Oral Epithelial Cell Line Research Articles

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.

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.

Design, Characterization, and Anticancer and Antimicrobial Activities of Mucoadhesive Oral Patches Loaded with Usnea barbata (L.) F. H. Wigg Ethanol Extract F-UBE-HPMC.

The oral cavity’s common pathologies are tooth decay, periodontal disease, and oral cancer; oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high mortality rate. Our study aims to formulate, develop, characterize, and pharmacologically investigate the oral mucoadhesive patches (F-UBE-HPMC) loaded with Usnea barbata (L.) F.H. Wigg dry ethanol extract (UBE), using HPMC K100 as a film-forming polymer. Each patch contains 312 µg UBE, with a total phenolic content (TPC) of 178.849 µg and 33.924 µg usnic acid. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were performed for their morphological characterization, followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Pharmacotechnical evaluation involved the measurement of the specific parameters for mucoadhesive oral patches as follows: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time. Thus, each F-UBE-HPMC has 104 ± 4.31 mg, a pH = 7.05 ± 0.04, a disintegration time of 130 ± 4.14 s, a swelling ratio of 272 ± 6.31% after 6 h, and a mucoadhesion time of 102 ± 3.22 min. Then, F-UBE-HPMCs pharmacological effects were investigated using brine shrimp lethality assay (BSL assay) as a cytotoxicity prescreening test, followed by complex flow cytometry analyses on blood cell cultures and oral epithelial squamous cell carcinoma CLS-354 cell line. The results revealed significant anticancer effects by considerably increasing oxidative stress and blocking DNA synthesis in CLS-354 cancer cells. The antimicrobial potential against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 was assessed by a Resazurin-based 96-well plate microdilution method. The patches moderately inhibited both bacteria strains growing and displayed a significant antifungal effect, higher on C. albicans than on C. parapsilosis. All these properties lead to considering F-UBE-HPMC suitable for oral disease prevention and therapy.

FOXN3 Expression Regulated by miR-299-5p Inhibiting the Proliferation, Migration and Invasion of Oral Squamous Cell Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is one of the commonest malignancies of the oral cavity. FOXN3 is a tumor suppressor that represses the progression of many tumors. Nonetheless, its role in OSCC has not been elucidated. This work is performed to probe the role and dysregulation mechanism of FOXN3 in OSCC. FOXN3 mRNA and miR-299-5p expressions were quantified by quantitative real-time polymerase chain reaction (qRT-PCR); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to detect OSCC cell growth; Transwell experiment was conducted to detect cell migration and invasion; dual-luciferase reporter experiment and bioinformatics were adopted to analyze the relationship between miR-299-5p and FOXN3; Western blot was implemented to detect FOXN3 protein expression. FOXN3 expression was remarkably down-modulated, and miR-299-5p expression was markedly up-modulated in OSCC tissues and cell lines compared with paracancerous tissues and normal oral epithelial cell line. FOXN3 overexpression impeded OSCC cell growth, migration and invasion. FOXN3 was proven to be a downstream target of miR-299-5p, and miR-299-5p mimics enhanced OSCC cell growth, migration and invasion. Moreover, FOXN3 overexpression partially reversed the promoting effects of miR-299-5p mimics on OSCC cell growth, migration and invasion. FOXN3 expression is remarkably down-modulated in OSCC tissues and cell lines, and miR-299-5p targets FOXN3 to facilitate OSCC cell growth, migration and invasion. These results imply that miR-299-5p/FOXN3 axis may be a potential target for OSCC treatment.

Apoptotic effect of crude scorpion venom on head and neck squamous cell carcinoma cell line versus normal human epithelial cell line

Aim: The aim of this study was to investigate and compare the apoptotic effect of Leiurus quinquestriatus (LQ) scorpion venom on oral squamous cell carcinoma (OSCC) and normal human epithelial cell lines, in an attempt to introduce a novel selective therapeutic modality for OSCC. Methodology: we explored the effect of LQ scorpion venom on OSCC cell line (HNO-97) compared to normal oral epithelial (OEC) cell line. The cell viability by MTT assay and the expression of PUMA by ELISA were analyzed. Cell cycle analysis was studied for both groups using flow cytometry. Finally, we compared the expression of ROS by ELISA. Results LQ scorpion venom showed selective cytotoxic and apoptotic effect on OSCC cells. In comparison to normal cells, significant dose dependent cell viability reduction, cell cycle arrest and increased expression of apoptotic marker PUMA as well as ROS were observed in cancer cell group in comparison to the control group. Conclusions: LQ crude venom exerts a strong cytotoxic effect against OSCC with minimal effect on normal epithelial cells, thus, providing a selective, effective and less toxic possible chemotherapeutic agent for such a malignancy.

