Oral Epithelial Cells Research Articles

Green Synthesis of Gold Nanoparticles by Aqueous Extract of Zingiber officinale: Characterization and Insight into Antimicrobial, Antioxidant, and In Vitro Cytotoxic Activities

The main challenge for researchers in the biomedical sectors concerns the development of new active compounds through cost-effectiveness, rapid, simple, and ecofriendly methods to overcome antibiotic resistance to pathogenic microbes. Herein, gold nanoparticles (AuNPs) were fabricated by a green approach through the reduction of chloroauric acid (HAuCl4) by harnessing the metabolites present in the aqueous extract of Zingiber officinale rhizome. UV-Vis spectroscopy, FT-IR, XRD, TEM, DLS, and zeta potential were used to characterize phytosynthesized AuNPs. In addition, the antimicrobial effect was investigated against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), unicellular fungi (Candida albicans), and multicellular fungi (Aspergillus brasiliensis). Antioxidant and in vitro cytotoxic activities were assessed. TEM and XRD showed the successful formation of spherical and crystalline structures, with sizes in the range of 5–53 nm (average size: 15.11 ± 8.5 nm). The zeta potential value (i.e., –28.8 mV) explains the high stability of synthesized AuNPs. AuNPs exhibit promising activity against prokaryotic and eukaryotic microorganisms with variable inhibition zones and low MIC values in the range of 25–6.25 µg mL−1. Phytosynthesized AuNPs exhibit DPPH scavenging activity with percentages of 87.6 ± 0.5% at a maximum concentration (1000 µg mL−1), which can be compared with ascorbic acid (97.3 ± 0.2%). Moreover, the AuNPs displayed a target-oriented effect for cancer cell lines HepG2 and MCF7 at low IC50 concentrations of 131.9 ± 9.34 and 288.23 ± 31.39 µg mL−1 compared to the normal oral epithelial OEC cell line (487.612 ± 3.53 µg mL−1). Overall, the phytosynthesized AuNPs show wide activities that enable their use in various biomedical and biotechnological applications.

Comments on "Sappanone A Aggrandises Ionising Radiation-induced Damage in Oral Epithelial Cells".

Comments on "Sappanone A Aggrandises Ionising Radiation-induced Damage in Oral Epithelial Cells".

Sappanone A Aggrandises Ionising Radiation-induced Damage in Oral Epithelial Cells.

To analyse the role played by Sappanone A, a bioactive ingredient isolated from the heartwood of Caesalpinia sappan, in the regulation of oral epithelial cell viability under radiation. Cell viability of human oral keratinocytes (HOKs) and mouse salivary gland cells under ionising radiation was analysed. Expression of Ki67 was measured by immunohistochemical staining. Fragmentation of deoxyribonucleic acid (DNA) was measured by comet assay. Cell death was analysed using trypan blue exclusion assay. Cell viability was measured using a Cell Counting Kit 8 (CCK8; Abcam, Cambridge, UK) assay. Sappanone A decreased cell viability of HOK cells and mouse salivary gland cells under ionising radiation. In addition, Sappanone A enhanced radiation-induced genomic DNA fragmentation, accompanied by impaired homologous recombination and non-homologous end joining DNA repair. Mechanistic evaluation revealed that Sappanone A counteracted radiation-induced inosine monophosphate dehydrogenase 2 (IMPDH2) activation, and that this effect could be abolished by reconstituted expression of a Sappanone A-binding defective IMPDH2 mutant. The present study highlights a novel role played by Sappanone A in the modulation of radiosensitivity of oral epithelial cells.

The Anti-Inflammatory Effects of Iberin on TNF-α-Stimulated Human Oral Epithelial Cells: In Vitro Research.

