Melbourne Dental School - Research Publications
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ItemTranscriptional regulation of glucose transporters in human oral squamous cell carcinoma cells(WILEY, 2022-09)The increased glucose uptake observed in cancer cells is mediated by glucose transporters (GLUTs), a class of transmembrane proteins that facilitate the transport of glucose and other substrates across the plasma membrane. Despite the important role of glucose in the pathophysiology of oral squamous cell carcinoma (OSCC), there is very limited data regarding the expression of GLUTs in normal or malignant cells from the oral mucosa. We analysed the messenger RNA (mRNA) expression of all 14 GLUTs in two OSCC (H357/H400) and one non-malignant oral keratinocyte (OKF6) cell line using a quantitative polymerase chain reaction. GLUT expression was evaluated at baseline and after treatment with two specific GLUT inhibitors, namely, BAY876 (GLUT1) and WZB117 (GLUT1, GLUT3 and GLUT4). Here, we show that GLUT1, GLUT3, GLUT4, GLUT5, GLUT6, GLUT8, GLUT12 and GLUT13 transcripts were measurably expressed in all cell lines while GLUT2, GLUT7, GLUT9, GLUT11 and GLUT14 were not expressed. GLUT10 was only found in H357. In the presence of BAY876 and WZB117, OSCC cells exhibited significant alterations in the transcriptional profile of GLUTs. In particular, we observed distinct proliferation-dependent changes of mRNAs to GLUT1, GLUT3, GLUT4, GLUT5 and GLUT6 in response to selective GLUT inhibitors. In summary, we documented for the first time the expression of GLUT5, GLUT6 and GLUT12 in normal and malignant oral keratinocytes. Whilst regulation of GLUT transcripts was cell line and inhibitor specific, GLUT3 was consistently upregulated in actively proliferating OSCC cell lines, but not in OKF6, regardless of the inhibitor used, suggesting that modulation of this transporter may act as one of the primary compensation mechanisms for OSCC cells upon inhibition of glucose uptake.
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ItemMetabolomic Profile of Indonesian Betel Quids(MDPI, 2022-10)Consumption of areca nut alone, or in the form of betel quid (BQ), has negative health effects and is carcinogenic to humans. Indonesia is one of the largest producers of areca nuts worldwide, yet little is known about the biomolecular composition of Indonesian areca nuts and BQs. We have recently shown that phenolic and alkaloid content of Indonesian BQs exhibits distinct geographical differences. Here, we profiled for the first time the metabolomics of BQ constituents from four regions of Indonesia using non-targeted gas chromatography-mass spectrometry (GC-MS) analysis. In addition to well-known alkaloids, the analysis of small-molecule profiles tentatively identified 92 phytochemicals in BQ. These included mainly benzenoids and terpenes, as well as acids, aldehydes, alcohols, and esters. Safrole, a potentially genotoxic benzenoid, was found abundantly in betel (Piper betle) inflorescence from West Papua and was not detected in areca nut samples from any Indonesian region except West Papua. Terpenes were mostly detected in betel leaves and inflorescence/stem. Areca nut, husk, betel leaf, the inflorescence stem, and BQ mixture expressed distinctive metabolite patterns, and a significant variation in the content and concentration of metabolites was found across different geographical regions. In summary, this was the first metabolomic study of BQs using GC-MS. The results demonstrate that the molecular constituents of BQs vary geographically and suggest that the differential disease-inducing capacity of BQs may reflect their distinct chemical composition.
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ItemCharacterization of a novel dual murine model of chemotherapy-induced oral and intestinal mucositis(NATURE PORTFOLIO, 2023-01-25)Oral and intestinal mucositis are debilitating inflammatory diseases observed in cancer patients undergoing chemo-radiotherapy. These are devastating clinical conditions which often lead to treatment disruption affecting underlying malignancy management. Although alimentary tract mucositis involves the entire gastrointestinal tract, oral and intestinal mucositis are often studied independently utilizing distinct organ-specific pre-clinical models. This approach has however hindered the development of potentially effective whole-patient treatment strategies. We now characterize a murine model of alimentary tract mucositis using 5-Fluorouracil (5-FU). Mice were given 5-FU intravenously (50 mg/kg) or saline every 48 h for 2 weeks. Post initial injection, mice were monitored clinically for weight loss and diarrhea. The incidence and extent of oral mucositis was assessed macroscopically. Microscopical and histomorphometric analyses of the tongue and intestinal tissues were conducted at 3 interim time points during the experimental period. Repeated 5-FU treatment caused severe oral and intestinal atrophy, including morphological damage, accompanied by body weight loss and mild to moderate diarrhea in up to 77.8% of mice. Oral mucositis was clinically evident throughout the observation period in 88.98% of mice. Toluidine blue staining of the tongue revealed that the ulcer size peaked at day-14. In summary, we have developed a model reproducing the clinical and histologic features of both oral and intestinal mucositis, which may represent a useful in vivo pre-clinical model for the study of chemotherapy-induced alimentary tract mucositis and the development of preventative therapies.
