Shiuan-Shinn Lee

The upregulation of heat shock protein 70 expression in areca quid chewing-associated oral squamous cell carcinomas

Heat shock protein 70 (HSP70) is an important stress-induced protein. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP70 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP70 expression. 41 OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N-acetyl-l-cysteine (NAC), AP-1 inhibitor curcumin, extracellular signal-regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. The results from immunohistochemistry demonstrated that HSP70 expression was significantly higher in OSCC specimens (p<0.05). No significant difference in HSP70 expression was observed with respect to age, sex, T category, and stage (p>0.05). The low HSP70 expression was associated with lymph node metastasis (p=0.005). The high HSP70 expression was found in poor differentiated tumor groups (p=0.036). Arecoline was found to elevate HSP70 expression in a dose- and time-dependent manner (p<0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline-induced HSP70 expression (p<0.05). Taken together, HSP70 expression is significantly upregulated in areca quid chewing-associated OSCC. HSP70 could be used clinically as a marker for tumors possessing the potential for differentiation as well as lymph node metastasis. In addition, arecoline-induced HSP70 expression was downregulated by NAC, curcumin, PD98059, and staurosporine.

Upregulation of Heme Oxygenase-1 Expression in Areca-quid-chewing-associated Oral Squamous Cell Carcinoma

BACKGROUND/PURPOSE Heme oxygenase-1 (HO-1) is known as an oxidative stress responsive protein that is upregulated by various physiologic and endogenous stimuli. HO-1 has been proposed to provide an important cellular response that protects cells against oxidative damage. Areca quid chewing is a major risk factor in the development and further progression of oral squamous cell carcinoma (OSCC). The aim of the present study was to investigate the difference in HO-1 expression in normal human oral epithelium and OSCC, and further explore the potential mechanism that may lead to HO-1 expression. METHODS Thirty-five OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by clinicopathologic profiles. The oral epithelial GNM cell line was challenged with arecoline, a major areca nut alkaloid, by reverse-transcriptase polymerase chain reaction. Furthermore, tobacco smoke carcinogen benzo[a]pyrene (BaP) and glutathione (GSH) precursor N-acetyl-L-cysteine were added to find the possible regulatory mechanisms. RESULTS HO-1 expression was significantly higher in OSCC specimens (p < 0.05). No significant difference in HO-1 expression was observed with respect to age, sex, T category, and stage (p > 0.05). The high HO-1 expression was associated with lymph node metastasis (p = 0.005). In addition, arecoline was found to elevate HO-1 mRNA in a dose-dependent manner (p < 0.05). The addition of BaP enhanced arecolineinduced HO-1 expression (p < 0.05). Moreover, addition of NAC markedly inhibited arecoline-induced HO-1 expression (p < 0.05). CONCLUSION Taken together, these results suggest that HO-1 expression is significantly upregulated in OSCC from areca quid chewers, and arecoline may be responsible for enhanced HO-1 expression in vivo. The compounds of cigarette smoke may act synergistically in the pathogenesis of areca-quid-chewing-associated OSCC. The regulation of HO-1 expression induced by arecoline is critically dependent on intracellular GSH concentration.

Protective Effect of Ginkgo biloba leaves extract, EGb761, on Endotoxin-Induced Acute Lung Injury via a JNK- and Akt-Dependent NFκB Pathway

Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway.

Cytotoxicity and genotoxicity of chlorhexidine on macrophages in vitro.

Chlorhexidine (CHX) is the most widely used antiseptic for wound, skin disinfection, and dental hygiene. The aim of this study is to investigate the possible correlation between CHX‐induced cytogenotoxicity and alterations in normal cell cycle on RAW264.7 macrophages. The cytotoxicity, mechanism of cell death, mitotic activity, and reactive oxygen species (ROS) generation were determined by tetrazolium bromide reduction assay, flow cytometry, cytokinesis‐block proliferation index, and superoxide dismutase‐inhibitable reduction of ferricytochrome c, respectively. The genotoxicity was measured using comet assay and cytokinesis‐block micronucleus assay. The cytotoxicity of CHX in RAW264.7 cells presented a dose‐ and time‐dependent manner (p < 0.05). The mode of cell death shifted from apoptosis to necrosis when the dosage of CHX increased. The genotoxicity of CHX in RAW264.7 cells had shown DNA damage in a dose‐dependent manner (p < 0.05). Prolongation of cell cycle and the increase of ROS generation also expressed in a dose‐dependent manner (p < 0.05). Taken together, the data suggested that CHX‐induced cytotoxicity and genotoxicity on macrophages may be via ROS generation.

