Dysplastic Oral Keratinocytes Research Articles

Effect of ALA-PDT on inhibition of oral precancerous cell growth and its related mechanisms.

Early treatment of oral precancerous lesions is considered as a key strategy for in oral carcinogenesis prevention. Increasing evidence has suggested that the transforming growth factor beta (TGF-β) signaling pathway is tightly involved in the process of oral-carcinogenesis. In this study, we investigated the inhibition effect and potential mechanism of 5-aminolaevulinic acid photodynamic therapy (ALA-PDT) in human oral precancerous cells via TGF-β pathway. Here, the dysplastic oral keratinocyte (DOK) cells were incubated with ALA concentration of 1mM/mL for 4h and then irradiated with a Helium-Neon (He-Ne) ion laser at 633nm (200mW/cm2). The control cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium. We analyzed the differentially expressed genes and correlated pathways in oral precancerous cells following ALA-PDT using Affymetrix microarrays. TGF-β pathway was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. Bioinformatics analysis was performed to evaluate the expression of TGF-β1 in human oral cancer samples and adjacent normal samples. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), flow cytometry, 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and wound healing assay were used to assess the effects of ALA-PDT plus TGF-β receptor inhibitor (LY2109761) in DOK cells. The TGF-β signaling could exert in suppressive effects on DOK cells after ALA-PDT. The cell proliferation and migration rate of DOK cells was significantly reduced and apoptosis and ROS generation induced more effectively by ALA-PDT combined with LY2109761. Furthermore, cell cycle analysis revealed that the combined treatment resulted in G0/G1 phase arrest. ALA-PDT suppresses the growth of oral precancerous cells by regulating the TGF-β signaling pathway, and its suppressive effect was enhanced using LY2109761. These results indicate that it could be a promising alternative treatment against oral precancerous lesions.

Peroxiredoxin 1 inhibits autophagy through interacting with Rab7 in human dysplastic oral keratinocyte cells

ObjectiveAs a major risk factor for oral leukoplakia (OLK), oxidative stress can induce intracellular reactive oxygen species (ROS), which is closely related to autophagy. Ras-related protein 7 (Rab7) is an important molecule involved in autophagy. Peroxiredoxin 1 (Prx1) is a key antioxidant protein, overexpressed in a variety of malignant tumors. We found that the expression of Prx1 in OLK was up-regulated, and Prx1 is associated with autophagy. This study aims to identify mechanisms of Prx1 in oxidative stress associated autophagy in human dysplastic oral keratinocyte (DOK) cells. DesignHydrogen peroxide (H2O2) was used to induce autophagy in DOK cells. CCK8 assay and flow cytometry were conducted to examine cell viability, cell apoptosis and intracellular ROS level. Autophagy-associated proteins were detected by western blot and immunofluorescence. MHY1485 was applied to investigate the role of Prx1 in autophagy. On the basis of online docking tool Zdock, Prx1 interacting with Rab7 was verified by immunofluorescence double-staining, Duolink PLA, and Co-immunoprecipitation (Co-IP). ResultsH2O2 induced autophagy and ROS increase in DOK cells, and the expression of Prx1 increased gradually according to H2O2 concentration. Prx1 knockdown attenuated the inhibition of MHY1485 on the expressions of autophagy-related proteins and the ratio of LC3 II/LC3 I induced by H2O2. Moreover, Prx1 interacted with Rab7 in DOK cells. ConclusionsPrx1 was involved in the regulation of H2O2-induced autophagy in DOK cells partly by physically interacting with Rab7. Prx1 knockdown promoted autophagy maybe by releasing more Rab7 into the autophagy process.

Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma

In an endeavour to understand metastasis from oral squamous cell carcinomas, we characterised the metastatic potential of a human tongue derived cell line (SCC-4 cells) and compared this phenotype to pre-cancerous dysplastic oral keratinocyte (DOK) cells derived from human tongue and primary gingival keratinocytes (PGK). We demonstrate that SCC-4 cells constitutively synthesize and release significant amounts of IL-6, a process that is enhanced by the addition of the TLR2/TLR6 agonist, Pam2CSK4. The expression of TLR2/6 and IL-6Ra/gp130 receptors was also confirmed in SCC-4 cells. Cancerous SCC-4 human tongue cells also have a classic EMT profile, unlike precancerous human tongue DOK cells. We also established that IL-6 is driving anoikis resistance in an autocrine fashion and that anti-IL-6 neutralising antibodies, anti-IL-6 receptor antibodies and anti-TLR2 receptor antibodies inhibit anoikis resistance in cancerous SCC-4 human tongue cells. The data suggest a promising role for anti-IL-6 receptor antibody and anti-TLR2 receptor antibody treatment for oral cancer.

Mucoadhesive Nucleoside-Based Hydrogel Delays Oral Leukoplakia Canceration

Some oral squamous cell carcinomas (OSCCs) originate from preexisting oral potentially malignant disorders (OPMDs). Oral leukoplakia (OLK) is the most common and typical OPMD in the clinic, so treatment for it is essential to reduce OSCC incidence. Local chemotherapy is an option other than surgery considering the superficial site of OLK. However, there are no standardized drugs applied to OLK, and traditionally used chemotherapeutic drugs revealed limited efficacy for lack of adhesion. Hence, there is a growing demand to prepare new agents that combine mucoadhesion with an anti-OLK effect. Here, an isoguanosine–tannic acid (isoG-TA) supramolecular hydrogel via dynamic borate esters was successfully fabricated based on isoG and TA. Previously reported guanosine-TA (G-TA) hydrogel was also explored for an anti-OLK effect. Both gels not only exhibited ideal adhesive properties but also integrated anti-OLK activities in one system. In vitro cell viability indicated that isoG and TA inhibited the proliferation of dysplastic oral keratinocytes (DOKs). The in vivo OLK model evidence revealed that both gels showed potential to prevent OLK canceration. In addition, the probable anti-DOK mechanisms of isoG and TA were investigated. The results indicated that isoG could bind to adenosine kinase (ADK) and then affected the mammalian target of rapamycin (mTOR) pathway to inhibit DOK proliferation. TA could significantly and continuously reduce reactive oxygen species (ROS) in DOKs through its antioxidant effect. ROS plays an important role in the progression of cell cycle. We proved that the low level of ROS may inhibit DOK proliferation by inducing G0/G1 arrest in the cell cycle. Altogether, this study innovatively fabricated an isoG-TA hydrogel with ideal adhesion, and both isoG and TA showed in vitro inhibition of DOKs. Moreover, both isoG-TA and G-TA hydrogels possessed potential in delaying the malignant transformation of OLK, and the G-TA hydrogel showed a better statistical effect, providing an effective strategy for controlling OLK.

Effects of laser photobiomodulation in the management of oral lichen planus: a literature review.

This review aims to understand whether Photobio-modulation (PBM) therapy is a valid aid in the management of Oral Lichen Planus (OLP) and its symptoms. Moreover, an analysis to determine whether it is a valid replacement for conventional therapies and whether standardized protocols can be used in PBM sessions or whether these should be changed depending on the type of injury has been made. Finally, an evaluation to determine whether PBM may induce transformation of dysplastic oral keratinocytes into squamous cell carcinoma has been made. Searches were conducted on two search databases for relevant publications released between 1992 and 2019. The databases used were: Pubmed "Medline", and Google Scholar. Forty-four articles complied with the inclusion criteria and were included for quality assessment and data extraction. All the studies reported positive effects of PBM; how-ever, there was wide heterogeneity in the laser parameters used in the management of the OLP. The effective dose ranges from 2 to 3 J/cm2, in order to see the desired biological effects. PBM is useful in controlling algal sensation and can be used in cases of OLP lesions that are not responsive to conventional therapies or when corticosteroid doses are too high for the patient, resulting in possible side effects. Standardized biostimulation protocols with further scientific insights are therefore required.

