Alcohol consumption modulates Candida albicans-induced oral carcinogenesis and progression.

OBJECTIVES: This study aimed to determine the impact of low levels of alcohol consumption on the interaction of the oral cavity with Candida albicans, a species that is commonly found at higher levels in the oral cavities of regular alcohol consumers, patients with pre-malignant diseases, and patients with existing oral cancer (OC). METHODS: The gingival squamous cell carcinoma cell line, Ca9-22, was subjected to low-level ethanol exposure before co-culture with heat-inactivated C. albicans (HICA). We performed cell viability assays, measured reactive oxygen species, and used Western blot analysis for cell death markers to examine the effect of ethanol and HICA on cells. Scratch assays and anchorage-independent growth assays were used to determine cell behavioral changes. RESULTS: The results showed that ethanol in combination with HICA exacerbated cell death and cell cycle disruption, delayed NF-κB signaling, increased TIMP-2 secretion, and subsequently decreased MMP-2 secretion when compared to exposure to HICA alone. Conversely, both ethanol and HICA independently increased proliferation of Ca9-22 cells in scratch assays, and in combination, increased their capacity for anchorage-independent growth. CONCLUSION: Low levels of ethanol may provide protective effects against Candida-induced inflammatory oral carcinogenesis or OC progression.

The two faces of autophagy in oral squamous cell carcinoma.

OBJECTIVE: To undertake a comprehensive review of the current knowledge and understanding of autophagy in oral squamous cell carcinoma (OSCC), focusing on putative roles in tumour suppression and survival along with the influence of this cell death pathway on the development of resistance to chemotherapeutic treatment. DESIGN: Several well utilised databases (PubMed, Medline, Google Scholar) were searched for the relevant literature using terms and keywords including but not limited too; autophagy and cancer, autophagy and OSCC, tumour survival, autophagy and oral microbiome, autophagy immunogenicity, OSCC chemoresistance. RESULTS: Up-regulation of autophagy has been shown to promote tumour cell survival in the tumour microenvironment while in healthy cells, autophagy induction acts to prevent severe DNA mutations that can lead to cancer. Cancers utilise the autophagy pathway to promote survival during the stress of chemotherapeutic treatment and can induce resistance to chemotherapeutic drugs CONCLUSION: The ambiguous role of autophagy within cancers is still problematic in clinical fields. Within OSCC understanding whether autophagy plays a preventive or causative role is essential and may be beneficial in determining how modulation of this pathway may impact on OSSC and oral cancer patients.

Cisplatin induces autophagy-associated apoptosis in human oral squamous cell carcinoma (OSCC) mediated in part through reactive oxygen species.

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. Chemoresistance is a critical problem in OSCC leading to therapeutic failure and tumour recurrence. Recently, autophagy has acquired an emerging interest in cancer as it has been shown to be frequently activated in tumour cells treated with chemotherapeutics. Whether drug-induced autophagy represents a mechanism that allows cancer cells to survive or a pro-death mechanism associated with apoptosis remains controversial. This study evaluated the cellular response to cisplatin and the role of autophagy in mediating cisplatin resistance in OSCC cells. Our results demonstrated that cisplatin concurrently induced apoptosis and autophagy in OSCC cell lines partially through the ROS/JNK pathway. Moreover, inhibition of cisplatin-induced apoptosis abrogated autophagy, indicating a complex interplay between these pathways. Cisplatin-induced autophagy does not appear to elicit a pro-survival effect in OSCC as early-stage autophagy inhibition, using either a pharmacological inhibitor or knockdown of the key autophagy protein ATG5, did not sensitise cells to cisplatin. Additionally, autophagy did not play a role in acquired resistance to cisplatin in our novel cisplatin-resistant OSSC cell line (SCC-4cisR) obtained by pulsed stepwise exposure of SCC-4 cells to cisplatin (~14-fold change in sensitivity). There was no change in the basal levels of autophagy in the SCC-4cisR cells compared to the SCC-4 cells. Furthermore, a significant increase in cisplatin-induced autophagy was observed only in the SCC-4 cells, but not in the derived SCC-4cisR cells. Collectively, these data indicate that autophagy may not be implicated in acquired cisplatin resistance in OSCC.

