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The present study aimed to explore the expression and regulatory role of FAM83D in the different developmental stages of esophageal squamous cell carcinoma (ESCC) to determine the effect of FAM83D on the proliferation, migration, and invasion of ESCC cells and to elucidate its underlying molecular mechanism. Immunohistochemistry (IHC) analysis revealed that the expression of FAM83D was obviously elevated in ESCC tissues compared to adjacent normal tissues. Furthermore, the FAM83D levels was positively correlated with tumor size, TNM stage, T stage, and N stage, while it was negatively correlated with FBXW7 expression, Karnofsky Performance Status (KPS) score, and survival rate. Subsequently, ESCC cell lines with low FAM83D expression were constructed using RNA interference technology to investigate the impact of FAM83D on the biological behavior of ESCC cells. Silencing of FAM83D inhibited the proliferation and migration of ESCC cells but promoted apoptosis. Furthermore, a reduction in FAM83D expression may also induce cell cycle arrest at the G0/G1 phase and regulate the expression of proteins related to epithelial-mesenchymal transition (EMT), the cell cycle, and apoptosis. Further research indicated that silencing FAM83D led to the upregulation of FBXW7 expression. These results suggested that FAM83D may exert its effects on ESCC by downregulating FBXW7. Additionally, using a 4NQO solution in the drinking water to establish an ESCC mouse model, IHC analysis revealed that FAM83D expression levels were positively correlated with the pathological grade of esophageal lesions in the mice and negatively correlated with the expression levels of FBXW7 and E-cadherin. The above results demonstrated that FAM83D may facilitate the progression of ESCC by negatively regulating FBXW7 expression and that FAM83D could represent a promising therapeutic target for ESCC.
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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.
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Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity and is associated with high morbidity due to local invasion and lymph node metastasis. Tumor infiltrating lymphocytes (TILs) are associated with good prognosis in oral cancer patients and dictate response to treatment. Ectopic sites for immune activation in tumors, known as tertiary lymphoid structures (TLS), and tumor-associated high-endothelial venules (TA-HEVs), which are specialized lymphocyte recruiting vessels, are associated with a favorable prognosis in OSCC. Why only some tumors support the development of TLS and HEVs is poorly understood. In the current study we explored the infiltration of lymphocyte subsets and the development of TLS and HEVs in oral epithelial lesions using the 4-nitroquinoline 1-oxide (4NQO)-induced mouse model of oral carcinogenesis. We found that the immune response to 4NQO-induced oral epithelial lesions was dominated by T cell subsets. The number of T cells (CD4+, FoxP3+, and CD8+), B cells (B220+) and PNAd+ HEVs increased from the earliest to the latest endpoints. All the immune markers increased with the severity of the dysplasia, while the number of HEVs and B cells further increased in SCCs. HEVs were present already in early-stage lesions, while TLS did not develop at any timepoint. This suggests that the 4NQO model is applicable to study the dynamics of the tumor immune microenvironment at early phases of oral cancer development, including the regulation of TA-HEVs in OTSCC.
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Introduction: Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. Methods: 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Results: Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Discussion: Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.
