Metabolomic analysis of human oral cancer cells with adenylate kinase 2 or phosphorylate glycerol kinase 1 inhibition.

The purpose of this study was to use liquid chromatography-mass spectrometry (LC-MS) with XCMS for a quantitative metabolomic analysis of UM1 and UM2 oral cancer cells after knockdown of metabolic enzyme adenylate kinase 2 (AK2) or phosphorylate glycerol kinase 1 (PGK1). UM1 and UM2 cells were initially transfected with AK2 siRNA, PGK1 siRNA or scrambled control siRNA, and then analyzed with LC-MS for metabolic profiles. XCMS analysis of the untargeted metabolomics data revealed a total of 3200-4700 metabolite features from the transfected UM1 or UM2 cancer cells and 369-585 significantly changed metabolites due to AK2 or PGK1 suppression. In addition, cluster analysis showed that a common group of metabolites were altered by AK2 knockdown or by PGK1 knockdown between the UM1 and UM2 cells. However, the set of significantly changed metabolites due to AK2 knockdown was found to be distinct from those significantly changed by PGK1 knockdown. Our study has demonstrated that LC-MS with XCMS is an efficient tool for metabolomic analysis of oral cancer cells, and knockdown of different genes results in distinct changes in metabolic phenotypes in oral cancer cells

Potential protein biomarkers for burning mouth syndrome discovered by quantitative proteomics.

Burning mouth syndrome (BMS) is a chronic pain disorder characterized by severe burning sensation in normal looking oral mucosa. Diagnosis of BMS remains to be a challenge to oral healthcare professionals because the method for definite diagnosis is still uncertain. In this study, a quantitative saliva proteomic analysis was performed in order to identify target proteins in BMS patients' saliva that may be used as biomarkers for simple, non-invasive detection of the disease. By using isobaric tags for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry to quantify 1130 saliva proteins between BMS patients and healthy control subjects, we found that 50 proteins were significantly changed in the BMS patients when compared to the healthy control subjects ( p ≤ 0.05, 39 up-regulated and 11 down-regulated). Four candidates, alpha-enolase, interleukin-18 (IL-18), kallikrein-13 (KLK13), and cathepsin G, were selected for further validation. Based on enzyme-linked immunosorbent assay measurements, three potential biomarkers, alpha-enolase, IL-18, and KLK13, were successfully validated. The fold changes for alpha-enolase, IL-18, and KLK13 were determined as 3.6, 2.9, and 2.2 (burning mouth syndrome vs. control), and corresponding receiver operating characteristic values were determined as 0.78, 0.83, and 0.68, respectively. Our findings indicate that testing of the identified protein biomarkers in saliva might be a valuable clinical tool for BMS detection. Further validation studies of the identified biomarkers or additional candidate biomarkers are needed to achieve a multi-marker prediction model for improved detection of BMS with high sensitivity and specificity

Characterization of Electronic Cigarette Aerosol and Its Induction of Oxidative Stress Response in Oral Keratinocytes.

In this study, we have generated and characterized Electronic Cigarette (EC) aerosols using a combination of advanced technologies. In the gas phase, the particle number concentration (PNC) of EC aerosols was found to be positively correlated with puff duration whereas the PNC and size distribution may vary with different flavors and nicotine strength. In the liquid phase (water or cell culture media), the size of EC nanoparticles appeared to be significantly larger than those in the gas phase, which might be due to aggregation of nanoparticles in the liquid phase. By using in vitro high-throughput cytotoxicity assays, we have demonstrated that EC aerosols significantly decrease intracellular levels of glutathione in NHOKs in a dose-dependent fashion resulting in cytotoxicity. These findings suggest that EC aerosols cause cytotoxicity to oral epithelial cells in vitro, and the underlying molecular mechanisms may be or at least partially due to oxidative stress induced by toxic substances (e.g., nanoparticles and chemicals) present in EC aerosols

E-cigarette aerosols induce unfolded protein response in normal human oral keratinocytes.

Objective: Since the introduction in 2004, global usage of e-cigarettes (ECs) has risen exponentially. However, the risks of ECs on oral health are uncertain. The purpose of this study is to understand if EC aerosol exposure impacts the gene pathways of normal human oral keratinocytes (NHOKs), particularly the unfolded protein response (UPR) pathway. Materials and methods: EC aerosols were generated reproducibly with a home-made puffing device and impinged into the culture medium for NHOKs. DNA microarrays were used to profile the gene expression changes in NHOKs treated with EC aerosols, and the Ingenuity Pathway Analysis (IPA) was used to reveal signaling pathways altered by the EC aerosols. Quantitative PCR was used to validate the expression changes of significantly altered genes. Results: DNA microarray profiling followed by IPA revealed a number of signaling pathways, such as UPR, cell cycle regulation, TGF-β signaling, NRF2-mediated oxidative stress response, PI3K/AKT signaling, NF-κB signaling, and HGF signaling, activated by EC aerosols in NHOKs. The UPR pathway genes, C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), X box binding protein 1 (XBP1), and inositol-requiring enzyme 1 alpha (IRE1α) were all significantly up-regulated in EC aerosol-treated NHOKs whereas immunoglobulin heavy-chain binding protein (BIP) and PRKR-like ER kinase (PERK) were slightly up-regulated. qPCR analysis results were found to be well correlated with those from the DNA microarray analysis. The most significantly changed genes in EC aerosol-treated NHOKs versus untreated NHOKs were CHOP, ATF4, XBP1, IRE1α and BIP. Meanwhile, Western blot analysis confirmed that CHOP, GRP78 (BIP), ATF4, IRE1α and XBP1s (spliced XBP1) were significantly up-regulated in NHOKs treated with EC aerosols. Conclusion: Our results indicate that EC aerosols up-regulate the UPR pathway genes in NHOKs, and the induction of UPR response is mediated by the PERK - EIF2α - ATF4 and IRE1α - XBP1 pathways