The novel therapeutic potential of bovine α-lactalbumin made lethal to tumour cells (BALMET) and oleic acid in oral squamous cell carcinoma (OSCC).

BACKGROUND: Since the serendipitous discovery of bovine α-lactalbumin made lethal to tumour cells (BAMLET)/human α-lactalbumin made lethal to tumour cells there has been an increased interest in the ability of the two components, oleic acid and α-lactalbumin, to form anti-cancer complexes. Here we have investigated the in-vitro efficacy of the BAMLET complex in killing oral cancer (OC) cells, determined the active component of the complex and investigated possible biological mechanisms. MATERIALS AND METHODS: Two OC cell lines (±p53 mutation) and one dysplastic cell line were used as a model of progressive oral carcinogenesis. We performed cell viability assays with increasing BAMLET concentrations to determine the cytotoxic potential of the complex. We further analysed the individual components to determine their respective cytotoxicities. siRNA knockdown of p53 was used to determine its functional role in mediating sensitivity to BAMLET. Cell death mechanisms were investigated by flow cytometry, confocal microscopy and the lactate dehydrogenase assay. RESULTS: Our results show that BAMLET is cytotoxic to the OC and dysplastic cell lines in a time and dose-dependent manner. The cytotoxic component was found to be oleic acid, which, can induce cytotoxicity even when not in complex. Our results indicate that the mechanism of cytotoxicity occurs through multiple simultaneous events including cell cycle arrest, autophagy like processes with a minor involvement of necrosis. CONCLUSION: Deciphering the mechanism of cytotoxicity will aid treatment modalities for OC. This study highlights the potential of BAMLET as a novel therapeutic strategy in oral dysplastic and cancerous cells.

The interplay of the oral microbiome and alcohol consumption in oral squamous cell carcinomas.

Oral cancer (OC) is among the top twenty occurring cancers in the world, with a mortality rate of 50%. A shift to a functionally inflammatory or a 'disease state' oral microbiome composition has been observed amongst patients with premalignant disorders and OC, with evidence suggesting alcohol could be exacerbating the inflammatory influence of the oral microorganisms. Alcohol dehydrogenase (ADH, EC 1.1.1.1) converts alcohol into a known carcinogenic metabolite, acetaldehyde and while ADH levels in oral mucosa are low, several oral commensal species possess ADH and could produce genotoxic levels of acetaldehyde. With a direct association between oral microbiome status, alcohol and poor oral health status combining to induce chronic inflammation with increased acetaldehyde levels - this leads to a tumour promoting environment. This new disease state increases the production of reactive oxygen species (ROS), while impairing anti-oxidant systems thus activating the redox signalling required for the promotion and survival of tumours. This review aims to highlight the evidence linking these processes in the progression of oral cancer.

Iron chelators in cancer therapy.

Iron chelators have long been a target of interest as anticancer agents. Iron is an important cellular resource involved in cell replication, metabolism and growth. Iron metabolism is modulated in cancer cells reflecting their increased replicative demands. Originally, iron chelators were first developed for use in iron overload disorders, however, their potential as anticancer agents has been gaining increasing interest. This is due, in part, to the downstream effects of iron depletion such as the inhibition of proliferation through ribonucleotide reductase activity. Additionally, some chelators form redox active metal complexes with iron resulting in the production of reactive oxygen species and oxidative stress. Newer synthetic iron chelators such as Deferasirox, Triapine and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicrbazone (Dp44mt) have improved pharmacokinetic properties over the older chelator Deferoxamine. This review examines and discusses the various iron chelators that have been trialled for cancer therapy including both preclinical and clinical studies. The successes and shortcomings of each of the chelators and their use in combination therapies are highlighted and future potential in the cancer therapy world is considered.

Methylxanthines Inhibit Primary Amine Oxidase and Monoamine Oxidase Activities of Human Adipose Tissue.

Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects.

Can ethanol affect the cell structure? A dynamic molecular and Raman spectroscopy study.

