Dysplastic Oral Keratinocytes Research Articles

Dental pulp stem cells promote malignant transformation of oral epithelial cells through mitochondrial transfer.

Oral epithelial dysplasia includes a range of clinical oral mucosal diseases with potentially malignant traits. Dental pulp stem cells (DPSCs) are potential candidates for cell-based therapies targeting various diseases. However, the effect of DPSCs on the progression of oral mucosal precancerous lesions remains unclear. Animal experiments were conducted to assess the effect of human DPSCs (hDPSCs). We measured the proliferation, motility and mitochondrial respiratory function of the human dysplastic oral keratinocyte (DOK) cells cocultured with hDPSCs. Mitochondrial transfer experiments were performed to determine the role mitochondria from hDPSCs in the malignant transformation of DOK cells. hDPSCs injection accelerated carcinogenesis in 4NQO-induced oral epithelial dysplasia in mice. Coculture with hDPSCs increased the proliferation, migration, invasion and mitochondrial respiratory function of DOK cells. Mitochondria from hDPSCs could be transferred to DOK cells, and activated mTOR signaling pathway in DOK cells. Our study demonstrates that hDPSCs activate the mTOR signaling pathway through mitochondrial transfer, promoting the malignant transformation of oral precancerous epithelial lesions.

The mechanism of low molecular weight fucoidan-incorporated nanofiber scaffolds inhibiting oral leukoplakia via SR-A/Wnt signal axis.

Oral leukoplakia (OLK) is the most common oral precancerous lesion, and 3%-17% of OLK patients progress to oral squamous cell carcinoma. OLK is susceptible to recurrence and has no effective treatment. However, conventional drugs have significant side effects and limitations. Therefore, it is important to identify drugs that target OLK. In this study, scavenger receptor A (SR-A) was found to be abnormally highly expressed in the oral mucosal epithelial cells of OLK patients, whereas molecular biology studies revealed that low molecular weight fucoidan (LMWF) promoted apoptosis of dysplastic oral keratinocytes (DOK) and inhibited the growth and migration of DOK, and the inhibitory effect of LMWF on OLK was achieved by regulating the SR-A/Wnt signaling axis and related genes. Based on the above results and the special situation of the oral environment, we constructed LMWF/poly(caprolactone-co-lactide) nanofiber membranes with different structures for the in-situ treatment of OLK using electrospinning technology. The results showed that the nanofiber membranes with a shell-core structure had the best physicochemical properties, biocompatibility, and therapeutic effect, which optimized the LMWF drug delivery and ensured the effective concentration of the drug at the target point, thus achieving precise treatment of local lesions in the oral cavity. This has potential application value in inhibiting the development of OLK.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence

BackgroundOral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear. MethodsThis study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H2O2. In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2. ResultsClinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H2O2-induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest. ConclusionPrx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target.

Curcumin is effective in managing oral inflammation: An in vitro study.

Oral inflammation is among the most prevalent oral pathologies with systemic health implications, necessitating safe and effective treatments. Given curcumin's documented anti-inflammatory and antioxidant properties, this study focuses on the potential of a curcumin-based oral gel in safely managing oral inflammatory conditions. This in vitro study utilized four human cell lines: oral keratinocytes (HOKs), immortalized oral keratinocytes (OKF6), periodontal ligament fibroblasts (HPdLF), and dysplastic oral keratinocytes (DOKs). The cells were treated with Lipopolysaccharides (LPS) and curcumin-based oral gel to simulate inflammatory conditions. A panel of cellular assays were performed along with antimicrobial efficacy tests targeting Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis. LPS significantly reduced proliferation and wound healing capacities of HOKs, OKF6, and HPdLF, but not DOKs. Treatment with curcumin-based oral gel mitigated inflammatory responses in HOKs and HPdLF by enhancing proliferation, colony formation, and wound healing, along with reducing apoptosis. However, its impact on OKF6 and DOKs was limited in some assays. Curcumin treatment did not affect the invasive capabilities of any cell line but did modulate cell adhesion in a cell line-specific manner. The curcumin-based oral gel showed significant antimicrobial efficacy against C. albicans and S. mutans, but was ineffective against P. gingivalis. This study demonstrates the potential of the curcumin-based oral gel as a safe and effective alternative to conventional antimicrobial treatments for managing cases of oral inflammation. This was achieved by modulating cellular responses under simulated inflammatory conditions. Future clinical-based studies are recommended to exploit curcumin's therapeutic benefits in oral healthcare.

