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miR-211 expression in human oral squamous cell carcinoma (OSCC) has been implicated in poor patient survival. To investigate the oncogenic roles of miR-211, we generated K14-EGFP-miR-211 transgenic mice tagged with GFP. Induction of oral carcinogenesis in transgenic mice using 4-nitroquinoline 1-oxide (4NQO) resulted in more extensive and severe tongue tumorigenesis compared with control animals. We found that 4NQO and arecoline upregulated miR-211 expression in OSCC cells. In silico and experimental evidence further revealed that miR-211 directly targeted transcription factor 12 (TCF12), which mediated suppressor activities in OSCC cells and was drastically downregulated in tumor tissues. We used GeneChip analysis and bioinformatic algorithms to identify transcriptional targets of TCF12 and confirmed through reporter and ChIP assays that family with sequence similarity 213, member A (FAM213A), a peroxiredoxin-like antioxidative protein, was repressed transcriptionally by TCF12. FAM213A silencing in OSCC cells diminished oncogenic activity, reduced the ALDH1-positive cell population, and increased reactive oxygen species. TCF12 and FAM213A expression was correlated inversely in head and neck carcinoma samples according to The Cancer Genome Atlas. OSCC patients bearing tumors with high FAM213A expression tended to have worse survival. Furthermore, 4NQO treatment downregulated TCF12 and upregulated FAM213A by modulating miR-211 both in vitro and in vivo. Overall, our findings develop a mouse model that recapitulates the molecular and histopathologic alterations of human OSCC pathogenesis and highlight a new miRNA-mediated oncogenic mechanism. Cancer Res; 76(16); 4872–86. ©2016 AACR.

Head and neck squamous cell carcinoma (HNSCC) is a frequently fatal disease due, in large part, to a high rate of second primary tumor (SPT) formation. The 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis provides a robust system in which to study chemopreventive agents in the context of chemically induced HNSCC tumors. STAT3 is a potent oncogene that is hyperactivated by tyrosine phosphorylation early in HNSCC carcinogenesis and is a rational therapeutic target. We recently reported that loss-of-function of the STAT3 phosphatase PTPRT promotes STAT3 activation in HNSCC tumors and preclinical models and may serve as a predictive biomarker of response to STAT3 inhibitors, including the small-molecule Stattic. We therefore investigated the hypothesis that Ptprt-knockout (KO) mice would be more susceptible to 4-NQO–induced oral carcinogenesis and more sensitive to Stattic-mediated chemoprevention compared with wild-type (WT) mice. Herein, we demonstrate that Ptprt WT and KO mice develop similar spectra of HNSCC disease severity upon 12 weeks of 4-NQO administration, with no apparent effect of Ptprt genotype on carcinogenesis or treatment outcome. Targeting of STAT3 with Stattic resulted in a chemopreventive effect against 4-NQO–induced oral cancer (P = 0.0402). While these results do not support a central role for PTPRT in 4-NQO–induced HNSCC carcinogenesis, further investigation of STAT3 as a chemoprevention target in this cancer is warranted. Cancer Prev Res; 9(8); 657–63. ©2016 AACR.

// Aixian Lan 1, * , Wenjun Li 1, * , Yao Liu 1 , Zhaohui Xiong 2 , Xinyan Zhang 1 , Shanshan Zhou 3 , Olesya Palko 2 , Hao Chen 2 , Mayanga Kapita 2 , Justin R. Prigge 4 , Edward E. Schmidt 4 , Xin Chen 3 , Zheng Sun 1 , Xiaoxin Luke Chen 2 1 Department of Oral Medicine, Beijing Stomatological Hospital & School of Stomatology, Capital Medical University, Beijing 100050, China 2 Cancer Research Program, JLC-BBRI, North Carolina Central University, Durham, NC 27707, USA 3 Department of Pharmaceutical Engineering, School of Pharmaceutical & Life Sciences, Changzhou University, Jiangsu 213164, China 4 Department of Immunology and Infectious Diseases, Montana State University, Bozeman, MT 59717, USA * These authors have contributed equally to this work Correspondence to: Xin Chen, email: xinchen@cczu.edu.cn Zheng Sun, email: sunzheng12@vip.126.com Xiaoxin Luke Chen, email: lchen@nccu.edu Keywords: oral cancer, chemoprevention, NRF2, sulforaphane, 4NQO Received: May 04, 2016 Accepted: June 03, 2016 Published: July 15, 2016 ABSTRACT Oxidative stress is known to play an important role in oral cancer development. In this study we aimed to examine whether a chemical activator of NRF2, sulforaphane (SFN), may have chemopreventive effects on oxidative stress-associated oral carcinogenesis. We first showed that Nrf2 activation and oxidative damage were commonly seen in human samples of oral leukoplakia. With gene microarray and immunostaining, we found 4-nitroquinoline 1-oxide (4NQO) in drink activated the Nrf2 pathway and produced oxidative damage in mouse tongue. Meanwhile whole exome sequencing of mouse tongue identified mutations consistent with 4NQO’s mutagenic profile. Using cultured human oral keratinocytes and 4NQO-treated mouse tongue, we found that SFN pre-treatment activated the NRF2 pathway and inhibited oxidative damage both in vitro and in vivo . On the contrary, a structural analogue of SFN without the isothiocyanate moiety did not have such effects. In a long-term chemoprevention study using wild-type and Nrf2 -/- mice, we showed that topical application of SFN activated the NRF2 pathway, inhibited oxidative damage, and prevented 4NQO-induced oral carcinogenesis in an Nrf2-dependent manner. Our data clearly demonstrate that SFN has chemopreventive effects on oxidative stress-associated oral carcinogenesis, and such effects depend on Nrf2 and the isothiocyanate moiety.

Mutagen sensitivity assay, which measures the enhanced cellular response to DNA damage induced in vitro by mutagens/carcinogens, has been used in the study of cancer susceptibility. 4-Nitroquinoline-1-oxide (4-NQO), an ultraviolet (UV) radiation-mimetic chemical, can produce chromosomal breaks in mammalian cells and induce cancer. Given the potential role of 4-NQO as the experimental mutagen substituting for UV as the etiological carcinogen of cutaneous melanoma (CM), we tested the hypothesis that cellular sensitivity to 4-NQO is associated with the risk of developing CM in a case–control study of 133 patients with primary CM and 176 cancer-free controls. Short-term blood cultures were treated with 4-NQO at a final concentration of 10 µmol/l for 24 h and scored chromatid breaks in 50 well-spread metaphases. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals. We found that the log-transformed frequency of chromatid breaks was significantly higher in 133 patients than in 176 controls (P = 0.004) and was associated with an increased risk for CM (adjusted odds ratio = 1.78, 95% confidence interval: 1.12–2.84) after adjustment for age and sex. Moreover, as the chromatid break values increased, the risk for CM increased in a dose-dependent manner (Ptrend = 0.003). Further analysis explored a multiplicative interaction between the sensitivity to 4-NQO and a family history of skin cancer (Pinteraction = 0.004) on the risk of CM. Therefore, our findings suggest that sensitivity to 4-NQO may be a risk factor for the risk of CM, which is more sensitive than UV-induced chromosome breaks.

