Human Oral Keratinocyte Cell Research Articles

Stress hormones promote DNA damage in human oral keratinocytes

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

Low-frequency electromagnetic field influences human oral mucosa keratinocyte viability in response to lipopolysaccharide or minocycline treatment in cell culture conditions

The aim of the current study was to investigate the influence of low-frequency electromagnetic field (LF-EMF) exposure on viability parameters of oral mucosa keratinocytes cultured in in vitro conditions. The effect of LF-EMF stimulation on cell viability was also specified in the simultaneous presence of lipopolysaccharide (LPS) infectious agent or minocycline (Mino) anti-inflammatory agent. Viability parameters such as early-, late apoptosis and necrosis of keratinocytes were analysed by the flow cytometry method (FCM).The exposure of human oral keratinocyte cell cultures to LF-EMF acting alone or combined with LPS/minocycline agents caused changes in the percentage of cells that undergo programmed or incidental cell death. The overall obtained results are compiled in a graphical form presented in Fig. 1.

Hsa_circ_0002162 has a critical role in malignant progression of tongue squamous cell carcinoma through targeting miR-33a-5p.

The aim of this study was to explore the effect of hsa_circ_0002162 on regulating cell proliferation, apoptosis, and invasion, and investigate its potential target microRNA (miRNA) in tongue squamous cell carcinoma (TSCC). Hsa_circ_0002162 expression was detected in human TSCC cell lines and human oral keratinocytes (HOK) cell line. Cell proliferation, apoptosis, invasion, and candidate target miRNA expressions were detected in hsa_circ_0002162 knockdown-treated CAL-27 cells and hsa_circ_0002162 overexpression-treated SCC-9 cells. In the rescue experiment, miR-33a-5p knockdown plasmid was transfected into hsa_circ_0002162 knockdown-treated CAL-27 cells, while miR-33a-5p overexpression plasmid was transfected into hsa_circ_0002162 overexpression-treated SCC-9 cells. Subsequently, cell proliferation, apoptosis, and invasion were detected, and then luciferase reporter assay was performed. Hsa_circ_0002162 expression was increased in human TSCC cell lines SCC-9, CAL-27, HSC-4, and SCC-25 compared with HOK. In CAL-27 cells, hsa_circ_0002162 knockdown inhibited cell proliferation and invasion and promoted apoptosis. In SCC-9 cells, hsa_circ_0002162 overexpression enhanced cell proliferation and invasion and suppressed apoptosis. Furthermore, a negative regulation of hsa_circ_0002162 on miR-33a-5p (but not miR-302b-5p and miR-545-5p) was observed. In the rescue experiment, miR-33a-5p knockdown increased cell proliferation and invasion, and decreased apoptosis in hsa_circ_0002162 knockdown-treated CAL-27 cells, whereas miR-33a-5p overexpression decreased cell proliferation and invasion, but increased apoptosis in hsa_circ_0002162 overexpression-treated SCC-9 cells. The luciferase reporter assay showed the direct binding of hsa_circ_0002162 to miR-33a-5p. In conclusion, hsa_circ_0002162 had an important role in malignant progression of TSCC through targeting miR-33a-5p.

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

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

Effects of an elemental diet, Elental®, may differ between healthy oral cells and oral cancer cells.

