Areca nut extract treatment elicits the fibroblastoid morphological changes, actin re-organization and signaling activation in oral keratinocytes

BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.     

Effects of areca nut extract on the apoptosis pathways in human neutrophils

Ho W‐H, Lee Y‐Y, Chang L‐Y, Chen Y‐T, Liu T‐Y, Hung S‐L. Effects of areca nut extract on the apoptosis pathways in human neutrophils. J Periodont Res 2010; 45: 412–420. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. Material and Methods: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. Results: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP‐ribose) polymerase, and of caspase‐3 and caspase‐8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase‐3α/β may be involved in the ANE‐modulated effects of neutrophils. Conclusion: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

三氧化二砷纳米粒对舌鳞癌细胞PI3K/AKT通路的影响

目的：探讨三氧化二砷固体脂质纳米粒对舌鳞癌Tca8113细胞作用前后PI3K/AKT信号通路关键因子表达情况。方法：使用MTT的方法观察三氧化二砷固体脂质纳米粒对舌鳞癌Tca8113细胞作用后的存活率情况,并用使用Western blot检测方法检测PI3K/AKT信号通路关键蛋白PI3K、p-AKT、AKT的表达和变化。结果：三氧化二砷固体脂质纳米粒对舌鳞癌细胞作用后,MTT显示舌鳞癌Tca8113细胞增殖抑制,且具有浓度依赖性,PI3K/AKT信号通路关键因子PI3K、p-AKT、AKT表达降低。结论：三氧化二砷纳米粒降低舌鳞癌细胞的PI3K/AKT通路信号传导。     

Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells

Wang C‐C, Chen T‐Y, Wu H‐Y, Liu T‐Y, Jan T‐R. Areca nut extracts suppress the differentiation and functionality of human monocyte‐derived dendritic cells. J Periodont Res 2012; 47: 198–203. © 2011 John Wiley & Sons A/S Background and Objective: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. Material and Methods: Human peripheral blood monocytes were cultured in the presence of granulocyte–monocyte colony‐stimulating factor and interleukin‐4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS) was examined. Results: Monocytes cultured in granulocyte–monocyte colony‐stimulating factor and interleukin‐4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)‐DR, CD11c and the co‐stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA‐DR and CD11c, but markedly attenuated the proportion of CD40‐positive cells and the mean fluorescence intensity of CD86. The expression of co‐stimulatory molecules in LPS‐activated dendritic cells was not affected, whereas the mRNA expression of interleukin‐12 induced by LPS was markedly suppressed by ANE treatment in a concentration‐dependent manner. Conclusion: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte‐derived dendritic cells was attenuated in the presence of ANE.     

PI3K/AKT/mTOR/p70S6K通路在人舌鳞癌细胞耐药中的作用

目的:通过抑制舌鳞癌细胞中PI3K/AKT/mTOR/p70S6K信号通路的表达,检测细胞凋亡与自噬的变化,探讨舌鳞癌耐药的机制。方法:以舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP为研究对象。分别用PI3K/AKT抑制剂LY294002、mTOR抑制剂Rapamycin、p70S6K抑制剂LY2584702作用该通路各个环节。Western blot检测通路抑制剂作用后相关蛋白的变化。Cyto-ID荧光染色检测自噬体的形成。流式细胞术检测细胞凋亡水平。结果:Western blot结果显示加入抑制剂后舌鳞癌细胞Beclin1表达分别高于对照组,LC3II与LC3I以及Bax与Bcl-2的比值均升高,该通路的p-AKT、p-mTOR、p-p70S6K等蛋白均有下降(P<0.05)。流式结果显示加入抑制剂后的舌鳞癌细胞凋亡率分别较对照组升高(P<0.05)。Cyto-ID荧光染色后结果显示加入抑制剂后的舌鳞癌细胞自噬小体的数目明显高于对照组(P<0.05)。对比两组细胞显示加入抑制剂后的Cal27细胞发生凋亡与自噬的水平高于Cal27/CDDP(P<0.05)。结论:抑制PI3K/AKT/mTOR/p70S6K信号通路可诱导舌鳞癌细胞Cal27与舌鳞癌耐顺铂细胞Cal27/CDDP发生凋亡与自噬,激活的PI3K/AKT/mTOR/p70S6K通路抑制细胞凋亡与自噬是Cal27/CDDP细胞产生顺铂耐药的原因之一。     

Inhibitory Effects of Areca Nut Extract on Expression of Complement Receptors and Fc Receptors in Human Neutrophils

Background: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement‐ and antibody‐opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F‐actin in ANE‐treated neutrophils is also analyzed. Methods: The viability of ANE‐treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G‐opsonized fluorescent beads was analyzed using flow cytometry. Expression of F‐actin was determined using confocal laser scanning microscopy. Results: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration‐dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F‐actin was inhibited after ANE treatment. Conclusions: ANE inhibits the complement‐ and IgG‐mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F‐actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.     

