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Although oxidative stress is closely associated with tumor invasion and metastasis, its' exact role and mechanism in the initial stage of oral cancer remain ambiguous. Glutamine uptake mediated by alanine-serine-cysteine transporter 2 (ASCT2) participates in glutathione synthesis to resolve oxidative stress. Currently, we firstly found that ASCT2 deletion caused oxidative stress in oral mucosa and promoted oral carcinogenesis induced by 4-Nitroquinoline-1-oxide (4-NQO) using transgenic mice of ASCT2 knockout in oral epithelium. Subsequently, we identified an upregulated gene Thbs1 linked to macrophage infiltration by mRNA sequencing and immunohistochemistry. Importantly, multiplex immunohistochemistry showed M1-like tumor-associated macrophages (TAMs) were enriched in cancerous area. Mechanically, targeted ASCT2 effectively curbed glutamine uptake and caused intracellular reactive oxygen species (ROS) accumulation, which upregulated Thbs1 in oral keratinocytes and then activated p38, Akt and SAPK/JNK signaling to polarize M1-like TAMs via exosome-transferred pathway. Moreover, we demonstrated M1-like TAMs promoted malignant progression of oral squamous cell carcinoma (OSCC) both in vitro and in vivo by a DOK transformed cell line induced by 4-NQO. All these results establish that oxidative stress triggered by ASCT2 deletion promotes oral carcinogenesis through Thbs1-mediated M1 polarization, and indicate that restore redox homeostasis is a new approach to prevent malignant progression of oral potentially malignant disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaoan Tao reports was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

miR-504 plays a pivotal role in the progression of oral cancer. However, the underlying mechanism remains elusive in vivo. Here, we find that miR-504 is significantly down-regulated in oral cancer patients. We generate miR-504 knockout mice (miR-504 -/- ) using CRISPR/Cas9 technology to investigate its impact on the malignant progression of oral cancer under exposure to 4-Nitroquinoline N-oxide (4NQO). We show that the deletion of miR-504 does not affect phenotypic characteristics, body weight, reproductive performance, and survival in mice, but results in changes in the blood physiological and biochemical indexes of the mice. Moreover, with 4NQO treatment, miR-504 -/- mice exhibit more pronounced pathological changes characteristic of oral cancer. RNA sequencing shows that the differentially expressed genes observed in samples from miR-504 -/- mice with oral cancer are involved in regulating cell metabolism, cytokine activation, and lipid metabolism-related pathways. Additionally, these differentially expressed genes are significantly enriched in lipid metabolism pathways that influence immune cell infiltration within the tumor microenvironment, thereby accelerating tumor development progression. Collectively, our results suggest that knockout of miR-504 accelerates malignant progression in 4NQO-induced oral cancer by regulating tumor cell proliferation and lipid metabolism, affecting immune cell infiltration., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in this work., (Copyright © 2024. Published by Elsevier Inc.)

Background and Objectives : PIN1 is overexpressed in several human cancers, including prostate cancer, breast cancer, and oral squamous carcinomas. Juglone (J), derived from walnut, was reported to selectively inhibit PIN1 by modifying its sulfhydryl groups. In this study, the potential effects of juglone, also known as PIN1 inhibitor, on oral cancer and carcinogenesis were investigated at the molecular level. Materials and Methods : 4-Nitroquinoline N-oxide (4-NQO) was used to create an oral cancer model in animals. Wistar rats were divided into five groups: Control, NQO, Juglone, NQO+J, and NQO+J*. The control group received the basal diet and tap water throughout the experiment. The NQO group received 4-NQO for 8 weeks in drinking water only. The Juglone group was administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). The NQO+J group received 4-NQO in drinking water for 8 weeks, starting 1 week after the cessation of 4-NQO treatment. They were then administered intraperitoneally in a juglone solution for 10 weeks. (1 mg/kg/day). NQO+J* group: received 4 NQO for 8 weeks in drinking water and administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). They were sacrificed at the end of the 22-week experimental period. The tongue tissues of the rats were isolated after the experiment, morphological changes were investigated by histological examinations, and the molecular apoptotic process was investigated by rt-qPCR and western blot. Results : Histological results indicate that tumors are formed in the tongue tissue with 4-NQO, and juglone treatment largely corrects the epithelial changes that developed with 4-NQO. It has been determined that apoptotic factors p53, Bax, and caspases are induced by the effect of juglone, while antiapoptotic factors such as Bcl-2 are suppressed. However, it was observed that the positive effects were more pronounced in rats given juglone together with 4-NQO. Conclusions : The use of PIN1 inhibitors such as juglone in place of existing therapeutic approaches might be a promising and novel approach to the preservation and treatment of oral cancer and carcinogenesis. However, further research is required to investigate the practical application of such inhibitors.

Autophagy has been proposed to play a dual role in cancer-as a tumor suppressor in early stages and oncogenic in late stages of tumorigenesis. This study investigated the role of autophagy in oral carcinogenesis using the model of oral squamous cell carcinoma (OSCC) induced by carcinogen 4-nitroquinoline 1-oxide (4NQO), mimicking molecular and histopathologic aspects of human OSCC. The induction of autophagy by spermidine (SPD) treatment reduced the severity of lesions and the incidence of OSCC in mice exposed to 4NQO. On the other hand, autophagy inhibition by chloroquine treatment had no protection. The comet assay indicated that SPD reduced 4NQO-induced DNA damage, likely related to the activation of DNA repair and the decrease of reactive oxygen species. As sphingolipid alterations have been reported in OSCC, sphingolipids in the tongue and plasma of animals were analyzed and plasma C16 ceramide levels were shown to increase proportionally to lesion severity, indicating its potential as a biomarker. Mice exposed to 4NQO plus SPD had lower levels of C16 ceramide than the 4NQO group, which indicated SPD's ability to prevent the 4NQO-induced carcinogenesis. Together, these data indicate that activation of autophagy has a tumor suppressor role during the early stages of oral carcinogenesis. Because of its ability to induce autophagy accompanied by reduced oxidative stress and DNA damage, SPD may have a protective action against chemically induced oral cancer., Competing Interests: Disclosure Statement None declared., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

