A method for scoring 4-nitroquinoline 1-oxide-induced murine esophageal squamous neoplasia.

A mouse model for esophageal squamous cell carcinoma (ESCC) is induced by oral administration of the carcinogen 4-nitroquinoline 1-oxide (4-NQO). There is not an objective method for determining histopathologic severity of disease in this model. We aim to create a clearly defined and easily applied scoring system that can quantify the severity of 4-NQO-induced murine ESCC. Fifteen wild-type C57BL/6J mice were treated with 4-NQO for 8 (n = 8) or 16 (n = 7) weeks, while the rest (n = 9) were treated with vehicle, as 8 weeks of 4-NQO typically results in dysplasia and 16 weeks in carcinoma. We identified histologic abnormalities of the esophagus in this model and developed metrics to grade severity of dysplasia, papillomas, and invasion. Scores were then calculated using quantitative digitized image analysis for measuring depth and extent of each feature within the entire sample. Each feature was also assigned a weight based on its relation to cancer severity. Histology scores were significantly different in the three groups, suggesting that this method can discriminate dysplasia from carcinoma. This model can be applied to any mouse treated with 4-NQO.

Investigating the impact of long term exposure to chemical agents on the chromosomal radiosensitivity using human lymphoblastoid GM1899A cells.

This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising radiation. Human lymphoblastoid GM1899A cells were cultured in the presence of 4-nitroquinoline N-oxide (4NQO), N-nitroso-N-methylurea (MNU) and hydrogen peroxide (H2O2) for up to 6 months at the highest non-(geno)toxic concentration identified in pilot experiments. Acute challenge doses of 1 Gy X-rays were given and chromosome damage (dicentrics, acentric fragments, micronuclei, chromatid gaps/breaks) was scored. Chronic exposure to 20 ng/ml 4NQO, 0.25 µg/ml MNU or 10 µM H2O2 hardly induced dicentrics and did not significantly alter the yield of X-ray-induced dicentrics. Significant levels of acentric fragments were induced by all chemicals, which did not change during long-term exposure. Fragment data in combined treatment samples compared to single treatments were consistent with an additive effect of chemical and radiation exposure. Low level exposure to 4NQO induced micronuclei, the yields of which did not change throughout the 6 month exposure period. As for fragments, micronuclei yields for combined treatments were consistent with an additive effect of chemical and radiation. These results suggest that cellular radiation responses are not affected by long-term low-level chemical exposure.

Genomic landscape and clonal architecture of mouse oral squamous cell carcinomas dictate tumour ecology.

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.

CD44(+) tumor cells promote early angiogenesis in head and neck squamous cell carcinoma.

The role of CD44 in progression of head and neck squamous cell carcinoma (HNSCC) has been controversial. The goal of this study was to study the effects of CD44(+) tumor cells on the initial stages of tumor angiogenesis and to evaluate CD44 as a potential marker of tumor angiogenesis. The CD44 gene expression was studied using the Cancer Genome Atlas (TCGA) Head and Neck Cancer data base. Expression levels of CD44 and of microvascular density (MVD) markers were assessed by immunohistochemistry performed with tissue microarrays in a cohort of 49 HNSCC patients, 11 patients with dysplasia and 12 control oral mucosa tissues. The 4-nitroquinoline-1-oxide oral carcinogenesis mouse model was used to study CD44 expression during carcinogenesis. Gelatin sponges seeded with CD44(+), CD44(-) and unsorted cancer cells suspended in Matrigel were implanted in NOD/SCID mice into a dorsal skinfold chamber and compared to non-seeded sponges as controls. Angiogenic response was assessed by intravital microscopy. In the TCGA analysis, CD44 gene expression correlated with various pro-angiogenic genes. In human HNSCC tissues, CD44 expression was upregulated and was associated with blood vessels, although no correlation between MVD and CD44 expression was found. During oral carcinogenesis CD44 expression was upregulated. In dorsal skinfold chambers, CD44(+) cells showed a significantly higher MVD than CD44(-) or unsorted cells (p < 0.001). The results indicate that CD44(+) cells contain pro-angiogenic factors and stimulate tumor angiogenesis in HNSCC. Thus, CD44 might emerge as a potential angiogenic biomarker and a therapeutic target for anti-angiogenic therapies.