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Carcinogénesis , Glicoproteínas , Ratones , Animales , Humanos , Vimentina , Carcinógenos/análisis , Hojas de la Planta/química , CadherinasRESUMEN
Background/purpose: 4-Nitroquinoline 1-oxide (4NQO)-induced tongue carcinoma and 7,12-dimethlybenz(a)anthracene (DMBA)-induced cheek pouch carcinoma are the most common and classical chemical carcinogen-induced animal models of oral carcinogenesis. The purpose of this study was to provide the research trends and characteristics of 4NQO- and DMBA-induced experimental oral carcinogenesis. Materials and methods: The papers on both 4NQO- and DMBA-induced experimental oral carcinogenesis were published since 1962. All the eligible papers were retrieved on 12 May 2023 from the Scopus database. Results: There were 506 and 349 papers on 4NQO- and DMBA-induced experimental oral carcinogenesis with 10,152 and 6306 citations, respectively. The common distinctive keywords such as rat, tongue neoplasms, drinking water, tumor microenvironment, and cyclooxygenase (COX)-2 were identified in the papers on 4NQO; and the common keywords such as hamster, cheek pouch, lipid peroxidation, glutathione, antioxidants, and topical drug administration were identified in the papers on DMBA. Importantly, 105 and 65 potential chemopreventive agents were identified from the papers on 4NQO and DMBA, respectively. Furthermore, 15 promising agents such as COX-2 inhibitor, curcumin, garlic were researched concurrently in both the two animal models. Conclusion: This study for the first time reports the scientometric characteristics of 4NQO- and DMBA-induced experimental oral carcinogenesis. Importantly, we identify a valuable profile for oral cancer chemopreventive agents, which will aid researchers and investigators in studying oral cancer chemoprevention.
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Purpose: Malignant head and neck squamous cell carcinoma (HNSCC) is characterized by a poor prognosis and resistance to conventional radiotherapy. Infiltrating myeloid-derived suppressive cells (MDSCs) is prominent in HNSCC and is linked to immune suppression and tumor aggressiveness. This study aimed to investigate the impact of boron neutron capture therapy (BNCT) on the MDSCs in the tumor microenvironment and peripheral blood and to explore the potential for MDSCs depletion combined with BNCT to reactivate antitumor immunity. Methods and materials: Carcinogen, 4-NQO, -induced oral tumors were irradiated with a total physical dose of 2 Gy BNCT in Tsing Hua Open Reactor (THOR). Flow cytometry and immunohistochemistry accessed the dynamics of peripheral MDSCs and infiltrated MDSCs within the tumor microenvironment. Mice were injected with an inhibitor of CSF-1 receptor (CSF-1R), PLX3397, to determine whether modulating M-MDSCs could affect mice survival after BNCT. Results: Peripheral CD11b+Ly6ChighLy6G- monocytic-MDSCs (M-MDSCs), but not CD11b+Ly6CloLy6Ghigh polymorphonuclear-MDSCs (PMN-MDSCs), increased as tumor progression. After BNCT treatment, there were temporarily decreased and persistent increases of M-MDSCs thereafter, either in peripheral blood or in tumors. The administration of PLX-3397 hindered BNCT-caused M-MDSCs infiltration, prolonged mice survival, and activated tumor immunity by decreasing tumor-associated macrophages (TAMs) and increasing CD8+ T cells. Conclusion: M-MDSCs were recruited into 4-NQO-induced tumors after BNCT, and their number was also increased in peripheral blood. Assessment of M-MDSCs levels in peripheral blood could be an index to determine the optimal intervention window. Their temporal alteration suggests an association with tumor recurrence after BNCT, making M-MDSCs a potential intervention target. Our preliminary results showed that PLX-3397 had strong M-MDSCs, TAMs, and TIL (tumor-infiltrating lymphocyte) modulating effects that could synergize tumor control when combined with BNCT.
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OBJECTIVES: To investigate the effects of dietary folate and sex on histopathology of oral squamous cell carcinoma in mice. MATERIALS AND METHODS: Mice (C57Bl/6, 30/sex) were fed either a deficient folate or sufficient folate diet. Vehicle or 4-nitroquinoline1-oxide (50 µg/mL) in vehicle were administered in drinking water for 20 weeks, followed by 6 weeks of regular drinking water. Oral lesions were observed weekly. Tongues were studied for histopathologic changes. Immunohistochemical techniques were used to measure cell proliferation (Ki67+), and to quantify expression of folate receptor, reduced folate carrier, and proton-coupled folate transporter. T cells, macrophages, and neutrophils were counted and normalized to area. RESULTS: All 4NQO-treated mice developed oral tumors. Dietary folate level did not affect tumor burden. More tumors were observed on the ventral aspect of the tongue than in other locations within the oral cavity. 4-nitroquinoline-1-oxide-treated mice displayed 27%-46% significantly lower expression of all three folate transport proteins; diet and sex had no effect on folate transporter expression. T-cell and neutrophil infiltration in tongues were 9.1-fold and 18.1-fold increased in the 4-nitroquinoline-1-oxide-treated mouse tongues than in controls. CONCLUSION: Treatment with 4NQO was the primary factor in determining cancer development, decreased folate transport expression, and lymphoid cell infiltration.
