Estimating Anticancer Effects of Yohimbine in DMBA-Induced Oral Carcinogenesis Hamster Model: Utilizing Biochemical and Immunohistochemical Techniques.

Yohimbine is a potent bioactive indole alkaloid, isolated from a variety of biological sources and has long been used as a natural stimulant and aphrodisiac, particularly to treat erectile dysfunction. However, some literature also points toward its anticancer effect in different experimental models. The current study aimed to address a clinical concern on the therapeutic utilization of yohimbine as a repurposed drug. We employed 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch carcinogenesis model juxtaposed with biochemical investigation of several detoxification and antioxidant markers, such as Cyt p450, Cyt b5, thiobarbituric acid reactive substance (TBARS), glutathione (GSH), glutathione reductase (GR), glutathione S transferase (GST), DT-diaphorase, vitamin C, vitamin E, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). The immunohistochemical assessment of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), proliferating cell nuclear antigen (PCNA), and cyclin D1 expression were also performed to observe the effect of yohimbine on these markers. The hamsters treated with DMBA presented the growth of tumors in the buccal pouches, accompanied by significant changes in the liver and buccal mucosa levels of Phase I & II detoxification enzymes and lipid peroxidation (LPO). A significant rise in the range of 2- to 3.5-fold was observed in Cyt p450, Cyt b5, and LPO in DMBA-treated animals. However, oral administration of yohimbine significantly restored the LPO, antioxidant, and detoxifying enzyme activities. Additionally, the levels of COX-2, IL-6, PCNA, and cyclin D1 were also found to be downregulated by yohimbine treatment. In conclusion, yohimbine improved the biochemical and immunohistochemical markers of DMBA-induced oral cancer and reverted to near normal values via ameliorating the underlying inflammation and oxidative stress conditions. Our study highlighted the potential of yohimbine as anticancer agent, especially against oral cancer and suggested its possible use as repurposed drug.

OSCC-derived EVs educate fibroblasts and remodel collagen landscape.

Cancer-associated myofibroblasts (mCAFs) represent a significant component of the tumor microenvironment due to their contributions to extracellular matrix (ECM) remodeling. The pro-tumor mechanisms of extracellular vesicles (EVs) by regulating mCAFs and related collagens remain poorly understood in oral squamous cell carcinoma (OSCC). In this study, through analysis of single-cell sequencing data and immunofluorescence staining, we confirmed the increased presence of mCAFs and enrichment of specific collagen types in OSCC tissues. Furthermore, we demonstrated that OSCC-derived EVs promote the transformation of fibroblasts into mCAFs, leading to tumor invasion. Proteomic analysis identified the presence of TGF-ß1 in EVs and revealed its role in inducing mCAFs via the TGF-ß1/Smad3 signaling pathway. Experiments in vivo confirmed that EVs, particularly those carrying TGF-ß1, trigger COL18high COL5high matrix deposition, thereby forming the pro-tumor ECM in OSCC. In summary, our investigation unveils the significant involvement of OSCC-derived EVs in orchestrating the differentiation of fibroblasts into mCAFs and modulating specific collagen types within the ECM. Therefore, this study provides a theoretical basis for targeting the EV-mediated TGF-ß1 signaling pathway as a potential therapeutic strategy for OSCC.

Decoding Oral Carcinogenesis and Tumor Progression in Whole Cigarette Smoke Exposure: A Systematic Review.

This systematic review aims to highlight the molecular mechanisms by which whole cigarette smoke affects oral carcinogenesis and its progression in human oral cells, based on evidence from original research articles published in the literature. A literature search was conducted using three databases: Web of Science, Scopus, and PubMed from May to June 2024. The articles were screened, and the data were extracted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines (2020). The included studies were subsequently evaluated using the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tool for bias factors. From the 14 included studies, two types of cell lines were frequently utilized: human oral mucosal epithelial cells or oral squamous cell carcinoma cells. In these cell lines, one of three forms of exposure was applied: cigarette smoke, its extract, or condensate. The mechanism of oral carcinogenesis and tumor progression includes aberrations in the heme metabolic pathway, modulation of miRNA-145, NOD1 and BiP expression, MMP-2, MMP-9, and cathepsin modulation, abnormal TSPO binding, RIP2-mediated NF-κB activation, MZF1-mediated VEGF binding, and activation of the RAGE signaling pathway. In conclusion, cigarette smoke significantly influences the development and progression of oral squamous cell carcinoma, based on the evidence highlighted in human oral cells. While previous studies have focused on specific carcinogens and pathways, this review added to our understanding of the overall impact of whole cigarette smoke on oral carcinogenesis at the molecular and cellular levels.