Iberin is a bioactive chemical found in cruciferous plants that has been demonstrated to have anticancer properties. However, there have been no reports on its effects on periodontal resident cells, and many questions remain unanswered. The aim of this study was to examine whether iberin had anti-inflammatory effects on human oral epithelial cells, including influences on signal transduction pathway activation in TNF-α-στιμυλατεd χελλσ. Iberin inhibited the production of interleukin (IL)-6 and C-X-C motif chemokine ligand 10 (CXCL10), as well as the expression of vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 in tumor necrosis factor (TNF)-α-stimulated TR146 cells, a human oral epithelial cell line. Moreover, iberin administration increased the expression of antioxidant signaling pathways, such as Heme Oxygenase (HO)-1 and NAD(P)H quinone dehydrogenase 1 (NQO1). Furthermore, we found that iberin could inhibit the activation of the nuclear factor (NF)-κB, signal transducer and activator of transcription (STAT)3, and p70S6 kinase (p70S6K)-S6 ribosomal protein (S6) pathways in TNF-α-stimulated TR146 cells. In conclusion, iberin reduced inflammatory mediator expression in human oral epithelial cells by preventing the activation of particular signal transduction pathways.

Do silver/hydroxyapatite and zinc oxide nano-coatings improve inflammation around titanium orthodontic mini-screws? In vitro study

Overcoming the failure percentage of orthodontic mini-screws (OMSs), which is about 30% of overall orthodontic cases, especially in malocclusion treatment that requires orthopaedic heavy forces, is a great challenge. Bacterial infections, soft tissue and bone inflammation, and weak connections between bones and the OMS surface are among the main causalities of this failure. The aim of the study is to evaluate in vitro the microbiological activities of the deposited nanomaterials (Silver/hydroxyapatite nanoparticles (Ag/HA NPs) and zinc oxide nanoparticles (ZnO NPs)) in terms of microbial inhibition. In addition, the in-vitro cytotoxicity and cytocompatibility of the synthesized nano-coatings prior to their in-vivo application in animal models were tested on four types of cells, namely, fibroblasts, osteocytes, osteoblasts, and oral epithelial cells. Ag/HA NPs and ZnO NPs were built up onto the surface of titanium OMSs by electrochemical deposition. This electrochemical deposition was performed on 50 orthodontic mini screws and the deposited materials were characterized with the aid of scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX) analysis, X-ray Diffraction (XRD) and nano-scratch test. In addition, the microbiological activities of the deposited nanomaterials were explored in vitro in terms of microbial inhibition. Furthermore, the cytotoxicity and cytocompatibility were tested on four types of cells, namely, fibroblasts, osteocytes, osteoblasts and oral epithelial cells. SEM images revealed spherical Ag NPs in the range of 40-70nm in diameter, rod-shaped HA NPs and porous scaly ZnO NPs on the surface of the OMSs. XRD analysis confirmed the crystal structures of AgNPs, HA NPs, and ZnO NPs. ZnO NPs coated OMS had the highest antimicrobial activity than Ag/HA coated OMS against Gram-positive, Gram-negative and fungal strains. Moreover, after incubation, the decrease in the number of bacterial colonies was significant with ZnO and Ag/HA nanoparticles (with the greatest decrease for the former), due to the potent antibacterial effect of nanoparticles against Escherichia coli and Enterococcus faecalis. Moreover, ZnO NPs-coated OMSs showed a better cytocompatibility with oral epithelium, bone cells, and fibroblasts compared to Ag/HA NPs. The suggested nanocoating is a promising strategy to overcome the development of an inflammatory zone around the fixed OMSs.

Evaluation & comparison of micronuclei in smokers with & without diabetes

Background: Micronuclei are chromatin masses that arise from chromosomal fragments or whole chromosome that lack behind at the anaphase stage of cell division. Their presence in cells means the number of chromosomal aberrations arising during mitosis. Cigarette smoke contains over 4000 chemical carcinogens, with 200 known carcinogens that show significant genotoxic effects in human cells. Chronic high levels of glucose during Diabetes Mellitus are thought to increase oxidative stress and the formation of free radicals that in turn damage cells. Reactive oxygen species chemically attack cellular components altering metabolism, inflammatory mediators, and antioxidant defense mechanisms, overall favoring the pathogenesis of the disease and the persistence of genetic damage. Scoring of micronuclei is considered a DNA damage biomarker for chromosomal damage and instability. Aim: Considering this, we decided to evaluate & compare the frequency of micronuclei in oral epithelial cells in smokers & in diabetics. Materials & Methods: A total number of eligible 150 individuals were included in the study; 50 smokers without diabetes, 50 smokers with diabetes & 50 age and sex matched non smoker healthy individuals group as controls. Oral epithelial cells were taken from buccal smears and subjected to PAP stain. Mean micronuclei were taken from all the subjects. Results: Least number of micronuclei was found in non smoker, non diabetic group & highest numbers were found in smoker, Diabetic group. Conclusion: Micronuclei assay is non-invasive technique that offers a very simple method for obtaining information on status of the epithelial cells, particularly DNA damage, proliferative potential of basal cells and cell death.