The effectiveness of an elemental diet(ED), Elental®, against radiotherapy‑ or chemoradiotherapy‑induced oral mucositis was previously reported. However, the administration of additional nutrition or an ED in patients with oral cancer may also provide extra nutrition for cancer cells, which could result in cancer development. At present, it remains unclear whether the beneficial effects of an ED are likely to surpass its potential harmful effects on oral cancer treatment. In the present study, we aimed to clarify whether Elental® has different effects on a healthy human oral keratinocyte(HOK) cell line compared with its effects on oral squamous cell carcinoma (OSCC) cell lines (HSC2, HSC3, HSC4). The efficacy of Elental® was compared in relation to the growth and migration ability of HOK and OSCC cell lines using MTT assay and migration assay, respectively. In addition, whole transcriptome analysis and network analysis were performed to determine the difference in the mechanism of action of Elental® between HOK and HSC2 cells. In addition, Elental® promoted growth and migration ability of‑malnourished and 5‑fluorouracil(5‑FU)‑treated damaged HOK cells cultured in low nutrition medium (0% growth supplement). However, Elental® did not affect the growth ability of 5‑FU‑treated damaged HSC2 cell line in low nutrition medium (0 or 1% fetal bovine serum (FBS), as well as the growth ability of HSC3 and HSC4 cell lines in medium containing 0%FBS. Elental® pre‑treatment also enhanced the apoptosis‑inducing effect of anticancer agents against OSCC cells. In addition, whole transcriptome analysis and Ingenuity Pathways Analysis(IPA) data suggested that Elental® may help in the proliferation and survival of HOK through the induction of ERK. Moreover, Elental® added stress to HSC2 cells through the induction of the endoplasmic reticulum stress response marker, BiP and GRP94. The results showed that Elental® may add stress to HSC2 cells and provide growth stimulation to HOK. These findings suggest that the effects of Elental® on healthy oral cells and oral cancer cells may differ.

Exosomal lncRNA HEIH promotes cisplatin resistance in tongue squamous cell carcinoma via targeting miR-3619-5p/HDGF axis

BackgroundAccumulating evidence has suggested that long noncoding RNAs (lncRNAs) are involved in the progression of types of human cancers. It has been known that exosomes can mediate cell-cell crosstalk by transferring lncRNAs in tumor progression. This study aimed to investigate the role of exosomal lncRNA HEIH on cisplatin (DDP) resistance in tongue squamous cell carcinoma (TSCC). MethodsThe expression of HEIH in human oral keratinocytes cell line (HOK), DDP-sensitive TSCC cell line (SCC4/S) and DDP-resistant TSCC cell line (SCC4/DDP) was measured. SCC4/S and SCC4/DDP cells were transfected with sh-HEIH to examine TSCC cell proliferation and apoptosis. The DDP-resistant exosomes were extracted and identified. The expression of miR‑3619-5p and TDGF in DDP-sensitive recipient cells was determined. The binding capacity between HEIH and miR‑3619-5p, along with miR‑3619-5p and TDGF was verified. ResultsHEIH expression was significantly upregulated in SCC4/DDP cells. Downregulation of HEIH inhibited DDP resistance and cell proliferation and promoted cell apoptosis. HEIH acted as a competing endogenous RNA (ceRNA) for miR‑3619-5p to upregulate HDGF expression. Exosomal HEIH promoted cell proliferation and drug resistance and inhibited cell apoptosis by sponging miR‑3169-5p and upregulating HDGF. ConclusionExosomal HEIH acted as a ceRNA for miR‑3619-5p to upregulate HDGF, thereby promoting DDP resistance in TSCC cells.

Elemental diet directly affects chemotherapy-induced dermatitis and raw wound areas.

Elental® is an L-glutamine-rich elemental diet (ED) that has been widely used in Japan as a nutritional supplement for malnourished patients. In addition, Elental® has been successfully used in the management of chemotherapy-induced mucositis in cancer patients. Recently, it was also reported that Elental® can effectively reduce chemotherapy-induced oral mucositis in patients with oral squamous cell carcinoma, and can also reduce mucositis and dermatitis in animal models. However, it is unclear whether oral intake or topical application of Elental® can act directly on chemotherapy-induced oral mucositis or dermatitis. The aim of the present study was to investigate the possible direct healing effect of Elental® on chemotherapy-induced dermatitis and raw wound areas in a mouse model. Dermatitis and raw wounds were induced in nude mice by administration of 5-fluorouracil (5-FU) (via gastric tube) and mechanical injury (using a metal brush or a surgical knife). We then compared the outcome following oral or topical application of Elental® in these mice. The effect of Elental® on the growth and migration ability of the human oral keratinocyte cell line, HOK, was also examined using MTT and migration assays, respectively. In the mouse model, both oral administration and topical application of Elental® reduced 5-FU-induced dermatitis and healed raw wound areas more effectively compared with the topical application of saline. The MTT assay revealed that Elental® exerted a growth-promoting effect on HOKs. In addition, Elental® enhanced the ability of HOKs to migrate, as demonstrated by the migration assay. These findings demonstrated that the topical application as well as the oral intake of Elental® exerted a direct healing effect on chemotherapy-induced dermatitis or raw wound areas. The data also indicated that oral intake of an ED may exert a direct healing effect on chemotherapy-induced oral mucositis.