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ItemAre There Betel Quid Mixtures Less Harmful than Others? A Scoping Review of the Association between Different Betel Quid Ingredients and the Risk of Oral Submucous Fibrosis(MDPI, 2022-05)Oral submucous fibrosis (OSF) is a potentially malignant condition of the oral cavity characterized by progressive fibrosis of the submucosal tissues. OSF is typically associated with the use of betel quid (BQ), a chewing package made of natural products (e.g., areca nut, betel leaves), with or without smokeless tobacco. BQ ingredients contain pro-carcinogenic bioactive compounds, but also potentially protective biomolecules, and we have shown recently that the chemical properties of different BQ recipes vary, which may explain the unequal prevalence of OSF and oral cancer in BQ users in different geographical regions. Hence, this scoping review was aimed at evaluating the existing literature regarding different BQ compounds and their association with OSF. The repository of the National Library of Medicine (MEDLINE/PubMed), medRxiv databases, Google scholar, Baidu scholar, CNKI, and EBSCO were used to search for publications that investigated the association between BQ chewing and OSF up to November 2021. The search terminology was constructed using the keywords "betel quid" and "oral submucous fibrosis", and their associated terms, with the use of Boolean operators. The search was conducted under Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines, together with clear inclusion and exclusion criteria. The review showed that the risk of developing OSF varied between different BQ recipes, and that chewing BQ mixtures containing betel inflorescence (BI) significantly increased the risk of OSF, as did the addition of tobacco. Conversely, the use of betel leaf in the mixture was likely to be protective, which may be due to the presence of polyphenols. Although further research is needed to determine the effect of individual BQ ingredients in the development of OSF, our pilot results provide the scope and rationale for informing future chemopreventive strategies for OSF and oral cancer in BQ chewers.
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ItemOral keratinocytes synthesize CTACK: A new insight into the pathophysiology of the oral mucosa(WILEY, 2018-02)The skin-associated chemokine CTACK plays a key role in many inflammatory conditions and could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP). In this study, we investigated, by RT-PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS), the production of CTACK in oral keratinocytes, its expression in tissues from normal and OLP patients, and its role in T-cell recruitment.CTACK was produced by the oral epithelium, and it affects chemotaxis of memory CLA+ cells to the oral epithelium. CTACK mRNA was expressed constitutively in primary oral epithelium and was increased during pro-inflammatory IFN-γ treatment. We found a constitutive production of CTACK at a protein level in oral primary cells that increased after IFN-γ treatment. Moreover, we confirmed that CTACK attracts memory T cells and those T cells that express CLA above the level of basal migration.
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ItemOral swirl samples - a robust source of microRNA protected by extracellular vesicles(WILEY, 2017-04)BACKGROUND: MicroRNAs are small non-coding RNAs which are dysregulated in disease states, such as oral cancer. Extracellular vesicles, a potential source of microRNA, are found in saliva. OBJECTIVE: To demonstrate that a quantifiable amount of microRNA can be isolated from oral swirl samples. Additionally, we hypothesized that extracellular vesicles may protect contained microRNA from degradation in these samples. METHOD: A polyethylene glycol-based precipitation was used for extracellular vesicle enrichment of oral swirl samples. Comparison was made between samples treated with and without RNase. Further, samples from three subjects were exposed to a range of conditions over 7 days and assessed for presence of microRNA by reverse-transcription quantitative PCR. Extracellular vesicles from samples were identified under transmission electron microscopy. RESULTS: An adequate quantity of microRNA for qPCR analysis was extractable from samples despite exposure to conditions under which degradation of RNA would be expected. CONCLUSION: A technique was developed to isolate an adequate quantity of microRNA for analysis from oral swirl samples. Extracellular vesicle-associated microRNA may be protected from degradation. This technique moves towards chairside application of translational microRNA research in the field of oral cancer prognostics.