Extracellular product of Pseudomonas aeruginosa in growth medium is involved in the pro-inflammatory cytokine response of human oral epithelial cells in vitro.

Epithelial cells are the first barrier to any microbial invasion. Finding a safe and affordable substance to stimulate the innate immune response of epithelial cells is one of the main challenges immunologists and vaccine manufacturers are facing. This study aimed to show the comparative effect of sterile bacterial secretion (SBS) and Pseudomonas aeruginosa bacterial cell isolates obtained from burn wound infections on the ability of human epithelial cells (HECs) to produce interleukin (IL)-1β and tumor necrosis factor alpha (TNF-α) in vitro. The HEC cultures were exposed to P. aeruginosa 8 (Pa 8), Pa 2 and Pa 1 bacterial cells (isolated from burn wound infections). The other 3 groups of HECs were exposed to 50 μL of sterile, endotoxin-free SBS of Pa 8, Pa 2 and Pa 1. The time course of changes in IL-1β mRNA, TNF-α mRNA, IL-1β, and TNF-α was examined. Moderate (p < 0.05) elevations of IL-1β mRNA in HECs and IL-1β protein in the supernatant of the HEC culture were observed following exposure to SBS of Pa 8, Pa 2 and Pa 1 at most time points. High elevation (p < 0.05) of IL-1β was seen in the supernatant of the HEC culture that was exposed to bacterial cells (Pa 8, Pa 2 and Pa 1). Similar results were found when TNF-α mRNA was measured in HECs and TNF-α in the supernatant of the HEC cultures after exposure to bacterial cells (Pa 8, Pa 2 and Pa 1) and the SBS of Pa 8, Pa 2 and Pa 1. This is the first time that the capacity of SBS to generate epithelial cell pro-inflammatory cytokines in vitro has been shown. In other words, SBS enhanced a nonspecific immune response, which opens the door to the possibility of using SBS from P. aeruginosa as an adjuvant in the future.

Salivary non-apoptotic tumoral microvesicles: A potential progressive marker in oral cancer patients.

Tumour cell-secreted microvesicles (MVs) contribute immensely to tumour progression. However, the role of tumoral salivary MVs in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we elucidated the role of non-apoptotic salivary tumoral MVs in OSCC development, especially relating to the migration ability. We purified and compared non-apoptotic salivary tumoral MVs from 63 OSCC patients and orthotopic OSCC mice model. Next, we compared the protein difference between apoptotic and non-apoptotic MVs by Western blot, proteomics and flow cytometry from saliva and CAL27 cells. Finally, we collected the non-apoptotic MVs and co-cultured with normal oral epithelial cells, the migration ability was examined by wound healing assay and Western blot assay. Our results indicated that the levels of non-apoptotic tumoral S-MVs were significantly higher in OSCC patients with T3 to T4 stages than in patients with T1 to T2 stages or healthy donors. In OSCC mice model, we found elevations of non-apoptotic tumoral MVs associated with tumoral volume. EGFR overexpression increased the generation of non-apoptotic tumoral MVs which could significantly promote normal epithelial cell migration. In conclusion, elevated levels of non-apoptotic tumoral S-MVs are associated with clinicopathologic features of OSCC patients, implying that non-apoptotic tumoral S-MVs are a potential progressive marker of OSCC.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Effects of E-cigarette on Oral Health – A Systematic Scoping Review