LncRNA MCM3AP-AS1 promotes the development of oral squamous cell carcinoma by inhibiting miR-363-5p.

The aim of the present study was to study the mechanism of the long non-coding (lnc)RNA MCM3AP-AS1 in the development of oral squamous cell carcinoma (OSCC). Patients with OSCC (n=36) volunteered to join the study, and their tumor/normal tissues were collected. MCM3AP-AS1 and microRNA (miR)-363-5p expression in tissues and cells was determined by reverse transcription-quantitative (RT-q)PCR. Following transfection, a CCK-8 assay and Transwell experiments were conducted to explore the effects of MCM3AP-AS1 on OSCC cell proliferation, migration and invasion. The interaction between MCM3AP-AS1 and miR-363-5p was detected by luciferase reporter gene assay. RT-qPCR analysis demonstrated significantly higher MCM3AP-AS1 expression in tumor tissues or OSCC cells compared with normal tissues or human oral keratinocytes cells (P<0.05). A high MCM3AP-AS1 level was associated with poor prognosis in OSCC patients (P<0.05 or P<0.01). Compared to the small interfering (si)-negative control (NC) group, OSCC cells of si-MCM3AP-AS1 group exhibited markedly lower optical density (at 450 nm) value and relative migration and invasion (P<0.05). miR-363-5p was directly inhibited by MCM3AP-AS1. OSCC cells of si-MCM3AP-AS1 + inhibitor-NC group exhibited clearly lower relative proliferation, migration and invasion compared with cells of si-NC + inhibitor-NC group and si-MCM3AP-AS1 + miR-363-5p inhibitor group (P<0.05). MCM3AP-AS1 promoted OSCC cells proliferation, migration and invasion by inhibiting miR-363-5p.

Differential Expression and Function of Bicellular Tight Junctions in Skin and Oral Wound Healing.

Bicellular tight junctions are multiprotein complexes that are required for maintenance of barrier function and fence function in epithelial tissues. Wound healing in the oral cavity leads to minimal scar formation compared to the skin, and the precise mechanisms for this regenerative response remain to be elucidated. We hypothesized that oral and skin tissues express a different tight junction repertoire both at baseline and during the wound healing response, and that these molecules may be critical to the differential repair between the two tissues. We re-analyzed a mouse skin and palate epithelium microarray dataset to identify the tight junction repertoire of these tissue types. We then re-analyzed a skin and tongue wound healing microarray dataset to see how expression levels of tight junction genes change over time in response to injury. We performed in vitro scratch assays on human oral and skin keratinocyte cell lines to assay for tight junction expression over time, tight junction expression in response to lipopolysaccharide and histamine treatment, and the effects of siRNA knockdown of claudin 1 or occludin on migration and proliferation. Our data showed that oral and skin epithelium expressed different tight junction genes at baseline and during the wound healing response. Knockdown of claudin 1 or occludin led to changes in proliferation and migration in human skin keratinocytes but not oral keratinocytes. Furthermore, we also showed that skin keratinocytes were more permeable than oral keratinocytes upon histamine treatment. In conclusion, this study highlights a specific subset of functional tight junction genes that are differentially expressed between the oral and skin tissues, which may contribute to the mechanisms leading to distinct healing phenotypes in response to injury in the two tissues.