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

OBJECTIVES Hypoxia inducible factor (HIF)-1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF-1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF-1α expression in normal human oral epithelium and areca quid chewing-associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF-1α expression. METHODS Twenty-five OSCC from areca quid chewing-associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N-acetyl-l-cysteine (NAC), AP-1 inhibitor curcumin, extracellular signal-regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. RESULTS Hypoxia inducible factor-1α expression was significantly higher in OSCC specimens than normal specimen (P<0.05). Arecoline was found to elevate HIF-1α expression in a dose- and time-dependent manner (P<0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline-induced HIF-1α expression (P<0.05). CONCLUSIONS Hypoxia inducible factor-1α expression is significantly upregulated in areca quid chewing-associated OSCC and HIF-1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.

Cytotoxicity of dentine bonding agents on human pulp cells is related to intracellular glutathione levels

AIM To evaluate ex vivo the mechanisms of cytotoxicity of dentine bonding agents in human pulp cells in vitro. METHODOLOGY Human pulp cells were obtained from impacted third molars with informed consent and then cultured using an explant technique. Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity was judged using an assay of tetrazolium bromide reduction. To determine whether glutathione (GSH) levels were important in the cytotoxicity of dentine bonding agents, cells were pretreated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Three replicates of each dentine bonding agents were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (anova). Tests of differences of the treatments were analysed by Duncans test. RESULTS Clearfil SE Bond, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (P<0.05). The cytotoxicity was upregulated by dentine bonding agents in the following order: PB>SB>CB. Addition of OTZ extracellularly protected the pulp cells from dentine bonding agents-induced cytotoxicity (P<0.05). Addition of BSO enhanced pulp cell death on dentine bonding agents-induced cytotoxicity (P<0.05). CONCLUSIONS Dentine bonding agents have significant potential for pulpal toxicity. GSH depletion could be the mechanism for dentine bonding agents-induced cytotoxicity.

Up-regulation of Receptor Activator Nuclear Factor–Kappa B Ligand Expression by Root Canal Sealers in Human Osteoblastic Cells

Histologic investigations have demonstrated that root canal sealers can induce mild to severe inflammatory alternations. A recently identified tumor necrosis factor family molecule, receptor activator of nuclear factor-kappaB ligand (RANKL), plays a critical role in the development of osteoclasts that result in bone resorption. The aim of this study was to investigate the effects of root canal sealers AH26, Canals, and N2 on the expression of RANKL in human osteoblast cell line U2OS cells. Freshly mixed materials were filled in glass rings (4 mm height and 10 mm in diameter) and eluted in 10 mL of culture medium for 1 day. Subsequently, various dilutions (final dilution: 1/2, 1/4, and 1/8) of these extraction media were prepared for this study. Cytotoxicity was measured by the propidium iodide fluorescence assay. The expression of RANKL was measured by reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that AH26, Canals, and N2 were cytotoxic to U2OS cells in a concentration-dependent manner (P < .05). The exposure of U2OS cells to root canal sealers resulted in the up-regulation of RANKL mRNA and protein expression (P < .05). The expression of RANKL was up-regulated by root canal sealers in the following order: N2 > AH26 > Canals. Taken together, the activation of RANKL might play an important role in the pathogenesis of periapical bone destruction induced by root canal sealers.