Oxidative stress promotes oral carcinogenesis via Thbs1-mediated M1-like tumor-associated macrophages polarization.

Although oxidative stress is closely associated with tumor invasion and metastasis, its' exact role and mechanism in the initial stage of oral cancer remain ambiguous. Glutamine uptake mediated by alanine-serine-cysteine transporter 2 (ASCT2) participates in glutathione synthesis to resolve oxidative stress. Currently, we firstly found that ASCT2 deletion caused oxidative stress in oral mucosa and promoted oral carcinogenesis induced by 4-Nitroquinoline-1-oxide (4-NQO) using transgenic mice of ASCT2 knockout in oral epithelium. Subsequently, we identified an upregulated gene Thbs1 linked to macrophage infiltration by mRNA sequencing and immunohistochemistry. Importantly, multiplex immunohistochemistry showed M1-like tumor-associated macrophages (TAMs) were enriched in cancerous area. Mechanically, targeted ASCT2 effectively curbed glutamine uptake and caused intracellular reactive oxygen species (ROS) accumulation, which upregulated Thbs1 in oral keratinocytes and then activated p38, Akt and SAPK/JNK signaling to polarize M1-like TAMs via exosome-transferred pathway. Moreover, we demonstrated M1-like TAMs promoted malignant progression of oral squamous cell carcinoma (OSCC) both in vitro and in vivo by a DOK transformed cell line induced by 4-NQO. All these results establish that oxidative stress triggered by ASCT2 deletion promotes oral carcinogenesis through Thbs1-mediated M1 polarization, and indicate that restore redox homeostasis is a new approach to prevent malignant progression of oral potentially malignant disorders.

Single-cell transcriptome dissecting the microenvironment remodeled by PD1 blockade combined with photodynamic therapy in a mouse model of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) stands as a predominant and perilous malignant neoplasm globally, with the majority of cases originating from oral potential malignant disorders (OPMDs). Despite this, effective strategies to impede the progression of OPMDs to OSCC remain elusive. In this study, we established mouse models of oral carcinogenesis via 4-nitroquinoline 1-oxide induction, mirroring the sequential transformation from normal oral mucosa to OPMDs, culminating in OSCC development. By intervening during the OPMDs stage, we observed that combining PD1 blockade with photodynamic therapy (PDT) significantly mitigated oral carcinogenesis progression. Single-cell transcriptomic sequencing unveiled microenvironmental dysregulation occurring predominantly from OPMDs to OSCC stages, fostering a tumor-promoting milieu characterized by increased Treg proportion, heightened S100A8 expression, and decreased Fib_Igfbp5 (a specific fibroblast subtype) proportion, among others. Notably, intervening with PD1 blockade and PDT during the OPMDs stage hindered the formation of the tumor-promoting microenvironment, resulting in decreased Treg proportion, reduced S100A8 expression, and increased Fib_Igfbp5 proportion. Moreover, combination therapy elicited a more robust treatment-associated immune response compared with monotherapy. In essence, our findings present a novel strategy for curtailing the progression of oral carcinogenesis.

4-nitroquinoline 1-oxide-induced oral epithelial lesions exhibit time- and stage-dependent changes in the tumor immune microenvironment.

Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Mass cytometry and transcriptomic profiling reveal PD1 blockade induced alterations in oral carcinogenesis.

Oral squamous cell carcinoma is the predominant subtype of head and neck squamous cell carcinoma, characterized by a challenging prognosis. In this study, we established a murine model of oral carcinogenesis using 4-nitroquinoline-1-oxide (4-NQO) induction to investigate the impact of immunotherapy on microenvironmental alterations. Mice in the precancerous condition were randomly divided into two groups: one receiving programmed death-1 (PD1) monoclonal antibody treatment and the other, control immunoglobulin G. Our observations showed that while PD1 blockade effectively delayed the progression of carcinogenesis, it did not completely impede or reverse it. To unravel the underlying reasons for the limited effectiveness of PD1 blockade, we collected tongue lesions and applied mass cytometry (CyTOF) and RNA sequencing (RNA-seq) to characterize the microenvironment. CyTOF analysis revealed an increased macrophage subset (expressing high levels of IFNγ and iNOS) alongside a diminished Th1-like subset (exhibiting low expression of TCF7) and three myeloid-derived suppressor cell subsets (displaying low expression of MHC Class II or IFNγ) following anti-PD1 treatment. Notably, we observed an increased presence of cancer-associated fibroblasts (CAFs) expressing collagen-related genes after PD1 blockade. Furthermore, we found a negative correlation between the infiltration levels of CAFs and CD8+ T cells. These findings were validated in murine tongue tissue slides, and publicly available multi-omics datasets. Our results suggest that CAFs may impair the therapeutic efficacy of PD1 blockade in oral carcinogenesis by the remodeling of the extracellular matrix.