4-Nitroquinoline N -oxide (4-NQO) and its derivatives react with genomic DNA to form stable quinolone monoadducts, which are highly mutagenic and genotoxic. While the chronic high-dose exposure of epithelial cells to a carcinogen such as 4-NQO leads to tumor development, its effect on other cells has not been explored yet. Since the immunosuppression due to aberrant immunological profile is recognized as a significant cause in tumors, here we determine the interaction between 4-NQO and immune cells both in vivo and in vitro , and its effect on oral squamous cell carcinoma (OSCC) progression in a murine model. Immune cell profiling of the spleen and peripheral blood revealed a significant decrease in the B-cell population in 4-NQO-exposed mice than the untreated group. Additionally, γδ T and CD5 + B lymphocyte populations decreased at both pre- and post-cancerous stages of OSCC. These results suggested that 4-NQO induced tumor transition from pre-malignant lesions to OSCC by altering certain immune cells systemically. Next, to establish the effect of 4-NQO on immune cells, human B- and T-cell lines were subjected to 4-NQO; the reduction in cell viability, increase in DNA damage response marker, and induction of apoptosis were more pronounced in B than T cells. Altogether, our results indicated that in addition to the genotoxicity of oral epithelial cells, 4-NQO potentiates long-range effects on specific immune cells to induce cell death to cause very-early immunosuppressive response during oral carcinogenesis, and thus immunosuppression and tumor development are coevolved., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Sahu, Thakur, Peroumal, Utkalaja, Dutta, Kumari, Subhadarsini and Acharya.)

An important rat model using the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) has been described for the study of the process of oral carcinogenesis. This model replicates the gradual progression seen in oral carcinoma patients. However, due to its high level of toxicity, its use in fundamental research is challenging. Here, we propose a secure and efficient modified protocol based on a lower dose of 4NQO concentration as well as an increased water supply and hypercaloric diet, in order to reduce the damage caused to the animals during the process of oral carcinogenesis. Twenty-two male Wistar rats were exposed to 4NQO, evaluated clinically once a week and euthanized at 12 and 20 weeks for histopathological analysis. The protocol involves a staggered dose of 4NQO up to a concentration of 25 ppm, associated with two days of pure water, a 5% glucose solution once a week and a hypercaloric diet. This modified protocol prevents the immediate consequences of the carcinogen. At week 7, all animals displayed clinically evident tongue lesions. From a histological perspective, after 12 weeks of 4NQO exposure, 72.7% of the animals developed epithelial dysplasia and 27.3% developed in situ carcinoma. In the group exposed for 20 weeks, epithelial dysplasia and in situ carcinoma were diagnosed in one case each, whereas invasive carcinoma was diagnosed in 81.8% of the cases. Nonsignificant modification of animal's behavior and weight was observed. This new proposed 4NQO protocol was secure and effective for studying oral carcinogenesis and can be used to conduct lengthy investigations., Competing Interests: Declaration of Competing Interest none., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Head and neck cancer is the sixth most common malignancy, and there is an urgent need to identify physiological processes contributing to tumorigenesis. Extracellular acidification caused by aerobic glycolysis within tumor microenvironments can stimulate proton-sensing receptors. GPR68, or ovarian cancer G protein-coupled receptor 1, responds to extracellular acidity and is highly expressed in head and neck squamous cell carcinoma (HNSCC) as well as normal esophageal tissue. To study the role of GPR68 in oral dysplasia, wild-type and GPR68 -/- mice were treated with 4-Nitroquinoline N-oxide (4NQO) in drinking water for 11-13 weeks, followed by normal water for 11-12 weeks. 4NQO treatment resulted in 45 percent of GPR68 -/- mice developing severe dysplasia or squamous cell carcinoma compared to only 10.5 percent of GPR68 +/+ mice. This correlated with increased frequencies of regulatory T cells in the spleens of male GPR68 -/- mice. Dysplastic regions of the tongue had increased CD31 staining compared to normal regions in both GPR68 -/- and GPR68 +/+ mice, suggesting that angiogenesis was GPR68-independent. RNA knockdown studies using HNSCC cell lines demonstrated no direct effect of GPR68 on survival or growth. Overall, we demonstrate that GPR68-deficiency worsens the severity of chemical-induced oral dysplasia, suggesting a protective role for this gene in tumorigenesis., (© 2023. The Author(s).)