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ItemAntimicrobial activity and regulation of CXCL9 and CXCL10 in oral keratinocytes(WILEY, 2016-10)Chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 are dysregulated in oral inflammatory conditions, and it is not known if these chemokines target microorganisms that form oral biofilm. The aim of this study was to investigate the antimicrobial activity of CXCL9 and CXCL10 on oral microflora and their expression profiles in oral keratinocytes following exposure to inflammatory and infectious stimuli. Streptococcus sanguinis was used as a model and Escherichia coli as a positive control. The antimicrobial effect of CXCL9/CXCL10 was tested using a radial diffusion assay. mRNA transcripts were isolated from lipopolysaccharide (LPS)-treated and untreated (control) oral keratinocyte cell lines at 2-, 4-, 6-, and 8-h time-points of culture. The CXCL9/10 expression profile in the presence or absence of interferon-γ (IFN-γ) was assessed using semiquantitative PCR. Although both chemokines demonstrated antimicrobial activity, CXCL9 was the most effective chemokine against both S. sanguinis and E coli. mRNA for CXCL10 was expressed in control cells and its production was enhanced at all time-points following stimulation with LPS. Conversely, CXCL9 mRNA was not expressed in control or LPS-stimulated cells. Finally, stimulation with IFN-γ enhanced basal expression of both CXCL9 and CXCL10 in oral keratinocytes. Chemokines derived from oral epithelium, particularly CXCL9, demonstrate antimicrobial properties. Bacterial and inflammatory-stimulated up-regulation of CXCL9/10 could represent a key element in oral bacterial colonization homeostasis and host-defense mechanisms.
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ItemThe Non-Conventional Effects of Glucocorticoids in Cancer(WILEY, 2016-11)
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ItemFunctional and molecular effects of a green tea constituent on oral cancer cells(WILEY, 2019-08)BACKGROUND: Green tea is heavily consumed on a global basis for its health benefits. The active ingredient, (-)-epigallocatechin gallate (EGCG), is a major polyphenol demonstrated to inhibit the growth of various non-oral cancer cell lines and interfere with the carcinogenic process, including downregulation of the epidermal growth factor receptor (EGFR). Our aim was to determine the phenotypic changes of oral cancer cells treated with EGCG and concurrently assess the effect on EGFR expression and activation. METHODS: Oral cancer cells (H400 and H357) were treated with 10 µg/mL and 20 µg/mL of EGCG for up to 72 hours. Phenotypic changes were assessed by performing cell proliferation analysis and cell migration (Transwell) assays. Expression of EGFR and its phosphorylated form (p-EGFR) was determined by Western blotting. RESULTS: Cell proliferation of both cell lines was significantly reduced at 48hrs when treated with 20 µg/mL EGCG. However, after 72 hours of treatment the effect of EGCG on cell proliferation ceased. Treatment of both cell lines with 10 µg/mL and 20 µg/mL of EGCG resulted in significant reduction in cell migration. Mechanistically, EGFR expression did not change significantly after treatment with EGCG; however, there was a reduction in its phosphorylated form. CONCLUSION: EGCG transiently inhibits both cell proliferation and migration of oral cavity cancer cells. This effect is associated with a decrease in the expression of phosphorylated EGFR. It is possible that more frequent bursts of EGCG could result in a persistent and sustained cancer inhibition, but this requires further research for clarification.
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ItemThe immunopathogenesis of oral lichen planus-Is there a role for mucosal associated invariant T cells?(WILEY, 2019-08)Oral lichen planus (OLP) is a chronic, T-cell-mediated, immune condition of unknown cause. OLP may present with painful symptoms requiring treatment, as well as lesions outside the oral cavity. It is likely that what initiates the OLP disease process is a complex interaction of host susceptibility and environmental triggers. While it is possible that OLP represents a true autoimmune condition against an epithelial autoantigen, the mechanisms that lead to this immune dysregulation are still poorly understood. In this review article, we discuss current concepts relating to the immunopathogenesis of OLP, as well as the potential contributory roles the oral microbiota and mucosal-associated invariant T (MAIT) cells.