The typical progression of oral cancer is from hyperplastic epithelial lesions through dysplasia to invasive carcinoma. It is important to investigate malignant oral cancer progression and development in order to determine useful approaches of prevention of dysplastic lesions. The present study aimed to gain insights into the underlying molecular mechanism of oral carcinogenesis by establishing a rat model of oral carcinogenesis using 4-nitroquino-line 1-oxide. Subsequently, transcription profile analysis using an integrating microarray was performed. The dynamic gene expression changes of the six stages of rat oral carcinogenesis (normal, mild epithelial dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and oral squamous cell carcinomas) were analyzed using component plane presentations (CPP)-self-organizing map (SOM). Six genes were verified by quantitative polymerase chain reaction, immunohistochemistry and succinate dehydrogenase (SDH) activity assay kit. Numerous differentially expressed genes (DEGs) were identified during rat oral carcinogenesis. CPP-SOM determined that these DEGs were primarily enriched during cell cycle, apoptosis, inflammatory response and tricarboxylic acid cycle, indicating the coordinated regulation of molecular networks. In addition, the expression of specific DEGs, such as janus kinase 3, cyclin-dependent kinase A-1, B-cell chronic lymphocytic leukaemia/lymphoma 2-like 2, nuclear factor-κB, tumor necrosis factor receptor superfamily member 1A, cyclin D1 and SDH were identified to have high concordance with the results from microarray data. The current study demonstrated that oral carcinogenesis is a multi-step and multi-gene process, with a distinct pattern alteration along a continuum of malignant transformation. In addition, this comprehensive investigation provided a theoretical basis for the understanding of the molecular alterations associated with oral carcinogenesis.

Background Atherosclerosis seriously threats human health. Homocysteine is an independent risk factor closely related to DNA methylation. MTHFR C667T loci polymorphism is closely associated with homocysteine level. This study aimed to investigate the relationship among MTHFR C667T loci polymorphism, genome-wide methylation, and atherosclerosis. Material/Methods Blood sample was collected from 105 patients with coronary atherosclerosis and 105 healthy controls. Pyrosequencing methylation was used to detect LINE-1 methylation level. Polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) was used to test MTHFR. Results LINE-1 methylation level in the patient group was significantly lower than in the controls (t=5.007, P

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.

Background The objective was to assess histopathological changes and the expression of proliferating cell nuclear antigen (PCNA), Bcl-2, suppressor of cytokine signaling (SOCS) 1 and 3, Vimentin, TWIST1, and Cdh 1 and 2 in early stages of experimental oral carcinogenesis process using a shorter period of exposure to 4-nitroquinoline oxide (4-NQO) model. Methods In this study, 20 rats were divided into control group (n = 10), sacrificed on the first day of the experiment, and experimental group (n = 10) treated with 50 ppm of 4-NQO solution dissolved in drinking water for 8 and 12 weeks. The histological sections were stained with H&E or subjected to immunohistochemistry for detecting PCNA, Bcl-2, SOCS 1 and 3, and STAT 3. Some specimens were used for verification of Vimentin expression, Cdh 1, Cdh 2, and TWIST1 by RT–qPCR. Results At both 8 and 12 weeks, morphological changes occurred mainly in the posterior portion of the tongue and were limited to the epithelial tissue, including moderate to severe dysplasia at 8 weeks, and severe dysplasia with exacerbation of atypical cells at 12 weeks. Expression of SOCS 1 and 3 increased from 8 to 12 weeks (P < 0.05), whereas STAT 3 expression was reduced mainly at 12 weeks (P < 0.05) in comparison with the control group. The expression of all epithelial–mesenchymal transition markers (EMT) was increased after 12 weeks, reaching statistical significance (P < 0.05) for Cdh 1 and 2. Conclusions Together, the results suggested that overexpression of Bcl-2, SOCS 1 and 3, and Cdh 1 and 2 is associated with the early neoplasic changes in modified 4-nitroquinoline 1-oxide-induced murine oral cancer model.

The aim of this study was to evaluate the antigenotoxic and antioxidant potential of shikonin (SH), acetylshikonin (ACS) and Arnebia euchroma callus extract (EXT). The antigenotoxic activity was investigated by the umu-test as the inhibition of the SOS system induction caused by genotoxic chemical agents - 4-nitroquinoline oxide and 2-aminoanthracene. Moreover the ability of SH, ACS and EXT to prevent photogenotoxicity triggered by chlorpromazine under UVA irradiation was measured. The cytotoxicity of EXT toward V79 Chinese hamster cell line was additionally assessed. Shikonin and acetylshikonin had no effect on 4-NQO induced genotoxicity whereas EXT demonstrated an unclear effect. The protection against 2AA induced genotoxicity was observed for all tested substances. The highest protection was demonstrated for EXT with inhibition of 66%. SH and ACS reduced 2AA genotoxicity with inhibition of about 60%. Under UVA the strongest and dose-dependent activity was observed for EXT. Acetylshikonin was a weak anti-photogenotoxin whereas shikonin had no clear effect. EXT was highly cytotoxic toward the V79 cell line - the cells' morphology was affected seriously and apoptosis was impacted. The antioxidant activity of SH, ACS and EXT was studied by means of electron paramagnetic resonance spectroscopy using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. All three samples exhibited radical scavenging properties.

Oral carcinogenesis involves the progression of the normal mucosa into potentially malignant disorders and finally into cancer. Tumors are heterogeneous, with different clusters of cells expressing different genes and exhibiting different behaviors. 4-nitroquinoline 1-oxide (4-NQO) and arecoline were used to induce oral cancer in mice, and the main factors for gene expression influencing carcinogenesis were identified through single-cell RNA sequencing analysis. Male C57BL/6J mice were divided into two groups: a control group (receiving normal drinking water) and treatment group (receiving drinking water containing 4-NQO (200 mg/L) and arecoline (500 mg/L)) to induce the malignant development of oral cancer. Mice were sacrificed at 8, 16, 20, and 29 weeks. Except for mice sacrificed at 8 weeks, all mice were treated for 16 weeks and then either sacrificed or given normal drinking water for the remaining weeks. Tongue lesions were excised, and all cells obtained from mice in the 29- and 16-week treatment groups were clustered into 17 groups by using the Louvain algorithm. Cells in subtypes 7 (stem cells) and 9 (keratinocytes) were analyzed through gene set enrichment analysis. Results indicated that their genes were associated with the MYC_targets_v1 pathway, and this finding was confirmed by the presence of cisplatin-resistant nasopharyngeal carcinoma cell lines. These cell subtype biomarkers can be applied for the detection of patients with precancerous lesions, the identification of high-risk populations, and as a treatment target.

Calcitriol, the active metabolite of vitamin D, has been widely studied for its preventive and therapeutic activity against several cancers including oral squamous cell carcinoma (OSCC). However, the impact of dietary vitamin D supplementation on initiation and progression of OSCC is unclear. To address this gap in knowledge, we conducted preclinical trials using the 4-nitroquinoline-1-oxide (4NQO) carcinogen model of oral carcinogenesis. Female C57BL/6 mice were maintained on one of three vitamin D diets [ 25 IU, 100 IU, 10,000 IU] and exposed to 4NQO in drinking water for 16 weeks followed by regular water for 10 weeks. Body weight measurements obtained through the study duration did not reveal any differences between the three diets. Animals on 100 IU diet showed lower incidence of high-grade dysplasia/OSCC and higher CD3+ T cells compared to animals on 25 IU and 10000 IU diets. Serum 25(OH)D(3) levels were highest in animals on 10000 IU diet at week 0 (prior to carcinogen exposure) but showed ~50% reduction at week 26. Histologic evaluation revealed highest incidence of OSCC in animals maintained on 10000 IU diet. Animals on 100 IU and 10000 IU diets showed higher vitamin D receptor (VDR) and CYP24A1 immunostaining in high-grade dysplastic lesions and OSCC compared to normal tongue. Validation studies performed in a 4NQO-derived OSCC model showed that short-term treatment of animals on a 25 IU diet with calcitriol significantly inhibited tumor growth compared to controls but did not affect tumor growth in animals on reference diet (1000 IU). Collectively, our results highlight the complex dynamics between vitamin D status and oral carcinogenesis. Our observations also suggest that therapeutic benefits of short-term calcitriol treatment may be more pronounced in vitamin D deficient hosts.