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.

Glucose upregulates amphiregulin in oral dysplastic keratinocytes: A potential role in diabetes-associated oral carcinogenesis.

BACKGROUND: Compelling evidence implicates diabetes-associated hyperglycemia as a promoter of tumor progression in oral potentially malignant disorders (OPMD). Yet, information on hyperglycemia-induced cell signaling networks in oral oncology remains limited. Our group recently reported that glucose-rich conditions significantly enhance oral dysplastic keratinocyte viability and migration through epidermal growth factor receptor (EGFR) activation, a pathway strongly linked to oral carcinogenesis. Here, we investigated the basal metabolic phenotype in these cells and whether specific glucose-responsive EGFR ligands mediate these responses. METHODS: Cell energy phenotype and lactate concentration were evaluated via commercially available assays. EGFR ligands in response to normal (5 mM) or high (20 mM) glucose were analyzed by quantitative real-time PCR, ELISA, and western blotting. Cell viability and migration assays were performed in the presence of pharmacological inhibitors or RNA interference. RESULTS: When compared to normal keratinocytes, basal glycolysis in oral dysplastic keratinocytes was significantly elevated. In highly glycolytic cells, high glucose-activated EGFR increasing viability and migration. Notably, we identified amphiregulin (AREG) as the predominant glucose-induced EGFR ligand. Indeed, enhanced cell migration in response to high glucose was blunted by EGFR inhibitor cetuximab and AREG siRNA. Conversely, AREG treatment under normal glucose conditions significantly increased cell viability, migration, lactate levels, and expression of glycolytic marker pyruvate kinase M2. CONCLUSION: These novel findings point to AREG as a potential high glucose-induced EGFR activating ligand in highly glycolytic oral dysplastic keratinocytes. Future studies are warranted to gain more insight into the role of AREG in hyperglycemia-associated OPMD tumor progression.

Vitamin D in the Prevention and Treatment of Oral Cancer: A Scoping Review.

Introduction: Oral cancer is a serious health problem with an increasing incidence worldwide. Researchers have studied the potential anti-cancerous action of vitamin D and its association with several cancers including oral cancer. The purpose of this scoping review is to synthesize the existing literature on the role of vitamin D on oral cancer. Methods: A scoping review of the literature was conducted using the framework developed by Arkey and O'Malley and the PRISMA-ScR guidelines. Nine databases were searched for peer-reviewed human studies published in English that either investigated the association of vitamin D with, or its impact on either the prevention or treatment of oral cancer. The authors then extracted data using a predefined form to summarize information about article type, study design, participant characteristics, interventions, and outcomes. Results: Fifteen articles met the review criteria. Among the 15 studies, 11 were case-control, 3 were cohort studies, and 1 was a clinical trial. In four studies, the evidence supported a preventive action of vitamin D against oral cancer and a reduction in the negative side effects associated with chemo- and radiotherapy. Several studies that focused on genetic polymorphisms and the expression of the 1,25 dihydroxyvitamin D3 receptor (VDR) suggested significant associations with vitamin D and increased oral cancer risk and worse survival rates. In contrast, two studies did not reveal a strong association between vitamin D and oral cancer. Conclusions: The current evidence suggests an association between vitamin D deficiency and an increased risk of oral cancer. VDR gene polymorphisms might also be a part of future preventive and therapeutic strategies against oral cancer. Carefully designed studies are required to explore and define what role, if any, vitamin D might play in the prevention and treatment of oral cancer.

PD-1 Carried on Small Extracellular Vesicles Leads to OSCC Metastasis.