Objective: This study evaluated the effect of laser photobiomodulation (PBM) on oral leukoplakia and squamous cell carcinomas (OSCC) in a model of oral carcinogenesis., Materials and Methods: Forty-one C57Bl/6 female mice were distributed in control group, 4-NQO group, Laser group 1.5 J and Laser group 9 J. Oral cancer was induced on the tongue by nitroquinoline oxide (4-NQO), diluted in the water for 16 weeks. In the 18th and 19th weeks, PBM with a diode laser, 0.028 cm 2 spot size, continuous emission mode, 660 nm wavelength was applied on the tongue of animals for seven sessions. Laser group 1.5 J received 30 mW power and 1.5 J energy. In the Laser group 9 J, 100 mW power, and 9 J energy were applied. In the 20th week the animals were euthanized., Results: All animals exposed to carcinogen developed clinical and histological alterations such as leukoplakia and OSCC on the tongue. There was no significant difference among Laser groups 1.5 and 9 J and 4-NQO group (not irradiated) regarding the area of leukoplakia and carcinomas (P > 0.05) or thickness of epithelial tissue and keratin (P > 0.05). There were also no association between PBM and histologic classification of the lesions (P = 0.87), frequency of OSCC (P = 0.57), grade of tumor differentiation (P = 0.88) or depth of invasion (P = 0.45)., Conclusion: Laser PBM, in both parameters used, does not influence on clinical and histological characteristics of oral leukoplakia and OSCC., Clinical Relevance: Results suggest that PBM may be a safe treatment for adverse effects of antineoplastic therapies in patients with leukoplakia and OSCC., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Our aim was to verify the modulative TP-4-ol capacity in 4-nitroquinoline-1-oxide induced oral rat cancer. The stereoisomers of TP-4-ol were used against the human tongue squamous cell line and the negative stereoisomer showed lower IC50. Thirty-one Holtzman rats (120-130 g) were cancer-induced by 4-nitroquinoline-1-oxide (4-NQO/8 weeks/25 ppm) and 32 Holtzman rats (120-130 g) were used to healthy and TP-4-ol toxicity experiments. Six groups were used, healthy, 0.1nL/g of TP-4-ol, 8nL/g of TP-4-ol, 4-NQO, 4-NQO + 0.1nL/g of TP-4-ol, and 4-NQO + 8nL/g of TP-4-ol. We performed the toxicity analysis by biochemical and histopathological analysis. The biochemistry analysis includes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), urea, and creatinine and the histopathology analysis includes the liver, kidney, lung, and spleen. Specifically, for malign modulation, we performed a macroscopic and microscopic analysis. The group exposed to 0.1nL/g of TP-4-ol demonstrated a reduced risk of malignancy in dysplasia considering the criteria of architecture and cytology. Similarly, a drop of percentual rats with SCC diagnosis was observed in 4-NQO + 0.1nL/g (41.6%) when compared to 4-NQO (87.5%). Moreover, the 4-NQO group presented a median of 2.62 SCC/rat and the 4-NQO + 0.1nL/g demonstrated a median of 0.75 SCC/rat. For toxicity analysis, 4-NQO + 0.1nL/g showed focal necrosis in the kidney and 4-NQO showed lung hemorrhagic areas. The concentration of 0.1nL/g was more effective in reducing the tongue induction of potentially malignant and malignant lesions by 4-NQO. A kidney toxicity was observed in healthy animals exposed to 0.1nL/g of TP-4-ol. The negative isoform of terpinen-4-ol negatively modulates the development of potentially malignant and malignant lesions in rats (Rattus nonverdicts albinos, Holtzman) exposed to 4-NQO. (-)-Terpinen-4-ol reduced the mice percentual with squamous cell carcinoma, 87.5 to 41.6%, and decreased the cancer/rat ratio of 2.62 in 4-NQO to 0.75 in 4-NQO + 0.1nL/g. This represents 52.4% by group and 71.3% in the cancer/rat ratio., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Oral cancer (OC), the most common cancer in the head and neck, which has a poor prognosis, histopathologically follows a stepwise pattern of hyperplasia, dysplasia, and cancer. Blocking the progression of OC in the precancer stage could greatly improve the survival and cure rates. AKT protein plays a critical role in the signal transduction of cancer cells, and we found that AKT was overexpressed in human OC samples through analysis of TCGA database. Therefore, this study is aimed at investigating the chemopreventive effect of an AKT inhibitor (MK2206 2HCl) on OC. In vivo , we established a 4-nitroquinoline-1-oxide- (4NQO-) induced mouse tongue carcinogenesis model to investigate the potential chemopreventive effect of MK2206 2HCl on mouse OC resulting from 4NQO. The results showed that MK2206 2HCl could significantly reduce the incidence rate and growth of OC, inhibit the transformation of dysplasia to cancer in the 4NQO-induced mouse tongue carcinogenesis model, and simultaneously markedly suppress cell proliferation, angiogenesis, and mast cell (MC) infiltration in 4NQO-induced mouse tongue cancers. In vitro , our results revealed that MK2206 2HCl could also inhibit oral squamous cell carcinoma (OSCC) cell malignant biological behaviors, including cell proliferation, colony formation, cell invasion, and migration, while promoting apoptosis. Mechanistic studies revealed that MK2206 2HCl suppressed matrix metalloproteinase 9 (MMP-9) and RhoC expression and promoted autophagy gene LC3 II expression. In summary, our findings demonstrated the chemopreventive effect of MK2206 2HCl on the 4NQO-induced mouse tongue carcinogenesis model, which likely has an underlying mechanism mediated by the MMP-9/RhoC signaling pathway and autophagy., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Panpan Yin et al.)

Objective: Based on a critical review of published studies, we aimed to develop a good practice guide for using 4-nitroquinoline-1-oxide (4NQO) as an inducer of oral carcinogenesis in Wistar rats., Design: A systematic search was performed on Medline Ovid, PubMed, Embase, Web of Science, and Scopus databases. The SYRCLE's risk of bias tool was used to assess the quality of the studies., Results: Thirty-five articles met the selection criteria; 22 (62.9%) of them administered 4NQO systemically in drinking water, with a mean concentration of 30.2 ppm (SD±15.9) and during a mean period of 20.8 (SD±7.8) weeks. The other 13 (37.1%) studies performed topical applications of 4NQO painting the oral mucosa of the animals three times a week (100%) with a mean period of administration of 16.8 (SD±7.0) weeks. Different 4NQO concentrations used for other periods achieved significant tumor development. Most studies didn't perform quantitative clinical analysis, and the histopathological diagnosis/grading criteria varied considerably., Conclusions: A poor description of solution care, adverse effects, and the number of losses were observed, and the reporting of these features needs to be improved. Suggestions to guide the development of future research are provided., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

For decades, the link between poor oral hygiene and the increased prevalence of oral cancer has been suggested. Most recently, emerging evidence has suggested that chronic inflammatory diseases from the oral cavity (e.g., periodontal disease), to some extent, play a role in the development of oral squamous cell carcinoma (OSCC). The present study aimed to explore the direct impact of biofilm‑induced periodontitis in the carcinogenesis process using a tobacco surrogate animal model for oral cancer. A total of 42 Wistar rats were distributed into four experimental groups: Control group, periodontitis (Perio) group, 4‑nitroquinoline 1‑oxide (4‑NQO) group and 4NQO/Perio group. Periodontitis was stimulated by placing a ligature subgingivally, while oral carcinogenesis was induced by systemic administration of 4NQO in the drinking water for 20 weeks. It was observed that the Perio, 4NQO and 4NQO/Perio groups presented with significantly higher alveolar bone loss compared with that in the control group. Furthermore, all groups receiving 4NQO developed lesions on the dorsal surface of the tongue; however, the 4NQO/Perio group presented larger lesions compared with the 4NQO group. There was also a modest overall increase in the number of epithelial dysplasia and OSCC lesions in the 4NQO/Perio group. Notably, abnormal focal activation of cellular differentiation (cytokeratin 10‑positive cells) that extended near the basal cell layer of the mucosa was observed in rats receiving 4NQO alone, but was absent in rats receiving 4NQO and presenting with periodontal disease. Altogether, the presence of periodontitis combined with 4NQO administration augmented tumor size in the current rat model and tampered with the protective mechanisms of the cellular differentiation of epithelial cells.