Lipopolysaccharide Enhances Wnt5a Expression through Toll-like Receptor 4, Myeloid Differentiating Factor 88, Phosphatidylinositol 3-OH Kinase/AKT and Nuclear Factor Kappa B Pathways in Human Dental Pulp Stem Cells

Introduction

Lipopolysaccharide (LPS) has been implicated in mesenchymal stem cell differentiation processes. Wnt5a, one of the “non-canonical” Wnt family members, is important in signaling stem cell differentiation and in the inflammatory responses of immune cells. Here we studied whether LPS can regulate the expression of Wnt5a in human dental pulp stem cells (hDPSCs) and investigated the intracellular signaling pathways activated by LPS.

Methods

Wnt5a mRNA and protein expression changes in hDPSCs were investigated by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay. In addition, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase activity assays were used to determine whether toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), nuclear factor kappa B (NF-kB), or the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathways are involved in LPS-induced Wnt5a expression. The activation of PI3K and AKT in hDPSCs was measured by Western blot analysis.

Results

Wnt5a mRNA and protein expression was rapidly increased in response to LPS in a time- and dose-dependent manner. LPS-induced Wnt5a expression was effectively attenuated by administration of a TLR4 neutralizing antibody, MyD88 inhibitory peptide, PI3-kinase inhibitors (LY294002 and wortmannin), an AKT inhibitor, or NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBa phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). Treatment of hDPSCs with LPS activated PI3-kinase (p85) and AKT signaling in a time-dependent manner. Moreover, LPS-mediated increases in κB-luciferase activity were diminished by the overexpression of dominant negative mutants of TLR4, MyD88, p85, AKT, and IκBa.

Conclusions

These results demonstrated that LPS-induced Wnt5a expression was mediated through the TLR4/MyD88/PI3-kinase/AKT pathway, which then initiated NF-κB activation in hDPSCs.     

SOX2通过PI3K/AKT通路调控口腔鳞癌细胞迁徙的机制研究

目的:研究过表达SOX2通过PI3K/AKT通路促进口腔鳞癌(oral squamous cell carcinoma,OSCC)细胞迁移及上皮间质转化(epithelial mesenchymal transition,EMT)的作用及机制.方法:收集OSCC组织及正常口腔黏膜组织,培养OSCC细胞株Tca83、SC...     

沉默法尼基转移酶对人涎腺腺样囊性癌细胞迁移、侵袭及上皮间充质转化的作用

目的　通过体外实验探讨法尼基转移酶（FTase）对涎腺腺样囊性癌SACC-LM和SACC-83细胞迁移侵袭及上皮间充质转化（EMT）的作用及其机制。方法　针对人FTase基因序列设计构建3条小干扰RNA（si-RNA）,采用处于对数生长期的SACC-LM及SACC-83细胞,经脂质体瞬时转染技术抑制细胞FTase表达,分别命名为FTase-siRNA-1组、FTase-siRNA-2组、FTase-siRNA-3组,同时设置阴性对照组（NC-siRNA）,空白对照组（仅加入转染试剂）。采用实时荧光定量逆转录聚合酶链反应检测FTase、HRAS的mRNA表达,选择沉默效率最高的FTase-siRNA进行后续实验;蛋白质免疫印迹法检测FTase、HRAS、蛋白激酶B（AKT）、磷酸化AKT、p65、磷酸化p65（Ser563）、上皮钙依赖黏附蛋白、波形蛋白、基质金属蛋白酶9（MMP-9）的蛋白表达以及HRAS膜蛋白表达;Transwell小室及细胞划痕实验检测细胞的侵袭和迁移能力。结果　与空白对照组及阴性对照组相比,FTase-siRNA-1组mRNA及蛋白相对表达量均降低（P<0.05）,HRAS mRNA和总蛋白表达的差异无统计学意义（P>0.05）,HRAS膜蛋白相对表达量降低（P<0.05）,上皮钙依赖黏附蛋白相对表达量升高（P<0.05）,波形蛋白相对表达量降低（P<0.05）,MMP-9蛋白相对表达量降低（P<0.05）,RAS/PI3K/AKT/核因子-κB信号通路相关蛋白AKT、p65相对表达量的差异无统计学意义（P>0.05）,但磷酸化AKT、磷酸化p65蛋白相对表达量降低;与空白及阴性对照组相比,FTase-siRNA-1组细胞侵袭及迁移能力显著下降（P<0.05）。结论　体外沉默FTase可有效抑制人涎腺腺样囊性癌细胞SACC-LM和SACC-83的侵袭、迁移能力,其作用可能是通过干扰HRAS膜蛋白的定位,调控RAS/PI3K/AKT/核因子-κB信号通路,介导EMT而实现。     