Immune checkpoint molecule PD-1, expressed on the cell surface, impairs antigen-driven activation of T cells and thus plays a critical role in tumorigenesis, progression, and the poor prognosis of oral squamous cell carcinoma (OSCC). In addition, increasing evidence indicates that PD-1 carried on small extracellular vesicles (sEVs) also mediates tumor immunity, although their contributions to OSCC are yet unclear. Here, we investigated the biological functions of sEV PD-1 in patients with OSCC. The cell cycle, proliferation, apoptosis, migration, and invasion of CAL27 cell lines treated with or without sEV PD-1 were examined in vitro. We performed mass spectrometry to investigate the underlying biological process, combined with an immunohistochemical study of SCC7-bearing mice models and OSCC patient samples. In vitro data demonstrated that sEV PD-1 induced senescence and subsequent epithelial-mesenchymal transition (EMT) in CAL27 cells by ligating with tumor cell surface PD-L1 and activating the p38 mitogen-activated protein kinase (MAPK) pathway. Comprehensive immunohistochemical analysis of the xenograft mice models and OSCC patient samples revealed a very close correlation between the level of circulating sEV PD-1 and lymph node metastasis. These results demonstrate that circulating sEV PD-1 triggers senescence-initiated EMT in a PD-L1-p38 MAPK-dependent manner, contributing to tumor metastasis. It also suggests that the inhibition of sEV PD-1 may be a promising therapeutic target for the treatment of OSCC.

TWIST1 regulates proliferation, migration, and invasion and is a prognostic marker for oral tongue squamous cell carcinoma.

BACKGROUND: Epithelial-mesenchymal transition is one of the main mechanisms for tumor progression and metastasis. Transcription factors such as TWIST1 are key regulators of the epithelial-mesenchymal transition and are regarded as potential therapeutic targets for the treatment of cancer. The purpose of this study was to examine TWIST1 as a possible epithelial-mesenchymal transition-related prognostic biomarker in oral epithelial dysplasia and oral tongue squamous cell carcinomas, as well as the biological behavior of TWIST1-silencing in oral tongue squamous cell carcinomas cell lines. METHODS: Immunohistochemical analysis of TWIST1, E-cadherin, and N-cadherin was carried out in 47 samples representing oral epithelial dysplasia and 41 oral tongue squamous cell carcinomas. The suppression of TWIST1 expression was performed using shRNA-expression vectors in HSC-3 and SCC-9 cells to investigate in vitro the impact of TWIST1 on proliferation, apoptosis, viability, migration, and invasion of SCC-9 and HSC-3 cells. RESULTS: The expression of nuclear TWIST1 was significantly higher in oral tongue squamous cell carcinomas than in oral epithelial dysplasis (p < 0.0001), whereas TWIST1 in the cytoplasm was more expressed in oral epithelial dysplasis (p = 0.012). The high cytoplasmic expression of TWIST1 was significantly associated with shortened overall survival (p < 0.05), and increased nuclear TWIST1 expression was significantly related to high risk of recurrence (p = 0.03). Knockdown of TWIST1 in oral tongue squamous cell carcinomas cells induced the expression of E-cadherin and inhibited N-cadherin, which were followed by decreased proliferation, migration, and invasion. CONCLUSIONS: Our research suggests that TWIST1 is linked to the development of oral tongue carcinogenesis and may be used as a prognostic indicator and therapeutic target for oral tongue squamous cell carcinomas patients.

Role of Candida albicans in Oral Carcinogenesis.

Oral carcinogenesis is also dependent on the balance of the oral microbiota. Candida albicans is a member oral microbiota that acts as an opportunistic pathogen along with changes in the epithelium that can predispose to premalignancy and/or malignancy. This systematic review uses the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines to analyze the role of Candida albicans in the process of oral carcinogenesis. Eleven articles qualified inclusion criteria, matched keywords, and provided adequate information about the carcinogenesis parameters of Candida albicans in oral cancer. Candida albicans in oral carcinogenesis can be seen as significant virulent factors for patients with oral squamous cell carcinoma (OSCC) or potentially malignant disorder (OPMD) with normal adjacent mucosa. Candida albicans have a role in the process of oral carcinogenesis concerning morphological phenotype changes in cell structure and genotype and contribute to the formation of carcinogenic substances that can affect cell development towards malignancy.

Laser photobiomodulation does not alter clinical and histological characteristics of 4-NQO-induced oral carcinomas and leukoplakia in mice.

