Human Oral Epithelium Research Articles

Four New Pregnane Steroids from Aglaia abbreviata and Their Cytotoxic Activities

AbstractFour new pregnane steroids, aglaiasterols A–D (1–4), have been isolated from the EtOH extract of stems of Aglaia abbreviata. They were identified as (3α,5α,17Z)‐3‐hydroxypregn‐17‐en‐16‐one (1), (3β,5α,17E)‐3‐hydroxypregn‐17‐en‐16‐one (2), (3β,5α,17Z)‐3‐hydroxypregn‐17‐en‐16‐one (3), and (3α,5α,20S*)‐3‐hydroxy‐16‐oxopregnan‐20‐yl acetate (4) on the basis of spectroscopic methods, including 1D‐ and 2D‐NMR techniques. Compounds 1–4 were evaluated for their cytotoxic activities against K562 (human leukemia), MCF‐7 (human breast cancer), and KB (human oral epithelium cancer) cells, and drug‐resistant cells of K562/A02, MCF‐7/ADM, and KB/VCR. These isolates showed weak to moderate inhibitory effects on the growth of the tested cell lines.

Elevated expression of JMJD6 is associated with oral carcinogenesis and maintains cancer stemness properties.

Cancer stem cells (CSCs) are defined as a small subpopulation of cancer cells within a tumor and responsible for initiation and maintenance of tumor growth. Thus, understanding of molecular regulators of CSCs is of paramount importance for the development of effective cancer therapies. Here, we identified jumonji domain-containing protein 6 (JMJD6) as a novel molecular regulator of oral CSCs. JMJD6 is highly expressed in CSC-enriched populations of human oral squamous cell carcinoma (OSCC) cell lines. Moreover, immunohistochemical staining revealed significantly high level of JMJD6 in OSCC tissues compared to normal human oral epithelia, suggesting that expression of JMJD6 positively correlates with oral carcinogenesis. Subsequent functional analysis showed that knockdown of endogenous JMJD6 in OSCC strongly suppressed self-renewal capacity, a key characteristic of CSCs, and anchorage-independent growth. Conversely, ectopic expression of JMJD6 enhanced CSC characteristics including self-renewal, ALDH1 activity, migration/invasion and drug resistance. Expression of CSC-related genes was also markedly affected by modulating JMJD6 expression. Mechanistically, JMJD6 induces interleukin 4 (IL4) transcription by binding to its promoter region. IL4 rescues self-renewal capacity in JMJD6- knocked down OSCC cells, suggesting the importance of JMJD6-IL4 axis in oral CSCs. Our studies identify JMJD6 as a molecular determinant of CSC phenotype, suggesting that inhibition of JMJD6 may offer an effective therapeutic modality against oral cancer.

Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium.

Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37°C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37°C at 5% CO2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.

Differential expression of stem cell-like proteins in normal, hyperplastic and dysplastic oral epithelium.

Objective The identification of stem cells (SC) remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche), but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia.Material and Methods Thirty oral epithelial dysplasia (OED), 25 oral lichen planus (OLP), 10 oral hyperkeratosis and 5 normal oral epithelium (OE) were immunohistochemically examined for four SC markers [integrin β1, neuron-glial-2 (NG2), notch 1 (N1) and keratin 15 (K15)].Results Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin β1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker.Conclusions Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further.

Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria

This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.

Cigarette Smoke Modulates NOD1 Signal Pathway and Human ß Defensins Expression in Human Oral Mucosa

Background/Aims: Nucleotide binding oligomerization domain 1 (NOD1) signal pathway and human ß defensins (hBDs) play crucial roles in innate immune. Cigarette smoke has been confirmed to dampen innate immune in some human tissues, such as oral mucosa. The aim of this study was to evaluate potential effects of smoking on NOD1 signaling and hBDs expression in oral mucosa. Methods: Tissue specimens of normal oral mucosa were collected from donors undergoing routine surgical treatment. All 20 participants were classified equally as two groups: non-smokers and smokers. By using Western blotting and immunohistochemistry, we investigated differential expression of crucial molecules in NOD1 signal pathway, hBD-1, -2, and -3 in oral mucosa tissues between non-smokers and smokers. Immortalized human oral mucosal epithelial (Leuk-1) cells were treated with various concentrations of cigarette smoke extract (CSE) for 24h. Western blotting and immunofluorescence assays were performed to study CSE-induced alteration of protein expression. Leuk-1 cells were treated with 4% CSE, iE-DAP (NOD1 agonist), CSE + iE-DAP, BAY 11-7082 (NF-κB inhibitor), 4% CSE + BAY 11-7082, respectively. Real-time PCR and ELISA were performed to detect the mRNA levels and secretion of hBD-1, -2, and -3, respectively. Results: The levels of NOD1, NF-κB, hBD-1 and hBD-3 significantly reduced in oral mucosa tissues of smokers compared with non-smokers. The levels of RIP2 (receptor-interacting protein 2), phospho-NF-κB (P-NF-κB) and hBD-2 remarkably enhanced in oral mucosal tissues of smokers. CSE treatment suppressed NOD1 and NF-κB expression and activated RIP2 and P-NF-κB expression in Leuk-1 cells. The mRNA and secretory levels of hBD-1 and -3 were down-regulated by CSE, while the mRNA and secretory level of hBD-2 were up-regulated by CSE. The iE-DAP or BAY 11-7082 treatment reversed the regulatory effects of CSE on levels of hBDs. Conclusion: The present study indicated that cigarette smoke could potentially modulate the expression of crucial molecules of NOD1 signal pathway and hBDs in human oral mucosal epithelium. NOD1 signal pathway could play an important role in the regulatory effects of CSE on hBDs levels in oral mucosal epithelial cells.