Chromatin conformation of human oral epithelium can identify orofacial cleft missing functional variants

Genome-wide association studies (GWASs) are the most widely used method to identify genetic risk loci associated with orofacial clefts (OFC). However, despite the increasing size of cohort, GWASs are still insufficient to detect all the heritability, suggesting there are more associations under the current stringent statistical threshold. In this study, we obtained an integrated epigenomic dataset based on the chromatin conformation of a human oral epithelial cell line (HIOEC) using RNA-seq, ATAC-seq, H3K27ac ChIP-seq, and DLO Hi-C. Presumably, this epigenomic dataset could reveal the missing functional variants located in the oral epithelial cell active enhancers/promoters along with their risk target genes, despite relatively less-stringent statistical association with OFC. Taken a non-syndromic cleft palate only (NSCPO) GWAS data of the Chinese Han population as an example, 3664 SNPs that cannot reach the strict significance threshold were subjected to this functional identification pipeline. In total, 254 potential risk SNPs residing in active cis-regulatory elements interacting with 1 718 promoters of oral epithelium-expressed genes were screened. Gapped k-mer machine learning based on enhancers interacting with epithelium-expressed genes along with in vivo and in vitro reporter assays were employed as functional validation. Among all the potential SNPs, we chose and confirmed that the risk alleles of rs560789 and rs174570 reduced the epithelial-specific enhancer activity by preventing the binding of transcription factors related to epithelial development. In summary, we established chromatin conformation datasets of human oral epithelial cells and provided a framework for testing and understanding how regulatory variants impart risk for clefts.

Evaluation of Usnea barbata (L.) Weber ex F.H. Wigg Extract in Canola Oil Loaded in Bioadhesive Oral Films for Potential Applications in Oral Cavity Infections and Malignancy.

Usnea lichens are known for their beneficial pharmacological effects with potential applications in oral medicine. This study aims to investigate the extract of Usnea barbata (L.) Weber ex F.H. Wigg from the Călimani Mountains in canola oil as an oral pharmaceutical formulation. In the present work, bioadhesive oral films (F-UBO) with U. barbata extract in canola oil (UBO) were formulated, characterized, and evaluated, evidencing their pharmacological potential. The UBO-loaded films were analyzed using standard methods regarding physicochemical and pharmacotechnical characteristics to verify their suitability for topical administration on the oral mucosa. F-UBO suitability confirmation allowed for the investigation of antimicrobial and anticancer potential. The antimicrobial properties against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 were evaluated by a resazurin-based 96-well plate microdilution method. The brine shrimp lethality assay (BSL assay) was the animal model cytotoxicity prescreen, followed by flow cytometry analyses on normal blood cells and oral epithelial squamous cell carcinoma CLS-354 cell line, determining cellular apoptosis, caspase-3/7 activity, nuclear condensation and lysosomal activity, oxidative stress, cell cycle, and cell proliferation. The results indicate that a UBO-loaded bioadhesive film’s weight is 63 ± 1.79 mg. It contains 315 µg UBO, has a pH = 6.97 ± 0.01, a disintegration time of 124 ± 3.67 s, and a bioadhesion time of 86 ± 4.12 min, being suitable for topical administration on the oral mucosa. F-UBO showed moderate dose-dependent inhibitory effects on the growth of both bacterial and fungal strains. Moreover, in CLS-354 tumor cells, F-UBO increased oxidative stress, diminished DNA synthesis, and induced cell cycle arrest in G0/G1. All these properties led to considering UBO-loaded bioadhesive oral films as a suitable phytotherapeutic formulation with potential application in oral infections and neoplasia.

