Normal Oral Keratinocyte Cell Lines Research Articles

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

Evaluation of the Anti-Carcinogenic Effect of Centella Asiatica on Oral Cancer Cell Line: In vitro Study.

To evaluate the anti-carcinogenic effect of Centella Asiatica on to evaluate the Anti-Carcinogenic Effect of Centella Asiatica on Oral Cancer Cell Line oral cancer cell line. Oral Cancer cell line and normal oral keratinocyte cell line were procured.Centella asiatica extract was prepared. The cells were then subjected to the test herbal specimens -Centella asiatica extract in succeeding concentrations of 25 µg/ml, 50 µg/ml, 100 µg/ml at time intervals of 24,48 and 72 hours. Cisplatin (2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml) was used as a positive control. This experiment was done in triplets. The study revealed that the p values were less than 0.05 at concentration 12.5µg/ml, 25µg/ml, 50 µg/ml,100 µg/ml and time period of 24hrs,48hrs,72hrs, thus implying that at these concentrations and time period, the obtained data were statistically significant, thus indicating that there is a statistically significantly decreases in the viable cells as the concentration of the drug as a time period increases The results reveals that centella asiatica possess potential effect of anti-carcinogenic, effect when compared to positive control (Cisplatin). The current study reveals that Centella asiatica has an potential anti-carcinogenic effect on oral cancer cell line. So this can be used to treat oral cancer with minimal crippling as compared with allopathic drugs.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes.

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Kava constituents exert selective anticancer effects in oral squamous cell carcinoma cells in vitro

Kava is a beverage made from the ground roots of the plant Piper Methysticum. Active compounds of Kava have previously been demonstrated to exert an antiproliferative effect through cell cycle arrest and promotion of apoptosis. Our aim was to investigate the in vitro effects of the main constituents derived from Kava on oral squamous cell carcinoma (OSCC) activity.Gas chromatography mass spectrometry (GCMS) was used to characterise the main constituents of two Kava preparations. Cell proliferation was assessed in two human OSCC cell lines (H400 and BICR56) and in normal oral keratinocytes (OKF6) treated with the identified Kava constituents, namely Flavokawain A (FKA), Flavokawain B (FKB), yangonin, kavain and methysticin using an MTS in vitro assay. Cell migration at 16 h was assessed using a Transwell migration assay. Cell invasion was measured at 22 h using a Matrigel assay. Cell adhesion was assessed at 90 min with a Cytoselect Adhesion assay. The two Kava preparations contained substantially different concentrations of the main chemical constituents. Treatment of malignant and normal oral keratinocyte cell lines with three of the identified constituents, 10 μg/ml FKA, 2.5 μg/ml FKB and 10 μg/ml yangonin, showed a significant reduction in cell proliferation in both H400 and BICR56 cancer cell lines but not in normal OKF6 cells. Remarkably, the same Kava constituents induced a significant reduction of OSCC cell migration and invasion. We have demonstrated, for the first time, that Kava constituents, FKA, FKB and yangonin have potential anticancer effects on OSCC. This highlights an avenue for further research of Kava constituents in the development of future cancer therapies to prevent and treat OSCC.

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.

The Antiviral Agent Cidofovir Induces DNA Damage and Mitotic Catastrophe in HPV-Positive and -Negative Head and Neck Squamous Cell Carcinomas In Vitro.

Cidofovir (CDV) is an antiviral agent with antiproliferative properties. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) cell lines and whether it is caused by a difference in response to DNA damage. Upon CDV treatment of HNSCC and normal oral keratinocyte cell lines, we carried out MTT analysis (cell viability), flow cytometry (cell cycle analysis), (immuno) fluorescence and western blotting (DNA double strand breaks, DNA damage response, apoptosis and mitotic catastrophe). The growth of the cell lines was inhibited by CDV treatment and resulted in γ-H2AX accumulation and upregulation of DNA repair proteins. CDV did not activate apoptosis but induced S- and G2/M phase arrest. Phospho-Aurora Kinase immunostaining showed a decrease in the amount of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe. In conclusion, CDV inhibits cell growth in HPV-positive and -negative HNSCC cell lines and was more profound in the HPV-positive cell lines. CDV treated cells show accumulation of DNA DSBs and DNA damage response activation, but apoptosis does not seem to occur. Rather our data indicate the occurrence of mitotic catastrophe.