Efficacy of argyrophilic nucleolar organizing region analysis using computer-assisted and manual in oral leukoplakia: A comparative study.

Nucleolar organizer regions are loops of DNA containing ribosomal RNA genes and presumably are associated with ribosomal RNA activity, protein synthesis, and cell proliferation. Argyrophilic nucleolar organizer region (AgNOR) count has been suggested as an objective method in differentiating dysplastic lesions from non-dysplastic lesions. This descriptive study was done on archival paraffin blocks (n = 60), consisting of 10 normal human oral epithelium, 22 cases of non-dysplastic leukoplakia (NDLK), and 28 cases of dysplastic leukoplakia (DLK). The AgNORs were counted with the aid of a manual using conventional light microscopy and photographs of the same were taken and analyzed using Image Pro Express 6.0 (Media Cybernetic Inc., USA) for windows. The mean AgNOR count per nucleus was found to be higher in patients with DLK as compared to NDLK and controls using both manual counting and image analysis method and on comparing both the techniques, image analysis provide a more accurate reflection of AgNOR counts than manual counting. To conclude, reliability of computerized image technique of AgNOR count is the most appropriate marker to differentiate between dysplastic and NDLK. Computer-assisted image analysis system was found to be an effective tool in achieving high reproducibility as compare to manual.

In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures

Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air–liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

Expression pattern of Notch intracellular domain (NICD) and Hes-1 in preneoplastic and neoplastic human oral squamous epithelium: their correlation with c-Myc, clinicopathological factors and prognosis in Oral cancer.

Notch pathway molecules crosstalk with Wnt/β-catenin signaling cascade in stem cells and tumors. However, the correlation between the expression pattern of Notch intracellular domain NICD, Hes-1 and c-Myc has not been studied in oral squamous cell carcinoma. The aim of this study is to investigate the correlation and prognostic significance of NICD, Hes-1 and c-Myc in oral cancer. Immunohistochemistry was used to study the expression pattern of NICD, Hes-1 and c-Myc in oral preneoplastic and neoplastic tissues. In addition, double immunofluorescence was used to examine the co-localization of NICD, Hes-1 and c-Myc in H314 cells. The expression pattern of NICD and Hes-1 was gradually increased from normal to dysplasia to carcinoma. Interestingly, statistically significant correlation was not observed between NICD, Hes-1 and c-Myc in oral squamous cell carcinoma. Furthermore, NICD+/c-Myc+ and Hes-1+/c-Myc+ double positive cases showed worst survival when compared with other cases in oral cancer. Notch signaling molecules, NICD and Hes-1, are found to be involved in the progression of oral squamous cell carcinoma. Interestingly, NICD, Hes-1 and c-Myc may have independent roles in oral cancer. On the other hand, we have demonstrated that NICD+/c-Myc+ and Hes-1+/c-Myc+ double positivity might be used as independent prognostic indicator of oral carcinoma.

Successful Application of Ex Vivo Expanded Human Autologous Oral Mucosal Epithelium for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

Ocular surface reconstruction with ex vivo expanded limbal stem cells (LSCs) is a widely used clinical treatment for patients with limbal stem cell deficiency (LSCD). This is not applicable to patients with bilateral LSCD where there are no remaining LSCs. Cultivated oral mucosa epithelium (OME) has been used as an alternative source of autologous epithelial stem cells for ocular reconstruction in few clinical trials. However, successful generation of stratified OME epithelium has only been achieved in the presence of animal feeder cells and/or animal-derived products in the culture media, likely to contribute to increased risk of pathogen transmission and graft rejection. In this study, we report generation of multilayered OME epithelium that shares many of the characteristics of corneal epithelium using a fully compliant good manufacturing practice, feeder- and animal product-free method. Proof of concept was achieved by transplantation of autologous ex vivo expanded OME in two patients with histologically confirmed bilateral total LSCD that resulted in successful reversal of LSCD in the treated eye up to 24 months.