Migration induced by epidermal and hepatocyte growth factors in oral squamous carcinoma cells in vitro: role of MEK/ERK, p38 and PI-3 kinase/Akt

J Oral Pathol Med (2012) 41 : 547–558 Background: Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti‐tumour signal inhibitors on the migratory activity. Methods: Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time‐lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. Results: In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose‐dependent activation of cell migration. Addition of the EGFR‐specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF‐induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI‐3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF‐induced migration was particularly sensitive to PI‐3 K‐inhibition, while in C12 cells, both HGF‐ and EGF‐induced migration were highly sensitive to p38‐blockade. Conclusion: The results demonstrate that the MEK/ERK, p38 and PI‐3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.     

Areca-treated fibroblasts enhance tumorigenesis of oral epithelial cells

Several hundred million Asians chew areca nut, which is strongly associated with oral carcinogenesis in people of this region. The impacts of areca nut extract on oral target cells are largely unclear. This study hypothesized an inductive role for areca-nut-exposed stromal cells in the progression of oral carcinomas in an at-risk population. Oral fibroblasts with chronic subtoxic areca nut extract treatment exhibited growth arrest and MMP-2 activation. The supernatant of arrested oral fibroblasts activated the AKT signaling pathway in oral carcinoma cells. The enhancement of proliferation, migration, and anchorage-independent growth of oral carcinoma cells elicited by such supernatant could be abrogated by blockers against MMP-2 or AKT. Subcutaneous co-injection of arrested oral fibroblasts into nude mice significantly enhanced the tumorigenicity of xenographic oral carcinoma cells. This study concludes that areca nut extract may impair oral fibroblasts and then modulate the progression of oral epithelial oncogenesis via their secreted molecules.     

甘草苷调控PI3K/Akt/m-TOR信号通路对口腔鳞状细胞癌细胞及裸鼠移植瘤小鼠的作用研究

目的:目的探讨甘草苷(Liquiritin,LIQ)对人口腔鳞状细胞癌细胞(OSCCs)的增殖抑制、凋亡诱导作用和分子机制并通过裸鼠模型进行验证。方法:CCK-8检测LIQ对口腔鳞状细胞癌细胞的增殖抑制作用;流式细胞术及TUNEL实验检测凋亡情况;Western blot检测蛋白表达变化;构建裸鼠瘤模型,连续给药20 d,绘制肿瘤生长曲线,并计算抑瘤率。结果:LIQ可以抑制口腔鳞状细胞癌细胞增殖及裸鼠瘤体积增长,并诱导细胞凋亡;LIQ抑制PI3K/Akt/m-TOR信号通路的过度激活。结论:LIQ能够在体内外对口腔鳞状细胞癌产生抗癌效果,其机制可能是通过抑制PI3K/AKT/m-TOR信号通路的过度激活。     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

Effects of Areca Nut Extract on Lipopolysaccharides‐Enhanced Adhesion and Migration of Human Mononuclear Leukocytes

Background: Areca chewers have a higher prevalence of periodontitis than non‐chewers. Cell adhesion and movement (migration) are important for leukocyte recruitment to inflammation sites. This study investigates the effects of areca nut extract (ANE) on the adhesion and migration abilities of the human immune cells, peripheral blood mononuclear cells (PBMCs). The combined effects of nicotine and lipopolysaccharides (LPS) were also analyzed. Methods: Purified PBMCs obtained from healthy adults were treated with ANE, nicotine, and/or LPS. Cell adhesion ability was examined using fibronectin‐coated microslides, Liu stain, and light microscopy. Cell migration ability was evaluated using the transwell system followed by staining and fluorescence microscopy. Statistical difference was analyzed using the Mann‐Whitney U test. Results: When compared with the media‐treated control samples, PBMCs treated with ANE for 4 hours showed a significant reduction of the adherent cells on the microslides. Interestingly, LPS treatment increased cell adhesion, which could be reduced by simultaneous ANE plus nicotine treatment. The chemotactic migration of PBMCs was reduced by ANE treatment for 1, 4, or 24 hours in a dose‐dependent manner. LPS treatment increased PBMC migration, which could be reduced by simultaneous treatment with ANE or with ANE plus nicotine. Conclusions: ANE reduced the adhesion and migration abilities of PBMC. ANEs, with or without nicotine, also attenuated the migration of LPS‐stimulated PBMCs. The results implicated that the immune cell functions were impaired in areca chewers, which might increase the host susceptibility to oral and periodontal infection.     