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models.

Simple SummaryOral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain.Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

AGING AND ORAL CANCER DEVELOPMENT

Background Cell aging or cellular senescence is a potent antitumor response characterized by an irreversible cell growth arrest. Paradoxically, aging is considered a risk factor for the development of cancer, with compelling evidence of senescent cells acting as cancer promoters. Senescent cells remain metabolically active and adopt a proinflammatory phenotype known as senescence-associated secretory phenotype (SASP) characterized by the secretion of different factors including IL-6 and IL-8, 2 well-known cancer-associated cytokines. Objective To study the senescence response of normal and dysplastic (OD) oral keratinocytes and to explore pharmacological ways to reduce its potential oncogenic effects. Methods Primary normal and dysplastic keratinocytes were grown until senescence was achieved and changes in gene and protein expression were analyzed. Senescent cells were treated with 2 different drugs (a Rho kinase and cGAS inhibitor) to explore possible ways to modify the SASP. To understand mechanisms regulating senescence, OD cells were genetically modified using the CRISPR/Cas9 system. Results Senescent normal oral keratinocytes and ODs develop a SASP characterized by a significant increase of IL-1α, IL-1β, IL-6, and IL-8, which is accompanied by a decrease in the IL-1 receptor antagonist (IL-1RA). Rock inhibitors are successful in reducing cytokine secretion by senescent cells, whereas cGAS inhibitors can only do it partially. Knock-out of IL-1RA suggests that IL-1RA has important functions in the regulation of senescence. Conclusions Our study shows that senescent oral keratinocytes adopt a proinflammatory state, rich in cytokines known to have oncogenic effects, which can be pharmacologically modified without affecting the senescence response.

N‐cadherin‐mediated Regulation of Metabolism in Oral Precancer

Cadherins are transmembrane proteins traditionally known to have a structural role in cellular adhesion. These proteins display tissue-specific changes in expression that have been correlated with poor prognosis in a variety of cancers. N-cadherin expression specifically has been associated with poor oral cancer prognosis. An estimated 80% of oral cancers exhibit aberrant N-cadherin expression. We have previously shown that cadherins can alter growth factor receptor signaling cascades, which have been shown in other studies to direct metabolic changes essential to oral cancer metastasis. The goal of the current study was to characterize the mechanism(s) by which abnormal expression of N-cadherin may alter metabolic activity in oral epithelia. We have utilized a dysplastic oral keratinocyte (DOK) line to develop retrovirally-transduced cells which express control levels or reduced levels of N-cadherin protein. Using these lines, we evaluated the effect of N-cadherin expression on EGF-dependent EGFR phosphorylation as well as downstream effectors within the mammalian target of rapamycin (mTOR) signaling pathway. Western blot analysis of our EGF-treated DOK lines suggested that N-cadherin expression suppressed phosphorylation of EGFR at Tyr 1068, which plays a role in EGFR degradation, and Tyr 1045, which can mediate cell survival through activation of signal transducer and activator of transcription 3 (STAT3). We also found minimal changes in phosphorylation of Tyr 992, a phosphosite associated with mitogenic signaling. EGFR activation has been shown to direct metabolism through phosphatidyl inositol-3 kinase (PI3K)/Akt/mTOR signaling. Our data indicated that N-cadherin expression is necessary for maximal EGF-directed phosphorylation of both Akt at Ser 473 and of mTORC1 at Ser 2448. mTOR has been shown to increase synthesis of the transcription factor hypoxia inducible factor-1 alpha (HIF-1 alpha) to redirect glycolytic intermediates from oxidative metabolism to anaerobic glycolysis by way of lactate dehydrogenase (LDH), a hallmark of the Warburg effect. We found that N-cadherin expression suppressed HIF-1 alpha synthesis but did not appear to alter lactate dehydrogenase expression. It is important to note that reduced expression of N-cadherin did not alter levels of either E- or P-cadherin expression. Taken together, these findings suggest a role for N-cadherin in the regulation of mTOR-related metabolic signaling, and in modulation of the Warburg effect in oral precancer. Given the high incidence of N-cadherin expression in oral cancer as well as its correlation with poor prognosis, elucidating the mechanisms by which N-cadherin regulates metabolism could reveal novel and promising targets for future therapeutic approaches.