Multiple modulatory activities of Andrographis paniculata on immune responses and xenograft growth in esophageal cancer preclinical models.

BACKGROUND: Esophageal cancer (EC) is a malignant gastrointestinal cancer with high morbidity worldwide and is the fourth leading cause of cancer-related deaths in China. Even though surgery and/or chemotherapy/chemoradiation might achieve good therapeutic response, recurrence rate is high due to cancer metastasis. Hence, the use of alternative adjuvant treatments, such as herbal medicines, for metastatic EC remains a great desire of the patients. Our previous studies have demonstrated the anti-metastatic efficacy of hot water extract of Andrographis paniculata (APW) in human esophageal cancer cells and tumor-bearing nude mice. PURPOSE: In the present study, the immunomodulatory activities of APW were further evaluated in human peripheral blood mononuclear cells (PBMCs) and in a carcinogen-induced esophageal tumorigenesis model using immune-competent C57BL/6 mice. Besides, the inhibitory effects of APW on esophageal cancer cell line-based xenografts and patient-derived xenografts (PDX) were examined so as to illustrate the potential multi-targeted efficacies of APW in esophageal cancer in pre-clinical models. RESULTS: In vitro results showed that APW could stimulate proliferation of PBMCs, as well as TNF-α and IFN-γproductions. In mice with 4-nitroquinoline 1-oxide-induced tumorigenesis, 21-day oral treatment with APW (1600 mg/kg) decreased the level of dysplasia in esophagus and significantly modulated the population of regulatory T cells. The cytokines productions by spleen lymphocytes of APW-treated mice were shifted towards normal resting state (i.e. unchallenged with carcinogen). Furthermore, APW treatment suppressed the growth of cell line-based xenografts by significantly increasing apoptosis in tumors, without causing severe body weight loss as chemotherapeutics did. Most importantly, the inhibitory effects of APW treatment on esophageal patient-derived xenografts growth were demonstrated for the first time. Besides, several diterpenes were detected in the plasma after oral administration of APW in mice, suggesting that multi-components of APW were bioavailable and might have contributed towards the varied pharmacological activities demonstrated in our studies. CONCLUSION: APW was shown to possess anti-tumor, anti-metastatic and immunomodulatory activities in esophageal cancer cell-based and animal models, including immunocompromised mice model and clinically relevant PDX model. Our findings illustrated the potential multi-targeted efficacies of APW in esophageal cancer management.

Autophagy is positively associated with the accumulation of myeloid­derived suppressor cells in 4­nitroquinoline­1­oxide­induced oral cancer.

It has previously been demonstrated that autophagy and inflammation act synergistically to promote carcinogenesis. However, the precise roles of autophagy in multistep oral carcinogenesis are still unclear, particularly regarding its association with tumor inflammation. The present study established a 4NQO­induced oral cancer mouse model and investigated autophagy status in the multistep process of oral carcinogenesis using immunohistochemistry, western blotting and immunofluorescence staining. Furthermore, the number of Gr­1+CD11b+ myeloid derived suppressor cells (MDSCs) and CD4+ Foxp3+ regulatory T cells (Tregs) during oral carcinogenesis and the association with autophagy status was also examined. The results revealed that the expression of autophagy biomarkers, including dihydrosphingosine 1-phosphate phosphatase LCB3 (LC3B), p62/SQSTM1 (p62) and Beclin 1 increased during 4NQO­induced carcinogenesis and in human oral cancer. The number of MDSCs and Tregs also increased during oral carcinogenesis. Furthermore, the expression of LC3B and p62 significantly correlated with the accumulation of MDSCs and the expression of Beclin 1 correlated with the increase of Tregs. These data indicated that autophagy may be activated by the tumor inflammation microenvironment during oral carcinogenesis.

A 4-Nitroquinoleneoxide-Induced Pleurotus eryngii Mutant Variety Increases Pin1 Expression in Rat Brain.