Angiopoietin-like 4 is upregulated by amphiregulin and activates cell proliferation and migration through p38 kinase in head and neck squamous cell carcinoma.

Angiopoietin-like 4 is a molecular hallmark that correlates with the growth and metastasis of head and neck squamous cell carcinoma, one of the most prevalent cancers worldwide. However, the molecular mechanisms by which angiopoietin-like 4 promotes head and neck squamous cell carcinoma tumorigenesis are unclear. Using well-characterized cell lines of head and neck squamous cell carcinoma development, including human normal oral keratinocytes, dysplastic oral keratinocytes, oral leukoplakia-derived oral keratinocytes, and head and neck squamous cell carcinoma cell lines, HN13, HN6, HN4, HN12, and CAL27, we investigated the signaling pathways upstream and downstream of angiopoietin-like 4-induced head and neck squamous cell carcinoma tumorigenesis. We found that both epidermal growth factor receptor ligands, epithelial growth factor, and amphiregulin led to angiopoietin-like 4 upregulation in normal oral keratinocytes and dysplastic oral keratinocytes and cooperated with the activation of hypoxia-inducible factor-1 in this effect. Interestingly, amphiregulin and angiopoietin-like 4 were increased in dysplastic oral keratinocytes and head and neck squamous cell carcinoma cell lines, and amphiregulin-induced activation of cell proliferation was dependent on angiopoietin-like 4. Although both p38 mitogen-activated protein kinases (p38 MAPK) and protein kinase B (AKT) were activated by angiopoietin-like 4, only pharmacological inhibition of p38 MAPK was sufficient to prevent head and neck squamous cell carcinoma cell proliferation and migration. We further observed that angiopoietin-like 4 promoted the secretion of interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin-1 alpha (IL-1α), vascular endothelial growth factor, platelet-derived growth factor (PDGF), and tumour necrosis factor alpha (TNF-α), cytokines and chemokines previously implicated in head and neck squamous cell carcinoma pathogenesis. Our results demonstrate that angiopoietin-like 4 is a downstream effector of amphiregulin and promotes head and neck squamous cell carcinoma development both through direct activation of p38 kinase as well as paracrine mechanisms.

Induction of Peroxiredoxin 1 by Hypoxia Promotes Cellular Autophagy and Cell Proliferation in Oral Leukoplakia via HIF-1α/BNIP3 Pathway.

Hypoxia is a key trigger in the transformation of oral leukoplakia into oral cancer. However, it is still too early to determine the role of hypoxia in the development of oral leukoplakia. Prx1, an antioxidant protein, upregulated by hypoxia, regulates cellular autophagy in leukoplakia. This study aimed to understand the mechanisms by which hypoxia induces Prx1 expression during autophagy in oral leukoplakia. We used an experimental model of tongue epithelial hyperplasia induced by 4-nitroquinoline-1-oxide (4NQO) and dysplastic oral keratinocytes. Prx1 knockdown DOK cells, Leuk-1 cells and control cells were harvested, and cell proliferation was assayed using the Cell Counting Kit-8. Several hypoxia and autophagy-related proteins were examined using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and western blotting in cells and mouse tongue tissues. In addition, the ultrastructure of the cells was observed by transmission electron microscopy. Hypoxia induces cell proliferation, autophagic vesicles and the expression of Prx1, BNIP3, LC3II/I and Beclin-1 in DOK and Leuk-1 cells. However, these effects were all attenuated by Prx1 knockdown. Histologically, 4NQO induced epithelial hyperplasia in the tongue mucosa. The expression of proliferation marker PCNA, autophagy-related proteins LC3B and Beclin-1, as well as HIF-1α/BNIP3 was significantly lower in the tongue tissues of Prx1flox/flox:Cre+ mice compared with Prx1flox/flox mice. In Prx1flox/flox:Cre+ mice, an increased expression of HIF-1α/BNIP3, LC3B and Beclin-1 was detected in epithelial hyperplasia tongue tissues compared to normal tissues. The current study suggests that Prx1 may promotes cell proliferation and autophagy in oral leukoplakia cells via the HIF-1α/BNIP3 pathway.