Long non-coding RNA SAMMSON as a novel potential diagnostic and prognostic biomarker for oral squamous cell carcinoma

Background/purposeOral squamous cell carcinoma (OSCC) is one of the most lethal malignancies which accounts for approximately 90% of all malignant oral tumours. SAMMSON is a lncRNA located on chromosome 3p13–3p14 and is known to act as an oncogene in several malignancies. However, its expression and clinical significance in oral squamous cell carcinoma (OSCC) remain mostly unclear. In this study, we investigated the expression and clinical relevance of lncRNA SAMMSON in human OSCC.Materials and methodsHuman OSCC cell lines (Tca8113, SCC9, SCC25, CAL27, HN12, HSU3, FADU) and a human normal oral keratinocyte cell line (HNOK) were used to detect the difference of SAMMSON expression. A total of 90 OSCC patients confirmed by pathological and clinical diagnoses at the Hospital of Stomatology, Department of Periodontology, Shandong University were enrolled. The mRNA expression level was analyzed by reverse transcription PCR (QRT-PCR). Statistical analyses including Student's t-test, chi-square method, Kaplan-Meier method, Univariate and, Multivariate Cox regression analysis were performed to analyse all data.ResultsThis study showed that the expression of SAMMSON was significantly increased in OSCC tissues and cell lines. High SAMMSON expression was significantly associated with TMN stage, tumour differentiation, lymph node metastasis distant metastasis and neighboring tissue infiltration. Patients with high expression of SAMMSON had poor overall survival and disease-free survival compared to those with low levels. Cox regression analysis showed that SAMMSON could act as an independent prognostic factor in OSCC.ConclusionSerum SAMMSON expression was associated with tumour SAMMSON expression. ROC curve analysis indicated the high diagnostic sensitivity and specificity of serum SAMMSON expression in OSCC patients as compared to other traditional serum biomarker SCCA, TSGF, and CEA. These results indicated that SAMMSON might play an essential role in OSCC progression and could serve as a novel prognostic and diagnostic biomarker in OSCC.

Effects of denture adhesives and mouth moisturizers to human oral fibroblast and human keratinocyte cells using direct and indirect cell culture systems.

The purpose of this study was to evaluate the effects of commercialized denture adhesives and mouth moisturizers using direct and indirect cell cultures for in vitro examinations with human fibroblast and epithelial cells. Denture adhesives (Faston, Poligrip Powder, New Poligrip Free, Tafugurippu Kurimu, Polident Adhesive, Tafugurippu Tomei) and mouth moisturizers (Concool Mouth Gel, Biotene Oral Balance Gel) were subjected to live and dead detection and pH level determination. The mouth moisturizers showed higher cytotoxicity effects comparing with control on every cell cultures and cells, and pH level did not show any significant differences. However, there was no correlation of type of denture adhesive or mouth moisturizer with cytotoxicity. We concluded that cytotoxicity affects human cells regardless of type of material, though some dependence was noted.

Quercetin Inhibits Cell Survival and Metastatic Ability via the EMT-mediated Pathway in Oral Squamous Cell Carcinoma.

This study aimed to investigate whether quercetin exerts anticancer effects on oral squamous cell carcinoma (OSCC) cell lines and to elucidate its mechanism of action. These anticancer effects in OSCC cells were assessed using an MTT assay, flow cytometry (to assess the cell cycle), wound-healing assay, invasion assay, Western blot analysis, gelatin zymography, and immunofluorescence. To investigate whether quercetin also inhibits transforming growth factor β1 (TGF-β1)-induced epithelial–mesenchymal transition (EMT) in human keratinocyte cells, HaCaT cells were treated with TGF-β1. Overall, our results strongly suggest that quercetin suppressed the viability of OSCC cells by inducing cell cycle arrest at the G2/M phase. However, quercetin did not affect cell viability of human keratinocytes such as HaCaT (immortal keratinocyte) and nHOK (primary normal human oral keratinocyte) cells. Additionally, quercetin suppresses cell migration through EMT and matrix metalloproteinase (MMP) in OSCC cells and decreases TGF-β1-induced EMT in HaCaT cells. In conclusion, this study is the first, to our knowledge, to demonstrate that quercetin can inhibit the survival and metastatic ability of OSCC cells via the EMT-mediated pathway, specifically Slug. Quercetin may thus provide a novel pharmacological approach for the treatment of OSCCs.