Risk factors for liver cancer include tobacco use, alcohol consumption, obesity, and male sex. Administration of 4-nitroquinonline-1-oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. This study compared the effects of diets high and low in saturated fat (HF and LF, respectively), and sex, on liver histopathology in 4NQO-treated mice and controls. We hypothesized that 4NQO would cause histopathological changes in liver, and that a HF diet would increase hepatic pathology when compared to the LF diet. Mice (C57Bl/6, 36/sex), were divided into a low fat (10 kcal% fat; LF) or high fat (60 kcal% fat, HF) diet. Mice were further subdivided into one of 3 water treatment groups for 17 weeks: water (control), vehicle (1.25% propylene glycol in water [PG]), or 4NQO in (50 μg/ml; 4NQO). All mice were subsequently given water alone for 6 more weeks. Upon euthanasia, livers were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E). H&E slides were graded for histopathology; frozen liver samples were analyzed for triglyceride content. Trichrome stained sections were graded for fibrosis. CD3+ T cells, CD68+ macrophages, and Ly6+ neutrophils were detected by immunohistochemistry. Compared to water controls, 4NQO-treatment caused mouse liver histopathological changes such as fibrosis, and increases in hepatic neutrophils, T cells, and macrophages. HF diet exacerbated pathological changes compared to LF diet. Male controls, but not females, demonstrated severe steatosis and increased triglyceride content. 4NQO treatment decreased hepatic fat accumulation, even in animals on a HF diet. In conclusion, this murine model of oral cancer may serve as a model to study the effects of tobacco and diet on liver., Competing Interests: The authors have declared that no competing interests exist.

Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent studies have reported dysbiotic oral microbiota and tumor-resident bacteria in human head and neck squamous cell carcinoma (HNSCC). We aimed to identify and validate oral microbial signatures in treatment-naïve HNSCC patients compared with healthy control subjects. We confirm earlier reports that the relative abundances of Lactobacillus spp. and Neisseria spp. are elevated and diminished, respectively, in human HNSCC. In parallel, we examined the disease-modifying effects of microbiota in HNSCC, through both antibiotic depletion of microbiota in an induced HNSCC mouse model (4-Nitroquinoline 1-oxide, 4NQO) and reconstitution of tumor-associated microbiota in a germ-free orthotopic mouse model. We demonstrate that depletion of microbiota delays oral tumorigenesis, while microbiota transfer from mice with oral cancer accelerates tumorigenesis. Enrichment of Lactobacillus spp. was also observed in murine HNSCC, and activation of the aryl-hydrocarbon receptor was documented in both murine and human tumors. Together, our findings support the hypothesis that dysbiosis promotes HNSCC development., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αβT cells capable of eliminating γH2AX + epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor., Competing Interests: Competing interest statement: D.M.L. is a scientific advisor to Constellation Pharma, a Science Partner of Nextech Ventures (Zurich, CH), a Science Advisory Board member of the Sidney Kimmel Cancer Center (Johns Hopkins School of Medicine), a Science Advisor to the Pezcoller Foundation (Trento, Italy), and a Special Advisor to the Director of Break Through Cancer, none of which poses a conflict of interest with the substance of this paper., (Copyright © 2021 the Author(s). Published by PNAS.)

HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The multistep process of oral carcinogenesis provides a biological rationale for the use of chemoprevention in individuals at increased risk of developing oral cancer. We aimed to determine if low doses of propranolol can prevent the development of oral cancer using a tobacco-relevant and p53-associated animal model of cancer initiation. Twenty-six Wistar rats were randomly allocated into two groups, vehicle, and propranolol. All animals received 4-nitroquinoline N-oxide (4NQO) at 25 ppm diluted in the drinking water for 20 weeks. Animals from the propranolol group received propranolol (0.1 mg/kg) 5 days per week by gavage for 18 weeks. The clinical analysis was performed by measuring the area of the lesion and assessment of scores based on lesion appearance (papule; plaque; nodule or ulcerated). Histopathological analysis was performed to determine the presence of epithelial dysplasia or invasive squamous cell carcinoma (SCC). The average lesion area in 4NQO + vehicle and in 4NQO + propranolol groups were 0.20  and 0.28 mm2, respectively (P = 0.53). The percentage of cases clinically graded as papules, thick plaques, nodular areas, and ulcerated lesions was similar between groups (P = 0.94). Histopathological diagnosis also did not differ between groups (P = 0.65), with 54.5 and 70% of cases being diagnosed as SCC in 4NQO and in 4NQO + propranolol groups, respectively. In conclusion, daily doses propranolol at 0.1 mg/kg were not as effective as a chemopreventive therapy in an animal model of 4NQO-induced carcinogenesis., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology., Materials and Methods: Male and female C57Bl/6 mice (36/sex) received a low-fat (10 kcal%) or high-fat (60 kcal%) diet. Water (control), vehicle, or 4-nitroquinoline-1-oxide in vehicle (50 μg/ml) was provided for 17 weeks followed by six additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined, and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified., Results: All 4-nitroquinoline-1-oxide-treated mice developed oral tumors. High-fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 ± 4.5 vs. 7.9 ± 2.5, mm/mouse, p < .05; high-fat diet vs. low-fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4-nitroquinoline-1-oxide-treated males displayed higher histopathology scores than females (4.2 ± 0.3 vs. 3.6 ± 0.2, respectively, p < .05). Lymphoid cell infiltration was greater in the 4-nitroquinoline-1-oxide mouse tongues than controls: T cells (14.0 vs. 0.96 cells/mm 2 ), macrophages (3.6 vs. 1.8 cells/mm 2 ), and neutrophils (12.0 vs. 0.38 cells/mm 2 )., Conclusion: High-fat diet and male sex increased the pathology of 4-nitroquinoline-1-oxide-induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Purpose: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents., Materials and Methods: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography., Results: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea., Conclusions: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