Background: E-cigarettes have been perceived as a safer product compared to conventional cigarettes and have often been used for the purpose of cigarette smoking cessation. The aim of this scoping review is to provide a systematic review of the existing literature on the effects of e-cigarettes on human oral health and oral tissues. Methodology: Systematic literature search was undertaken and reviewed independently by two experts in online databases till May 2019. Both human and invitro studies were included. Guidelines of preferred reporting items for systematic reviews and meta-analyses for scoping reviews (PRISMA-ScR) were followed for reporting of the data. Results: A total of 24 studies were included following removal of duplicates, title and abstract screening and; full text screening. E-cigarettes have deleterious effects on the oral cavity tissues comprising of clinical findings of periodontium, gingiva, oral hygiene and micro-organism growth. In addition, there is a threat of serious injuries associated with e-cigarette explosion. In-vitro studies reveal e-cigarette use may lead to oxidative damage, altered morphology, reduced cell viability and cytotoxicity of oral epithelial and fibroblast cells. Conclusions: Though e-cigarettes have been found to be less harmful than the conventional cigarette smoking, there is a need to exercise caution while considering it as a smoking cessation tool. Longitudinal population-based studies are required for analyzing its in-vivo effect on oral tissues.

Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease.

The interactions between bacteria, epithelium, and neutrophilic polymorphonuclear leukocytes (neutrophils) are the key to the initiation and progression of many chronic inflammatory-immune diseases. In addition, all can be influenced by external factors, such as micronutrients, thereby providing potentially novel approaches to therapy. This chapter will therefore provide detailed methods for core techniques involved in studying cellular and molecular epithelial responses to a bacterial challenge in relation to chronic inflammatory disease pathogenesis and therapy.

Improving the Corrosion Resistance of Magnesium Alloy by Magnesium Phosphate/Glass Composite Coatings Using Sol–Gel Method

Coating of Mg alloys with Mg-phosphate is usually performed by complex and costly methods. This work was mainly aimed at using Mg-phosphate ceramic for Mg metal implants by simple and cost-effective spin coating combined with a sol–gel approach. Where, Mg-phosphate ceramic particles were dispersed with different percentages (0, 10, and 30 wt. %) in the glass sol (85 SiO2 – 10 CaO – 5 P2O5 system) as a coating solution. The coated substrates were characterized by TGA, XRD, FTIR, contact angle, and SEM/EDX analyses, and the in vitro bioactivity test was performed in revised simulated body fluid (rSBF). The results showed the coating thickness was 8.8 ± 0.8, 5.4 ± 0.6, and 5 ± 0.7 μm for MP0, MP10, and MP30, respectively. Moreover, the coatings increased the hydrophilicity of the metal surface. All coatings enhanced the formation of an apatite-bone like layer on the Mg metal surface, and they were viable with oral epithelial cells at a concentration ≤ 125 μg/ml. Moreover, MP0 and MP10 coatings significantly enhanced the corrosion resistance of the metal, while; MP30 coating did not show a significant effect on it. Thus, the percentage of Mg-phosphate in the coating was valuable for corrosion resistance when it was ≤ 10 wt. %. As a result, the composite coatings showed promising coatings for Mg metal substrate to enhance its corrosion resistance at low percentages of Mg-phosphate ceramic.

Assessment of SARS-CoV-2 entry in gingival epithelial cells expressing CD147.

SARS-CoV-2, the causative agent of the debilitating COVID-19, is mainly transmitted by first infecting nose and lung epithelial cells. The mouth is also believed to be a viral portal site since certain types of oral epithelial cells were shown to express ACE2 receptor. However, it is unclear whether oral epithelial cells are directly infected by SARS-CoV-2. In this study, we addressed whether epithelial cells of the oral gingiva were susceptible to infection. Interestingly, we found that KRT5+ and KRT18+ gingival epithelial cells do not express ACE2 but highly express TMPRSS2 and Furin as well as CD147, which was proposed to be an alternative receptor for SARS-CoV-2. However, using SARS-CoV-2 pseudoviruses containing the spike protein, we observed that gingival epithelial cells were not susceptible to infection due to the lack of ACE2 expression and the inability of CD147 to mediate viral entry. These results strongly suggest that epithelial cells from the gingiva are not susceptible to SARS-CoV-2 and CD147 is not a receptor for the SARS-CoV-2 virus. The susceptibility of oral cells from other oral structures under healthy and pathological conditions still needs to be confirmed to better understand the role of the oral cavity in COVID-19 infection and transmission.