To develop Pleurotus eryngii varieties with improved medicinal qualities, protoplasts of P. eryngii were mutagenized using 4-nitroquinoleneoxide. The effects of the resulting variant mushrooms on a human cell were evaluated by applying their aqueous extracts to the human hepatoma cell line, HepG2, in vitro and examining any alteration in the proteomes of the treated HepG2. The P. eryngii mutant, NQ2A-12, was selected for its effects on increasing the expression level of Pin1 in HepG2. Pin1 is one of the peptidyl-prolyl cis-trans isomerases known to play an important role in repressing Alzheimer's disease pathogenesis. Validity of NQ2A-12 related to Alzheimer's disease was shown with an enhanced expression of Pin1 in a mouse brain tissue by injecting the NQ2A-12 extract. The mutant mushroom, NQ2A-12, could be developed as a new variety of P. eryngii with potential to protect against Alzheimer's disease.

Down-regulation of ß-catenin and the associated migration ability by Taiwanin C in arecoline and 4-NQO-induced oral cancer cells via GSK-3ß activation.

Cancer is one of the leading causes of death worldwide, and oral squamous cell carcinoma (OSCC) accounts for almost a sixth of all reported cancers. Arecoline, from areca nut is known to enhance carcinogenesis in oral squamous cells. The objective of this study is to determine the effect of Taiwanin C, from Taiwania cryptomerioides Hayata against Arecoline-associated carcinogenesis. An OSCC model was created in C57BL/6J Narl mice by administrating 0.5 mg mL-1 arecoline with 0.2 mg mL-1 4-NQO carcinogen for 8 and 28 wk to mimic the etiology of oral cancer patients in Asia. Mice were sacrificed and two cell lines, T28 from the tumor and N28 cancerous cell line from the surrounding non tumor area, were established. Taiwanin C showed effective anti-tumor activity in nude mice models. Taiwanin C significantly inhibited the cell viability of T28 cells in a dose dependent manner, but did not inflict any effect on N28 normal cells. Taiwanin C treatment inhibited the migration ability of T28 cells in a dose dependent manner as determined by wound healing and migration assays. Taiwanin C also reduced the levels of ß-catenin and its downstream metastatic proteins, Tbx3 and c-Myc. Besides, Taiwanin C inhibited the nuclear accumulation of ß-catenin and induced ß-catenin degradation via proteasome-mediated pathway. Moreover, Taiwanin C enhanced GSK-3ß and reduced the p-ser9 GSK-3ß protein level to inactivate Wnt signaling. Taken together, Taiwanin C blocked the cell migration effects of T28 cells mediated through the activation of GSK-3ß to enhance protein degradation and reduce nuclear accumulation of ß-catenin. © 2016 Wiley Periodicals, Inc.

Modulation of IL-1ß reprogrammes the tumor microenvironment to interrupt oral carcinogenesis.

Head and neck squamous cell carcinoma (HNSCC) development is a multistage process includes the normal, dysplasia and squamous cell carcinoma (SCC) stages. Recently, increasing evidence has suggested that the tumor microenvironment (TME) is an integral part of malignant transformation. Exploring certain key node genes in TME for future intervention in dysplasia to interrupt oral carcinogenesis was the primary goal of this research. To achieve this goal, systems biology approaches were first applied to the epithelia and fibroblasts collected at sequential stages in a 4-nitroquinoline-1-oxide (4NQO) -induced rat oral carcinogenesis model. Through bioinformatics network construction, IL-1ß was identified as one of the key node genes in TME during carcinogenesis. Immunohistochemical staining of human and rat samples demonstrated that IL-1ß expression patterns were parallel to the stages of malignant transformation. Silencing IL-1ß with lentivirus-delivered shRNA significantly inhibited oral squamous cell carcinoma cell growth both in vivo and in vitro. Based on these findings, we hypothesized that IL-1ß may be a chemoprevention target in TME during oral carcinogenesis. Therefore, we targeted IL-1 in the TME by oral mucosal injection of an IL-1 receptor antagonist in 4NQO rats. The results demonstrated that targeting IL-1 could interrupt oral carcinogenesis by reprogramming the TME.

Establishment of a highly metastatic buccal squamous cell carcinoma cell line from a Sprague-Dawley Rat.