Antibacterial Activity of Fruiting Body Extracts from Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes) against Oral Pathogen Streptococcus mutans.

The edible medicinal mushroom Flammulina velutipes (enokitake) has many applications as food and medicine, but its application in dentistry is unknown. This study aims to investigate the inhibitory effect of fruiting body extracts from F. velutipes on the growth and adhesion of Streptococcus mutans, the main cause of human caries, in vitro. Of the four extracts (named TG01 from water, TG02 from 95% ethanol, TG03 from 50% ethanol, and TG04 from ethyl acetate), TG03 had significant antibacterial activity (MIC = 10 mg/mL; MBC = 20 mg/mL). Planktonic growth and biofilm formation in S. mutans was repressed by TG03 at 5 mg/mL and above. Meanwhile, cytotoxicity analysis showed that TG03 was not toxic to human oral keratinocyte cells. HPLC-QQQ-MS analysis showed that the TG03 extract contained a large amount of arabitol, a sucrose substitute that reduces the development of caries. Thus, F. velutipes extracts can effectively inhibit the growth of the oral pathogen S. mutans without cytotoxicity against human oral keratinocytes. Therefore, F. velutipes is a good candidate for the development of oral hygiene agents to control dental caries.

Metal nanoparticles-induced activation of NLRP3 inflammasome in human oral keratinocytes is a possible mechanism of oral lichenoid lesions

The NLRP3 inflammasome has been implicated in the pathogenesis of various inflammatory diseases and is activated by particulate stimulants. Oral epithelial keratinocytes are frequently exposed to metal nanoparticles. In this study, we examined the effects of gold, silver, and palladium nanoparticles, which are frequently used for dental metal alloys on cell proliferation, cytotoxicity, autophagy, lysosomal functions, and NLRP3 inflammasome activation using the immortalized human oral keratinocyte cell line RT-7. The metal nanoparticles were agglomerated in the membrane vesicles in RT-7 cells and suppressed cell proliferation and increased lactate dehydrogenase activity as well as the proportion of apoptotic cells. Silver and palladium nanoparticles induced autophagy and lysosomal dysfunctions and all metal nanoparticles tested triggered the secretion of IL-1β through caspase-1 activation. Furthermore, the epithelium obtained from patients with oral lichenoid lesions (OLLs) had robust NLRP3, ASC, caspase-1, and IL-1β-positive keratinocytes and cDNA microarray showed significant elevation in the mRNA levels of NLRP3. These results suggest that internalized metal nanoparticles in oral mucosal epithelial cells activate the NLRP3 inflammasome through the induction of lysosomal damage and autophagy dysfunction. This process may be involved in the pathogenesis of OLL and suggest its potential as an alternative target for OLL therapy.

Proteome profiling to identify peroxiredoxin 1 interacting protein partners in nicotine-associated oral leukoplakia