Microscopic observations of SARS-CoV-2 like particles in different oral samples.

The emerging coronavirus pneumonia epidemic caused by the SARS-CoV-2 infection has spread rapidly around the world. The main routes of transmission of SARS-CoV-2 are currently recognised as aerosol/droplet inhalation. However, the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly known. The current data indicates the presence of viral RNA in oral samples, suggesting the implication of saliva in SARS-CoV-2 transmission, however, no direct observation of SARS-CoV-2 particles in different oral samples has been reported. In this study, we investigated whether particles of SARS-CoV-2 were present in oral samples collected from three symptomatic COVID-19 patients. Using scanning electron microscopy (SEM), the correlative strategy of light microscopy and electron microscopy and immunofluorescence staining, we showed the presence of SARS-like particles in RT-qPCR SARS-CoV-2-positive saliva, dental plaque and gingival crevicular fluid (GCF) samples. In the saliva samples, we demonstrated the presence of epithelial oral cells with morphogenetic features of SARS-CoV-2 infected cells. Inside those cells, vacuoles filled with nascent particles were observed, suggesting the potential infection and replication of SARS-CoV-2 in oral tissues. Our results corroborate previous studies and confirm that the oral cavity may be a potential niche for SARS-CoV-2 infection and a potential source of transmission.

Long-term outcomes of regenerative treatment by endoscopic oral mucosal epithelial cell sheet transplantation for the prevention of esophageal stricture after endoscopic resection

IntroductionWe reported the short-term outcomes of a regenerative medical treatment to prevent esophageal stricture after endoscopic submucosal dissection (ESD) using cultured tis oral mucosal epithelial cell sheets. This study investigated the long-term outcomes of this treatment. MethodsEpithelial cells, isolated from the patient's own oral mucosal tissue, were cultured for 16 days using temperature-responsive culture dishes. Then, the autologous cell sheets were endoscopically transplanted onto the bed of the esophageal ulcer after endoscopic mucosal resection (EMR) and ESD. Results of 10 patients who underwent endoscopic transplantation of oral mucosal epithelial cell sheets from April 2008 through February 2022 were recorded. We analyzed the outcome, the cause, and the endoscopic findings. ResultsThe median period of observation was 3761 days. No stricture was detected in any of the patients long-term. Two patients died because of pancreatic cancer and brainstem hemorrhage. One patient underwent chemo-radiotherapy for further treatment. The patients underwent surgery due to metastasis to the lymph nodes. Only the lymph nodes were dissected, and the esophagus remained intact. From the endoscopic findings: Melanosis was found at the transplanted site in a patient. Strong iodine staining was shown at the transplanted site in a patient. Transplantation of cultured oral mucosal epithelial cell sheets to prevent esophageal stricture has been proven to be a safe treatment. All patients showed no controlled esophageal stricture in the long term.

The critical role of IFNγ in the epidermotropic migration of lymphocytes in oral lichen planus.

The chemokines play a crucial role in the recruitment of lymphocytes in oral lichen planus, and the activated epithelial cells are the main producers of the chemokines. However, the signals provoking chemokine secretion still remain to be elucidated. The global expression profile of chemokines in oral epithelial cell line induced by IFNγ was determined by microarray analysis. The gene and protein expression was validated in primary culture of oral epithelial cells, and the effects of IFNγ on regulating chemokine production were compared with that of TNFα and IL2. Moreover, the capability of primary culture of oral epithelial cells to attract peripheral lymphocytes in response to IFNγ was investigated in oral lichen planus patients, and the cell phenotype of the recruited lymphocytes was analyzed using flow cytometry. IFNγ triggered the expression of multiple chemokines in the oral epithelial cells. The expression pattern of the chemokines closely resembled that in the epithelial cell layer of oral lichen planus lesions. Compared with IL2 and TNFα, IFNγ demonstrated a distinct maximal effect on the chemokines secretion in primary culture of oral epithelial cells. The migration of peripheral lymphocytes toward the culture supernatant of IFNγ-treated primary culture of oral epithelial cells was significantly enhanced in the oral lichen planus group compared to that in the healthy control group. IFNγ plays an important role in the chemokine secretion and epidermotropic migration of lymphocytes in oral lichen planus.