Biosynthesis of silver nanoparticles using Sambucus nigra L. fruit extract for targeting cell death in oral dysplastic cells

The study aims to evaluate the impact of silver nanoparticles, phytosynthesized with polyphenols from Sambucus nigra L. (SN) fruit extract (AgSN), on dysplastic oral keratinocytes (DOK) and human gingival fibroblasts (HGF) in terms of cell viability and apoptosis. The morphology and ultrastructure of treated cells as well as the mechanisms involved in cell death induction were investigated in DOK cultures.The structure of AgSN was studied by using the appropriate analysis tools such as UV–Vis, transmission electron microscopy, Raman spectroscopy, dynamic light scattering (DLS) and zeta potential assessment. DOK and HGF were treated either with silver nanoparticles capped with Sambucus nigra L. extract or with SN extract. Untreated cells were used as controls. Viability was determined by MTS assay. Transmission electronic microscopy (TEM) was used to evaluate the intracellular localization of the nanoparticles at 4 and 24 h. Annexin V-FITC/propidium iodide staining and the expressions of p53, BAX, BCL2, NFkB, phosphorylated NFkB (pNFkB), pan AKT, pan phosphoAKT, LC3B and ɣH2AX were evaluated to quantify the cell death. ELISA measurements of TNF-α and TRAIL was used for the study of the inflammatory response. Oxidative stress damage induced by nanoparticles was assessed by the malondialdehyde (MDA) level. Silver nanoparticles stimulated HGF proliferation and significantly diminished DOK viability at doses higher than 20 μg/ml. TEM analysis demonstrated the internalization of silver nanoparticles and showed ultrastructural changes of cells such as the appearance of vacuoles, autophagosomes, endosomes. AgSN inhibited the pro-survival molecules and regulators of apoptosis, diminished oxidative stress and inflammation and induced cell death through various mechanisms: necrosis, autophagy and DNA lesions. SN extract had antioxidant and anti-inflammatory effect and increased the DNA lesions and autophagy in DOK cells. Silver nanoparticles protected the normal cells and induced cell death in dysplastic cells by different mechanisms thus offering beneficial effects in the treatment of oral dysplasia.

The role of icIL-1RA in keratinocyte senescence and development of the senescence-associated secretory phenotype.

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. Interleukin-1 (IL-1) is a key mediator of this cytokine network, but the control of its activity in the senescence programme has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro We demonstrate that icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR/Cas9-mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from RhoA-activated kinase inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and deregulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.

Novel system in vitro of classifying oral carcinogenesis based on feature extraction for gray-level co-occurrence matrix using scanned laser pico projector.

Oral cancer is among the top 10 causes of death due to cancer worldwide. The prognosis for oral cancer patients is not good, with a 5-year survival rate of only 50%. Earlier and more precise classification will help clinicians make a diagnosis and patients survive. With the advancement of technology, computer-aided detection methods are used to help clinicians form therapy strategies. Gray-level co-occurrence matrix (GLCM) feature extraction of images describing the spatial distribution of gray levels is widely used in medical imaging analysis. Scanned laser pico projector (SLPP) has advantages such as high intensity, directivity, coherence, and mono-color with low bandwidth. In this study, GLCM feature extraction and SLPP reflex images were combined to make a small, non-staining, noninvasive classification system. According to the various image characteristics in oral carcinogenesis, SLPP reflex images better define the borders and three-dimensional structures and provide effective GLCM features such as contrast, energy, and homogeneity to classify carcinogenesis in dysplastic oral keratinocyte (DOK) and normal oral keratinocyte (NOK) cells. Moreover, it also reliably classifies highly metastatic (HSC-3) and tongue cancer (CAL-27) cells. A promising computer-aided classification system for oral cancer was developed to build a reliable intraoral examination system for in situ computer-aided diagnosis in normal clinics.