Microbiota has been widely considered to play a critical role in human carcinogenesis. Human papilloma virus, hepatitis B and C virus, and Helicobacter pylori are implicated in the pathogenesis of cancer of uterine cervix, liver, and stomach, respectively. However, whether Porphyromonas gingivalis (P. gingivalis), a common Gram negative oral bacteria, is associated with oral carcinogenesis still remains unclear and its underlying mechanism needs to be addressed. Here, we established a combined experimental system of 4NQO-induced oral carcinoma model and chronic periodontitis model and investigated the effects of P. gingivalis infection on oral carcinogenesis and fatty acid metabolism during oral carcinogenesis. The data showed that in this animal model, P. gingivalis infection induced mice periodontitis, increased the tongue lesion size and multiplicity of each mouse and promoted oral cancer development. P. gingivalis treatment significantly increased the level of free fatty acids and altered the fatty acid profile in tongue tissues and the serum of mice. And P. gingivalis induced the formation of fatty liver of the mice. Besides, immunohistochemical analysis and qRT-PCR showed that the expression of fatty-acid synthase and acetyl-CoA carboxylase 1 were increased in the tongue and liver tissues of 4NQO-treated mice infected with P. gingivalis. These results showed that P. gingivalis promoted oral carcinogenesis and aggravated disturbance of fatty acid metabolism, indicating a close association among P. gingivalis, lipid metabolic and oral carcinogenesis.

To identify potential biomarkers of lingual cancer, 75 female C57BL/6J mice were subjected to 16-week oral delivery of 4-nitroquinoline-1-oxide (4NQO; 50 mg/L), with 10 mice used as controls. Lingual mucosa samples representative of normal tissue (week 0) and early (week 12) and advanced (week 28) tumorigenesis were harvested for microarray and methylated DNA immunoprecipitation sequencing (MeDIP-Seq). Combined analysis with Short Time-series Expression Miner (STEM), the Cytoscape plugin cytoHubba, and screening of differentially expressed genes enabled identification of 63 hub genes predominantly altered in the early stage rather than the advanced stage. Validation of microarray results was carried out using qRT-PCR. Of 63 human orthologous genes, 35 correlated with human oral squamous cell carcinoma. KEGG analysis showed “pathways in cancer”, involving 13 hub genes, as the leading KEGG term. Significant alterations in promoter methylation were confirmed at Tbp, Smad1, Smad4, Pdpk1, Camk2, Atxn3, and Cdh2. HDAC2, TBP, and EP300 scored ≥10 on Maximal Clique Centrality (MCC) in STEM profile 11 and were overexpressed in human tongue cancer samples. However, expression did not correlate with smoking status, tumor differentiation, or overall survival. These results highlight potentially useful candidate biomarkers for lingual cancer prevention, diagnosis, and treatment.

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.

Notch signaling functions in diverse developmental and homeostatic processes, including stem cell self-renewal and cell fate determination. Notch1-inactivating mutations are frequently detected in skin and oro-esophageal cancers, suggesting a role for Notch1 as a tumor suppressor. Here, we clarify the contribution of Notch1 deficiency to oro-esophageal tumorigenesis using a physiological experimental model. Tongue and esophageal tumors induced in mice by 4-nitroquinoline-1-oxide (4-NQO) showed pathophysiological similarities to human tumors, including decreased Notch1 expression in the basal cells. We created mutant mice (N1cKO), in which the Notch1 gene was disrupted specifically in the squamous epithelium. The epithelium formed normally in N1cKO mice, and although multiple skin tumors were detected at 65 weeks, no tumors developed in the tongue and esophagus. However, 4-NQO-induced tumorigenesis assays revealed that tumor onset occurred earlier in N1cKO mice than in wild-type littermates, and the tumors arose preferentially from the Notch1-negative epithelium, indicating the tumor susceptibility of Notch1-deficient epithelium. Notch1 regulates the expression of TERT, and age-related telomere erosion was more rapid in Notch1-deficient basal cells. Our results indicated that although Notch1 deficiency had little effect on squamous epithelium formation, it predisposed the affected epithelium to tumor development, at least in part through accelerated telomere erosion.

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 4NQO-induced 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 Gr-1+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 4NQO-induced 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.

Made available in DSpace on 2018-12-11T17:18:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 The aim of this study was to evaluate the chemopreventive potential of purple carrot extract following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). For this purpose, histopathological analysis, proliferative status, antioxidant activity and inflammatory status were investigated in this setting. A total of 20 male rats were distributed into four groups as follows (n = 5 per group): Group 1—free access to water and commercial diet for 12 weeks; Group 2—received 4NQO at 50 ppm dose in drinking water daily and commercial diet for 12 weeks; Group 3—free access to water and received diet supplemented with purple carrot extract (0.1 g/kg) for 12 weeks; and Group 4—received 4NQO at 50 ppm dose in drinking water daily and diet supplemented with purple carrot extract (0.1 g/kg) for 12 weeks. Histopathological analysis revealed that animals treated with purple carrot extract reduced the oral lesions such as dysplasia and squamous cell carcinoma. Animals with oral pre-neoplastic lesions and treated with purple carrot extract decreased ki-67 and 8-OHdG immunoexpression. Moreover, pNFκBp50 and MyD88 protein expressions were decreased after purple carrot treatment associated or not with 4NQO exposure. SOD-Mn mRNA levels increased with treatment with purple carrot extract as well. In conclusion, our results demonstrated that purple carrot extract was able to protect oral lesions induced by 4NQO in Wistar rats as a result of antioxidant activity, anti-inflammatory potential and antiproliferative and antimutagenic actions. Department of Biosciences Federal University of São Paulo (UNIFESP), Rua Silva Jardim, 136, Vila Mathias Sao Paulo State University (UNESP), Campus Litoral Paulista Sao Paulo State University (UNESP), Campus Litoral Paulista

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development in vivo., Author summary The DEK oncogene is overexpressed in nearly all human cancers and portends a poor prognosis for many cancer types. High DEK expression causes cancer related phenotypes such as increased cellular proliferation, migration, and invasion in vitro. Despite the well documented link between high DEK expression and cancer, the consequences of Dek overexpression in vivo are poorly understood. To determine if Dek contributes to carcinogenesis in vivo, we generated a Dek inducible transgenic mouse model wherein Dek can be overexpressed in specific tissues and inhibited with the drug doxycycline. We targeted Dek overexpression to keratinocytes, the cell of origin for squamous cell carcinoma, and exposed the mice to the chemical carcinogen 4NQO to induce oral cavity and esophageal carcinogenesis. We found that DEK overexpression was sufficient to increase gross esophageal squamous cell carcinoma incidence and caused a trend toward increased cellular proliferation in adjacent non-tumor tissue. Importantly, we demonstrate for the first time that Dek overexpression specifically targeted to basal keratinocytes is sufficient to promote cellular and squamous cell carcinoma growth in vivo.

The majority of cases of oral squamous cell carcinoma (OSCC) develop from oral potentially malignant disorders, which have been confirmed to be involved in chronic oxidative stimulation. However, no effective treatment approaches have been used to prevent the development of dysplasia into cancerous lesions thus far. In the present study, a well-established OSCC model was used to detect proteomics profiles at different stages during oral malignant transformation. Of the 15 proteins that were found to be upregulated in both the dysplasia and carcinoma stages, the oxidative stress-associated proteins, thioredoxin-1 (Trx-1), glutaredoxin-1 and peroxiredoxin-2 were note as the proteins with significant changes in expression Trx-1 was identified to be the most significantly upregulated protein in the precancerous stage. Validation experiments confirmed that Trx-1 was overex-pressed both in dysplasia and cancerous tissue samples, and the inhibition of Trx-1 was able to promote the apoptosis of OSCC cells under hypoxic conditions. Furthermore, the experimental application of a Trx-1-specific inhibitory agent in an animal model led to a lower cancerization rate and a delay in tumor formation. The possible mechanisms were associated with the increased apoptosis via a reactive oxygen species (ROS)-dependent pathway. Taken together, our findings indicate that Trx-1 may be an important target for delaying oral malignant transformation, which provides a novel therapeutic strategy for the prevention and treatment of OSCC.