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

SOX11 Promotes Head and Neck Cancer Progression via the Regulation of SDCCAG8

The overall goal of this project is to gain insight into the role of the enhanced expression of SOX11, a member of the SOX transcription factor family, and SDCCAG8, a tumor antigen, in oral/head and neck cancer. We hypothesize that over-expression of SOX11, an embryonic development related gene, leads to an upregulation of SDCCAG8, promoting a malignant phenotype in oral/head and neck cancer. To test this hypothesis, we have first demonstrated that knockdown of SOX11 expression inhibits the proliferation, migration and invasion of oral/head and neck cancer cells. Next, we have confirmed that SOX11 binds to the promoter of SDCCAG8 by using ChIP and luciferase assays and proven that up-regulation (or down-regulation) of SOX11 induces (or inhibits) the expression of SDCCAG8 in oral/head and neck cancer cells. To further investigate the clinical significance of SDCCAG8 over-expression in oral/head and neck cancer, we have utilized the deep sequencing data from the TCGA database and performed a correlation analysis of SDCCAG8 gene expression with clinicopathological parameters of oral/head and neck cancer patients. The results show that high expression of SDCCAG8 is significantly associated with overall survival, tumor size and stage of the cancer patients. Taken together, our findings indicate that SDCCAG8 is a prognostic biomarker in oral/head and neck cancer and SOX11 may promote the progression of oral/head and neck cancer via the regulation of SDCCAG8

Mass Spectrometry-based Proteomic Analysis of Oral Cancer Cells

Mass spectrometry (MS), especially tandem mass spectrometry (MS/MS), is a powerful tool for proteomic and metabolomics applications. Untargeted metabolomics results can be well visualized and interpreted by using the cloud plot with XCMS Online software. The first objective of this study is to perform a comprehensive metabolomics analysis of oral cancer cells and identify metabolites altered by the knockdown of either adenylate kinase 2 (AK2) or phosphorylate glycerol kinase 1 (PGK1). UM1 and UM2 oral cancer cells were treated with siRNA to knockdown AK2 or PGK1. MS/MS and XCMS were performed to compare the metabolite profiles between the cells with siRNA knockdown and with scrambled siRNA control. Our studies confirmed the utility of XCMS to interpret the metabolomic results from oral cancer cells. When AK2 or PGK1 was knocked down in the UM1 or UM2 cells, more metabolites were found to be down-regulated than up-regulated. Heat map analysis indicates that a common group of metabolites were altered by AK2 knockdown between the UM1 and UM2 cells, and similar finding was observed for the PGK1 knockdown study. Tracer-based metabolomics, a subset of metabolomics with a labeled substrate, is a new platform that would help researchers understand the metabolic phenotype of cancer cells. The second objective of this study is to develop the novel methodology which combines the tracer-based metabolomics, immunoprecipitation (IP), and MS-based proteomics to detect the metabolic labeling of a specific protein from the entire protein complex in oral cancer cells. [U-13C6]-glucose was introduced into the UM1 and UM2 cells, and the labeled proteins were analyzed by liquid chromatography (LC) with MS/MS. We found that UM1 and UM2 cells displayed different types of 13C labeled peptide mass isotopomer distribution patterns. Mass isotopomer distribution pattern decayed faster and the intensities of each isotopic peak were lower for the UM2 cells than those for the UM1 cells. We also demonstrated that a specific labeled protein, e.g., 78kDa glucose-regulated protein (GRP 78), can be pulled down with IP and analyzed by LC-MS/MS. Our results indicated that the UM1 cells utilize more glucose than the UM2 cells possibly to maintain their invasive and metastatic phenotypes. Also, the methodologies were able to identify any single 13C-labeled protein from the whole cell lysate if antibody is commercially available. Therefore, using XCMS and our newly developed tracer-based metabolomics, we may have an improved understanding of the metabolic phenotype of oral cancer cells

SOX11 Promotes Head and Neck Cancer Progression via the Regulation of SDCCAG8

The overall goal of this project is to gain insight into the role of the enhanced expression of SOX11, a member of the SOX transcription factor family, and SDCCAG8, a tumor antigen, in oral/head and neck cancer. We hypothesize that over-expression of SOX11, an embryonic development related gene, leads to an upregulation of SDCCAG8, promoting a malignant phenotype in oral/head and neck cancer. To test this hypothesis, we have first demonstrated that knockdown of SOX11 expression inhibits the proliferation, migration and invasion of oral/head and neck cancer cells. Next, we have confirmed that SOX11 binds to the promoter of SDCCAG8 by using ChIP and luciferase assays and proven that up-regulation (or down-regulation) of SOX11 induces (or inhibits) the expression of SDCCAG8 in oral/head and neck cancer cells. To further investigate the clinical significance of SDCCAG8 over-expression in oral/head and neck cancer, we have utilized the deep sequencing data from the TCGA database and performed a correlation analysis of SDCCAG8 gene expression with clinicopathological parameters of oral/head and neck cancer patients. The results show that high expression of SDCCAG8 is significantly associated with overall survival, tumor size and stage of the cancer patients. Taken together, our findings indicate that SDCCAG8 is a prognostic biomarker in oral/head and neck cancer and SOX11 may promote the progression of oral/head and neck cancer via the regulation of SDCCAG8