Novel Nystatin A₁ derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

Nano-hydroxyapatite and nano-titanium dioxide exhibit different subcellular distribution and apoptotic profile in human oral epithelium.

Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2.

Successful culture and characterisation of ex vivo expanded human autologous oral mucosa epithelium using a feeder- and animal product- free method for the treatment of total bilateral limbal stem cell deficiency

Successful culture and characterisation of ex vivo expanded human autologous oral mucosa epithelium using a feeder- and animal product- free method for the treatment of total bilateral limbal stem cell deficiency

Reciprocal Response of Human Oral Epithelial Cells to Internalized Silica Nanoparticles

Silica nanoparticles (SiO2 NPs) are one of the most widely used engineered nanoparticles and can been found in a wide range of consumer products. Despite their massive global production scale, little is known about their potential effects in the context of unintended exposure or ingestion. Using TR146 cells as an in vitro model of the human oral buccal mucosa, the uptake, spatial intracellular distribution, reactive oxygen species (ROS) production, inflammatory response, and cytotoxic effects of commercial SiO2 NPs are examined. SiO2 NPs are shown to dock and cross the cellular membrane barrier in a dose–time‐dependent manner. Confocal sectioning reveals translocation of SiO2 NPs into the cell nucleus after 12 h of exposure. A concentration threshold of more than 500 × 10−6 m is observed, above which SiO2 NPs are shown to exert significant oxidative stress with concomitant upregulation of inflammatory genes IL6 and TNFA. Further analysis of the p53 pathway and a series of apoptotic and cell cycle biomarkers reveals intracellular accumulation of SiO2 NPs exert marginal nanotoxicity. Collectively, this study provides important information regarding the uptake, intracellular distribution, and potential adverse cellular effects of SiO2 NPs commonly found in consumer products in the human oral epithelium.

Inherent phenotypic plasticity facilitates progression of head and neck cancer: Endotheliod characteristics enable angiogenesis and invasion

The presence of the EMT (epithelial-mesenchymal transition), EndMT (endothelial-mesenchymal transition) and VM (vasculogenic mimicry) demonstrates the multidirectional extent of phenotypic plasticity in cancers. Previous findings demonstrating the crosstalk between head and neck squamous cell carcinoma (HNSCC) and vascular endothelial growth factor (VEGF) imply that HNSCC cells share some functional commonalities with endothelial cells. Our current results reveal that cultured HNSCC cells not only possess endothelial-specific markers, but also display endotheliod functional features including low density lipoprotein uptake, formation of tube-like structures on Matrigel and growth state responsiveness to VEGF and endostatin. HNSCC cell subpopulations are also highly responsive to transforming growth factor-β1 and express its auxiliary receptor, endoglin. Furthermore, the endotheliod characteristics observed in vitro recapitulate phenotypic features observed in human HNSCC tumors. Conversely, cultured normal human oral keratinocytes and intact or ulcerated human oral epithelia do not express comparable endotheliod characteristics, which imply that assumption of endotheliod features is restricted to transformed keratinocytes. In addition, this phenotypic state reciprocity facilitates HNSCC progression by increasing production of factors that are concurrently pro-proliferative and pro-angiogenic, conserving cell energy stores by LDL internalization and enhancing cell mobility. Finally, recognition of this endotheliod phenotypic transition provides a solid rationale to evaluate the antitumorigenic potential of therapeutic agents formerly regarded as exclusively angiostatic in scope.

Evaluation of the efficacy of AgNOR as a proliferative marker in oral leukoplakia: A morphometric analysis

Background:Silver stainable nucleolar organizer regions (AgNORs) are replicatory markers which may have a place in objectively characterizing dysplasia.Materials and Methods:A study of various morphometric parameters related to AgNORs was performed in basal and parabasal layers of normal human oral epithelium, nondysplastic leukoplakia, and dysplastic leukoplakia employing photomicrographs of silver stained paraffin embedded sections using image analysis, to assess the usefulness of these parameters in distinguishing dysplastic leukoplakia from nondysplastic oral leukoplakia.Results:Out of various mean AgNOR related parameters, AgNOR count, area, perimeter, and proportion were found to be higher in dysplastic leukoplakia as compared to nondysplastic leukoplakia. On statistical analysis, AgNOR count showed statistically significant differentiation between dysplastic and nondysplastic leukoplakia. While other parameters can distinguish normal oral epithelium from dysplastic and nondysplastic leukoplakia.Conclusion:To conclude, the AgNOR count is the most appropriate marker to differentiate between dysplastic and nondysplastic leukoplakia.