Analysis of differentially expressed genes in dysplastic oral keratinocyte cell line and their role in the development of HNSCC

Dysplasia is the presence of abnormal types of cells in a tissue precipitated by over or diminished expression of certain genes. These cells act as a precursor to cancer. Dysplastic oral keratinocyte (DOK) cell lines have an aneuploid complex karyotype. They provide an opportunity to study the action of specific carcinogens on malignant transformations. This study aimed to identify the differentially expressed genes in dysplastic cells and their possible association with head and neck squamous cell carcinoma (HNSCC). These genes can be developed as diagnostic, prognostic, or therapeutic leads.The list of genes related to oral keratinocyte dysplasia and head and neck cancer was accessed from the GEO (Gene Expression Omnibus) database. Gene expression profiling was done between dysplastic oral keratinocytes and normal human oral keratinocytes. Gene expression and Kaplan Meier survival analysis were performed using the UALCAN database to assess the correlations between dysregulated genes identified in dysplastic keratinocytes and primary tumors of HNSCC. The GEO omnibus dataset identified numerous differentially expressed genes of which the top 10 up and downregulated genes in dysplastic oral keratinocytes were curated for further analysis. The expression profile of these genes was assessed using the HNSCC dataset (TCGA, Firehose Legacy). Among all the genes assessed, only one gene, the OLR1 gene encoding oxidized low-density lipoprotein, was found to be overexpressed in both the groups viz., dysplastic keratinocytes and HNSCC cases with a strong correlation with the survival status of patients. There was significant correlation between the gene expression pattern observed in dysplastic keratinocytes and the primary tumor of the HNSCC group, with an exotic gene that was seldom discussed in association with cancer, viz., OLR1. Exploration into other top-ranking differentially expressed genes in dysplastic cases would aid in identifying the candidate gene associated with both phenotypes.

Calcitriol Treatment Decreases Cell Migration, Viability and β-Catenin Signaling in Oral Dysplasia.

Nearly 90% of oral cancers are characterized as oral squamous cell carcinoma (OSCC), representing the sixth most common type of cancer. OSCC usually evolves from oral potentially malignant disorders that, in some cases, are histologically consistent with a oral dysplasia. The levels of 1α,25 dihydroxyvitamin D3 (1,25-(OH)2D3; calcitriol), the active form of vitamin D3, have been shown to be decreased in patients with oral dysplasia and OSCC. Moreover, treatment with 1,25-(OH)2D3 has been proven beneficial in OSCC by inhibiting the Wnt/β-catenin pathway, a signaling route that promotes cell migration, proliferation, and viability. However, whether this inhibition mechanism occurs in oral dysplasia is unknown. To approach this question, we used dysplastic oral keratinocyte cultures and oral explants (ex vivo model of oral dysplasia) treated with 1,25-(OH)2D3 for 48 h. Following treatment with 1,25-(OH)2D3, both in vitro and ex vivo models of oral dysplasia showed decreased levels of nuclear β-catenin by immunofluorescence (IF) and immunohistochemistry (IHC). Consistently, reduced protein and mRNA levels of the Wnt/β-catenin target gene survivin were observed after treatment with 1,25-(OH)2D3. Moreover, 1,25-(OH)2D3 promoted membranous localization of E-cadherin and nuclear localization of vitamin D receptor (VDR). Functionally, DOK cells treated with 1,25-(OH)2D3 displayed diminished cell migration and viability in vitro.

Abstract 310: Cadherin-dependent modulation of chemosensitivity in oral tumor progression