Background Head and neck squamous cell carcinoma (HNSCC) is a cancer that is characterized by its high morbidity and mortality rates. While tobacco use and alcohol consumption are 2 major contributing factors for HNSCC carcinogenesis, how the combination of tobacco and alcohol increases HNSCC risk is not understood. Methods We combined the 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis and Meadows–Cook alcohol mouse models to elucidate the molecular events and to identify the novel biomarkers associated with oral cancer development. Results By genome-wide RNA-seq of tongue samples (3 mice per group), we identified changes in transcripts that mediate alcohol metabolism and oxidative stress (Aldh2, Aldh1a3, Adh1, Adh7, and Cyp2a5) in mice treated with 4-NQO followed by ethanol (4-NQO/EtOH) as compared to the vehicle control/untreated (V.C./Untr.) samples. We measured major, global increases in specific histone acetylation and methylation epigenetic marks (H3K27ac, H3K9/14ac, H3K27me3, and H3K9me3) in the oral cavities of V.C./EtOH, 4-NQO/Untr., and 4-NQO/EtOH treatment groups compared to the V.C./Untr. group. We detected changes in histone epigenetic marks near regulatory regions of genes involved in ethanol metabolism by chromatin immunoprecipitation. For instance, the Aldh2 promoter showed increased H3K27me3 marks, and Aldh2 mRNA levels were reduced by 10-fold in 4NQO/EtOH versus V.C./Untr. tongue samples. 4-NQO/EtOH treatment also caused increases in markers of oxidative stress, including 4-HNE, MCT4/SLC16a3, and TOM20, as measured by immunohistochemistry. Conclusions We delineate a mechanism by which 4-NQO and ethanol can regulate gene expression during the development of HNSCC and suggest that histone epigenetic marks and oxidative stress markers could be the novel biomarkers and targets for the prevention of HNSCC.

Several studies have shown that apple (Malus sp.) has many components able to exert chemopreventive activity. The aim of this study was to evaluate the chemopreventive potential of apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline 1-oxide (4NQO) by means of histopathological analysis and gene expression of antioxidant enzymes, such as CuZnSOD, MnSOD and catalase. A total of 30 male Wistar rats were distributed into five groups, as follows (n = 6 per group): Group 1 - negative control group (non-treated group); Group 2 - received 4NQO during 8 weeks in drinking water and treated with apple extract by gavage between the 1st and 4th weeks daily (initiation phase); Group 3 - received 4NQO for 8 weeks in drinking water and treated with apple extract by gavage between the 5th and 8th weeks daily (promotion phase); Group 4 - received apple extract by gavage for eight consecutive weeks only; and Group 5 - received 4NQO for 8 weeks in drinking water daily. Histopathological analysis revealed that apple extract protect oral lesions induced by 4NQO at initiation or promotion phase. Higher gene expression of CuZnSOD and MnSOD enzymes were noticed in groups treated with apple extract as well. Taken together, our results demonstrate that the apple extract is able to modulate medium-term oral carcinogenesis assay as a result of antioxidant activity.

// Xiao-Han Tang 1 , Alison M. Urvalek 1,* , Kwame Osei-Sarfo 1,* , Tuo Zhang 2 , Theresa Scognamiglio 3 and Lorraine J. Gudas 1 1 Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA 2 Genomics Resources Core Facility, Weill Cornell Medical College, New York, NY, USA 3 Department of Pathology, Weill Cornell Medical College, New York, NY, USA * These authors have contributed equally to this work Correspondence to: Lorraine J. Gudas, email: // Keywords : cancer diagnosis, squamous cell carcinoma, tongue lesions, RNA-seq Received : February 10, 2015 Accepted : May 31, 2015 Published : June 10, 2015 Abstract We compared the changes in global gene expression between an early stage (the termination of the carcinogen treatment and prior to the appearance of frank tumors) and a late stage (frank squamous cell carcinoma (SCC)) of tongue carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral cavity and esophageal squamous cell carcinoma. Gene ontology and pathway analyses show that increases in “cell cycle progression” and “degradation of basement membrane and ECM pathways” are early events during SCC carcinogenesis and that changes in these pathways are even greater in the actual tumors. Myc, NFκB complex (NFKB1/RELA), and FOS transcription networks are the major transcriptional networks induced in early stage tongue carcinogenesis. Decreases in metabolism pathways, such as in “tricarboxylic acid cycle” and “oxidative phosphorylation”, occurred only in the squamous cell carcinomas and not in the early stages of carcinogenesis. We detected increases in ALDH1A3, PTGS2, and KRT1 transcripts in both the early and late stages of carcinogenesis. The identification of the transcripts and pathways that change at an early stage of carcinogenesis provides potentially useful information for early diagnosis and for prevention strategies for human tongue squamous cell carcinomas.

The aim of this study was to highlight the role of 1α,25-dihydroxyvitamin D3 (calcitriol) in esophageal squamous cell carcinoma (SCC). The human esophageal SCC cell lines CE81T and TE2 were selected for cellular and animal experiments to investigate the changes in tumor behavior after calcitriol supplementation and the underlying mechanisms. Moreover, we evaluated the relationship between calcitriol supplementation, myeloid-derived suppressor cell (MDSC) recruitment, IL6 levels, and tumor progression by a 4-nitroquinoline 1-oxide (4-NQO)–induced esophageal tumor animal model. In this study, we demonstrated that calcitriol supplementation inhibited aggressive tumor behavior both in vitro and in vivo. The underlying changes included increased cell death, a lower degree of epithelial–mesenchymal transition, and inhibited IL6 signaling. In the 4-NQO–induced esophageal tumor animal model, increased IL6 and MDSC recruitment were linked with invasive esophageal tumors. Supplementation with calcitriol attenuated the level of IL6, the induction of MDSCs, and the incidence of 4-NQO–induced invasive tumors. Moreover, the IL6-induced changes in C57 mice, including augmented MDSC recruitment, increased levels of ROS and p-Stat3 in MDSCs, and higher suppressive function of MDSCs in T-cell proliferation, which were abrogated by calcitriol supplementation. On the basis of our results, we concluded that calcitriol abrogated the IL6-induced aggressive tumor behavior and MDSC recruitment to inhibit esophageal tumor promotion. Therefore, we suggest that supplementation with vitamin D3 may be a promising strategy for the prevention and treatment of esophageal SCC. Mol Cancer Ther; 14(6); 1365–75. ©2015 AACR.

Oral and oropharyngeal cancers are the sixth most common cancers worldwide. Despite intensive investigation, oral squamous cell carcinomas (OSCC) represent a clinical challenge resulting in significant morbidity and mortality. Resistance to cell death is common in OSCC and is often mediated by the Bcl-2 family proteins. Among all anti-apoptotic Bcl-2 family members, Mcl-1 functions as a major survival factor, particularly in solid cancers. Despite the confirmed importance of Mcl-1 in several neoplasms, the role of Mcl-1 in OSCC survival has yet to be explored. In this study, we found that knocking down Mcl-1 sensitized OSCC cells to ABT-737, which binds to Bcl-2/Bcl-xL but not Mcl-1. We report for the first time that a BH3 mimetic, Sabutoclax, which functions as an inhibitor of all anti-apoptotic Bcl-2 proteins, induced cancer-specific cell death in an Mcl-1-dependent manner through both apoptosis and toxic mitophagy. In vivo studies demonstrated that Sabutoclax alone decreased tumor growth in a carcinogen-induced tongue OSCC mouse model. In a combination regimen, Sabutoclax and COX-2 inhibitor, Celecoxib, synergistically inhibited the growth of OSCC in vitro and also significantly reduced OSCC tumor growth in vivo. Overall, these results identify Mcl-1 as a therapeutic prospective target in OSCC.