We have previously demonstrated that PD-1 blockade decreased the incidence of high-grade dysplasia in a carcinogen-induced murine model of oral squamous cell carcinoma (OSCC). It remains unknown, however, whether there are additional factors involved in escape from immune surveillance that could serve as additional targets for immunoprevention. We performed this study to further characterize the immune landscape of oral premalignant lesions (OPL) and determine the impact of targeting of the PD-1, CTLA-4, CD40, or OX40 pathways on the development of OPLs and oral carcinomas in the 4-nitroquinoline 1-oxide model. The immune pathways were targeted using mAbs or, in the case of the PD-1/PD-L1 pathway, using PD-L1-knockout (PD-L1 ko ) mice. After intervention, tongues and cervical lymph nodes were harvested and analyzed for malignant progression and modulation of the immune milieu, respectively. Targeting of CD40 with an agonist mAb was the most effective treatment to reduce transition of OPLs to OSCC; PD-1 alone or in combination with CTLA-4 inhibition, or PD-L1 ko , also reduced progression of OPLs to OSCC, albeit to a lesser extent. Distinct patterns of immune system modulation were observed for the CD40 agonists compared with blockade of the PD-1/PD-L1 axis with or without CTLA-4 blockade; CD40 agonist generated a lasting expansion of experienced/memory cytotoxic T lymphocytes and M1 macrophages, whereas PD-1/CTLA-4 blockade resulted in a pronounced depletion of regulatory T cells among other changes. These data suggest that distinct approaches may be used for targeting different steps in the development of OSCC, and that CD40 agonists merit investigation as potential immunoprevention agents in this setting. PREVENTION RELEVANCE: PD-1/PD-L1 pathway blockade, as well as activation of the CD40 pathway, were able to prevent OPL progression into invasive OSCC in a murine model. A distinct pattern of immune modulation was observed when either the CD40 or the PD-1/PD-L1 pathways were targeted., (©2020 American Association for Cancer Research.)

Oral cancer patients often have severe, chronic, and mechanically induced pain at the site of the primary cancer. Oral cancer pain is initiated and maintained in the cancer microenvironment and attributed to release of mediators that sensitize primary sensory nerves. This study was designed to investigate the histopathology associated with painful oral cancers in a preclinical model. The relationship of pain scores with pathologic variables was also investigated in a cohort of 72 oral cancer patients. Wild-type mice were exposed to the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Nociceptive (pain) behavior was measured with the dolognawmeter, an operant device and assay for measuring functional and mechanical allodynia. Lesions developed on the tongues and esophagi of the 4NQO-treated animals and included hyperkeratoses, papillomas, dysplasias, and cancers. Papillomas included lesions with benign and dysplastic pathological features. Two histologic subtypes of squamous cell carcinomas (SCCs) were identified-SCCs with exophytic and invasive components associated with papillary lesions (pSCCs) and invasive SCCs without exophytic histology (iSCCs). Only the pSCC subtype of tongue cancer was associated with nociceptive behavior. Increased tumor size was associated with greater nociceptive behavior in the mouse model and more pain experienced by oral cancer patients. In addition, depth of invasion was associated with patient-reported pain. The pSCC histology identifies 4NQO-induced tongue cancers that are expected to be enriched for expression and release of nociceptive mediators.

Social isolation has affected a large number of people and may lead to impairment of physical and mental health. Although stress resulting from social isolation may increase cancer progression, its interference on tumorigenesis is poorly known. In this study, we used a preclinical model to evaluate the effects of social isolation stress on chemically induced oral carcinogenesis. Sixty-two 21-day-old male Wistar rats were divided into isolated and grouped groups. After 90 days of age, the rats from both groups underwent oral carcinogenesis with 4-nitroquinoline 1-oxide (4NQO) for 20 weeks. All rats were assessed for depressive-like behavior and euthanized for oral squamous cell carcinoma (OSCC) diagnosis and measurement of inflammatory mediators in the tumor microenvironment. Social isolation stress increased the OSCC occurrence by 20.4% when compared to control. Isolated rats also showed higher tumor volume and cachexia than the grouped rats. Social isolation did not induce changes in the depressive-like behavior after carcinogenic induction. Tumors from stressed rats had increased levels of the inflammatory mediators, TNF-alpha, IL1-beta and MCP-1. The concentrations of TNF-alpha and MCP-1 were significantly increased in the large tumors from isolated animals. Higher tumor levels of TNF-alpha, IL-6, IL1-beta and MCP-1 were positively correlated with OSCC growth. This study provides the first evidence that social isolation stress may facilitate OSCC occurrence and tumor progression, an event accompanied by increased local levels of inflammatory mediators., Competing Interests: The authors have declared that no competing interests exist.

The early detection and management of oral premalignant lesions (OPMDs) improve their outcomes. Animal models that mimic histological and biological processes of human oral carcinogenesis may help to improve the identification of OPMD at-risk of progression into oral squamous cell carcinoma and to develop preventive strategies for the entire field of cancerization. No animal model is perfectly applicable for investigating human oral carcinogenesis. However, the 4-nitroquinoline 1-oxide (4-NQO) mouse model is well established and mimics several morphological, histological, genomic and molecular features of human oral carcinogenesis. Some of the reasons for the success of this model include its reproducible experimental conditions with limited variation, the possibility of realizing longitudinal studies with invasive intervention or gene manipulation, and sample availability for all stages of oral carcinogenesis, especially premalignant lesions. Moreover, the role of histological and molecular alterations in the field of cancerization (i.e., macroscopically healthy mucosa exposed to a carcinogen) during oral carcinogenesis can be easily explored using this model. In this review, we discuss the advantages and drawbacks of this model for studying human oral carcinogenesis. In summary, the 4-NQO-induced murine oral cancer model is relevant for investigating human oral carcinogenesis, including the immune microenvironment, and for evaluating therapeutic and chemoprevention agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the most common subsite of head and neck cancer, with a 5-year survival rate of only 50%. There is a pressing need for animal models that recapitulate the human disease to understand the factors driving OSCC carcinogenesis. Many laboratories have used the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) to investigate OSCC formation. The importance of the 4NQO mouse model is that it mimics the stepwise progression observed in OSCC patients. The 4NQO carcinogen model has the advantage that it can be used with transgenic mice with genetic modification in specific tissue types to investigate their role in driving cancer progression. Herein, we describe the basic approach for administering 4NQO to mice to induce OSCC and methods for assessing the tissue and disease progression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