OBJECTIVES: The incidence of buccal squamous cell carcinoma (buccal SCC) is considered to be the second highest out of all oral cancers, but the unsatisfactory in vivo tumorigenicity and metastatic potential of the widely used cell lines have greatly delayed studies on the mechanisms of tumor progression. This study aimed to establish a highly metastatic buccal SCC cell line, which may serve a useful tool in buccal SCC research. MATERIALS AND METHODS: Buccal SCC was induced by 4-nitroquinoline-1-oxide (4NQO) in Sprague-Dawley rats. The cancer samples were collected, and the tumor cells were purified in vitro. A highly aggressive cell line termed "Rca-B" was established by an invasion assay. Its proliferative ability, cell cycle distribution, baseline level of apoptosis, carcinogenicity and metastatic behavior in nude mice were investigated. RESULTS: To date, Rca-B cells have been stably cultured in vitro for more than 180 passages. These cells were polygonal or spindle-shaped, grew adhesively, and exhibited a stable epithelial phenotype as characterized by positive expression of cytokeratin. The population doubling time was 25.09 h. Cells in S-phase of the cell cycle accounted for 31.17% of the total number of cells, and the baseline level of apoptosis was 8.52%. The in vitro migration and invasion assays revealed highly aggressive features of Rca-B cells. In addition, the rate of xenograft formation was 100%, and the incidence of experimental lung metastasis was 81.8% in immunodeficient nude mice. CONCLUSION: The Rca-B cell line was established as a highly metastatic rat buccal SCC cell line, and its in-depth characterization, which includes malignant behaviors, allows for a wealth of functional studies on the molecular mechanisms of buccal SCC progression and targeted therapy.

Inhibition of 4NQO-Induced Oral Carcinogenesis by Dietary Oyster Shell Calcium.

Oyster has gained much attention recently for its anticancer activity but it is unclear whether calcium, the major antitumor ingredient in oyster shell, is responsible for the anticarcinogenic role of the oyster. To address this issue, C57BL/6 mice were fed with the carcinogen 4-nitroquinoline-1-oxide (4NQO, 50 µg/mL) and normal diet or a diet containing oyster powder, oyster calcium, or calcium depleted oyster powder. The tongue tissue specimens isolated from these mice were histologically evaluated for hyperplasia, dysplasia, and papillary lesions, and then analyzed for proliferation and differentiation markers by immunohistochemistry. The results showed that mice on the diet containing oyster calcium significantly reduced rates of tumors in the tongue and proliferation and enhanced differentiation in the oral epithelium compared with the diet containing calcium depleted oyster powder. These results suggest that calcium in oyster plays a critical role in suppressing formation of oral squamous cell carcinoma and proliferation and promoting differentiation of the oral epithelium.

Inhibition of 4-nitroquinoline-1-oxide-induced oral carcinogenesis by dietary calcium.

Calcium is a strong inducer of keratinocyte differentiation. We have previously demonstrated that extracellular calcium promotes keratinocyte differentiation via E-cadherin-catenin complex-mediated phospholipase C-γ1 (PLC-γ1) activation in the plasma membrane. However, it is unclear whether dietary calcium regulates keratinocyte proliferation, differentiation or carcinogenesis. To address this issue, the rates of oral tumor and levels of proliferation and differentiation in the oral epithelium were assessed in mice on different calcium diets and the carcinogen 4-nitroquinoline-1-oxide. The results showed that mice on the high calcium diet had lower rates of oral tumors, lower levels of proliferation and higher levels of differentiation in the normal oral epithelium than those on the normal calcium diet. Higher levels of E-cadherin, ß-catenin, p120-catenin (p120), epidermal growth factor receptor (EGFR), and calcium and lower levels of PLC-γ1 were also noted in the normal oral epithelium in mice on high calcium diet than the control mice. In contrast, mice on low calcium diet had opposite effects. However, dietary calcium had no effect on the proliferation, differentiation or the levels of E-cadherin, ß-catenin, p120, PLC-γ1 and EGFR in oral tumors. These data indicate that dietary calcium increases calcium levels in oral epithelium, suppresses oral carcinogenesis, inhibits proliferation and promotes differentiation of normal oral epithelium. Increased E-cadherin, ß-catenin, p120 and EGFR and decreased PLC-γ1 may participate in the inhibitory effect of dietary calcium in oral carcinogenesis.