Tumor Microenvironment in Oral Squamous Cell Carcinoma: Special Stains and Scanning Electron Microscopic Study

Abstract Background: Effect of the oral cancerous epithelial cells on the extracellular matrix (ECM) and vice versa is also responsible for the growth and progression of cancer. The effect of inflammation on the stroma and its association with progression has also been stated in literature. Materials and Methods: The control group consisted of 10 patients with apparently normal mucosa with no history of habits, the study group consisted of 30 patients with oral squamous cell carcinoma (OSCC) confirmed by examination and incisional biopsy. H and E staining, Masson’s Trichrome Staining, and Verhoeff’s Van Gieson staining were used to study the inflammation, collagen fibers, and elastic fiber, respectively. A few selected specimens were studied under the scanning electron microscope. The grade of inflammation was statistically correlated with collagen and elastic fibers. Observation: Grade of inflammation was closely associated with lymphadenopathy and the density of collagenous and elastic components of the ECM. The scanning electron microscopic evaluation of elastic fibers revealed irregular fragmented elastic fibers. Conclusion: The stroma is the key to complete removal and treatment of OSCC; however, more studies are needed to define the role of each component, therefore improving the prognosis of a patient.

Characterization of Leuconostoc mesenteroides MJM60376 as an oral probiotic and its antibiofilm activity.

Lactic acid bacteria have been widely used as probiotics for improving gut health. However, studies on oral probiotics were very limited. In this study, 67 lactic acid bacteria (LAB) were isolated from fermented food and screened for antagonistic activity against Streptococcus mutans, the causative pathogen of dental caries. Leuconostoc mesenteroides MJM60376 showed the highest antagonistic activity against S. mutans KCTC3065. L. mesenteroides MJM60376 also showed oral probiotic characteristics including weak acid production, lysozyme tolerance, adhesion to oral epithelial cell (YD-38), antibiotic susceptibility, and good coaggregation ability with S. mutans. Furthermore, the biofilm formation of S. mutans was significantly reduced when cocultured with L. mesenteroides. Scanning electron microscopy analysis showed that amounts of attached bacteria of S. mutans and network-like structures were significantly reduced by L. mesenteroides MJM60376. Cell-free supernatant (CFS) of L. mesenteroides MJM60376 also greatly inhibited biofilm formation of S. mutans from the adherent stage, the activity remained even after it was treated with catalase, trypsin, or pH neutralized. Expression levels of biofilm formation-related genes were significantly reduced in S. mutans when it was treated with the CFS of L. mesenteroides MJM60376. Therefore, L. mesenteroides MJM60376 has great potential to be used as a multifunctional ingredient.

HIV-1 Proteins gp120 and Tat Promote Epithelial-Mesenchymal Transition and Invasiveness of HPV-Positive and HPV-Negative Neoplastic Genital and Oral Epithelial Cells.