Oral cancer is one of the most common and lethal cancers in the world. Combination chemotherapy coupled with nanoparticle drug delivery holds substantial promise in cancer therapy. This study aimed to evaluate the efficacy and safety of two dosages of our novel pH and temperature sensitive doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NPs) with attention to the MMP-2 mRNA profile in a 4-nitroquinoline-1-oxide induced oral squamous cell carcinoma (OSCC) model in the rat. Our results showed that both IV and oral dosages of DOX-MTX NP caused significant decrease in mRNA levels of MMP-2 compared to the untreated group (p0.003). Surprisingly, MMP-2 mRNA was not affected in DOX treated compared to cancer group (p0.05). Our results indicated that IV dosage of MTX-DOX is more effective than free DOX (12 fold) in inhibiting the activity of MMP-2 in OSCCs (P0.001). Furthermore, MMP-2 mRNA expression in the DOX-MTX treated group showed a significant relation with histopathological changes (P=0.011). Compared to the untreated cancer group, we observed no pathological changes and neither a significant alteration in MMP-2 amount in either of healthy controls that were treated with oral and IV dosages of DOX-MTX NPs whilst cancer group showed a high level of MMP-2 expression compared to healthy controls (p0.001).Taking together our results indicate that DOX- MTX NPs is a safe chemotherapeutic nanodrug that its oral and IV forms possess potent anti-cancer properties on aggressive tumors like OSCC, possibly by affecting the expression of genes that drive tumor invasion and metastasis.

Background: Squamous cell carcinoma (SSC) of the head and neck is the sixth most common cancer and is rarely diagnosed in early stages. The transcription factor Krvppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation. Inducible mice carrying an oral-specific ablation of Klf4 (K14-CreERtam/Klf4flox/flox) develop mild dysplastic lesions and abnormal differentiation in the tongue. Aiming to analyze whether Klf4 cooperate in oral chemical carcinogenesis,we applied 4-nitroquinoline 1-oxide (4NQO), a tobacco surrogate, to this conditional Klf4 knockout mice. Methods: K14-CreERtam/Klf4flox/flox and control mice were treated with 4NQO for 16 weeks and monitored until week 30. Histopathological samples were used for diagnostic purposes and immunofluorescence detection of epithelial differentiation markers. Results: 4NQO-treated K14-CreERtam/Klf4flox/flox mice (Klf4KO 4NQO) showed a significant weight loss and developed more severe dysplastic lesions than control mice with 4NQO (P < 0.005). The Klf4KO 4NQO showed a tendency to higher incidence of oral SCC and a marked keratinization pattern in dysplasias, in situ carcinomas and SCC. Also, tongues derived from Klf4KO 4NQO mice exhibited reduced terminal differentiation as judged by cytokeratin 1 staining when compared with 4NQO-treated controls. Conclusions: Klf4 ablation results in more severe dysplastic lesions in oral mucosa, with a tendency to higher incidence of SCC, after chemical carcinogenesis. We show here, in a context similar to the human carcinogenesis, that absence of Klf4 accelerates carcinogenesis and correlates with the absence of cytokeratin 1 expression. These results suggest a potential role for KLF4 as a tumor suppressor gene for the tongue epithelium. Fil: Paparella, María L.. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Anatomía Patológica; Argentina Fil: Abrigo, Marianela. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentina Fil: Bal de Kier Joffe, Elisa. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentina Fil: Raimondi, Ana Rosa. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Heat shock protein 90 (Hsp90), a potential therapeutic target, has been widely recognized in vitro and in vivo in immunodeficient mice. Here, we aimed to evaluate the role of Hsp90 in an immunocompetent mouse model of esophageal squamous cell cancer (ESCC).The carcinogen 4-nitroquinoline 1-oxide (4NQO) was used to induce ESCC in C57BL/6 mice. Cancer progression was analyzed through observation of appearance, hematoxylin-eosin staining, immunohistochemical detection, and terminal dUTP nick-end labeling analysis.4NQO led to the progressive appearance of preneoplastic and tumoral lesions in the esophagus, with 100 % incidence of ESCC in situ occurring only after 16 weeks of carcinogen exposure. Most of these lesions evolved spontaneously into highly invasive ESCC even after 4NQO withdrawal (weeks 16-22). Interestingly, there was marked upregulation of Hsp90 and its client proteins in tumoral lesions at 22 weeks. Hsp90 inhibition by intraperitoneal injection of SNX-2112 over the following 2 weeks downregulated AKT and cyclin D1 expression, leading to significant reduction in tumor incidence and prevention of ESCC progression. Moreover, SNX-2112 treatment decreased proliferating cell nuclear antigen expression and increased the number of apoptotic cells in ESCC tissues.Our in vivo findings support the contribution of Hsp90 to ESCC progression, which was achieved by stimulating apoptosis and inhibition of cell proliferation, and provide a strong rationale for further evaluation of Hsp90 inhibitors for treating ESCC.

Lactic acid bacteria (LAB) and yeasts, representing the prevailing microbiota associated with different foods generally consumed without any cooking, were identified and characterized in vitro for some functional properties, such as acid-bile tolerance and antigenotoxic activity. In particular, 22 Lactobacillus plantarum strains and 14 yeasts were studied. The gastro-intestinal tract tolerance of all the strains was determined by exposing washed cell suspensions at 37°C to a simulated gastric juice (pH 2.0), containing pepsin (0.3% w/v) and to a simulated small intestinal juice (pH 8.0), containing pancreatin (1 mg mL-1) and bile extract (0.5%), thus monitoring changes in total viable count. In general, following a strain-dependent behaviour, all the tested strains persisted alive after combined acid-bile challenge. Moreover, many strains showed high in vitro inhibitory activity against a model genotoxin, 4-nitroquinoline-1-oxide (4-NQO), as determined by the short-term method, SOS-Chromotest. Interestingly, the supernatants from bacteria- or yeasts-genotoxin co-incubations exhibited a suppression on SOS-induction produced by 4-NQO on the tester strain Escherichia coli PQ37 (sfiA::lacZ) exceeding, in general, the value of 75%. The results highlight that food associated microorganisms may reach the gut in viable form and prevent genotoxin DNA damage in situ. Our experiments can contribute to elucidate the functional role of food-associated microorganisms general recognized as safe (GRAS) ingested with foods as a part of the diet.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and tobacco is one of the most common factors for HNSCC of the oral cavity. We have previously observed that bitter melon (Momordica charantia) extract (BME) exerts antiproliferative activity against several cancers including HNSCC. In this study, we investigated the preventive role of BME in 4-nitroquinoline 1-oxide (4-NQO) carcinogen-induced HNSCC. We observed that BME feeding significantly reduced the incidence of 4-NQO–induced oral cancer in a mouse model. Histologic analysis suggested control 4-NQO–treated mouse tongues showed neoplastic changes ranging from moderate dysplasia to invasive squamous cell carcinoma, whereas no significant dysplasia was observed in the BME-fed mouse tongues. We also examined the global transcriptome changes in normal versus carcinogen-induced tongue cancer tissues, and following BME feeding. Gene ontology and pathway analyses revealed a signature of biological processes including “immune system process” that is significantly dysregulated in 4-NQO–induced oral cancer. We identified elevated expression of proinflammatory genes, s100a9, IL23a, IL1β and immune checkpoint gene PDCD1/PD1, during oral cancer development. Interestingly, BME treatment significantly reduced their expression. Enhancement of MMP9 (“ossification” pathway) was noted during carcinogenesis, which was reduced in BME-fed mouse tongue tissues. Our study demonstrates the preventive effect of BME in 4-NQO–induced carcinogenesis. Identification of pathways involved in carcinogen-induced oral cancer provides useful information for prevention strategies. Together, our data strongly suggest the potential clinical benefits of BME as a chemopreventive agent in the control or delay of carcinogen-induced HNSCC development and progression. Cancer Prev Res; 11(4); 191–202. ©2017 AACR. See related editorial by Rao, p. 185