The role that tobacco consumption plays in the etiology of oral cancer carcinogenesis, and of alcohol consumption acting as a co-factor, have been well established. However, in recent years, the contribution of alcohol consumption alone to oral cancer has been proposed. In fact, a high percentage of patients who develop oral cancer have both habits (tobacco and alcohol consumption), and other small patient groups only consume alcohol or do not have any other identifiable bad habits. In the present study we demonstrate, using a combination of dynamic molecular modelling and Raman spectroscopy, that ethanol has a significant effect on oral cells in vitro, mainly interacting with the lipids of the cell membrane, changing their conformation. Thus, it is possible to conclude that ethanol can affect the cell permeability, and by consequence serve as a possible trigger in oral carcinogenesis.

Salivary N-glycosylation as a biomarker of oral cancer: A pilot study.

Reliable biomarkers for oral cancer (OC) remain scarce, and routine tests for the detection of precancerous lesions are not routine in the clinical setting. This study addresses a current unmet need for more sensitive and quantitative tools for the management of OC. Whole saliva was used to identify and characterize the nature of glycans present in saliva and determine their potential as OC biomarkers. Proteins obtained from whole saliva were subjected to PNGase F enzymatic digestion. The resulting N-glycans were analyzed with weak anion exchange chromatography, exoglycosidase digestions coupled to ultra-high performance liquid chromatography and/or mass spectrometry. To determine N-glycan changes, 23 individuals with or without cancerous oral lesions were analyzed using Hydrophilic interaction ultra performance liquid chromatography (HILIC-UPLC), and peak-based area relative quantitation was performed. An abundant and complex salivary N-glycomic profile was identified. The main structures present in saliva were neutral oligosaccharides consisting of high mannose, hybrid and complex structures, followed by smaller fractions of mono and di-sialylated structures. To determine if differential N-glycosylation patterns distinguish between OC and control groups, Mann-Whitney testing and principle component analysis (PCA) were used. Eleven peaks were shown to be statistically significant (P ≤ 0.05), while PCA analysis showed segregation of the two groups based on their glycan profile. N-glycosylation changes are active in the oral carcinogenic process and may serve as biomarkers for early detection to reduce morbidity and mortality. Identifying which N-glycans contribute most in the carcinogenic process may lead to their use in the detection, prognosis and treatment of OC.

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents.

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors.

Raman spectroscopic analysis of oral cells in the high wavenumber region.

Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with submicrometer spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400-1800cm-1) has been shown to be very promising for optical biopsy purposes. However, limitations for discrimination of dysplastic and inflammatory processes based on the fingerprint region have been demonstrated. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2800-3600cm-1) provides more specific information based on NH, OH and CH vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the high-wavenumber spectral region in this context by collecting Raman spectra of nucleolus, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, in water immersion, which were then analyzed by principal components analysis as a method to discriminate the spectra. Analysis was performed before and after digital subtraction of the bulk water signal. In the normal cell line, the three subcellular regions are well differentiated before water subtraction, although the discrimination of the two nuclear regions is less well defined after water subtraction. Comparing the respective subcellular regions of the three cell lines, before water subtraction, the cell lines can be discriminated using sequential PCA and Feature Discriminant Analysis with up to ~100% sensitivity and 97% specificity for the cytoplasm, which is improved to 100% sensitivity and 99% specificity for the nucleus. The results are discussed in terms of discrimination comparing the CH vibrational modes of nucleic acids, proteins and lipids. The potential role of the OH vibrations, considering free water and confined water, in the discrimination of cell cultures and pathological processes are also discussed.

Oral cancer: Deregulated molecular events and their use as biomarkers.