Abstract The progression of healthy oral epithelia to an oral carcinoma often involves aberrant expression of a group of calcium-dependent transmembrane proteins called cadherins. Histological studies of human tumors have demonstrated increased expression of the epithelial-resident placental (P-cadherin) and ectopic expression of the mesenchymal neuronal (N-cadherin) cadherin in oral dysplasia. We hypothesized that these changes in cadherin expression during early oral tumor development could facilitate increases in survival signaling. We genetically modified dysplastic oral keratinocytes to overexpress P-cadherin or suppress endogenous expression of N-cadherin. P-cadherin overexpression and N-cadherin suppression independently elicited a 3-6-fold decrease in apoptosis levels in response to docetaxel treatment compared to similarly treated controls. We found that in untreated dysplastic cells, P- and N-cadherin expression had positive and negative correlations, respectively, with the activation of the survival-associated enzymes focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AKT kinase. The total levels of nuclear factor kappa B (NFkb) were positively correlated with P-cadherin overexpression and reciprocally correlated with levels of N-cadherin. In docetaxel-treated dysplastic cells, we observed the reverse effect compared to untreated cells: the increased level of N-cadherin was positively correlated with FAK activity whereas FAK activity was suppressed by P-cadherin. P-cadherin expression in docetaxel-treated cells also increased activation of both MAPK and AKT, whereas alteration of N-cadherin levels had no effect. Because N-cadherin levels are known to increase in late-stage oral tumors, we extended these signaling studies to oral squamous carcinoma cells (OSCCs) expressing low or high levels of N-cadherin. In contrast to its suppression of survival pathways in docetaxel-treated dysplastic cells, in docetaxel-treated OSCCs, N-cadherin expression strongly enhanced phosphorylation of MAPK and AKT, and increased expression of NFkb. N-cadherin overexpression during this treatment also increased expression of the antiapoptotic proteins Mcl-1 and suppressed phosphorylation of the apoptotic regulator signal transducer and activator of transcription 3 (pSTAT3). Increase cadherin expression in all untreated cell lines increased expression of an inducer of epithelial to mesenchymal transition, the transmembrane protein mucin-1 (MUC1). N-cadherin overexpression also increased motility in both cell lines. Taken together, our data demonstrate antiapoptotic signaling directed by increased levels of P-cadherin in oral dysplasia and N-cadherin in OSCC, whereas increased levels of N-cadherin in dysplastic cells directed cellular signaling that were associated with a proapoptotic phenotype. Citation Format: Aaron Mody, Cassandra Decker, Kathryn R. Lawson. Cadherin-dependent modulation of chemosensitivity in oral tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 310.

A Curcumin-Based Oral Gel Has Potential Protective Efficacy against Oral Mucositis: In Vitro Study.

Oral mucositis is a common distressing complication of cancer therapy, characterised by painful sores within the oral cavity. Current management options offer limited symptomatic relief. Curcumin, a natural polyphenolic compound with recognised anti-inflammatory and antioxidant properties, has emerged as a potential protective agent against oral mucositis. This study explores the therapeutic potential of curcumin in mitigating the impact of oral mucositis by investigating a commercially available curcumin-based oral gel, PerioGold®. Liquid chromatography-tandem mass spectrometry was used to characterise the main constituents of PerioGold®. The cytotoxicity of curcumin constituent was investigated in four cell lines: primary oral keratinocytes (HOKs), immortalised oral keratinocytes (OKF6), dysplastic oral keratinocytes (DOKs), and oral squamous cell carcinoma cells (PE/CA-PJ15). Concentrations of hydrogen peroxide were optimised to develop in vitro models that mimic oral mucositis. The inhibitory effects of PerioGold® against the production of reactive oxygen species were assessed using a designated kit. OKF6 cells were the most sensitive to oxidative stress, while PE/CA-PJ15 cells showed the highest resistance. Pretreatment of the investigated cells for 24 h with PerioGold® demonstrated a significant antioxidative effect in all cells based on a dose-response pattern. PerioGold® exhibits clinical potential for protecting against oxidative stress, warranting further individualised clinical investigations.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/β-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear β-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

Stability and cytotoxicity of biopolymer-coated liposomes for use in the oral cavity

This study investigates the stability and cytotoxicity of biopolymer-coated liposomes for use in the oral cavity. Liposomes (3 mM and 6 mM) were prepared by the thin film method and hydrated with phosphate buffer (PB) or glycerol phosphate buffer (G-PB). For coating, liposomes were added to a biopolymer solution of opposite charge. Particle stability was evaluated by measuring the size, polydispersity index, and zeta potential for up to 60 weeks. In vitro interaction of fluorescent-labelled biopolymer-coated liposomes and dysplastic oral keratinocytes was analyzed by confocal microscopy. Potential cytotoxicity was assessed in dysplastic oral keratinocytes by cell proliferation and cell viability. All three biopolymers showed good coating abilities for both concentrations and hydration media. The alginate coated liposomes in PB, 3 mM chitosan-coated liposomes in PB, and chitosan-coated liposomes in G-PB were stable for up to 60 weeks. In vitro studies demonstrated low cytotoxicity for all coated liposomes and non-specific cellular uptake of biopolymer-coated liposomes, independent of biopolymer, surface charge, lipid concentration and hydration media. All three formulations demonstrated low cytotoxicity and were considered safe. Alginate- and chitosan-coated liposomes demonstrated good stability over time and may be promising agents for use in the oral cavity and should be investigated further.