It has been hypothesized that high glucose concentrations might contribute to the overall intracellular oxidative stress either by the direct generation of reactive oxygen species (ROS) or by altering the redox balance. Moreover, it has also been suggested that high glucose concentration can increase the susceptibility of DNA to genotoxic effects of xenobiotics. The aim of this approach was to test high glucose concentrations for pro-genotoxicity in human liver cells by setting up an in vitro model for hyperglycaemia. The experimental design included performing of tests on both human HepG2 tumour cells and HepaRG immortalized cells. Increased cell susceptibility to genotoxic xenobiotics was tested by challenging cell cultures with 4-nitroquinoline-N-oxide (4NQO) and evaluating the extent of primary DNA damage by comet assay. Moreover, we evaluated the relationship between glucose concentration and intracellular ROS, as well as the effects of glucose concentration on the induction of Nrf2-dependent genes such as Glutathione S-transferases, Heme‑oxygenase-1, and Glutathione peroxidase-4. To investigate the involvement of ROS in the induced pro-genotoxic activity, parallel experimental sets were set up by considering co-treatment of cells with the model mutagen 4NQO and the antioxidant, glutathione precursor N-acetyl-L-cysteine. High glucose concentrations caused a significant increase in the levels of primary DNA damage, with a pro-genotoxic condition closely related to the concentration of glucose in the culture medium when cells were exposed to 4NQO. High glucose concentrations also stimulated the production of ROS and down-regulated genes involved in contrasting of the effects of oxidative stress. In conclusion, in the presence of high concentrations of glucose, the cells are in unfavourable conditions for the maintenance of genome integrity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Tongue tumors, which are oropharyngeal tumors, are increasing in frequency. Pyrrolidine dithiocarbamate (PDTC) is a powerful antioxidant and antitumoral agent., Objectives: To evaluate the protective and therapeutic effects of PDTC in a tongue cancer model induced with 4-nitroquinoline 1-oxide (4-NQO)., Material and Methods: We included 40 rats in the trial and assigned them randomly to 5 groups. Group 1 (cancer, n = 7): 4-NQO (0-12 weeks); group 2 (protection, n = 8): 4-NQO (0-12 weeks) + PDTC (300 mg/kg/day, 0-12 weeks); group 3 (therapy-high dose, n = 10): 4-NQO (0-12 weeks) + PDTC (600 mg/kg/day, weeks 12-30); group 4 (therapy-low dose, n = 10): 4-NQO (0-12 weeks) + PDTC (300 mg/kg/day, weeks 12-30); and group 5 (control). Cardiac blood samples were taken to analyze oxidative stress parameters (total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI)). Histopathological assessment was performed under a light microscope., Results: The results of the histopathological assessment showed that the model we used in group 1 was successful, which was consistent with the literature. The PDTC dose administered in group 2 could not prevent tumor formation. Group 3 demonstrated that PDTC in high doses is effective as a therapeutic agent. Group 4 indicated that PDTC in low doses has no therapeutic effect. The results of the biochemical assessment showed that in group 3, TOS and OSI values were significantly lower than in groups 1, 2 and 4. No significant difference was found in the TOS and OSI values between groups 5 and 3., Conclusions: Our study demonstrated histopathologically that in an experimentally generated tongue cancer model, application of 600 mg/kg/day of PDTC led to a significant reduction in the size of the tumor. This was supported by the biochemical parameters.

Purpose: Beta-adrenergic signaling can influence cancer progression and the use of beta blockers as adjuvant drugs in oncologic patients has been suggested. However, the involvement of beta-adrenergic blockers in tumorigenesis is poorly understood. This study investigated the action of beta-adrenergic blocker propranolol on tumor onset using a preclinical model of chemically induced oral cancer., Methods: Thirty-two male Wistar rats were subjected to daily subcutaneous injection of beta-blocker propranolol (10 mg/kg; SubQ), while another 32 rats received only a PBS injection (sham group). One week after starting propranolol treatment, all rats were submitted to chemical induction of oral carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). After 16 weeks, they were assessed for occurrence of oral squamous cell carcinoma (OSCC), in addition to measurement of tumor volume and thickness, and tissue levels of cytokines IL-6, TNF-alpha and IL-10 in the tumor microenvironment., Results: Propranolol treatment reduced the occurrence of OSCC by 31%, 95% CI ( - 127, 216). Beta-adrenergic blocker significantly decreased thickness of OSCC when compared with PBS. Rats treated with propranolol exhibited a lower tumor volume when compared with control rats, but this result did not reach statistical significance. Tumors from propranolol-treated rats exhibited reduced concentrations of pro-inflammatory cytokines IL-6 and TNF-α. There was no difference in the IL-10 levels between tumors from propranolol- and sham-treated rats., Conclusion: Beta-adrenergic signaling may be one of the mechanisms associated with chemically induced oral carcinogenesis.

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&#95;targets&#95;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.