Oral Cancer (OC) is a subset of head and neck cancer (HNC) with an annual worldwide incidence of 275,000 cases. OC remains a significant burden worldwide in terms of diagnosis, treatment and prognosis. Despite desirable outcomes in early diagnosed OCs and treatment advances most OCs are detected in advanced stages. The 5-year survival rate of early-stage disease is ∼80% and that of late-stage disease is only ∼20%. Recurrence and chemoresistance from a treatment point of view and pain and disfiguration are important factors contributing to the high morbidity and mortality of OC. Furthermore the process of oral carcinogenesis is complex and not yet fully understood. Consequently numerous potential biomarkers have been hypothesised though controversial results across the board hamper their clinical implementation. Of greatest advantage would be biomarkers signalling early events preceeding OC. Biomarker targets predominately involve deregulated molecular events that participate in cell signalling, growth, survival, motility, angiogenesis and cell cycle control but can also use changes in metabolic genes to discriminate healthy form disease state. Promising potential biomarkers include the growth signalling oncogenes, Epidermal Growth Factor Receptor and Cyclin D1, the anti-growth signalling components p53 and p21, apoptotic effectors such as Bcl-2 and also components involved in immortalisation, angiogenesis, invasion and metastasis processes. Translation of these potential biomakers to the patients is closer than ever though few issues remain to be resolved. Firstly large clinical trials are needed to validate their clinical applicability but also standardised methods of collection, storage and processing methods are needed to minimise variability.

Induction of apoptosis in oral squamous carcinoma cells by pyrrolo-1,5-benzoxazepines.

Oral cancer (OC) is a largely asymptomatic disease, resulting in one of the highest mortality rates of any cancer. OC is currently ranked as the sixth most common cancer in the world, according to a recent World Health Organization analysis, and its prevalence is increasing, both in western and developing regions. Depending on the stage of OC, treatment strategies include surgery, radiation therapy and chemotherapy, or a combination thereof. As with numerous other types of cancer, resistance to conventional chemotherapeutic drugs is increasing in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the use of a novel group of compounds, the pyrrolo­1,5­benzoxazepines (PBOXs), as a therapeutic alternative for the treatment of OC. PBOXs are microtubule­targeting agents that are able to induce apoptosis in numerous cancer cell types, thereby preventing tumour cell proliferation. Ca9.22 gingival and TR146 buccal cell lines were used as models for OSSC. Cell viability and proliferation in the presence of two PBOXs: PBOX­6 and PBOX­15, was monitored using an AlamarBlueTM assay. Flow cytometric analysis of propidium iodide­stained cells was used to determine the DNA content, and therefore the percentage of cells in each phase of the cell cycle. Microtubule disruption was determined by indirect immunofluorescence staining. Changes in protein expression and degradation were determined by western blotting. The results of the present study indicated that both PBOX­6 and ­15 were able to induce apoptotic cell death by disrupting the microtubule network in both cell lines. The EC50 values were subsequently calculated for both PBOX­6 and ­15, and PBOX­15 was shown to possess a higher potency. Both compounds displayed anti­proliferative effects mediated through sustained G2/M arrest accompanied by tubulin disruption, and a decrease in DNA repair protein poly (ADP ribose) polymerase expression. These findings suggest that PBOXs may prove useful, either alone or in combination with other agents, in the treatment of chemotherapeutic resistant OSCC.

Raman micro-spectroscopy for rapid screening of oral squamous cell carcinoma.

Raman spectroscopy can provide a molecular-level fingerprint of the biochemical composition and structure of cells with excellent spatial resolution and could be useful to monitor changes in composition for dysplasia and early, non-invasive cancer diagnosis (carcinoma in situ), both ex-vivo and in vivo. In this study, we demonstrate this potential by collecting Raman spectra of the nucleoli, nuclei and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (pre-cancerous, DOK) cell lines and from normal oral epithelial primary cell cultures, in vitro, which were then analysed by principal component analysis (PCA) as a multivariate statistical method to discriminate the spectra. Results show significant discrimination between cancer and normal cell lines. Furthermore, the dysplastic and cancer cell lines could be discriminated based on the spectral profiles of the cytoplasmic regions. The principal component loading plot, which elucidates the biochemical features responsible for the discrimination, showed significant contributions of nucleic acid and proteins for nucleolar and nuclear sites and variation in features of lipids for the cytoplasmic area. This technique may provide a rapid screening method and have potential use in the diagnosis of dysplasia and early, non-invasive oral cancer, the treatment of which involves much less extensive and complex surgery and a reduction in associated co-morbidity for the patient.