Made available in DSpace on 2018-12-11T17:16:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-01 Evidence show that stress hormones can influence cancer progression, but its role in carcinogenesis is poorly understood. In this study, we used a new method based on oral carcinogenesis model in rats to test the hypothesis that physiological levels of stress hormones in the normal tissue microenvironment would have significant predictive value for chemically induced cancer occurrence. Male Wistar rats were submitted to a tongue biopsy for measuring not-stress induced levels of norepinephrine, corticosterone, adrenocorticotropic hormone (ACTH) and brain-derived neurotrophic factor (BDNF) in the tissue before carcinogenic induction. Rats were treated with the 4-nitroquinoline-1-oxide (4NQO) chemical carcinogen for twenty weeks and then euthanized for microscopic evaluation of the tongue lesions. Increased pre-carcinogen norepinephrine concentrations and reduced basal corticosterone levels in the normal tissue microenvironment were predictive for oral squamous cell carcinoma (OSCC) occurrence. Likewise, increased pre-carcinogen norepinephrine levels in the normal microenvironment were associated a lower expression of pCDKN2a-p16 in OSCCs. Post-carcinogen levels of corticosterone and BDNF in oral leukoplakia tissues (precursor lesion of OSCC) and post-carcinogen corticosterone concentrations in OSCCs were higher than basal levels in the normal mucosa. Increased norepinephrine concentrations in OSCCs were associated to a greater tumor volume and thickness. Furthermore, higher levels of norepinephrine, ACTH and BDNF in OSCCs were associated to a lesser intensity of the lymphoplasmocytic infiltrate. This study shows that pre-carcinogen stress hormones levels in the normal microenvironment may be predictive for chemically induced cancer in rats. Moreover, chemical carcinogenesis can promote stressor-like effects with hormonal changes in the tissue microenvironment, which may be associated to tumor progression. Psychoneuroimmunology Research Center Oral Oncology Center São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Department of Animal Clinic Surgery and Reproduction São Paulo State University (Unesp) School of Veterinary Medicine, 793 Clóvis Pestana St. Department of Statistics Oral Oncology Center São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Department of Pathology and Clinical Propedeutics São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Department of Biomedical Statistics and Informatics Mayo Clinic College of Medicine, 200 First St. SW Immunopharmacology Laboratory Department of Basic Sciences São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Psychoneuroimmunology Research Center Oral Oncology Center São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Department of Animal Clinic Surgery and Reproduction São Paulo State University (Unesp) School of Veterinary Medicine, 793 Clóvis Pestana St. Department of Statistics Oral Oncology Center São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Department of Pathology and Clinical Propedeutics São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St. Immunopharmacology Laboratory Department of Basic Sciences São Paulo State University (Unesp) School of Dentistry, 1193 José Bonifácio St.

This study evaluated the effect of an experimental carcinogenic, 4-Nitroquinoline 1-oxide (4NQO), in the spontaneous alveolar bone loss (ABL) in an animal model.Twenty-two male Wistar rats were included in this study. They were randomly divided into two groups: the control group (n = 10) received food and water ad libitum, and the test group (n = 12) receive the same food; however, 25 ppm of 4NQO was diluted in the drinking water. All animals were euthanized after 20 weeks, and the tongues were removed and analyzed macroscopically to determine the presence of oral mucosal lesions. All specimens were paraffin-embedded and histological sections were obtained. The microscopic analysis was based on routine procedure (haematoxylin and eosin stain). The analysis of spontaneous ABL was performed by a calibrated examiner using standardized photographs and imaging software. Differences in spontaneous ABL were assessed among the three resulting groups: control, 4NQO with oral squamous cell carcinoma (OSCC), and 4NQO without OSCC.In the 4NQO-treated group, nine animals developed OSCC. The animals in the 4NQO with OSCC group presented significantly more spontaneous ABL (0.65 ± 0.21 mm) than the control group (0.34 ± 0.05) (p 0.001). The animals in the 4NQO without OSCC group showed a mean spontaneous ABL of 0.47 ± 0.13 mm, which was not statistically significant different when compared to the control group (p = 0.096).It was concluded that the presence of OSCC enhanced spontaneous ABL in Wistar rats when compared to control animals. Additionally, it was shown that, solely, administration of 4NQO may not be considered responsible for alveolar bone destruction.

Cancer arising in the oral cavity is one of the major causes of mortality worldwide and demands immediate attention. Regardless of the use of advanced treatment for oral cancer, successful treatment resulting in cancer survival is low. Currently available drugs are ineffective and are toxic. Therefore, successful treatment without toxic effects remains essential. This is quite challenging, leading to the identification of natural bioactive compounds for oral cancer treatment. Thus, a plant extract rich in phenolics is preferred for studying the cellular, biochemical and molecular changes associated with oral carcinogenesis.The present study aims to deal with the above need using Acacia nilotica (L.) leaf extract (AN) and ethyl gallate (EG), a phenolic compound present in AN against oral carcinogenesis. Extension of a tumor cell line to a mouse model was investigated with 4-nitroquinoline 1-oxide (4-NQO) as carcinogen, a surrogate for tobacco. The progression of squamous cell carcinoma (SCC) was achieved through hyperplasia and dysplasia after 4-NQO induction in Swiss albino mice. Administration of AN and EG to animals undergoing dysplasia led to the inhibition of SCC, thereby reducing the tumor burden. The antioxidant capacity of AN and EG was also brought out via biochemical analysis. Further investigation of biomarkers in tongue tissues revealed the involvement of apoptosis in vivo Moreover, no adverse or toxic effect was observed earlier in rats upon oral administration of AN and EG. Thus, AN and EG shows strong hope as drugs against oral cancer progression.

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Oral squamous cell carcinoma (OSCC) is preceded by progressive oral premalignant lesions (OPL). Therefore, therapeutic strategies that prevent malignant progression of OPLs are expected to reduce the incidence of OSCC development. Immune checkpoint inhibitors that target the interaction of programmed death receptor 1 (PD-1) on T cells with the PD-1 ligand PD-L1 on cancer cells have been shown to extend the survival of patients with advanced OSCC. Here, we used the 4-nitroquinoline-1-oxide (4-NQO) mouse model of oral carcinogenesis to test the hypothesis that PD-1 blockade may control the progression of OPLs. Mice were exposed to 4-NQO in their drinking water and then randomly assigned to two treatment groups that received either a blocking antibody for PD-1 or a control IgG. We found that anti–PD-1 treatment significantly reduced the number of oral lesions that developed in these mice and prevented malignant progression. Low-grade dysplastic lesions responded to PD-1 blockade with a significant increase in the recruitment of CD8+ and CD4+ T cells and the accumulation of CTLA-4+ T cells in their microenvironment. Notably, PD-1 inhibition was accompanied by induction of IFNγ, STAT1 activation and the production of the T-cell effector granzyme B in infiltrating cells, and by the induction of apoptosis in the epithelial cells of the oral lesions, suggesting that T-cell activation mediates the immunopreventive effects of anti–PD-1. These results support the potential clinical benefit of PD-1 immune checkpoint blockade to prevent OSCC development and progression and suggest that CTLA-4 inhibitors may enhance the preventive effects of anti–PD-1. Cancer Prev Res; 10(12); 684–93. ©2017 AACR. See related editorial by Gutkind et al., p. 681