ObjectiveTobacco smoking is one of the main risk factors for oral squamous cell carcinoma (OSCC) and can induce generation of reactive oxygen species (ROS). In our previous studies, we demonstrated that nicotine, the major ingredient in tobacco, can upregulate an important antioxidant enzyme Peroxiredoxin 1 (Prx1), in oral leukoplakia (OLK), an oral precancerous lesion. The underlying regulatory mechanisms, however, remain unclear. This study aims to identify regulatory mechanisms of nicotine and identify Prx1 interacting proteins in nicotine-associated OLK. DesignLiquid chromatography-tandem mass spectrometry (LC–MS/MS) combined with bioinformatics analysis was conducted to profile Prx1 binding proteins in human dysplastic oral keratinocyte (DOK) cells. Candidate interaction proteins were further verified using Co-immunoprecipitation (Co-IP), Western blot or Duolink assay in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK in mice and human OLK tissues. ResultsWe identified Thioredoxin (Trx), Nucleolar GTP-binding protein 1 (GTPBP4), GTP-binding protein Di-Ras2 (DIRAS2) and apoptosis signal-regulating kinase 1 (ASK1) as key Prx1 interacting proteins regulated by nicotine. Our data showed that nicotine upregulated Trx, GTPBP4, DIRAS2, and downregulated ASK1 in 4NQO-induced OLK in mice, at least in part dependent on Prx1. The modulations of Trx, GTPBP4, DIRAS2 and ASK1 by nicotine were also found in OLK smokers compared to OLK non-smokers. The in-situ interaction of Trx, GTPBP4, DIRAS2 and ASK1 with Prx1 were validated in human OLK tissues. ConclusionNicotine may promote OLK development via regulating Prx1 binding proteins Trx, GTPBP4, DIRAS2 and ASK1. The results of this study will help to develop therapeutic approaches for OLK in humans targeting Prx1 interacting protein network.

Regulation of IL-24 in human oral keratinocytes stimulated with Tannerella forsythia.

Interleukin-24 is a pleiotropic immunoregulatory cytokine and a member of the IL-20R subfamily of the IL-10 family. The aim of this study was to investigate the regulation of IL-24 in the human oral keratinocyte cell line HOK-16B following infection with Tannerella forsythia, a major periodontal pathogen. T. forsythia induced the expression of IL-24 mRNA and the secretion of glycosylated IL-24 in HOK-16B cells. Glycosylation of IL-24 is linked to its solubility and bioavailability. T. forsythia-stimulated reactive oxygen species (ROS) induced the expression of IL-24, which was regulated by IL-6. The ROS inhibitor N-acetylcysteine and MAPK inhibitors significantly reduced the expression of IL-6 and IL-24 induced by T. forsythia. Recombinant human IL-24 significantly enhanced the expression of IL-1α, IL-8, CXCL10, and MCP-1 in HOK-16B cells. Together, these results indicate that ROS, MAPKs, and IL-6 comprise the axis of IL-24 expression in HOK-16B cells stimulated with T. forsythia. Thus, IL-24 may be involved in inflammation in oral keratinocytes.

E-cigarette Aerosol Condensate Enhances Metabolism of Benzo(a)pyrene to Genotoxic Products, and Induces CYP1A1 and CYP1B1, Likely by Activation of the Aryl Hydrocarbon Receptor.

E-cigarette aerosol contains lower levels of most known carcinogens than tobacco smoke, but many users of e-cigarettes are also smokers, and these individuals may be vulnerable to possible promoting and/or cocarcinogenic effects of e-cigarettes. We investigated the possibility that a condensate of e-cigarette aerosol (EAC) enhances the metabolism of the tobacco carcinogen, benzo(a)pyrene (BaP), to genotoxic products in a human oral keratinocyte cell line. Cells were pretreated with EAC from two popular e-cigs and then with BaP. Metabolism to its ultimate carcinogenic metabolite, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro B[a]P (BPDE), was assayed by measuring isomers of its spontaneous hydrolysis products, BaP tetrols. The pretreatment of cells with EAC enhanced the rate of BaP tetrol formation several fold. Pretreatment with the e-liquid resulted in a smaller enhancement. The treatment of cells with EAC induced CYP1A1/1B1 mRNA and protein. The enhancement of BaP tetrol formation was inhibited by the aryl hydrocarbon receptor (AhR) inhibitor, α-napthoflavone, indicating EAC likely induces CYP1A1/1B1 and enhances BaP metabolism by activating the AhR. To our knowledge, this is first report demonstrating that e-cigarettes can potentiate the genotoxic effects of a tobacco smoke carcinogen.

Decreased expression of hsa_circ_0072387 as a valuable predictor for oral squamous cell carcinoma.