Peroxiredoxin1 Knockdown Inhibits Oral Carcinogenesis via Inducing Cell Senescence Dependent on Mitophagy.

PurposeCellular senescence is a physiological phenomenon by which cells irreversibly lose their proliferative potential. It is not clear whether senescent cells are related to malignant transformation in oral precancerous lesions. The role of peroxiredoxin1 (Prx1)-induced cell senescence in OLK malignant transformation has not been reported. The aim of this study is to investigate the role and mechanism of cell senescence in oral carcinogenesis.MethodsIn this study, 4-nitro-quinoline-1-oxide (4NQO) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/- mice and dysplastic oral keratinocyte (DOK) were used. Prx1 knockdown DOK cells were harvested with shRNA injection, and cell senescence was detected via the senescence-associated β-galactosidase (SA β-gal) assay. The senescence and mitophagy-related proteins were observed by immunohistochemistry (IHC), Western blot and qRT-PCR. The binding of Prx1 with prohibitin 2 (PHB2) and light chain 3 (LC3) was predicted via ZDOCK and measured in mice by Duolink analysis.ResultsHistologically, 4NQO treatment induced epithelial hyperplasia, dysplasia (mild, moderate and severe), carcinomas in situ and oral squamous cell carcinoma (OSCC) in mouse tongue mucosa. The malignant transformation rate in Prx1+/- mice (37.5%) was significantly lower compared with Prx1+/+ mice (57.1%). In Prx1+/+ mice, a higher number of senescent cells and greater expression of p53 and p21 were observed in hyperplastic and dysplastic tongue tissues when compared with those in OSCC tissues. Prx1 knockdown induced a greater number of senescent cells in hyperplastic tissues, and DOK cells accompanied cell cycle arrest at the G1 phase and PHB2/LC3II downregulation. Prx1 was predicted to dock with PHB2 and LC3 via ZDOCK, and the interactions were confirmed by in situ Duolink analysis.ConclusionPrx1 silencing inhibits the oral carcinogenesis by inducing cell senescence dependent on mitophagy.

Nicotine promotes the development of oral leukoplakia via regulating peroxiredoxin 1 and its binding proteins.

Tobacco can induce reactive oxygen species (ROS) production extensively in cells, which is a major risk factor for oral leukoplakia (OLK) development. Peroxiredoxin 1 (Prx1) is a key antioxidant protein, upregulated in a variety of malignant tumors. We previously found that nicotine, the main ingredient of tobacco, promotes oral carcinogenesis via regulating Prx1. The aim of the present study was to screen and identify the Prx1 interacting proteins and investigate the mechanisms of nicotine on the development of OLK. Through liquid chromatography-tandem mass spectrometry combined with bioinformatics analysis, the candidate Prx1 interacting proteins of cofilin-1 (CFL1), tropomyosin alpha-3 chain (TPM3), and serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform (PPP2R1A) were screened in human dysplastic oral keratinocyte cells treated with nicotine. CFL1, TPM3, and PPP2R1A were highly expressed in human OLK tissues. The expression of CFL1 increased and the expression of PPP2R1A decreased in OLK of smokers compared to that in OLK of non-smokers. Nicotine upregulated CFL1 and downregulated PPP2R1A in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK tissues in mice in part dependent on Prx1. Furthermore, the in-situ interaction of CFL1, TPM3, and PPP2R1A with Prx1 were validated in human OLK tissues. Our results suggested that tobacco might promote the development of OLK via regulating Prx1 and its interacting proteins CFL1 and PPP2R1A.

ROCK inhibition modulates the senescence-associated secretory phenotype (SASP) in oral keratinocytes.