OBJECTIVE: This study evaluated the effect of laser photobiomodulation (PBM) on oral leukoplakia and squamous cell carcinomas (OSCC) in a model of oral carcinogenesis. MATERIALS AND METHODS: Forty-one C57Bl/6 female mice were distributed in control group, 4-NQO group, Laser group 1.5 J and Laser group 9 J. Oral cancer was induced on the tongue by nitroquinoline oxide (4-NQO), diluted in the water for 16 weeks. In the 18th and 19th weeks, PBM with a diode laser, 0.028 cm2 spot size, continuous emission mode, 660 nm wavelength was applied on the tongue of animals for seven sessions. Laser group 1.5 J received 30 mW power and 1.5 J energy. In the Laser group 9 J, 100 mW power, and 9 J energy were applied. In the 20th week the animals were euthanized. RESULTS: All animals exposed to carcinogen developed clinical and histological alterations such as leukoplakia and OSCC on the tongue. There was no significant difference among Laser groups 1.5 and 9 J and 4-NQO group (not irradiated) regarding the area of leukoplakia and carcinomas (P > 0.05) or thickness of epithelial tissue and keratin (P > 0.05). There were also no association between PBM and histologic classification of the lesions (P = 0.87), frequency of OSCC (P = 0.57), grade of tumor differentiation (P = 0.88) or depth of invasion (P = 0.45). CONCLUSION: Laser PBM, in both parameters used, does not influence on clinical and histological characteristics of oral leukoplakia and OSCC. CLINICAL RELEVANCE: Results suggest that PBM may be a safe treatment for adverse effects of antineoplastic therapies in patients with leukoplakia and OSCC.

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats.

BACKGROUND: On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose transporters (GLUT1/GLUT3/GLUT4), if any, in such putative association. METHODS AND RESULTS: Oral carcinogenesis was induced by 4-nitroquinoline N-oxide in 10 non-diabetic and 10 diabetic rats; 8 non-diabetic and 7 diabetic rats served as controls. Expressions of selected GLUTs at mRNA and protein levels were analyzed in oral tissue (normal/lesion) by quantitative real-time PCR and immunohistochemistry respectively. Premalignant lesions (hyperplasia/dysplasia/carcinoma-in-situ) appeared on tongues of carcinogen-treated animals. Significant increase of GLUT1mRNA level was seen from normal to lesion tongues, along increasing lesion grades (from hyperplasia/mild dysplasia to moderate/severe dysplasia) and in lesions induced under hyperglycemic condition than that induced under normoglycemic one; a similar trend was found in transcript variant-1 of GLUT1, but not in variant-2. GLUT3 and GLUT4 mRNA levels were comparable among lesions irrespective of grades and glycemic status. Concordant to mRNA level, overall expression of GLUT1 protein was higher in tongue lesions in presence of hyperglycemia than in absence of such condition; non-lesion portions of tongues exposed to carcinogen showed a similar trend. Moreover in carcinogen-treated groups, non-lesion and lesion portions of tongues under hyperglycemic condition showed predominantly membranous expression for GLUT1 which was again significantly higher than equivalent portions of tongue under normoglycemic condition. CONCLUSION: Hyperglycemia seemed to favor GLUT1 over-expression and membrane localization of the protein during oral carcinogenesis. GLUT1 transcript variant-1 appeared to be more important than variant-2 in disease pathogenesis.

Breast cancer susceptibility gene 1 regulates oxidative damage via nuclear factor erythroid 2-related factor 2 in oral cancer cells.

OBJECTIVE: To determine whether the breast cancer susceptibility gene 1 (BRCA1) regulates oxidative damage in oral cancer cells by interacting with nuclear factor erythroid 2-like 2 (NRF2). DESIGN: The BRCA1 gene was silenced in CAL-27 and DOK cells using specific shRNA, and NRF2 was activated with sulforaphane. The expression levels of BRCA1, NRF2 and its target genes were assessed by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8 assay was used to detect cell proliferation, apoptosis was detected by flow cytometry, and 8-OXo-2'-deoxyguanosine level was measured by enzyme-linked immunosorbent assay. The expression of BRCA1 and NRF2 in patients with oral leukoplakia and oral squamous cell carcinoma were evaluated by immunohistochemistry. RESULTS: BRCA1 knockdown downregulated NRF2 and its target genes, increased proliferation rates, reduced apoptosis, and increased 8-OXo-2'-deoxyguanosine levels compared to the control. Activation of NRF2 by sulforaphane significantly upregulated NRF2 levels in the BRCA1-depleted cells, and restored proliferation, apoptosis and 8-OXo-2'-deoxyguanosine level in a dose-dependent manner. Compared with patients with leukoplakia, BRCA1 and NRF2 expression were increased in patients with oral squamous cell carcinoma. CONCLUSIONS: BRCA1 depletion increases oxidative damage and promotes the malignant phenotype, which may eventually promote oral carcinogenesis. The NRF2-activator sulforaphane is a potential chemo-preventive agent for oral cancer.