Though therapy that promotes anti-tumor response about CD8 + tumor-infiltrating lymphocytes (TILs) has shown great potential, clinical responses to CD8 + TILs immunotherapy vary considerably, largely because of different subpopulation of CD8 + TILs exhibiting different biological characters. To define the relationship between subpopulation of CD8 + TILs and the outcome of antitumor reaction, the phenotype and function of CD103 + CD8 + TILs in esophageal squamous cell carcinoma (ESCC) were investigated. CD103 + CD8 + TILs were presented in ESCC, which displayed phenotype of tissue-resident memory T cells and exhibited high expression of immune checkpoints (PD-1, TIM-3). CD103 + CD8 + TILs were positively associated with the overall survivals of ESCC patients. This population of cells elicited potent proliferation and cytotoxic cytokine secretion potential. In addition, CD103 + CD8 + TILs were elicited potent anti-tumor immunity after anti-PD-1 blockade and were not affected by chemotherapy. This study emphasized the feature of CD103 + CD8 + TILs in immune response and identified potentially new targets in ESCC patients.

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Chalcones are chemically defined as α,β-unsaturated ketones with a 1,3-diphenyl-2-propen-1-one nucleus. These compounds occur naturally in plants and are considered precursors of flavonoids. Given that evaluating genetic toxicology tests is essential in investigating the safe use and chemopreventive potential of different natural and synthetic compounds, this study aimed to assess the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activity of the chalcone 1E,4E-1-(4-chlorophenyl)-5-(2,6,6-trimethylcyclohexen-1-yl)penta-1,4-dien-3-one (CAB7β). The CAB7β was synthesized via Claisen-Schmidt reaction. The Ames test was applied using the co-treatment model as well as a micronucleus assay of mouse bone marrow with co-, pre- and post-treatment models. Our results indicate no genotoxic effect for CAB7β in any of the tests applied. At all the concentrations used, CAB7β showed a significant DNA protective effect against the mutagenic action of 4-nitroquinoline-1-oxide and sodium azide according to the Ames test, and against doxorubicin in the co-, pre- and post-treatment models of the micronucleus assay. CAB7β alone displayed cytotoxic activity in the micronucleus test. At concentrations of 12,5 and 50 µg/plate, CAB7β showed a moderate cytotoxic profile only in Salmonella typhimurium strain TA98. However, an anticytotoxic effect was observed against S. typhimurium strain TA100 for all the concentrations tested and during co-, pre- and post-treatment in the micronucleus assay. It was concluded that CAB7β exhibited a slightly cytotoxic effect in S. typhimurium strain TA98 and significant antigenotoxic and anticytotoxic effects in cells of mouse, making it a promising candidate in chemoprevention and possibly in the development of new cancer treatments.

The PI3K signaling pathway is frequently mutated in head and neck squamous cell carcinoma (HNSCC), often via gain-of-function (GOF) mutations in the PIK3CA gene. Here, we present novel genetically engineered mouse models (GEMM) carrying a GOF allele Loxp-STOP-Loxp(LSL)-PIK3CA H1047R (E20) alone or in combination with heterozygous LSL - p53 +/R172H (p53) mutation with tissue-specific expression to interrogate the role of oncogenic PIK3CA in transformation of upper aerodigestive track epithelium. We demonstrated that the GOF PIK3CA mutation promoted progression of 4-nitroquinoline 1-oxide-induced oral squamous cell carcinoma (OSCC) in both E20 single mutant and E20/p53 double mutant mice, with frequent distal metastasis detected only in E20/p53 GEMM. Similar to in human OSCC, loss of p16 was associated with progression of OSCC in these mice. RNA-seq analyses revealed that among the common genes differentially expressed in primary OSCC cell lines derived from E20, p53, and E20/p53 GEMMs compared with those from the wild-type mice, genes associated with proliferation and cell cycle were predominantly represented, which is consistent with the progressive loss of p16 detected in these GEMMs. Importantly, all of these OSCC primary cell lines exhibited enhanced sensitivity to BYL719 and cisplatin combination treatment in comparison with cisplatin alone in vitro and in vivo , regardless of p53 and/or p16 status. Given the prevalence of mutations in p53 and the PI3K pathways in HNSCC in conjunction with loss of p16 genetically or epigenetically, this universal increased sensitivity to cisplatin and BYL719 combination therapy in cancer cells with PIK3CA mutation represents an opportunity to a subset of patients with HNSCC. IMPLICATIONS: Our results suggest that combination therapy of cisplatin and PI3K inhibitor may be worthy of consideration in patients with HNSCC with PIK3CA mutation., (©2020 American Association for Cancer Research.)

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role., Competing Interests: The authors have declared that no competing interests exist.

B Cell-Specific Moloney Murine Leukemia Virus Integration Site 1 (Bmi-1, Bmi1), an epigenetic protein, is necessary for normal stem cell self-renewal in adult animals and for cancer stem cell (CSC) functions in adult animals. To elucidate the functions of Bmi-1 in the oral cavity we created a transgenic mouse line (KrTBmi-1) that expresses ectopic, Flag-tagged Bmi-1 in tongue basal epithelial stem cells only upon doxycycline (DOX) treatment. Genome wide transcriptomics and Ingenuity Pathway Analysis identified several pathways altered by exogenous Bmi-1 expression in the normal tongue epithelium, including EIF2 signaling ( P value = 1.58 x 10 -49 ), mTOR signaling ( P value = 2.45 x 10 -12 ), oxidative phosphorylation ( P = 6.61 x 10 -3 ) and glutathione redox reactions I ( P = 1.74 x 10 -2 ). Overall, our data indicate that ectopic Bmi-1 expression has an impact on normal tongue epithelial homeostasis. We then assessed the KrTBmi-1 mice in the 4-nitroquinoline 1-oxide (4-NQO) model of oral carcinogenesis. We found that 80% of mice expressing exogenous Bmi-1 (+DOX, +4-NQO KrTBmi-1; N = 10) developed tumors classified as grade 3 or higher, compared to 60% and 40% of mice expressing just endogenous Bmi-1 (+DOX, +4-NQO Kr and -DOX, +4-NQO KrTBmi-1 groups, respectively; N = 10/group; P value = <0.0001); and 30% of mice expressing ectopic Bmi-1 mice developed 20 or more lesions compared to 10% of mice expressing only endogenous Bmi-1 ( P = .009). This demonstrates that exogenous Bmi-1 expression increases the susceptibility of mice to 4-NQO-induced oral carcinogenesis, strengthening the evidence for Bmi-1 as a therapeutic target in human oral squamous cell carcinoma.