Senescent cells accumulate in different organs and develop a senescence-associated secretory phenotype (SASP), associated with the development of age-related pathologies. The constitution of the SASP varies among cell types and with the method of senescence induction; nevertheless, there is substantial overlap among SASPs, especially the presence of pro-inflammatory cytokines such as IL-1β, IL-1α, IL-6 and IL-8. These cytokines are highly conserved among SASPs and are implicated in the development of several cancers. Here, we report that ROCK inhibition by Y-27632 reduces levels of IL-1α, IL-1β, IL-6 and IL-8 secreted by senescent normal and dysplastic oral keratinocytes without affecting the permanent cell growth arrest. The data indicate some inflammatory genes downregulated by Y-27632 remain downregulated even after repeated passage in the absence of Y-27632. We propose ROCK kinase inhibition as a novel alternative to current strategies to modulate the inflammatory components of the SASP, without compromising the permanent cell growth arrest. This observation potentially has wide clinical applications, given the involvement of senescence in cancer and a wide range of age-related disease. It also suggests care should be exercised when using Y-27632 to facilitate cell expansion of primary cells, as its effects on gene expression are not entirely reversible.

ANALYSIS OF PI3 K/MTOR PATHWAY, CELL CYCLE, AND APOPTOSIS IN PATIENTS WITH EVEROLIMUS-ASSOCIATED STOMATITIS

Objectives: Describe the epidemiologic profile of patients who used everolimus and identify possible alterations, through the analysis of PI3 K/mTOR proteins, cell cycle, and apoptosis, which may be associated with the development of everolimus-associated stomatitis. Study Design: Medical records from patients who used everolimus were analyzed. In the prospective study, patients with breast, kidney, and neuroendocrine cancer were evaluated. Patients with everolimus-associated stomatitis underwent biopsy for histologic, immunohistochemical, and immunofluorescence analysis. In parallel, dysplastic oral keratinocytes (DOK) were cultured and assays were conducted. Results: A total of 129 medical records were analyzed. Ninety-seven patients were female. The mean age was 57.02 years. The prevalence of stomatitis was 33.3%. Patients with breast cancer (everolimus plus exemestane) had a 2.34-fold increased risk of developing stomatitis. In the prospective study, 11 patients were female. The mean age was 59.09 years. Nine patients developed stomatitis. Three biopsies were performed. All sections revealed epithelial tissue with ulcerated areas and intense inflammatory infiltrates. In vitro experiments showed that DOK treated with everolimus plus exemestane reduced cell viability and inhibited cell migration. Conclusion: Everolimus plus exemestane increases the chances to develop stomatitis and the in vitro experiments suggest a greater damage to the oral epithelium when there is an association of these drugs.

Evaluation of Surface Characteristics and Cytotoxicity of Dental Composites

The purpose of this study was to evaluate the surface and in vitro cytotoxicity on human dysplastic oral keratinocytes (DOK) of four commercial resins-based dental composites commonly used in prosthodontics dental therapies: two indirect composites for crown and bridges—SR Adoro (IvoclarVivadent GmbH) and Solidex (Shofu Dental GmbH); and two dual-curing luting resin cements—RelyxUnicem (3M ESPE Dental Products) and Variolink Esthetic DC (IvoclarVivadentGmbH). A complex assessment of surface characteristics of the four materials was conducted before and after the exposure to artificial saliva through various analyses, such as Scanning Electron Microscopy, Atomic Force Microscopy and Cross Polarized Light Microscopy (PLM). The results showed that DOK viability was not severely affected by exposure to any of these materials; however, Variolink expressed higher values but still above the toxicity level of the rest of the composites. The analysis of the surface structure between initial and artificial saliva exposed specimens returned a compact aspect in both categories and although Variolink and Relyx were subjected to increased roughness after saliva exposure, no damage of the internal compactness was recorded, demonstrating a fair behavior of the luting cements in contact with the saliva.

Systemic and Local Biocompatibility Assessment of Graphene Composite Dental Materials in Experimental Mandibular Bone Defect.