The influence of sleep restriction on expression of apoptosis regulatory proteins p53, Bcl-2 and Bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

PURPOSE: The aim of this study was to evaluate whether sleep restriction (SR) could affect the mechanisms and pathways' essentials for cancer cells in tongue cancer induced by 4-nitroquinoline 1-oxide in Wistar rats. METHODS: The animals were distributed into 4 groups of 5 animals each treated with 50 ppm 4 NQO solution through their drinking water for 4 and 12 weeks. The animals were submitted to sleep restriction for 21 days using the modified multiple platform method, which consisted of placing 5 rats in a cage (41 × 34 × 16 cm) containing 10 circular platforms (3.5 cm in diameter) with water 1 cm below the upper surface. The investigations were conducted using immunohistochemistry of p53, Bax and Bcl-2 proteins related to apoptosis and its pathways. RESULTS: Although no histopathologic abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre-neoplastic lesions. Data analysis revealed statistically significant differences (P < 0.05) in 4 weeks group for p53, and for bcl-2. Following 12 weeks of 4NQO administration, we found significant differences between SR and control groups in p53, bax, and bcl-2 immunoexpression. CONCLUSION: Our results reveal that sleep restriction exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis.

Diffusion Reflection: A Novel Method for Detection of Oral Cancer.

Intraoperative detection of residual disease in oral cancer may reduce the high rate of recurrences. The aim of the present study was to evaluate the detection sensitivity of diffusion reflection (DR) measurements of bioconjugated gold nanorods (GNRs) to cancerous sites in a rat model of oral squamous cell carcinoma. We used hyperspectral spectroscopy and DR measurements of GNRs bioconjugated to slide specimens of rat tongues where squamous carcinoma was induced by 4NQO (4-nitroquinoline-N-oxide). Wistar-derived male rats were used: 6 were sacrificed at wk 32 to 37 following 4NQO administration (experimental rats), as were 2 control rats at wk 32 and 36. The detection results were compared with histopathology: 19 sites of cancerous changes were identified microscopically (11 invasive cancer and 8 carcinoma in situ [CIS]). The GNRs attached selectively to areas of carcinomatous changes with an intensity exceeding 17 intensity units at 780 nm (overall specificity, 97%; overall sensitivity, 87%) when the hyperspectral spectroscopy system was used. The resulting DR slopes of the reflected intensity showed an increase of >80% in areas of invasive cancer and an increase of >30% in the CIS sites. The resulting intensity units of the hyperspectral spectroscopy system in the invasive cancer significantly exceed those of the CIS (t test, p = .0002; Mann-Whitney, p = .0024). The results demonstrate a great potential of the direct DR scanning as a new and simple tool for detecting residual disease intraoperatively.

Comparable molecular alterations in 4-nitroquinoline 1-oxide-induced oral and esophageal cancer in mice and in human esophageal cancer, associated with poor prognosis of patients.