Towards a defined, serum- and feeder-free culture of stratified human oral mucosal epithelium for ocular surface reconstruction

Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration. In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.

VEGF expression from human dysplastic or malignant oral epithelium may be related to mast cell density and the subsequent angiogenetic phenomena

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine and mast cells play a role in neoangiogenesis in various malignancies. The aim of the present study was to elucidate the role of VEGF and mast cells in the early stages of tumorigenesis in oral squamous cell carcinoma (OSCC). Immunohistochemistry was conducted to study VEGF expression and microvessel density (MVD) in 49 tissue samples, 31 OSCCs, 13 leukoplakias (8 with and 5 without dysplasia) and 5 samples from normal oral tissue. Counterstaining with tolouidine blue was conducted to reveal mast cells. The number of microvessels and mast cells were counted at the same optical field. A gradually increased VEGF expression was observed from normal oral epithelium to leukoplakia and OSCC. MVD was found to increase significantly between normal oral tissue and OSCC (p=0.000). The number of mast cells was found to increase significantly between normal oral tissue, dysplasia (p=0.012) and OSCC (p=0.000). In the early stages of tumorigenesis in OSCC, VEGF, which is secreted by the epithelium, is gradually increased immediately affecting the population of mast cells, which are then related to the increase of microvessels.

Acute effects of cigarette smoke on three-dimensional cultures of normal human oral mucosa

Context: Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects.Objective: The present work aimed at evaluating from a morphological point of view whole smoke early effects on epithelial intercellular adhesion and keratinocyte terminal differentiation in a three-dimensional model of human oral mucosa.Materials and methods: Biopsies of keratinized oral mucosa of healthy nonsmoking women (n = 5) were collected. After culturing in a Transwell system, one fragment of each biopsy was exposed to the smoke of one single cigarette; the remnant represented the internal control. The distribution of epithelial differentiation markers (keratin-10, K10, and keratin-14, K14, for suprabasal and basal cells respectively), desmosomes (desmoglein-1, desmoglein-3), tight junctions (occludin), adherens junctions (E-cadherin, β-catenin), and apoptotic cells (p53, caspase 3) were evaluated by immunofluorescence.Results: Quantitative analysis of K14 immunolabeling revealed an overexpression in the suprabasal layers as early as 3 h after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction.Discussion and conclusion: These results suggested that the first significant response to cigarette smoke came from the basal and suprabasal layers of the human oral epithelium. The considered model maintained the three-dimensional arrangement of the human mucosa in the oral cavity and mimicked the inhalation/exhalation cycle during the exposure to cigarette smoke, offering a good possibility to extrapolate the reported observations to humans.

β-catenin expression in areca quid chewing-associated oral squamous cell carcinomas and upregulated by arecoline in human oral epithelial cells

Nuclear localization of β-catenin is known to associate with malignant transformation of many squamous cell carcinomas. The aim of this study was to compare β-catenin expression in normal human oral epithelium and areca quid chewing associated oral squamous cell carcinomas (OSCCs) and further to explore the potential mechanisms that may lead to induce β-catenin expression. A total of 40 areca quid chewing-associated OSCCs 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, extracellular signal-regulated protein kinase inhibitor PD98059, glutathione precursor N-acetyl-l-cysteine (NAC), tyrosine kinase inhibitor herbimycin-A, p38 inhibitor SB203580, and phosphatidylinositaol 3-kinase inhibitor LY294002 were added to find the possible regulatory mechanisms. β-catenin expression was significantly higher in OSCC specimens than that in normal oral epithelial specimens (p < 0.05). It was demonstrated that normal oral epithelium showed only membranous staining for β-catenin, and membranous staining was lost or reduced with an increase in cytoplasmic/nuclear staining in OSCCs. Arecoline was found to elevate β-catenin expression in a dose-dependent manner (p < 0.05). The addition of PD98059, NAC, herbimycin-A, SB203580, and LY294002 markedly inhibited the arecoline-induced β-catenin expression (p < 0.05). β-catenin expression is significantly upregulated in areca quid chewing-associated OSCC. The localization of β-catenin expression is correlated with the tumor size and clinical stage. In addition, β-catenin expression induced by arecoline is downregulated by PD98059, NAC, herbimycin-A, SB203580, and LY294002.