Knockdown of PGC1α suppresses dysplastic oral keratinocytes proliferation through reprogramming energy metabolism

Oral potentially malignant disorders (OPMDs) are precursors of oral squamous cell carcinoma (OSCC). Deregulated cellular energy metabolism is a critical hallmark of cancer cells. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC1α) plays vital role in mitochondrial energy metabolism. However, the molecular mechanism of PGC1α on OPMDs progression is less unclear. Therefore, we investigated the effects of knockdown PGC1α on human dysplastic oral keratinocytes (DOKs) comprehensively, including cell proliferation, cell cycle, apoptosis, xenograft tumor, mitochondrial DNA (mtDNA), mitochondrial electron transport chain complexes (ETC), reactive oxygen species (ROS), oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and glucose uptake. We found that knockdown PGC1α significantly inhibited the proliferation of DOKs in vitro and tumor growth in vivo, induced S-phase arrest, and suppressed PI3K/Akt signaling pathway without affecting cell apoptosis. Mechanistically, downregulated of PGC1α decreased mtDNA, ETC, and OCR, while enhancing ROS, glucose uptake, ECAR, and glycolysis by regulating lactate dehydrogenase A (LDHA). Moreover, SR18292 (an inhibitor of PGC1α) induced oxidative phosphorylation dysfunction of DOKs and declined DOK xenograft tumor progression. Thus, our work suggests that PGC1α plays a crucial role in cell proliferation by reprograming energy metabolism and interfering with energy metabolism, acting as a potential therapeutic target for OPMDs.

Red Light and 5% Aminolaevulinic Acid (5%) Inhibit Proliferation and Migration of Dysplastic Oral Keratinocytes via ROS Production: An In Vitro Study.

Undiagnosed and untreated oral precancerous lesions often progress into malignancies. Photodynamic therapy (PDT) might be a minimally invasive alternative to conventional treatments. 5-aminolevulinic acid (5-ALA) is one of the most commonly used photosensitizers in PDT, and it is effective on many cancer types. However, its hydrophilic characteristic limits cell membrane crossing. In the present study, the effect of a newly formulated gel containing 5% 5-ALA in combination with red light (ALAD-PDT) on a premalignant oral mucosa cell line was investigated. The dysplastic oral keratinocyte (DOK) cells were incubated with ALAD at different concentrations (0.1, 0.5, 1, and 2 mM) at two different times, 45 min or 4 h, and then irradiated for 7 min with a 630 nm LED (25 J/cm2). MTT assay, flow cytometry, wound healing assay, and quantitative PCR (qPCR) were performed. ALAD-PDT exerted inhibitory effects on the proliferation and migration of DOK cells by inducing ROS and necrosis. mRNA analysis showed modulation of apoptosis-related genes' expression (TP53, Bcl-2, survivin, caspase-3, and caspase-9). Furthermore, there was no difference between the shorter and longer incubation times. In conclusion, the inhibitory effect of the ALAD-PDT protocol observed in this study suggests that ALAD-PDT could be a promising novel treatment for oral precancerous lesions.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

Angiopoietin-like 4 induces head and neck squamous cell carcinoma cell migration through the NRP1/ABL1/PXN pathway

ObjectivesThe molecular mechanisms whereby angiopoietin-like 4 (ANGPTL4), a pluripotent protein implicated in cancer development, contributes to head and neck squamous cell carcinoma (HNSCC) growth and dissemination are unclear. Materials and MethodsWe investigated ANGPTL4 expression in human normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), oral leukoplakia cells (LEUK1), and HNSCC cell lines, as well as in tissue biopsies from patients with oral dysplasia, and primary and metastatic HNSCC. We further examined the contribution of ANGPTL4 cancer progression in an HNSCC orthotopic floor-of mouth tumor model and the signaling pathways linking ANGPTL4 to cancer cell migration. ResultsANGPTL4 expression was upregulated in premalignant DOKs and HNSCC cell lines compared to NOKs and was increased in tissue biopsies from patients with oral dysplasia, as well as in primary and metastatic HNSCC. We also observed that downregulation of ANGPTL4 expression inhibited primary and metastatic cancer growth in an HNSCC orthotopic tumor model. Interestingly, ANGPTL4 binding to the neuropilin1 (NRP1) receptor led to phosphorylation of the focal adhesion protein, paxillin (PXN), and tumor cell migration; this was dependent on the tyrosine kinase ABL1. Treatment with the ABL1 inhibitor, dasatinib and small interfering RNA silencing of NRP1 or ABL1 expression blocked PXN phosphorylation and tumor cell migration. ConclusionOur findings suggest an early, sustained, and angiogenesis-independent autocrine role for ANGPTL4 in HNSCC progression and expose ANGPTL4/NRP1/ABL1/PXN as an early molecular marker and vulnerable target for the prevention of HNSCC growth and metastasis.