BACKGROUND: The murine model of 4-nitroquinoline 1-oxide (4-NQO)-induced oral and esophageal cancer is frequently used to assess the effects of different cancer prevention/ therapy agents in vivo, but the molecular mechanisms in those 4-NQO-induced carcinogenesis are unknown. This study investigated aberrant expression of cell growth-critical genes in 4-NQO-induced oral and esophageal cancer tissues in mice compared to those present in the human disease for association with survival of patients. MATERIALS AND METHODS: C57LB6/129Sv mice were given 4-NQO in their drinking water to induce oral and esophageal cancer. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry were used to detect gene expression in the cancer tissues from mice and in 4-NQO-treated human esophageal cancer cell lines and esophageal cancer tissues. Methylation-specific PCR and DNA sequencing were performed to assess methylation of the Rarb2 promoter in murine tissues. Kaplan-Meier analysis was performed to associate gene expression in esophageal cancer tissues with survival data for patients with esophageal cancer. RESULTS: 4-NQO dose-dependently induced pre-malignant and malignant lesions in the oral cavity and esophagus in mice that pathologically and morphologically mimicked human oral and esophageal cancer. Molecularly, 4-NQO inhibited Rarß2 but induced expression of phosphorylated extracellular-signal-regulated kinase-1 and -2 (p-ERK1/2) and Cox2 proteins and Rarß2 gene promoter methylation in murine tumors. In vitro treatment with 4-NQO altered expression of RARß2, p-ERK1/2, and COX2 in human esophageal cancer cells. In tissues from 90 patients with esophageal cancer, expression of p-ERK1/2 and COX2 was up-regulated, and p-ERK1/2 expression was associated with advanced clinical tumor stage and consumption of hot beverages, while COX2 expression was associated with tumor de-differentiation in esophageal cancer. Furthermore, expression of p-ERK1/2 was associated with a worse overall survival rate of patients (p=0.014), whereas the association of COX2 expression with worse overall survival rate did not reach statistical significance (p=0.19). Knockdown of COX2 expression using transient transfection of a COX2 antisense expression vector inhibited Ki67 expression, an indicator of cell proliferation, in human esophageal cancer cells. CONCLUSION: 4-NQO-induced cancer in oral cavity and esophagus of mice not only pathologically and morphologically mimicked human oral and esophageal cancer, but also shared some molecular alterations (e.g. aberrant expression of Rarb2, p-ERK1/2, and Cox2). This study further demonstrated that targeting of the altered RARß2-led gene pathway could effectively suppress the development of this deadly type of cancer.

Effect of topical 5-ALA mediated photodynamic therapy on proliferation index of keratinocytes in 4-NQO-induced potentially malignant oral lesions.

Fractionation can improve photodynamic therapy (PDT) efficacy for potentially malignant oral lesion treatment. The aim of this study was to demonstrate the apoptosis/proliferation index of oral keratinocytes after two sessions of topical 5-ALA-mediated PDT in 4-Nitroquinoline-1-oxide-induced potentially malignant oral lesion, and to suggest the ideal interval between PDT sessions. Immuno-histochemical tests for proliferating cell nuclear antigen and caspase-3, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed at 6h, 24h, 48h, and 72h time intervals after PDT. The number of positive cells showing caspase-3 expression was significantly higher, mainly at 6h after PDT. In the first cycle of PDT, the highest frequency of positive cells for TUNEL was found at 24h. At 72h after PDT, proliferating cell nuclear antigen positive cells increased significantly, indicating that there was an epithelial response in direction towards DNA repair and cell proliferation at this time. Because cell proliferation increases and cell death index decreases at 72h after PDT, it is recommended that the interval between the PDT sessions must not be longer than 2days up to total lesion remission.

Basal stem cells contribute to squamous cell carcinomas in the oral cavity.

The cells of origin of oral cavity squamous cell carcinoma (OCSCC) are unknown. We used a cell lineage tracing approach (adult K14-CreER(TAM); ROSA26 mice transiently treated with tamoxifen) to identify and track normal epithelial stem cells (SCs) in mouse tongues by X-gal staining and to determine if these cells become neoplastically transformed by treatment with a carcinogen, 4-nitroquinoline 1-oxide (4-NQO). Here, we show that in normal tongue epithelia, X-gal(+) cells formed thin columns throughout the entire epithelium 12 weeks after tamoxifen treatment, indicating that the basal layer contains long-lived SCs that produce progeny by asymmetric division to maintain homeostasis. Carcinogen treatment results in a ~10-fold reduction in the total number of X-gal(+) clonal cell populations and horizontal expansion of X-gal(+) clonal cell columns, a pattern consistent with symmetric division of some SCs. Finally, X-gal(+) SCs are present in papillomas and invasive OCSCCs, and these long-lived X-gal(+) SCs are the cells of origin of these tumors. Moreover, the resulting 4-NQO-induced tumors are multiclonal. These findings provide insights into the identity of the initiating cells of oral cancer.