In Vitro Study of the Potential Role of Olive Oil Oleuropein in Modulating the 5-FU Cytotoxic Efficacy against the Tongue Squamous Cell Carcinoma

BACKGROUND: 5-fluorouracil (5-FU) is an anticancer drug used to inhibit the proliferation of many different tumor cells. Since severe side effects are associated with this drug, its combination with different natural compounds would allow the use of a significantly lower dose of 5-FU. Oleuropein (OLEU), has been shown to have inhibitory effects on various types of cancers. AIM: The main objective of the current study was to assess the cytotoxic effect of OLEU and the chemotherapeutic drug 5-FU on Human Tongue Carcinoma Cancer Cell Line (HNO-97) and Human Normal Oral Epithelial Cell Line (OEC) either independently or combinatory effect. MATERIALS AND METHODS: 3-(4,5- dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability, and half-maximal inhibitory concentration (IC50) was calculated. Flowcytometry for cell cycle analysis was performed. Also, in vitro scratch assay was done to assess the inhibitory effects of OLEU on the migration of cells.RESULTS: MTT assay study demonstrated that OLEU and 5-FU alone or in combinations have produced a significant inhibitory effect on both normal and cancer cell lines with a favorable impact for OLEU on cancer cell lines rather than the normal one. A significant increase in the cell inhibitory % was reported between the single and the combinations treated groups as compared to the non-treated control group. Cell cycle analysis via flowcytometry showed that OLEU had induced cell cycle arrest at G0/1 phase, decreased S phase and G2/M phase either independently or in combination for 24h and 48h when compared with a non-treated control group. A Scratch assay test showed that OLEU could induce delayed wound healing. CONCLUSIONS: The findings of the present study suggest that OLEU can exert an anti-cancer effect on HNO-97 and may have the potential for potentiation of 5-FU cytotoxic effects and reduction of its adverse effects. In addition, OLEU could inhibit cancer progression and expansion from the initial tumor.

6-(Methylsulfinyl) Hexyl Isothiocyanate Inhibits IL-6 and CXCL10 Production in TNF-α-Stimulated Human Oral Epithelial Cells.

6-(Methylsulfinyl) hexyl isothiocyanate (6-MSITC) is a bioactive substance found in wasabi (Wasabia japonica) and has been reported to have some bioactive effects including anticancer and antioxidant effects. However, there are no reports on its effects on periodontal resident cells, and many points remain unclear. In this study, we aimed to investigate whether 6-MSITC exerts anti-inflammatory effects on human oral epithelial cells, including effects on signal transduction pathway activation. 6-MSITC inhibited interleukin (IL)-6 and C-X-C motif chemokine ligand 10 (CXCL10) production in TNF-α-stimulated TR146 cells, which are a human oral epithelial cell line. Moreover, we found that 6-MSITC could suppress signal transducer and activator of transcription (STAT)3, nuclear factor (NF)-κB, and p70S6 kinase (p70S6K)-S6 ribosomal protein (S6) pathways activation in TNF-α-stimulated TR146 cells. Furthermore, STAT3 and NF-κB inhibitors could suppress IL-6 and CXCL10 production in TNF-α-treated TR146 cells. In summary, 6-MSITC could decrease IL-6 and CXCL10 production in human oral epithelial cell by inhibiting STAT3 and NF-κB activation.

An Unexpected Role for Mediator of ErbB2 Induced Cell Migration, MEMO1, During Amelogenesis

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel related disorders or more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we identified that Memo1 is expressed in ameloblasts and that conditional deletion of Memo1 from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal associated genes and a reduction in late stage ameloblast markers, relative to controls. Histology revealed that the molecular defects correlated with a disruption of the apical Tome’s process and the basolateral interacting, papillary layer, in Memo1 mutant ameloblasts. Finally, deletion of Memo1 from an oral epithelial ameloblast cell line, followed by cellular and molecular analyses, revealed altered cytoskeletal networks, relative to control cells. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide both in vivo and in vitro systems to further interrogate the relationship of cytoskeletal and amelogenesis‐related defects.

Ferrocene-based modulators of cancer-associated tumor pyruvate kinase M2

We report the design and synthesis of fifteen novel ferrocene-based triazolopyrimidines and several other studies that establish these molecules as PKM2 modulators. Our ferrocene-based triazolopyrimidines (FTPs) were subjected to interaction with Pyruvate Kinase isoform, PKM2, which is vehemently overexpressed in oral, lung, breast, and colorectal cancers. The in silico outcomes and lactate dehydrogenase (LDH) coupled enzyme assay results revealed many of these molecules can be groomed into potential anticancer agents. Additionally, cyclic voltammetry disclosed that these species are redox-active, one of the criteria for metallocenes to act against cancer in general. Single-crystal X-Ray diffraction crystallography studies fully uncovered the 3D crystal lattice structure and their three-dimensional orientation in depth. Successively, in vitro cell viability studies were performed. Some compounds showed higher potency than cisplatin on CAL 27 cells representing human oral squamous cell carcinoma (OSCC). On top of that, these compounds turned out to be cancer-selective as the normal oral epithelial cell line was unaffected even at higher concentrations than the CAL 27 cell line.