We report the mutational spectra in a segment of the E. coli rpoB gene of bleomycin (BLEO), 4-nitroquinoline-1-oxide (NQO), and hydrogen peroxide (H 2 O 2 ). We compare these spectra with those of other mutagens and repair deficient strains in the same rpoB system, and review the key elements determining mutational hotspots and outline the questions that remain unanswered. We consider three tiers of hotspots that derive from 1) the nature of the sequence change at a specific base, 2) the direct nearest neighbors and 3) some aspect of the larger sequence context or the local 3D-structure of segments of DNA. This latter tier can have a profound effect on mutation frequencies, even among sites with identical nearest neighbor sequences. BLEO is dependent on the SOS-induced translesion Pol V for mutagenesis, and has a dramatic hotspot at a single mutational site in rpoB. NQO is not dependent on any of the translesion polymerases, in contrast to findings with plasmids treated in vitro and transformed into E. coli. The rpoB system allows one to monitor both G:C -> A:T transitions and G:C -> T:A transversions at the same site in 11 cases, each site having the identical sequence context for each of the two mutations. The combined preference for G:C -> A:T transitions at these sites is 20-fold. Several of the favored sites for hydrogen peroxide mutagenesis are not seen in the spectra of BLEO and NQO mutations, indicating that mutagenesis from reactive oxygen species is not a major cause of BLEO or NQO mutagenesis, but rather specific adducts. The variance in mutation rates at sites with identical nearest neighbors suggests that the local structure of different DNA segments is an important factor in mutational hotspots., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 10,000 IU diets. Serum 25OHD 3 levels were highest in animals on 10,000 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 10,000 IU diet. Animals on 100 IU and 10,000 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., Competing Interests: Declaration of Competing Interest The authors do not have any conflicts to disclose. The funding sponsors had no role in the design of the study, collection, analyses or interpretation of the data, writing of the manuscript and the decision to publish the results., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine 10 -H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G 2 M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents., Competing Interests: Declaration of Competing Interest All authors are employees of Philip Morris International., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.

We have previously showed that a transgenic (Tg) mouse model with cytokeratin 14 promoter (K14)-driven expression of E6 and E7 from beta-3 HPV49 in the basal layer of the epidermis and of the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7 Tg mice) are highly susceptible to upper digestive tract carcinogenesis upon exposure to 4-nitroquinoline 1-oxide (4NQO). Using whole-exome sequencing, we show that in K14 HPV49 E6/E7 Tg mice, development of 4NQO-induced cancers tightly correlates with the accumulation of somatic mutations in cancer-related genes. The mutational signature in 4NQO-treated mice was similar to the signature observed in humans exposed to tobacco smoking and tobacco chewing. Similar results were obtained with K14 Tg animals expressing mucosal high-risk HPV16 E6 and E7 oncogenes. Thus, beta-3 HPV49 share some functional similarities with HPV16 in Tg animals., (Copyright © 2019. Published by Elsevier Inc.)

Oral carcinoma (OSCC) is one of the most important causes of cancer death worldwide. OSCC cell lines and preclinical rodent models are crucial to addressing the mechanisms of OSCC and helping the development of new therapeutic strategies and interventions. The establishment of murine OSCC cell lines and syngeneic models are necessary to allow concordant investigation of both in vitro and in vivo pathogenesis. In this study, we established two murine tongue squamous cell carcinoma cell lines, designated MTCQ1 and MTCQ2, from 4NQO-induced OSCC using C57BL/6 mice. These cell lines express a variety of epithelial markers but produce only a tiny amount of E-cadherin. The expression of mesenchymal and stemness regulators are evident, and this is associated with the high mobility in these cell lines. MTCQ1 also shows high Ki67 and PCNA expression, and complicated alterations in p53 expression, which may underlie its high clonogenic potential and rapid orthotopic tumor induction. Using the MTCQ1 cell subclone tagged with GFP (MTCQ1-GFP), extensive neck nodal metastasis and lung metastasis were identified by immunostaining and fluorescence imaging. Inhibition of oncogenic miRNAs, particularly miR-134, was able to attenuate the oncogenicity of MTCQ1-GFP. Cisplatin treatment inhibited both in vitro and in vivo growth of MTCQ1-GFP, and it was found to decrease miR-134 expression in this subclone. The anti-PD-L1 treatment enhanced the inhibitory effects of cisplatin against tumorigenesis. This syngeneic preclinical model should help provide valuable mechanistic insights into OSCC, as well as helping with the development of new approaches to treating this disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Oral squamous cell carcinoma (OSCC) is the most common and most malignant disorder of the oral cavity. Standard cancer treatments have many complications for patients. Nausea, vomiting, and perturbation in blood cells are the most common side effects when using Doxorubicin (Dox) for the treatment of OSCC. Use of Doxorubicin-loaded nano-particles (n-Dox) give rise to increase its biological efficacy and the rapeutic effects. This study assessed the efficacy of the injectable form of the n-Doxon blood parameters and cardiac and liver enzymes compared to the commercial form of Dox in OSCC-induced by 4NQO in rats. Methods: 4-nitroquinoline-1-oxideas was used as a solution in drinking water for inducing OSCC during 14 weeks in male Sprague-Dawley rats. Four groups of animals were categorized randomly: first (OSCC+Dox), second (OSCC+n-Dox), third (OSCC) and, last, healthy animals. Results: Using n-Dox had no harmful effect on the number of white and red blood cells. Thrombocytopenia and leukopenia in animals treated with n-Dox was less than the other groups. Hemoglobin and hematocrit in all treated groups did not differ and were similar to the healthy control. Hepatic and cardiac enzymes did not show any significant difference in any of the groups. Conclusion: The results of this research showed that significant decreases in haematological changes occurred, including leukopenia and anemia, in an animal model of OSCC induced by 4-NQO following use of n-Dox with compare to Dox. Use of n-Dox is better than of Dox for treatment of OSCC.