GALECTIN-1 INHIBITION OF ORAL CANCER IN VITRO

Galectin-1 is a carbohydrate-binding molecule that has been shown to be over-expressed in many types of cancer, including oral squamous cell carcinoma (OSCC). The higher the level of expression of galectin-1 by OSCC cells the greater the likelihood of invasion, distant metastasis and a poor survival rate. Objectives Investigation of the effect of galectin-1 in OSCC invasion, migration and epidermal-mesenchymal transition in vitro,and the effect of inhibition of galectin-1 using a small-molecule inhibitor (OTX008). Results One normal oral keratinocyte (NOK) cell line and three OSCC cell lines were cultured and the expression of galectin-1 protein in each quantified using an ELISA. All cell lines were found to express galectin-1, and one of the OSCC lines produced significantly more galectin-1 than the NOK cell line at 6, 24 and 48 hours. All four cell lines were cultured with three concentrations of galectin-1 (50, 100 and 150 ng/mL) and four concentrations of OTX008 (12.5, 25, 50 and 100 μg/mL), and cell viability was assayed at 24, 48, 72 and 96 hours. Galectin-1 decreased cell viability at 24 hours in two of the OSCC lines, had no effect on the third, and increased cell viability in the NOK cells at 72 hours. OTX008 reduced cell viability in a dose-dependent manner in all cell lines, and this effect increased at each time point during a 96 hour culture period. OTX008 had the least effect on cell viability of the OSCC line with the highest galectin-1 levels compared to the other cell lines. Conclusions Galectin-1 is expressed by NOK and OSCC cell lines in vitro. OTX008 decreases the cell viability of OSCC and NOK cells in a dose-dependent manner, however this effect is reduced by higher endogenous levels of galectin-1.

TUNICAMYCIN-INDUCED ENDOPLASMIC RETICULUM STRESS UP-REGULATES TUMOUR-PROMOTING CYTOKINES IN ORAL SQUAMOUS CELL CARCINOMA

Objectives Signal transducer and activator of transcription (STAT)-3 lies at the convergence point of key pathways involved in many malignancies including oral squamous cell carcinoma (OSCC). Endoplasmic reticulum stress (ERS) and the unfolded protein response promote either survival or apoptosis in different cancers. We investigated the expression of STAT3 pathway-related genes and proteins under ERS in OSCC. Three normal oral keratinocyte (NOK) and three OSCC cell lines were subjected to tunicamycin to induce ERS for 24 hours or to the vehicle medium as control. A pathway-focussed array was used to analyse the modulation of STAT3 pathway gene expression under ERS using qPCR. The expression of key regulated proteins was investigated in the cell lines using immunocytochemistry and in 76 OSCC and 9 normal oral mucosa (NOM) tissue samples using tissue microarray technology and immunohistochemistry. Findings ERS resulted in up-regulation of interleukin-6 receptor (IL6R) gene in NOK cell lines (p = 0.001) and IL5 (p = 0.005) and IL22 (p = 0.024) in OSCC cell lines. Greater STAT3 (p = 0.019) and leukaemia inhibitory factor receptor (p = 0.042) protein expression was observed in treated than untreated NOK cell lines. Conclusions The gene and protein regulation patterns show that ERS plays a role in modifying the tumour microenvironment in OSCC by up-regulating tumour-promoting cytokines.

Tunicamycin-induced endoplasmic reticulum stress up-regulates tumour-promoting cytokines in oral squamous cell carcinoma