Anticancer and antioxidant profiling effects of Nerolidol against DMBA induced oral experimental carcinogenesis.

The objective of this study is to examine the chemopreventive effects of Nerolidol (NER) on hamster buccal pouch carcinogenesis (HBC) induced by 7,12-dimethylbenz(a)anthracene (DMBA) in male golden Syrian hamsters. In this study, oral squamous cell carcinoma was developed in the buccal pouch of an oral painted hamster with 0.5% DMBA in liquid paraffin three times weekly for 12 weeks. To assess DMBA-induced hamster buccal tissue carcinogenesis, biochemical endpoints such as Phase I and II detoxification enzymes, antioxidants, lipid peroxidation (LPO) by-products, and renal function markers, as well as histopathological examinations, were used. Furthermore, the immunohistochemical studies of interleukin-6 were investigated to find the inflammatory link in the HBC carcinogenesis. In our results, DMBA alone exposed hamsters showed 100% tumor growth, altered levels of antioxidants, detoxification agents, LPO, and renal function identifiers as compared to the control hamsters. The outcome in  present biochemical, histopathological, and immunohistochemistry studies has been found a reverse in NER-treated hamsters against the tumor. This study concluded that NER modulated the biochemical profiles (antioxidants, detoxification, LPO, and renal function markers) and inhibited tumor development in DMBA induced oral carcinogenesis.

Molecular Mechanisms of Malignant Transformation of Oral Submucous Fibrosis by Different Betel Quid Constituents-Does Fibroblast Senescence Play a Role?

Betel quid (BQ) is a package of mixed constituents that is chewed by more than 600 million people worldwide, particularly in Asia. The formulation of BQ depends on a variety of factors but typically includes areca nut, betel leaf, and slaked lime and may or may not contain tobacco. BQ chewing is strongly associated with the development of potentially malignant and malignant diseases of the mouth such as oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively. We have shown recently that the constituents of BQ vary geographically and that the capacity to induce disease reflects the distinct chemical composition of the BQ. In this review, we examined the diverse chemical constituents of BQ and their putative role in oral carcinogenesis. Four major areca alkaloids-arecoline, arecaidine, guvacoline and guvacine-together with the polyphenols, were identified as being potentially involved in oral carcinogenesis. Further, we propose that fibroblast senescence, which is induced by certain BQ components, may be a key driver of tumour progression in OSMF and OSCC. Our study emphasizes that the characterization of the detrimental or protective effects of specific BQ ingredients may facilitate the development of targeted BQ formulations to prevent and/or treat potentially malignant oral disorders and oral cancer in BQ users.

Identification of a Gene-Expression-Based Surrogate of Genomic Instability during Oral Carcinogenesis.

BACKGROUND: Our goal was to identify a gene-expression-based surrogate of genomic instability (GI) associated with the transformation of oral potentially malignant disorder (OPMD) into oral squamous cell carcinoma (OSCC). METHODS: GI was defined as the fraction of genome altered (FGA). Training sets included the CCLE and TCGA databases. The relevance of the enrichment score of the top correlated genes, referred to as the GIN score, was evaluated in eight independent public datasets from the GEO repository, including a cohort of patients with OPMD with available outcome. RESULTS: A set of 20 genes correlated with FGA in head and neck SCC were identified. A significant correlation was found between the 20-gene based GIN score and FGA in 95 esophagus SCC (r = 0.59) and 501 lung SCC (r = 0.63), and in 33 OPMD/OSCC (r = 0.38). A significantly increased GIN score was observed at different stages of oral carcinogenesis (normal-dysplasia -OSCC) in five independent datasets. The GIN score was higher in 10 OPMD that transformed into oral cancer compared to 10 nontransforming OPMD (p = 0.0288), and was associated with oral-cancer-free survival in 86 patients with OPMD (p = 0.0081). CONCLUSIONS: The GIN score is a gene-expression surrogate of GI, and is associated with oral carcinogenesis and OPMD malignant transformation.