Homeobox C5 expression is associated with the progression of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

BACKGROUND: Aberrant expression of homeobox genes (HOX), normally required for the differentiation of a particular tissue, has been reported in several types of cancer, but poorly addressed in oral squamous cell carcinoma (OSCC). The present study investigated the expression of HOXC5 in OSCC and identified molecular biomarker whose expression is associated with the multistep oral carcinogenesis. METHODS: The expression of HOXC5, proliferation cell nuclear antigen (PCNA), and Bcl-2 was examined by RT-PCR and Western blot analysis and confirmed by immunohistochemistry and transferase-mediated dUTP nick end-labeling (TUNEL) assay in a 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis model. RESULTS: Homeobox genes C5 was overexpressed in SCC tissues, but not in normal tissues by RT-PCR and Western blot analysis. Along with the progress of multistep carcinogenesis, the levels of HOXC5 expression of mRNA and protein significantly increased during the dysplasia (moderate to severe dysplasia) when compared with normal and hyperplasia. The levels of PCNA and Bcl-2 were sequentially increased from hyperplasia to dysplasia and SCC. By immunohistochemistry, HOXC5 expression was significantly increased in dysplasia, whereas PCNA expression was gradually increased during tongue carcinogenesis. TUNEL-positive cells were increased until dysplasia, but reduced in SCC. CONCLUSIONS: These results indicate that overexpression of HOXC5 is correlated with oral carcinogenesis and strongly contributed to the development of OSCC. HOXC5 may be a useful biomarker and has an emerging therapeutic target of OSCC.

Histological and immunohistochemical evaluation of the chemopreventive role of lycopene in tongue carcinogenesis induced by 4-nitroquinoline-1-oxide.

OBJECTIVE: Increasing attention has been given to the potential protective roles of specific antioxidant nutrients found in fruits and vegetables. However, there are relatively few reports on the cancer chemopreventive effects of lycopene or tomato carotenoids in animal models. Therefore, the chemopreventive efficacy of lycopene with regard to oral carcinogenesis was investigated using 4-nitroquinoline-1-oxide (4-NQO) induced tongue squamous cell carcinoma. MATERIALS AND METHODS: Twenty albino rats were equally divided into 2 groups. 4-NQO was dissolved in the drinking water (20 ppm) for rats of both groups. Rats in group 2 were administered lycopene at a dose of 2.5mg/kg body weight by intragastric intubation once a day. Incidence of oral neoplasms and histopathological changes were microscopically evaluated after 32 weeks of administration of the specific treatments. Moreover, immunohistochemical expressions of proliferating cell nuclear antigen (PCNA), E-cadherin and ß-catenin were analysed in tongue specimens using an image analyser computer system. RESULTS: Lycopene treatment significantly decreased the incidence of 4-NQO induced tongue carcinogenesis. A decreased percentage of PCNA-positive nuclei was associated with lycopene treatment. Proliferating cells were mainly confined to the basal and parabasal epithelial cell layers. Increased E-cadherin and ß-catenin immunoexpression was recorded in the lycopene treated group in comparison to the carcinogen group. CONCLUSION: Results of the present study indicate that lycopene can exert protective effects against 4-NQO induced tongue carcinogenesis through reduction in cell proliferation and enhanced cellular adhesion, suggesting a new mechanism for the anti-invasive effect of lycopene. Further studies are needed to provide more definitive answers to the question of the anticancer effects of lycopene.

Budding yeast Mph1 promotes sister chromatid interactions by a mechanism involving strand invasion.

Stalling of replication forks at lesions is a serious threat to genomic integrity and cell viability. Cells have developed a variety of pathways that allow continuation of synthesis, including translesion synthesis, postreplication repair and homologous recombination. We have devised a sensitive genetic system for detection of sister chromatid interactions in Saccharomyces cerevisiae. A 266bp sequence duplication in the KanMX4 module was generated and reversions were scored via G418 resistant colonies. Both 4-NQO induced and spontaneous reversions are strictly dependent on RAD52. Damage-induced reversions are also largely dependent on RAD51. Thus, most damage-induced events require a strand invasion step. Induced reversions were not affected in rev3 mutants and partially reduced in rad30 mutants indicating an involvement of Pol η. In cells lacking Mph1, a member of the FANCM family of DNA helicases, that has been implicated in a pathway for fork reactivation involving homologous recombination, damage-induced events are significantly reduced. Together with the spontaneous mutator phenotype of mph1 mutants this data strongly suggest that Mph1 has an additional function in recombination besides its previously described ability to disrupt D-loops. We propose that Mph1 promotes D-loop formation.