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Agradecemos a compreensão. on 2017-04-19T13:25:38Z (GMT) Submitted by JOSÉ NUNES CARNEIRO NETO null (jnunes.neto@yahoo.com.br) on 2017-04-19T20:06:34Z No. of bitstreams: 2 Dissertação.pdf: 1588988 bytes, checksum: a12b89295d9e4983ec344310cf458241 (MD5) Unlock-Dissertação.pdf: 1240866 bytes, checksum: a818a052abc232b34707b0fda4478055 (MD5) Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-19T20:19:37Z (GMT) No. of bitstreams: 1 carneironeto_jn_me_arafo_par.pdf: 376680 bytes, checksum: 0ea3c86490741209e38474ee45e1835c (MD5) Made available in DSpace on 2017-04-19T20:19:37Z (GMT). No. of bitstreams: 1 carneironeto_jn_me_arafo_par.pdf: 376680 bytes, checksum: 0ea3c86490741209e38474ee45e1835c (MD5) Previous issue date: 2017-03-14 O terpinen-4-ol (TP-4-ol) é um monoterpeno que constitui de 30-48% dos quimiotipos de óleos essenciais de plantas, como a Melaleuca alternifólia, laranjeira, pimenteira entre outras, apresentando propriedade anti-inflamatória, anticancerígena entre outras, caracterizadas como agentes quimiopreventivos e citoprotetores, respectivamente, contra as lesões com potencial de malignização e/ou malignas (LPMM) de língua e as hepato ou nefrotoxicidade. Sendo assim, o objetivo de nosso estudo foi avaliar o TP-4-ol quanto às propriedades quimiopreventivas sobre LPMM de língua e citoprotetoras ou citotóxicas sobre o fígado e rim de ratos quimicamente induzidos por óxido de 4-nitroquinolina (4-NQO). Além disso, avaliamos o TP-4-ol quanto ao seu potencial indutor de hepato e nefrotoxicidade em ratos saudáveis. Os 64 ratos foram divididos aleatoriamente para o experimento de 16 semanas, nos grupo 1 ratos saudáveis ou grupo 2 ratos oncológicos de língua (n=8) e os grupos de intervenções com [g/Kg de TP-4-ol] ratos saudáveis tratados [0,035] do grupo 3 e [0,0035] grupo 4, ratos oncológicos tratados [0,035] do grupo 5 e [0,0035] grupo 6 (n=12). Foram analisados os parâmetros biométricos (peso do corpo, língua, fígado e rim; consumo de água e ração); bioquímicos do fígado (alanina aminotransferase, aspartato aminotransferase, fosfatase alcalina) e rim (ureia e creatinina); macroscópicos da língua (tamanho e lesões fundamentais), fígado e rim (tamanho); e microscópicos da língua (alterações histopatológicas, risco de malignização da displasia moderada, invasão tumoral e infiltrado inflamatório), fígado e rim (alterações citotóxicas). Os resultados foram comparados entre médias e desvio-padrão por ANOVA com nível de significância de 5%. As análises dos 63 ratos demonstraram aumento do peso corporal e consumo de água com 0,0035g/Kg de TP-4-ol do grupo 6, que também reduziu os parâmetros bioquímicos, lesões fundamentais, alterações histopatológicas, o alto risco de malignização, carcinoma lingual em mais de 70% (in situ, micro-invasivo e invasivo) e o infiltrado inflamatório. A dose de 0,035g/Kg de TP-4-ol do grupo 3 é tóxica nos parâmetros analisados com exceção do microscópico do fígado. Conclui-se que o TP-4-ol é eficaz na quimioproteção (0,035/0,0035g/Kg - quimioprevenção) de lesões com potencial de malignização e/ou malignas de língua, citoprotetora (0,0035g/Kg) de hepato e nefrotoxicidade de ratos oncológicos e indutora de leve nefrotoxicidade (0,035g/Kg) em ratos saudáveis. Terpinen-4-ol is a monoterpene constituting 30-48% of the plants essential oils chemotypes, such as Melaleuca alternifolia, orange tree, peppermint among others, presenting anti-inflammatory, anticancer property among others, characterized as chemopreventive agents and cytoprotectants, respectively, against lesions with malignant and/or malignization potential of tongue and hepato or nephrotoxicity. Therefore, the aim of our study was to evaluate TP-4-ol for chemopreventive properties on tongue and cytoprotective or cytotoxic LPMM on the liver and kidney of 4-nitroquinoline 1-oxide (4-NQO) chemically induced rats. In addition, we evaluated TP-4-ol as to its potential for inducing hepate and nephrotoxicity in healthy rats. The 64 rats were randomly divided to the 16-week experiment, in group 1 healthy rats or group 2 oncological rats of tongue (n = 8) and groups of interventions with [g/kg of TP-4-ol] treated healthy rats [0.035] of group 3 and [0.0035] group 4, treated oncological rats [0.035] of group 5 and [0.0035] group 6 (n = 12). Were analyzed the biometric parameters (weight of body, tongue, liver and kidney, water and feed intake); biochemical of liver (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase) and kidney (urea and creatinine); macroscopic of tongue (size and fundamental lesions), liver and kidney (size); and microscopic of the tongue (histopathological alteration, risk of malignization of moderate dysplasia, tumor invasion and inflammatory infiltrate), liver and kidney (cytotoxic alterations). The results were compared between means and standard deviation by ANOVA with a significance level of 5%. The analysis of the 63 rats showed an increase in body weight and water consumption with 0.0035g/kg of TP-4-ol from group 6, which also reduced biochemical parameters, fundamental lesions, histopathological alterations, high risk of malignancy, lingual carcinoma in more than 70% (in situ, micro-invasive and invasive) and inflammatory infiltrate. The dose of 0.035g/kg of TP-4-ol of group 3 is toxic in the analyzed parameters except for the microscopic of the liver. It is concluded that TP-4-ol is effective in chemoprotection (0.035/0.0035g/kg - chemoprevention) of lesions with malignant and/or malignization potential of tongue, cytoprotective (0.0035g/kg) of hepate and nephrotoxicity of oncological rats and inductor of mild nephrotoxicity (0.035g/kg) in healthy rats.

OBJECTIVE:Areca nut chewing is carcinogenic to humans. However, little is known about the impact of areca nut chewing on esophageal squamous cell carcinoma (ESCC). METHODS:We retrospectively reviewed 286 ESCC patients who received surgery or preoperative chemoradiotherapy followed by surgery at our institution. Background characteristics including areca nut chewing history were analyzed. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine ESCC model was used to test the impact of arecoline, a main constituent of areca nut, on ESCC. RESULTS:Compared to patients without areca nut chewing history, patients with areca nut chewing history had overall a younger age of onset (Mean age: 56.75 versus 52.68 yrs, P

The management of epithelial dysplastic spread around an oral squamous cell carci-noma is very important, particularly intraoperatively. Both cytokeratin (CK) 14 and CK19 are believed to be involved in the development of precancerous lesions, and their expression profiles are quite specific in these and early cancer lesions. Here, expression of CK13, 14, 17, and 19 was investigated in a rat model of 4-nitroquinoline-1-oxide-induced tongue cancer during a series of carcinogenetic processes to determine their value in assessing the features of epithelial dysplastic spread around a cancer. Based on tissue conditions, the results showed that expression levels of CK13 and 14 decreased in the order of no change, dysplasia, and cancer, whereas those of CK17 and 19 increased in the same order. Expression of CK13 showed a significant difference among no change, dysplasia, and cancer. This indicates that comparing the immunohistochemical staining profiles of CKs, especially CK13, could help in assessing the characteristics of epithelial dysplastic spread around a cancer.