Procyanidin B2 inhibits angiogenesis and cell growth in oral squamous cell carcinoma cells through the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) pathway

ABSTRACT This study aimed to explore the therapy role of procyanidin B2 (PB2) in inhibiting angiogenesis and cell growth in oral squamous cell carcinoma. After oral mucosa epithelial cell (OMEC) and human oral squamous cell carcinoma (OSCC) cell line (SCC-25) were treated with PB2 or SCC-25 were treated with PB2 and rhVEGF, cell counting kit-8 (CCK-8) assay was used to determine the cell viability. The apoptosis, migration, invasion and angiogenesis of SCC-25 after indicated treatment were detected by Tunel, wound healing, transwell and tube formation assays. The protein expression related to apoptosis, metastasis and epithelial-mesenchymal transition (EMT) and changed expression of vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling was analyzed by Western blot. As a result, PB2 inhibited viability, invasion, migration and EMT and promoted apoptosis of SCC-25 cells. In addition, PB2 inhibited VEGF/VEGFR2 signaling and tumor itangiogenesis in OSCC. As expected, activation of VEGF/VEGFR2 signaling suppressed the effect of PB2 on growth and metastasis of OSCC cells. In conclusion, PB2 inhibited the VEGF/VEGFR2 pathway to suppress the angiogenesis and cell growth of SCC-25 cells.

Spent Coffee Grounds Valorization as Bioactive Phenolic Source Acquired Antifungal, Anti-Mycotoxigenic, and Anti-Cytotoxic Activities.

Spent coffee grounds (SCGs), which constitute 75% of original coffee beans, represent an integral part of sustainability. Contamination by toxigenic fungi and their mycotoxins is a hazard that threatens food production. This investigation aimed to examine SCGs extract as antimycotic and anti-ochratoxigenic material. The SCGs were extracted in an eco-friendly way using isopropanol. Bioactive molecules of the extract were determined using the UPLC apparatus. The cytotoxicity on liver cancer cells (Hep-G2) showed moderate activity with selectivity compared with human healthy oral epithelial (OEC) cell lines but still lower than the positive control (Cisplatin). The antibacterial properties were examined against pathogenic strains, and the antifungal was examined against toxigenic fungi using two diffusion assays. Extract potency was investigated by two simulated models, a liquid medium and a food model. The results of the extract showed 15 phenolic acids and 8 flavonoids. Rosmarinic and syringic acids were the most abundant phenolic acids, while apigenin-7-glucoside, naringin, epicatechin, and catechin were the predominant flavonoids in the SCGs extract. The results reflected the degradation efficiency of the extract against the growth of Aspergillus strains. The SCGs recorded detoxification in liquid media for aflatoxins (AFs) and ochratoxin A (OCA). The incubation time of the extract within dough spiked with OCA was affected up to 2 h, where cooking was not affected. Therefore, SCGs in food products could be applied to reduce the mycotoxin contamination of raw materials to the acceptable regulated limits.

Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC.

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

Up-regulated SUMO1 expression by inhibition of miRNA-548m as a potential cause of congenital orofacial cleft in a fetus

To explore the genetic etiology for a fetus with congenital orofacial cleft. Single nucleotide polymorphism microarray (SNP array) was carried out on skin tissues sampled from the fetus following induced abortion for the detection of copy number variation (CNVs). Pathogenicity of the candidate gene was validated through experiment. SNP array revealed that the fetus has carried a hemizygous 9.23Mb deletion at Xq21.31-q22.1(91 063 807-100 293 555), which was inherited from its mother. The region contained 13 OMIM genes and 1 ncRNA coding gene(MIR548M). Inhibiting of the expression of the MIR548M gene in oral epithelial celllines has resulted in up-regulation of the expression of SUMO1 gene which was known to involve in the pathogenesis of orofacial cleft. Dosage insufficiency of the MIR548M gene may underlie the etiology of orofacial cleft in this fetus.