Elevated Snail Expression Mediates Tumor Progression in Areca Quid Chewing-Associated Oral Squamous Cell Carcinoma via Reactive Oxygen Species

Background Snail is an important transcription factor implicated in several tumor progression and can be induced by reactive oxygen species (ROS). Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). Therefore, we hypothesize that the major areca nut alkaloid arecoline may induce Snail via ROS and involve in the pathogenesis of areca quid chewing-associated OSCC. Methodology/Principal Finding Thirty-six OSCC and ten normal oral epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. Cytotoxicity, 2′, 7′-dichlorofluorescein diacetate assay, and western blot were used to investigate the effects of arecoline in human oral keratinocytes (HOKs) and oral epithelial cell line OECM-1 cells. In addition, antioxidants N-acetyl-L-cysteine (NAC), curcumin, and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. Initially, Snail expression was significantly higher in OSCC specimens (p<0.05). Elevated Snail expression was associated with lymph node metastasis (p = 0.031) and poor differentiation (p = 0.017). Arecoline enhanced the generation of intracellular ROS at the concentration higher than 40 µg/ml (p<0.05). Arecoline was also found to induced Snail expression in a dose- and time-dependent manner (p<0.05). Treatment with NAC, curcumin, and EGCG markedly inhibited arecoline induced Snail expression (p<0.05). Conclusion/Significance: Our results suggest that Snail overexpression in areca quid chewing-associated OSCC is associated with tumors differentiation and lymph node metastasis. Arecoline-upregulated Snail expression may be mediated by ROS generation. In addition, arecoline induced Snail expression was downregulated by NAC, curcumin, and EGCG.

Cytotoxicity and genotoxicity of triethyleneglycol-dimethacrylate in macrophages involved in DNA damage and caspases activation

Triethyleneglycol‐dimethacrylate (TEGDMA) is a monomer and widely used in dental composite resins. TEGDMA has been found to exhibit cytotoxicity and genotoxicity on many cells. However, little is known about the potential toxicological implications of TEGDMA on murine macrophage cell line RAW264.7. In this study, TEGDMA demonstrated a cytotoxic effect to RAW264.7 cells in a concentration‐ and time‐dependent manner (p < 0.05). TEGDMA was found to induce two modes of cell death in a concentration‐dependent manner (p < 0.05). TEGDMA‐induced cell apoptosis was demonstrated by the increase in the portion of sub‐G0/G1 phase and DNA ladder formation. In addition, TEGDMA exhibited genotoxicity via a dose‐related increase in the numbers of micronucleus and DNA strand breaks (p < 0.05). Furthermore, the activation of caspase‐3, −8, and −9 were generated by TEGDMA in a dose‐dependent manner (p < 0.05). These results indicated that cytotoxicity and genotoxicity induced by TEGDMA in macrophages may be via DNA damage and caspase activation.

Rutin improves endotoxin‐induced acute lung injury via inhibition of iNOS and VCAM‐1 expression

Endotoxins exist anywhere including in water pools, dust, humidifier systems, and machining fluids. The major causal factor is endotoxins in many serious diseases, such as fever, sepsis, multi‐organ failure, meningococcemia, and severe morbidities like neurologic disability, or hearing loss. Endotoxins are also called lipopolysaccharide (LPS) and are important pathogens of acute lung injury (ALI). Rutin has potential beneficial effects including anti‐inflammation, antioxidation, anti‐hyperlipidemia, and anti‐platelet aggregation. Pre‐treatment with rutin inhibited LPS‐induced neutrophil infiltration in the lungs. LPS‐induced expression of vascular cell adhesion molecule (VCAM)‐1 and inducible nitric oxide synthase (iNOS) was suppressed by rutin, but there was no influence on expression of intercellular adhesion molecule‐1 and cyclooxygenase‐2. In addition, activation of the nuclear factor (NF)κB was reduced by rutin. Furthermore, we found that the inhibitory concentration of rutin on expression of VCAM‐1 and iNOS was similar to NFκB activation. In conclusion, rutin is a potential protective agent for ALI via inhibition of neutrophil infiltration, expression of VCAM‐1 and iNOS, and NFκB activation.