrix metalloproteinase-9 in inflamed human dental pulps.

The matrix metalloproteinases (MMPs) participate in a wide variety of extracellular matrix degradation. Detailed knowledge of MMPs may be important for understanding the pathogenesis of pulpal inflammation. The purpose of this study was to compare MMP-9 expression in clinically healthy human pulp and inflamed human pulp tissue specimens. We compared the levels of MMP-9 between clinically healthy pulp and inflamed pulp tissues by using the semi-quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of MMP-9 expression in pulp specimens. MMP-9 mRNA gene was found to be increased in inflamed pulps as compared with clinically healthy pulp tissues (p < 0.05). The results from immunohistochemistry demonstrated that MMP-9 expression was significantly higher in the inflamed pulps than clinically healthy pulps (p < 0.05). MMP-9 stain was detected in the odontoblasts, fibroblasts, inflammatory infiltrates, and endothelial cells. Taken together, MMP-9 may play an important role in the pathogenesis of pulpal inflammation.

The upregulation of insulin-like growth factor-1 in oral submucous fibrosis.

Insulin-like growth factor-1 (IGF-1) is a member of a family of two interacting polypeptide hormone ligands with close homology to proinsulin. IGF-1 can influence mesenchymal cell migration, proliferation, and extracellular matrix deposition, thus implicating it in the progression of fibrotic disorders. Currently, there is limited information about the regulation of IGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of this study was to compare IGF-1 expression in normal human buccal mucosa and OSF specimens and further explore the potential mechanism that may lead to induce IGF-1 expression. Twenty OSF specimens and 10 normal buccal mucosa were examined by immunohistochemistry. The activity of IGF-1 from cells cultured from OSF and normal buccal mucosa were by using reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Furthermore, the effect of arecoline, the major areca nut alkaloid, was added to explore the potential mechanism that may lead to induce IGF-1 expression. IGF-1 expression was significantly higher in OSF specimens (p<0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher IGF-1 protein expression than normal buccal mucosa fibroblast (BMF) both in mRNA and protein levels (p<0.05). In addition, arecoline was also found to elevate IGF-1 mRNA and protein expression in a dose-dependent manner (p<0.05). Taken together, the data presented here demonstrated that IGF-1 expression is significantly upregulated in OSF from areca quid chewers and arecoline may be responsible for the enhanced IGF-1 expression in vivo.

Raised keratinocyte growth factor-1 expression in oral submucous fibrosis in vivo and upregulated by arecoline in human buccal mucosal fibroblasts in vitro.

BACKGROUND: Keratinocyte growth factor-1 (KGF-1) is the seventh member of the fibroblast growth factor family. KGF-1 is produced by mesenchymal cells such as fibroblasts and upregulated in a variety of hyperplastic tissues. Currently, there is limited information about the regulation of KGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of the study was to compare KGF-1 expression in normal human buccal mucosa and OSF specimens and further to explore the potential mechanism that may lead to induce KGF-1 expression. METHODS: The expression of KGF-1 from fibroblasts cultured from OSF and normal buccal mucosa were using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether KGF-1 expression could affect by arecoline. Furthermore, 25 OSF specimens and six normal buccal mucosa specimens were examined by immunohistochemistry. RESULTS: Fibroblasts derived from OSF were found to exhibit higher KGF-1 expression than BMFs both in mRNA and protein levels (P < 0.05). In addition, upregulation of KGF-1 mRNA gene and protein expression were found in BMFs stimulated by arecoline (P < 0.05). From the results of immunohistochemistry, KGF-1 expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, inflammatory cells, and epithelial cells. CONCLUSIONS: Taken together, these results suggest that KGF-1 expression is significantly upregulated in OSF tissues from areca quid chewers and arecoline may be responsible for the enhanced KGF-1 expression in vivo.

Immunohistochemical localization of tissue-type plasminogen activator and type I plasminogen activator inhibitor in radicular cysts.

BACKGROUND: The plasminogen/plasmin proteolytic system participates in a wide variety of extracellular matrix degradation. Detailed knowledge of plasminogen activators (PAs) and their inhibitors may be important for understanding the pathogenesis of radicular cysts. The purpose of this study was to investigate the in situ localization of tissue-type PA (t-PA) and type I PA inhibitor (PAI-1) in radicular cysts. METHODS: Thirty formalin-fixed, paraffin-embedded specimens of radicular cysts were examined using immunohistochemistry. In addition, another section from each radicular cyst specimen was stained with hematoxylin and eosin to assess the presence of inflammatory infiltrates. Differences in t-PA and PAI-1 expression between tissues with low and high levels of inflammation were subsequently analyzed using Fisher's exact test. RESULTS: Both t-PA- and PAI-1-positive cells were detected in the lining epithelium, connective tissue, inflammatory infiltrates, and endothelium. In addition, the t-PA signal was mainly expressed in epithelial cells. However, the PAI-1 signal was mainly expressed in fibroblasts. Moreover, significantly greater t-PA as well as PAI-1 expression was noted in radicular cysts with high levels of inflammation as compared to tissues with low levels of inflammatory cell infiltrates (P < 0.05). CONCLUSIONS: The present study confirms earlier indications of local production of PA and its inhibitor in radicular cysts. In addition, this study further shows the tissue localization of the antigens for t-PA as well as PAI-1, and demonstrates that the expression of both t-PA and PAI-1 increases with the grade of inflammation in radicular cysts.