Inflammatory Cells Can Alter the Levels of H3K9ac and γH2AX in Dysplastic Cells and Favor Tumor Phenotype.

Oral potentially malignant disorders (OPMD) are clinical presentations that carry an increased risk of cancer development. Currently, epithelial dysplasia grade is based on architectural and cytological epithelial changes and is used to predict the malignant transformation of these lesions. However, predicting which OPMD will progress to a malignant tumor is very challenging. Inflammatory infiltrates can favor cancer development, and recent studies suggest that this association with OPMD lesions may be related to the etiology and/or aggressive clinical behavior of these lesions. Epigenetic changes such as histone modifications may mediate chronic inflammation and also favor tumor cells in immune resistance and evasion. This study aimed to evaluate the relationship between histone acetylation (H3K9ac) and DNA damage in the context of dysplastic lesions with prominent chronic inflammation. Immunofluorescence of "low-risk" and "high-risk" OPMD lesions (n = 24) and inflammatory fibrous hyperplasia (n = 10) as the control group was performed to assess histone acetylation levels and DNA damage through the phosphorylation of H2AX (γH2AX). Cell co-culture assays with PBMCs and oral keratinocyte cell lines (NOK-SI, DOK, and SCC-25) were performed to assess proliferation, adhesion, migration, and epithelial-mesenchymal transition (EMT). Oral dysplastic lesions showed a hypoacetylation of H3K9 and low levels of γH2AX compared to control. The contact of dysplastic oral keratinocytes with PBMCs favored EMT and the loss of cell-cell adhesion. On the other hand, p27 levels increased and cyclin E decreased in DOK, indicating cell cycle arrest. We conclude that the presence of chronic inflammation associated to dysplastic lesions is capable of promoting epigenetic alterations, which in turn can favor the process of malignant transformation.

Expression of Tyrosine Kinase Receptor 2 in Oral Squamous Cell Carcinoma and the Effect on Cell Proliferation and Migration and Epithelial-Mesenchymal Transition Process

To study the expression of tyrosine kinase receptor 2 (Tie2) in oral squamous cell carcinoma (OSCC) and its effect on cell proliferation and migration and the epithelial-mesenchymal transition (EMT) process. Immunohistochemistry (IHC) tests were conducted to examine the expression of Tie2 in OSCC tissues and normal oral mucosa tissues. Western blot was performed to examine the expression of Tie2 in dysplastic oral keratinocyte (DOK) cell line and OSCC cell lines, and the cell line with high Tie2 expression was selected as the experimental cell line. The Tie2-silenced lentiviral vector was successfully transfected onto the experimental cell line for subsequent experiments. Cell proliferation and cloning abilities were examined with CCK-8 and clone formation assays. Cell migration ability was examined with scratch and Transwell assays. The remodeling ability of cytoskeletal F-actin and the expressions of E-cadherin and N-cadherin were examined with confocal laser scanning microscope. Western blot was performed to examine the expression of EMT-related signature proteins, including E-cadherin, N-cadherin, and vimentin, and the expression of the protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). IHC results showed that the Tie2-positive rate of the OSCC group (74.5%) was significantly higher than that of the control group (19.4%) ( P<0.0001). The expression of Tie2 was higher in HSC-4 and SCC-9 cell lines compared to that in DOK cells. The lentiviral shRNA-162 group showed the best silencing effect, which was used as the experimental group and applied in subsequent experiments. Compared with those of the control group, the proliferation, cloning and migration capacities of the cells of the experimental group were significantly reduced. Furthermore, the green fluorescence intensity of the cytoskeleton F-actin was reduced, the number of filamentous pseudopods at the leading edge of the cells decreased and their length was shortened, and the expression of E-cadherin was significantly increased, while the expression of N-cadherin and vimentin was significantly reduced in the experimental group in comparison with those of the control group. The expression of p-AKT and p-ERK proteins decreased, while AKT and ERK protein expression increased. Tie2 was highly expressed in most OSCC cells. Silencing Tie2 can inhibit the proliferation, cloning, and migration ability of OSCC cells, inhibit F-actin remodeling, and alter the expression of its EMT-related signature proteins by regulating AKT and ERK signaling pathway, which suggests that Tie2 may be involved in the growth, metastasis and EMT process of OSCC.