The expression of the GATA6 gene in oral carcinoma cell lines

BackgroundThis study aimed to investigate the expression level of the GATA6 gene in different oral cancer cells.MethodsIn this study, we sub-cultured normal oral epithelial cell lines HOK, human tongue squamous cell carcinoma cell lines CAL-27 and SCC-4, and human salivary gland adenoid cystic carcinoma cell lines SACC-LM and SACC-83. Subsequently, we used reverse transcription-polymerase chain reaction RT-PCR and Western blot methods to detect the mRNA and the protein expressions of GATA6 in normal oral epithelial cells, human tongue squamous cell carcinoma cells, and human salivary gland adenoid cystic carcinoma cells.ResultsThe results of this study showed that the mRNA expression levels of GATA6 in CAL-27, SCC-4, and SACC-LM cells were significantly increased when compared with the HOK cells. However, the mRNA expression level of GATA6 in the SACC-83 cells had no significant difference compared with the HOK cells. The protein expression levels of GATA6 in the SCC-4 and SACC-LM cells were, however, significantly increased whereas the protein expression levels of GATA6 in the CAL-27 and SACC-83 cells had no significant difference when compared with the HOK cells.ConclusionThe GATA6 gene may be related to the occurrence and progression of certain oral cancers.

Characterization of Wolf‐Hirschhorn Syndrome Candidate 1 ( Whsc1 ) in Pitx2 ‐ miR‐23/24 ‐mediated Tooth Development

Objective Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate (CLP), hypodontia and taurodontism. However, the causative factors and underlying mechanisms of these oral anomalies are relatively unknown. Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a H3K36-specific methyltransferase that is frequently deleted in WHS. This gene has been associated with craniofacial defects including CLP and defects in occipital ossification. In our study, we aim to understand the role of WHSC1 in tooth development. Methods To characterize the role of Whsc1 in tooth development, we profiled the Whsc1 expression pattern during mouse tooth development by immunofluorescence staining (IF). To investigate the regulatory effects between Whsc1 and Pitx2, we overexpressed either Whsc1 or Pitx2 in oral epithelial cell line LS8 and detected their expression. Then, ChIP-PCR and luciferase assays were performed to confirm the binding on the promoter regions. To determine the negative regulation of Whsc1 by miR-23-3p and miR-24-3p, miRs were inhibited by our Plasmid-based microRNA inhibition system in PMIS-miR-23-27-24 embryos and LS8 cells followed by detecting the mRNA and protein level of Whsc1. Luciferase assays were also conducted to confirm the direct binding between miRs and Whsc1 3’UTR. Results Whsc1 expresses from early stage of tooth development and restricts to stem cell niches in homeostatic tooth germ. Whsc1 and Pitx2 reciprocally activate each other's expression through chromosomal and transcriptional regulation. miR-23-3p and miR-24-3p, two miRs that regulated by Pitx2, directly inhibit Whsc1. Conclusion Whsc1 expresses in the developing tooth germ and participates the Whsc1-Pitx2-miR23/24 regulatory network in oral epithelium cells. Our observations provide new insights into the potential role of Whsc1 in regulating tooth development and a possible causer of the dental defects in WHS.

Defining the Role of MEMO1 in Craniodental Mineralization

Defects in the mineralization of craniofacial tissues are an underlying pathology in a variety of human congenital disorders. Our understanding of these developmental processes is confounded by the complex intertwinement of different tissue types (e.g. ectoderm, neural crest and mesoderm) and mineralization methods (e.g. endochondral and intramembranous ossification). Understanding the unique cellular and molecular mechanisms driving the development and mineralization of craniodental tissues (e.g. bone and teeth) is critical to explain, prevent, and treat associated genetic disorders. Our lab has identified the protein Mediator of Cell Motility 1 (MEMO1)—initially found to associate with breast cancer metastasis—is critical for bone and enamel mineralization. However, MEMO1’s biological and molecular function in these processes is unknown.We are using in vivo mouse-based and in vitro cell culture-based models to address this knowledge gap. With our in vivo models, we have utilized immunofluorescence, scanning electron microscopy, µCT, single cell RNA-sequencing, and basic histological examination to define the craniodental defects upon loss of Memo1 in the ectoderm or oral epithelium. With our in vitro models, we have utilized an oral epithelial cell line, along with CRISPR/Cas9 genome editing, Western Blot analysis, bulk RNA-sequencing and immunofluorescence to gain insight into the cellular and molecular function of MEMO1 within oral epithelial cells. Analyses of the in vivo Memo1 ectoderm or oral epithelial mutants, relative to controls, identifies an important role for MEMO1 in enamel development. Conditional mutants display chalky enamel associated with a significant decrease in enamel density (p-value < 0.03). Compromised enamel in conditional mutants is further supported by alterations in gene and protein expression within ameloblasts and may include defects in the cytoskeleton. Supporting this observation, our oral epithelial in vitro Memo1 loss-of-function model displays changes in cytoskeletal dynamics, including increased cellular contractility.These findings provide novel insight into how MEMO1 contributes to mineralization, potentially deploying its breast cancer metastatic function (i.e., cytoskeletal regulation), within a novel biological setting—amelogenesis. The oral epithelial specific role of MEMO1 expands our understanding of craniodental mineralization and MEMO1’s molecular control of these processes. Furthermore, this provides novel insight into genetic diseases of mineralization, including those impacting the craniodental complex. Additional investigation into our Memo1 loss-of-function models will progress both the specific and general knowledge of tissue mineralization—providing novel opportunities to exploit this protein, or its interacting partners, in a clinical or therapeutic setting.