Expression and alterations of the PTEN / AKT / mTOR pathway in ameloblastomas

Objectives:  Recently, an allelic loss of phosphatase and tensin homologue (PTEN) was shown to occur in ameloblastomas. In carcinogenesis, loss of PTEN allows for overactivity of the phosphatidylinositol-3-kinase/protein kinase B (PI3K / AKT) pathway inducing an upregulation of mammalian-target of rapamycin (mTOR) and its downstream effector ribosomal-subunit-6 kinase (S6K); allowing for uncontrolled cell proliferation, apoptosis inhibition and cell cycle deregulation.
Methods:  Thirty ameloblastomas and five dental follicles were studied, looking at the immunohistochemical expression of total PTEN and AKT, as well as their phosphorylated (p) active forms, and the downstream effector and indicator of mTOR activity p70 ribosomal-subunit-6 kinase (pS6K). Also assessed was the expression of extracellular-signal-regulated kinase (ERK), which cross talks with AKT.
Results:  Total PTEN was absent in 33.3% of ameloblastomas, while its stabilized, phosphorylated^{ser380 / thr382 / thr383} form was absent in 83.3% of tumors. In contrast, AKT was expressed in 83.3% of ameloblastomas, showing high expression of the p-thr³⁰⁸AKT and p-ser⁴⁷³ AKT forms in 93.3% and 56.6% of cases, respectively. Further, the mTOR activated pS6K^{ser240 / 244} was detected in 86.7% of ameloblastomas, while ERK was overexpressed in 70.0% of the cases.
Conclusion:  Immunohistochemical analysis of aberrant signaling in the PI3K/AKT/mTOR pathway in ameloblastomas may represent a valuable tool for elucidating pathogenesis, aggressiveness and selecting optimal therapeutics.     

Heat shock protein 47 expression in oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

J Oral Pathol Med (2010) 40 : 390–396 Background: Heat shock protein 47 (HSP47) is a product of CBP2 gene located at chromosome 11q13.5, a region frequently amplified in human cancers. Areca quid chewing is a major risk factor of oral squamous cell carcinoma (OSCC). The aim of this study was to compare HSP47 expression in normal human oral epithelium and OSCC and further to explore the potential mechanisms that may lead to induce HSP47 expression. Methods: Thirty‐two OSCC specimens and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line OC2 cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002, cyclooxygenase‐2 inhibitor NS‐398, and tyrosine kinase inhibitor herbimycin A were added to find the possible regulatory mechanisms. Results: HSP47 expression was significantly higher in OSCC specimens than normal epithelium (P < 0.05). No significant difference in HSP47 expression was observed with respect to age, sex, T category, stage, and differentiation (P > 0.05). The lower HSP47 expression was associated with lymph node metastasis (P = 0.015). Arecoline was found to elevate HSP47 expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, PD98059, LY294002, NS398, and herbimycin A markedly inhibited the arecoline‐induced HSP47 expression (P < 0.05). Conclusion: Our findings demonstrated that HSP47 expression is significantly upregulated in areca quid chewing‐associated OSCCs. HSP47 could be used clinically as a marker for lymph node metastasis of oral carcinogenesis. In addition, arecoline‐induced HSP47 expression was downregulated by NAC, PD98059, LY294002, NS398, and herbimycin A.     

Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2

Background:  Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis.
Methods:  Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2.
Results:  Chronic subtoxic (<10 μg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated β-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody.
Conclusions:  This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers.     

PI3K/AKT信号通路在2型糖尿病患者种植体骨结合中作用机制的研究进展

口腔种植治疗目前被认为是缺牙患者的首选治疗方案,但糖尿病仍然是种植治疗的相对禁忌证。糖尿病患者的高血糖环境以及微血管病变等可能会影响成骨细胞及破骨细胞的功能,影响骨代谢,导致种植失败。目前,对于糖尿病影响种植体骨结合的确切机制尚未阐明。PI3K/AKT信号通路在骨组织代谢中起重要作用,可促进成骨细胞的增殖和分化;此外,PI3K/AKT/mTOR也是响应胰岛素信号传导的经典途径。该文分别对PI3K/AKT信号通路在2型糖尿病和种植体骨结合中作用机制的研究进展进行综述,为今后促进糖尿病患者种植体周围骨再生等研究奠定基础。