Sprouty 4 expression in human oral squamous cell carcinogenesis

Background/purposeReviewing literature, sprouty 4 (SPRY4) has not been studied in human oral squamous cell carcinomas (OSCCs). The study aimed to examine SPRY4 expression in human oral squamous cell carcinogenesis. Materials and methodsA total of 95 OSCCs, 10 OPMDs with malignant transformation (MT), 17 OPMDs without MT, and six normal oral mucosa (NOM) samples were recruited for immunohistochemical staining; three OSCC tissues with normal tissue counterpart NOM were employed for Western blotting. Three human oral cancer cell lines (OCCLs), an oral precancer cell line (dysplastic oral keratinocyte, DOK), and a primary culture of normal oral keratinocytes (HOK) were used for Western blotting; OCCLs and HOK were employed for real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated in terms of proliferation, migration, and invasion assays. ResultsSPRY4 protein expression was significantly increased in OSCCs compared with NOM. Protein and mRNA SPRY4 expression in OCCLs were significantly elevated compared with HOK. Significant increases in the degrees of proliferation, migration, and invasion were noted in OCCLs with SPRY4 siRNA transfection compared with those without transfection. SPRY4 protein level was increased in OPMD with MT compared to OPMD without MT. SPRY4 protein was significant increase in DOK in comparison with HOK. SPRY4 protein expression was significantly increased from NOM and OPMD without MT to OSCC. SPRY4 protein expression in OCCLs was significantly enhanced compared with DOK and HOK respectively. ConclusionOur results indicate that SPRY4 expression is possibly involved in human oral squamous cell carcinogenesis.

Characterization of DJ-1, PTEN, and p-Akt as Prognostic Biomarkers in the Progression of Oral Squamous Cell Carcinoma.

Objective Oral cancer has a five-year survival rate of 68%, and the methods used to assess it still relyheavily on morphology. Protein biomarkers can potentially increase the predictive power of histopathological evaluation. This study aims to examine the expression of three closely linked proteins implicated in the pathogenesis of oral squamous cell carcinoma (OSCC); protein deglycase (DJ-1), an oncogene, phosphatase, and tensin homolog (PTEN), a tumor suppressor gene, and phosphorylated protein kinase B (p-Akt), the activated form of a vital serine/threonine kinase, which is involved in the oncogenesis of several human malignancies, throughout the tumor progression steps to establish their potential as prognostic biomarkers. Study design Western blot analysis was carried out using four different cell lines representing the successive steps of OSCC progression, including normal oral keratinocytes, dysplastic oral keratinocytes, locally invasive OSCC, and metastatic OSCC. Results DJ-1 expression was found to be upregulated gradually throughout the successive steps of OSCC progressionfrom normal to dysplastic to locally invasive to metastatic OSCC. PTEN expression showed an overall opposite trend. Interestingly, a significant downregulation of p-Akt was seen in the locally invasive OSCC cells, although it was followed by a significant increase in p-Akt expression in the metastatic OSCC cell line, which is consistent with the role of p-Akt in the motility and migration of cancer cells. Conclusion This study documented trends in expression patterns of three important signaling molecules, DJ-1, PTEN, and p-Akt, in normal, premalignant, and malignant oral keratinocytes. The oncogenic DJ-1 and tumor suppressor PTEN were expressed in a manner consistent with their respective roles in tumorigenesis, while p-Akt only showed a significant upregulation in the metastatic OSCC cells. Overall, all three proteins exhibited unique trends throughout the progressive stages of OSCC tumor progression, thereby adding to their potential utility as prognostic biomarkers for oral cancer patients.