A green tea extract and epigallocatechin-3-gallate attenuate the deleterious effects of irinotecan in an oral epithelial cell model

ObjectiveTo investigate the ability of a green tea extract and epigallocatechin-3-gallate (EGCG) to protect oral epithelial cells against the deleterious effects of the chemotherapeutic agent irinotecan, with respect to cytotoxicity; reactive oxygen species (ROS) generation; cytokine and matrix metalloproteinase (MMP) production; and cell proliferation and migration. MethodsThe B11 oral keratinocyte and GMSM-K oral epithelial cell lines were used in this study. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. A fluorometric assay was used to quantify ROS production. Cell proliferation was assessed using a fluorescent cell tracker dye, while a migration assay kit was used to monitor cell migration. Cytokine and MMP secretion was quantified by an enzyme-linked immunosorbent assay. ResultsThe green tea extract and EGCG reduced the cytotoxicity of irinotecan toward oral keratinocyte and epithelial cell lines. Irinotecan-induced intracellular ROS generation by oral keratinocytes was reduced by the green tea extract and EGCG. Irinotecan negatively affected the proliferation and migration of oral keratinocytes in a dose-dependent manner. However, these effects were not neutralized by the green tea extract, while EGCG showed a trend to attenuate the irinotecan-induced decrease in cell migration. The green tea extract and EGCG also had a dose-dependent inhibitory effect on irinotecan-induced secretion of interleukin-6 and interleukin-8 by oral epithelial cells. Lastly, the irinotecan-induced decrease in the secretion of MMP-2 and MMP-9 by oral epithelial cells was partially restored by the green tea extract and EGCG. ConclusionsThe green tea extract and EGCG, through anti-cytotoxic, anti-oxidative, and anti-inflammatory properties, may protect the oral mucosa against the deleterious effects of the chemotherapeutic agent irinotecan and may be of interest for treating oral mucositis.

Isolation, Characterization and Biosafety Evaluation of Lactobacillus Fermentum OK with Potential Oral Probiotic Properties.

It has been reported that certain probiotic bacteria have inhibitory effects against oral pathogens. Lactobacillus spp. have been studied and used as probiotics globally, but due to difficulties with laboratory cultivation and experimentation with oral microorganisms, there are few studies on Lactobacillus spp. isolated from the oral cavity being used against oral pathogens. The purpose of this study was to evaluate the biosafety and inhibitory effects of Lactobacillus fermentum OK as a potential oral biotherapeutic probiotic against oral pathogens. L. fermentum OK was evaluated based on microbial and genetic characteristics. A 5% dilution of L. fermentum OK culture supernatant showed that 60% inhibition against the growth of S. mutans and L. fermentum OK displayed significant inhibitory effects against the growth of Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus gordonii, and Streptococcus sanguinis. However, proliferation of L. fermentum OK, when co-cultured with harmful oral bacteria, was retarded. L. fermentum OK was shown to produce 1130μmol/L hydrogen peroxide, aggregate efficiently with Streptococcus sobrinus, S. gordonii, S. mutans, S. sanguinis, and P. gingivalis, and reduce S. mutans that produced artificial dental plaque by 97.9%. The in vitro cell adhesion capacity of L. fermentum OK to an oral epithelial cell line was 3.1 cells per cell and the cell adhesion of F. nucleatum and S. mutans decreased strongly in protection and displacement assays. L. fermentum OK was evaluated for safety using ammonia production, biogenic amine production, hemolytic property, mucin degradation testing, antibiotic susceptibility, and whole genome sequencing (WGS). Based on this study, L. fermentum OK appears to be a safe and bioactive lactobacterial food ingredient, starter culture, and/or probiotic microorganism for human oral health.