AimsSignal transducer and activator of transcription (STAT)-3 lies at the convergence point of key pathways involved in many malignancies including oral squamous cell carcinoma (OSCC). Endoplasmic reticulum stress (ERS) and the unfolded protein response have been shown to be involved in the pathogenesis and progression of different cancers by influencing key cellular processes such as apoptosis. We investigated the differential expression of STAT3 pathway-related genes and proteins under ERS in OSCC. MethodsThree normal oral keratinocyte (NOK) and three OSCC cell lines were subjected to tunicamycin to induce ERS for 24 h or to the vehicle medium as control. A pathway-focussed array was used to analyse the modulation of STAT3 pathway gene expression under ERS using qPCR. The expression of key regulated proteins was investigated in the cell lines using immunocytochemistry and in 76 OSCC and 9 normal oral mucosa (NOM) tissue samples using tissue microarray technology and immunohistochemistry. ResultsERS resulted in up-regulation of interleukin-6 receptor (IL6R) gene in NOK cell lines (p = 0.001) and IL5 (p = 0.005) and IL22 (p = 0.024) in OSCC cell lines. Greater STAT3 (p = 0.019) and leukaemia inhibitory factor receptor (p = 0.042) protein expression was observed in treated than untreated NOK cell lines. ConclusionsThe gene and protein regulation patterns show that ERS plays a role in modifying the tumour microenvironment in OSCC by up-regulating tumour-promoting cytokines.

Abstract 95: Targeting BRD4 overcomes cetuximab resistance in HNSCC

Abstract Background: Nearly 600,000 people are diagnosed with head and neck cancer worldwide and 60% succumb to the disease within 5 years. The epidermal growth factor receptor (EGFR) is a major driver of HNSCC and in 2006 the EGFR monoclonal antibody cetuximab was FDA-approved for HNSCC treatment. However, cetuximab has not conferred significant long-term benefit compared to chemoradiation in patients with poorly differentiated recurrent-metastatic HNSCC. We and others have demonstrated that cetuximab treatment activates alternative receptor tyrosine kinases (RTKs) including Fibroblast Growth Factor (FGF) receptors, Met and Axl in HNSCC. To circumvent administration of multiple RTK inhibitors in combination with cetuximab, we sought to identify a common molecular target that regulates the expression of these RTKs. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of RTKs in breast cancer models. Hypothesis: We hypothesized that targeting BRD4 will overcome cetuximab resistance by depleting the expression of alternative RTKs in HNSCC. We further hypothesize that genetic and pharmacological targeting of BRD4 will synergize with cetuximab. Results: Using a phospho-RTK array, 72-hour cetuximab treatment increased or sustained phosphorylated levels of EGFR, HER2, HER3, MET, and AXL in several HNSCC cell lines. Treatment with the BRD4 inhibitor JQ1 abrogated both phosphorylated and total RTK abundance in the presence of cetuximab. Cetuximab and JQ1 robustly decreased phosphorylated Src and induced the senescence marker p21. Phenotypically, cetuximab and JQ1 significantly decreased survival and increased apoptosis in 6 HNSCC cell line models, while the normal oral keratinocyte cell line NOKsi had approximately 10-fold higher IC50s for the BRD4 inhibitors, JQ1 and I-BET762, relative to HNSCC cell lines. Importantly, two HNSCC models of acquired cetuximab resistance exhibited robust sensitivity to pharmacological (JQ1, IBET-762) and genetic (RNAi) BRD4 targeting strategies. Moreover, exogneous overexpression of different RTKs (HER3, ALK, and ROR1) resulted in cetuximab resistance that was reversed upon BRD4 targeting (RNAi and JQ1). Combination of cetuximab and JQ1 in co-culture experiments with T cells decreased the CD4+/CD25+ Treg population and PD-L1 expression on HNSCC cell lines. Further, preliminary findings indicate that JQ1 treatment prevents outgrowth of cetuximab-treated HNSCC patient-derived xenograft models. Conclusion: Our findings indicate that targeting BRD4 decreases the activation and expression of multiple RTKs that mediate resistance to the FDA-approved EGFR inhibitor cetuximab. Furthermore, BRD4 abrogates expression of immunosuppressive markers, making it a promising tumor intrinsic and extrinsic therapeutic strategy for HNSCC. Citation Format: Toni Brand, Yan Zeng, Brandon Leonard, Rachel O' Keefe, Hua Li, Daniel Johnson, Jennifer Grandis, Neil E. Bhola. Targeting BRD4 overcomes cetuximab resistance in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2017-95

A dual specificity kinase, DYRK1A, as a potential therapeutic target for head and neck squamous cell carcinoma.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Silencing of high-mobility group box 2 (HMGB2) modulates cisplatin and 5-fluorouracil sensitivity in head and neck squamous cell carcinoma.

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).

Cigarette smoke induces methylation of the tumor suppressor gene NISCH

We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (< 20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients’ plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.