O câncer bucal representa um problema de saúde pública mundial classificado como o sexto tipo de câncer mais comum. Apresenta índices de mortalidade em cinco anos preocupantes nas últimas décadas. Estima-se que aproximadamente 15% dos canceres bucais tenham causa desconhecida. Estudos publicados recentemente têm sugerido que a doença periodontal pode representar um risco aumentado para o desenvolvimento de câncer bucal. Entretanto, o impacto da doença periodontal sobre a patogênese do câncer bucal in vivo é quase inexistente na literatura. Esta tese avaliou, através de dois artigos, a relação entre a doença periodontal e o câncer bucal. O primeiro artigo da presente tese trata de uma revisão de estudos epidemiológicos sobre a associação da doença periodontal e câncer bucal, assim como estudos atuais a respeito de mecanismos biológicos. Os estudos epidemiológicos na sua maioria apontam a periodontite como um fator de risco independente para o câncer bucal após ajustes estatísticos para diferentes fatores confudindores, como o fumo. Os estudos biológicos tem na sua grande maioria a Porphyromonas gingivalis como agente etiológico para a doença periodontal e estaria envolvida em várias etapas de carcinogênese bucal como a proliferação, migração e invasão celular, assim como a metástase e resistência a agente quimioterapêuticos em estudos in vitro e in vivo a periodontite. O segundo artigo da presente tese teve como objetivo avaliar o efeito da periodontite induzida por meio de ligadura sobre a carcinogênese bucal através da exposição ao carcinógeno 4-Nitroquinolina 1-Óxido (4NQO) em ratos Wistar. Os resultados mostram o aumento da incidência de carcinoma espinocelular na língua de ratos quando na presença de periodontite previa. Além disso, houve um maior tamanho tumoral e uma menor diferenciação celular através da expressão de citoqueratina 10 nas línguas dos animais. Os resultados desta tese vêm ao encontro do que se encontra na literatura reforçando que a presença da doença periodontal é um importante modificador no desenvolvimento de câncer bucal. Oral cancer is a worldwide public health problem rated as the sixth most common cancer. Its survival rates have remained worrisome in the last decades. It is estimated that approximately 15% of oral cancer have an unknown etiology. Recently published studies have suggested that periodontal disease may represent an increased risk for oral cancer development. However, the impact of periodontal disease on oral carcinogenesis in vivo is almost non-existent. The current thesis evaluated, through two articles, the relationship between periodontal disease and oral cancer. The first article was a review of epidemiological studies about the association of periodontal disease and oral cancer, as well as current studies about biologic mechanisms. Periodontal disease and oral cancer share many common established risk factors, but after adjusting for different confounding factors, periodontal disease seems to be an independent risk for oral cancer in most of epidemiologies studies. Studies focusing in biological mechanisms of the relationship between both diseases mainly focus in P.gingivalis. P. gingivalis seems to be involved with various steps of oral carcinogenesis as proliferation, migration invasion, aggressiveness and also metastasis and resistance to anti-cancer agents in vitro and in vivo studies. The second article of the current thesis aimed to evaluate the effect of induced-periodontitis by ligature on the experimental oral carcinogenesis induced by the chemical 4- nitroquinoline 1-oxide (4NQO) in Wistar rats. The results showed an increased incidence of squamous cell carcinoma in the rats’ tongues when in presence of previous periodontitis. The results of this thesis corroborate what we may find in the literature, reinforcing that the presence of periodontal disease act as an important modifier to oral cancer.

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

The aim of the present study was to identify time-dependent changes in the expression of metabolic biomarkers during the various stages of oral carcinogenesis to provide an insight into the sequential mechanism of oral cancer development. An 1H nuclear magnetic resonance (NMR)-based metabolomics approach was used to analyze the blood plasma samples of Sprague-Dawley rats exhibiting various oral lesions induced by the administration of 4-nitroquinoline-1-oxide (4NQO) in drinking water. The 1H NMR spectra were processed by principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) to determine the metabolic differences between the three developmental stages of oral mucosa cancer (health, oral leukoplakia [OLK] and oral squamous cell carcinoma [OSCC]). The variable importance in projection (VIP) score derived from the PLS-DA model was used to screen for important metabolites, whose significance was further verified through analysis of variance (ANOVA). Data from the present study indicated that 4NQO-induced rat oral carcinogenesis produced oral pre-neoplastic and neoplastic lesions and provided an effective model for analyzing sequential changes in the 1H NMR spectra of rat blood plasma. The 1H NMR-based metabolomics approach clearly differentiates between healthy, OLK and OSSC rats in the PCA and PLS-DA models. Furthermore, lactic acid, choline, glucose, proline, valine, isoleucine, aspartic acid and 2-hydroxybutyric acid demonstrated VIP>1 in the PLS-D model and P

1 Pathology, 2 Psichobiology, 3 ABSTRACT Background: The aim of this study was to evaluate whether paradoxical sleep deprivation could affects the mechanisms and pathways essentials for cancer cells in tongue cancer induced by 4‑nitroquinole 1‑oxide in Wistar rats. Materials and Methods: For this purpose, 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 paradoxical sleep deprivation (PSD) for 72 h using the modified multiple platform method, which consisted of placing 5 mice 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 histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre‑neoplasic lesions. Data analysis revealed statistically significant differences (P < 0.05) in 4 weeks group for p53 and for bcl‑2 and for all immunomarkers after 12 weeks of 4NQO administration. Conclusion: Our results reveal that sleep deprivation exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis. AQ1

Oral squamous cell carcinoma (OSCC) remains as one of the most difficult malignancies to control because of its high propensity for local invasion and cervical lymph node dissemination. The aim of present study was to evaluate the efficacy of novel pH and temperature sensitive doxorubicin-methotrexate- loaded nanoparticles (DOX-MTX NP) in terms of their potential to change the VEGF-C expression profile in a rat OSCC model.120 male rats were divided into 8 groups of 15 animals administrated with 4-nitroquinoline-1-oxide to induce OSCCs. Newly formulated doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NP) and free doxorubicin were IV and orally administered.RESULTS indicated that both oral and IV forms of DOX-MTX- nanoparticle complexes caused significant decrease in the mRNA level of VEGF-C compared to untreated cancerous rats (p0.05) . Surprisingly, the VEGF-C mRNA was not affected by free DOX in both IV and oral modalities (p0.05). Furthermore, in DOX-MTX NP treated group, less tumors characterized with advanced stage and VEGF-C mRNA level paralleled with improved clinical outcome (p0.05). In addition, compared to untreated healthy rats , the VEGF-C expression was not affected in healthy groups that were treated with IV and oral dosages of nanodrug (p0.05).VEGF-C is one of the main prognosticators for lymph node metastasis in OSCC. Down-regulation of this lymph-angiogenesis promoting factor is a new feature acquired in group treated with dual action DOX-MTX-NPs. Beside the synergic apoptotic properties of concomitant use of DOX and MTX on OSCC, DOX-MTX NPs possessed anti-angiogenesis properties which was related to the improved clinical outcome in treated rats. Taking together, we conclude that our multifunctional doxorubicin-methotrexate complex exerts specific potent apoptotic and anti-angiogenesis properties that could ameliorate the clinical outcome presumably via down-regulating dissemination factor-VEGF-C expression in a rat OSCC model.