Increasing evidence points toward the key function of circular RNAs (circRNAs) in various carcinomas. This study aimed to identify aberrant expression of hsa_circ_0072387 in oral squamous cell carcinoma and probe its clinical significance. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to assess hsa_circ_0072387 expression levels in 63 paired OSCC tissues and three OSCC cell lines. The area under the receiver operator characteristic (ROC) curve was plotted to assess any potential clinical significance. Our data showed that hsa_circ_0072387 expression in OSCC was significantly downregulated compared with adjacent normal tissues (p<0.001). Compared to human normal oral keratinocyte cell, the levels of hsa_circ_0072387 were lower in three OSCC cell lines (SCC25, SCC15, CAL27). More significantly, hsa_circ_0072387 expression was associated with the TNM stage in OSCC (p=0.050). The area under the ROC curve reached up to 0.746. Based on bioinformatics, hsa-miR-129-3p, hsa-miR-141-3p, and hsa-miR-29-3p were predicted to be potential miRNAs binding with hsa_circ_0072387. Furthermore, hsa-miR-129-3p, hsa-miR-141-3p, and hsa-miR-29-3p were involved in multiple tumor-related signaling pathways. Our finding suggested that lower expression of has_circ_0072387 could be a key circRNA in OSCC and serve as a potential biomarker in OSCC diagnosis and therapeutic targets.

Role of mammary serine protease inhibitor on the inflammatory response in oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory condition with an unclear pathological mechanism. IκB kinase α (IKKα)-regulated mammary serine protease inhibitor (MASPIN) has been shown to mediate inflammation, particularly in cancers. Here, we explored the expression of MASPIN in OLP and its role in the inflammatory response. Immunohistochemistry staining and reverse transcription-polymerase chain reaction assays were used to detect the subcellular localization and expression of MASPIN and IKKα in OLP and healthy control tissues. Levels of the inflammatory factors were compared with enzyme-linked immunosorbent assays. MASPIN and IKKα were overexpressed and silenced, respectively, in an inflammation model of human oral keratinocytes (HOKs) stimulated with lipopolysaccharide (LPS). Mammary serine protease inhibitor expression was down-regulated, whereas IKKα expression was up-regulated in OLP tissues (p<0.01). The levels of tumour necrosis factor-alpha and interleukin-6 in OLP tissues were increased compared to those of healthy controls (p<0.01). MASPIN overexpression in LPS-stimulated HOK cells inhibited the levels of IKKα and the secretion of inflammatory cytokines. By contrast, IKKα silencing promoted the expression of MASPIN and inhibited the secretion of inflammatory cytokines. Both MASPIN and IKKα are involved in the inflammatory process of OLP, suggesting potential therapeutic targets.

Multiomic analysis of oral keratinocytes chronically exposed to shisha.

Tobacco is smoked in different form including cigarettes and water pipes. One popular form of water pipe smoking especially in Middle Eastern countries is shisha smoking. Shisha has been associated with various diseases including oral cancer. However, genomic alterations and gene expression changes associated with chronic shisha exposure have not been previously investigated. Whole-exome sequencing and gene expression profiling of immortalized human oral keratinocytes (OKF6/TERT1) cells chronically treated with 0.5% shisha extract for a period of 8months was undertaken to characterize molecular alterations associated with shisha exposure. Genomic DNA and RNA were extracted and preprocessed as per manufacturer's instruction and subjected to whole-exome and transcriptome sequencing using Illumina HiSeq2500 platform. Exome was analyzed using GATK pipeline whereas RNA-Seq data was analyzed using HiSat2 and HTSeq along with DESeq to elucidate differentially expressed genes. Whole-exome sequence analysis led to identification of 521 somatic missense variants corresponding to 389 genes RNA-Seq data revealed 247 differentially expressed genes (≥2-fold, P-value<0.01) in shisha treated cells compared to parental cells. Pathway analysis of differentially expressed genes revealed that interferon-signaling pathway was significantly affected. We predict activation of MAPK1 pathway which is known to play a key role in oral cancer. We also observed allele specific expression of mutant LIMA1 based on RNA-Seq dataset. Our findings provide insights into genomic alterations and gene expression pattern associated with oral keratinocytes chronically exposed to shisha.