The incidence of human papillomavirus (HPV)-associated anogenital and oropharyngeal cancer in human immunodeficiency virus (HIV)-infected individuals is substantially higher than in HIV-uninfected individuals. HIV may also be a risk factor for the development of HPV-negative head and neck, liver, lung, and kidney cancer. However, the molecular mechanisms underlying HIV-1-associated increase of epithelial malignancies are not fully understood. Here, we showed that HPV-16-immortalized anal AKC-2 and cervical CaSki epithelial cells that undergo prolonged exposure to cell-free HIV-1 virions or HIV-1 viral proteins gp120 and tat respond with the epithelial-mesenchymal transition (EMT) and increased invasiveness. Similar responses were observed in HPV-16-infected SCC-47 and HPV-16-negative HSC-3 oral epithelial cancer cells that were cultured with these viral proteins. EMT induced by gp120 and tat led to detachment of poorly adherent cells from the culture substratum; these cells remained capable of reattachment, upon which they coexpressed both E-cadherin and vimentin, indicative of an intermediate stage of EMT. The reattached cells also expressed stem cell markers CD133 and CD44, which may play a critical role in cancer cell invasion and metastasis. Inhibition of transforming growth factor (TGF)-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasive activity of HPV-16-immortalized anal and cervical epithelial cells. Collectively, our results suggest that these approaches along with HIV viral suppression with antiretroviral therapy (ART) might be useful to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia. IMPORTANCE HPV-16-immortalized genital and oral epithelial cells and HPV-negative oral cancer cells that undergo prolonged contact with cell-free HIV-1 virions or with viral proteins gp120 and tat respond by becoming more invasive. EMT cells induced by HIV-1 in cultures of HPV-16-immortalized anal and cervical epithelial cells express the stem cell markers CD133 and CD44. These results suggest that the interaction of HIV-1 with neoplastic epithelial cells may lead to their de-differentiation into cancer stem cells that are resistant to apoptosis and anti-cancer drugs. Thus, this pathway may play a critical role in the development of invasive cancer. Inhibition of TGF-β1 and MAPK signaling and vimentin expression, and restoration of E-cadherin expression reduced HIV-induced EMT and the invasiveness of HPV-16-immortalized anal and cervical epithelial cells. Taken together, these results suggest that these approaches might be exploited to limit the role of HIV-1 infection in the acceleration of HPV-associated or HPV-independent epithelial neoplasia.

Flavor Classification/Categorization and Differential Toxicity of Oral Nicotine Pouches (ONPs) in Oral Gingival Epithelial Cells and Bronchial Epithelial Cells.

Oral nicotine pouches (ONPs) are a modern form of smokeless tobacco products sold by several brands in the U.S., which comprise a significant portion of non-combustible nicotine-containing product (NCNP) sales to date. ONPs are available in various flavors and may contain either tobacco-derived nicotine (TDN) or tobacco-free nicotine (TFN). The growth in popularity of these products has raised concerns that flavored ONPs may cause adverse oral health effects and promote systemic toxic effects due to nicotine and other ONP by-products being absorbed into the circulatory system through oral mucosa. We hypothesized that flavored ONPs are unsafe and likely to cause oral and pulmonary inflammation in oral and respiratory epithelial cells. Before analyzing the effects of ONPs, we first classified ONPs sold in the U.S. based on their flavor and the flavor category to which they belonged using a wheel diagram. Human gingival epithelial cells (HGEP) were treated with flavored ONP extracts of tobacco (original, smooth), menthol (wintergreen and cool cider), and fruit flavor (americana and citrus), each from the TDN and TFN groups. The levels of ONP-induced inflammatory cytokine release (TNF-α, IL-6, and IL-8) by ELISA, cellular reactive oxygen species (ROS) production by CellRox Green, and cytotoxicity by lactate dehydrogenase (LDH) release assay in HGEP cells were assessed. Flavored ONP extracts elicited differential toxicities in a dose- and extract-dependent manner in HGEP cells 24 h post-treatment. Both fruit TDN and TFN extracts resulted in the greatest cytotoxicity. Tobacco- and fruit-flavored, but not menthol-flavored, ONPs resulted in increased ROS production 4 h post-treatment. Flavored ONPs led to differential cytokine release (TNF-α, IL-6, and IL-8) which varied by flavor (menthol, tobacco, or fruit) and nicotine (TDN vs. TFN) 24 h post-treatment. Menthol-flavored ONPs led to the most significant TNF-α release; fruit TFN resulted in the most significant IL-6 release; and fruit TDN and tobacco TFN led to the highest release of IL-8. Subsequently, human bronchial epithelial cells (16-HBE and BEAS-2B) were also treated with flavored ONP extracts, and similar assays were evaluated. Here, the lowest concentration treatments displayed increased cytotoxicity. The most striking response was observed among cells treated with spearmint and tobacco flavored ONPs. Our data suggest that flavored ONPs are unsafe and likely to cause systemic and local toxicological responses during chronic usage.