The main objective of this research is to demonstrate the biocompatibility of two experimental graphene dental materials by in vitro and in vivo tests for applications in dentistry. The novel graphene dental materials, including one restorative composite and one dental cement, were subjected to cytotoxicity and implantation tests by using a rat model of a non-critical mandibular defect. In vitro cytotoxicity induced by materials on human dental follicle stem cells (restorative composite) and dysplastic oral keratinocytes (dental cement) was investigated at 37 °C for 24 h. After in vivo implantation, at 7 weeks, bone samples were harvested and subjected to histological investigations. The plasma biochemistry, oxidative stress, and sub-chronic organ toxicity analysis were also performed. The resulting cytotoxicity tests confirm that the materials had no toxic effects against dental cells after 24 h. Following graphene dental materials implantation, the animals did not present any symptoms of acute toxicity or local inflammation. No alterations were detected in relative organ weights and in correlation with hepatic and renal histological findings. The materials’ lack of systemic organ toxicity was confirmed. The outcomes of our study provided further evidence on the graphene dental materials’ ability for bone regeneration and biocompatibility.

Porphyromonas gingivalis Promotes Oral Squamous Cell Carcinoma Progression in an Immune Microenvironment

Increasing evidence has revealed a significant association between microorganisms and oral squamous cell carcinoma (OSCC). Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, is considered an important potential etiologic agent of OSCC, but the underlying immune mechanisms through which P. gingivalis mediates tumor progression of the oral cancer remain poorly understood. Our cohort study showed that the localization of P. gingivalis in tumor tissues was related to poor survival of patients with OSCC. Moreover, P. gingivalis infection increased oral lesion multiplicity and size and promoted tumor progression in a 4-nitroquinoline-1 oxide (4NQO)–induced carcinogenesis mouse model by invading the oral lesions. In addition, CD11b+ myeloid cells and myeloid-derived suppressor cells (MDSCs) showed increased infiltration of oral lesions. Furthermore, in vitro observations showed that MDSCs accumulated when human-derived dysplastic oral keratinocytes (DOKs) were exposed to P. gingivalis, and CXCL2, CCL2, interleukin (IL)–6, and IL-8 may be potential candidate genes that facilitate the recruitment of MDSCs. Taken together, our findings suggest that P. gingivalis promotes tumor progression by generating a cancer-promoting microenvironment, indicating a close relationship among P. gingivalis, tumor progression of the oral cancer, and immune responses.

Effects of Low Molecular Weight Fucoidan on the Proliferation and Apoptosis of Dysplastic Oral Keratinocyte and Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors with high incidence, rapid progress, and high mortality. There are still some defects in the treatments of OSCC, which seriously affect the quality of patients’ life. Therefore, it is urgent to find a safer and more effective treatment for OSCC. Low molecular weight fucoidan (LMWF) has various biological activities, such as antitumor, anti-inflammatory, and antithrombotic, and has no obvious side effects, but the effect of LMWF on OSCC has not been reported. In this study, the effects of LMWF on dysplastic oral keratinocyte (DOK) and OSCC cells SCC-9 and SCC-25 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, TUNEL, colony formation, and wound healing assay, and the expression levels of Notch-1 and Jagged-1 treated with LMWF were detected by RT-qPCR. The results showed that LMWF could inhibit the proliferation and migration of DOK and SCC-9, and promote apoptosis. Low molecular weight fucoidan upregulated the expression of Notch-1 and Jagged-1 in DOK and SCC-9 cells. It indicated that LMWF might promote the apoptosis of DOK and SCC-9 by upregulating the Notch signal pathway, thereby inhibiting cell proliferation and playing an antitumor role. It provided theoretical basis to develop LMWF as a novel therapeutic drug for oral cancer.

Nuclear accumulation of β-catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia.

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of β-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of β-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the β-catenin destruction complex allows nuclear accumulation ofβ-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes(OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of β-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3β, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3β, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the β-catenin destruction complex leads to stabilization and nuclear accumulation of β-catenin in oral dysplasia.