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Oral cancer is primarily squamous-cell carcinoma with a 5-year survival rate of approximately 50%. Lysyl oxidase (LOX) participates in collagen and elastin maturation. The propeptide of LOX is released as an 18 kDa protein (LOX-PP) in the extracellular environment by procollagen C-proteinases and has tumor-inhibitory properties. A polymorphism in the propeptide region of LOX (rs1800449, G473A) results in a single amino acid substitution of Gln for Arg. Here we investigated the frequency of rs1800449 in OSCC employing TCGA database resources and determined the kinetics and severity of precancerous oral lesion development in wildtype and corresponding knockin mice after exposure to 4-nitroquinoline oxide (4 NQO) in drinking water. Data show that the OSCC is more common in humans carrying the variant compared to the wildtype. Knockin mice are more susceptible to lesion development. The immunohistochemistry of LOX in mouse tissues and in vitro studies point to a negative feedback pathway of wildtype LOX-PP on LOX expression that is deficient in knockin mice. Data further demonstrate modulations of T cell phenotype in knockin mice toward a more tumor-permissive condition. Data provide initial evidence for rs1800449 as an oral cancer susceptibility biomarker and point to opportunities to better understand the functional mechanism of LOX-PP cancer inhibitory activity.
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Neoplasias de la Boca , Proteína-Lisina 6-Oxidasa , Animales , Humanos , Ratones , Carcinógenos , Colágeno/genética , Neoplasias de la Boca/genética , Polimorfismo Genético , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.
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OBJECTIVES: Diets and nutritional habits are critical during carcinogenic processes, where a diet poor in fruits and vegetables and rich in meat and other foods of animal origin facilitates carcinogenesis. In this study, we aimed at investigating the possible involvement of vitamin D deficiency (VDD) and high cholesterol (HC) together in oral squamous cell carcinoma (OSCC) through modulating glycolysis. SUBJECTS AND METHODS: We compared total cholesterol, LDL, HDL, triglycerides, LDH, and vitamin D levels of OSCC patients and control individuals. We used GEO datasets for gene set enrichment and 4-nitroquinoline-1-oxide induced in vivo oral carcinogenesis models to investigate contribution of VDD and HC during carcinogenesis via possible modulation of glycolysis. RESULTS: We found that VDD and HC co-exist in OSCC patients, and deregulation of cholesterol and vitamin D levels results in enrichment of genes related to glycolysis. We, then, demonstrated that VDD and HC on their own and together facilitated the formation of larger tumors in 4NQO-induced in vivo cancer models, which are suppressed by glycolysis inhibition. CONCLUSION: We reported collaborative contribution of HC and VDD during oral carcinogenesis, which is mainly carried out via altering energy metabolism in tumor cells.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Deficiencia de Vitamina D , Ratas , Animales , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias de la Boca/genética , Carcinogénesis/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Vitamina D , Glucólisis , Deficiencia de Vitamina D/complicacionesRESUMEN
OBJECTIVES: Oral squamous cell carcinoma (OSCC) is the predominant type of oral cancer. Its incidence is high in certain geographic regions, and it is correlated with chewing tobacco. Epidermal growth factor receptor (EGFR), induced by tobacco carcinogens, is overexpressed in OSCC, leading to poor prognosis. Thus, EGFR inhibitors are promising agents against OSCC. High cost and toxicity of existing EGFR inhibitors necessitate alternative EGFR-targeted therapy. Here, we tested the antitumor potential of ethyl acetate fraction of an ethnomedicinal tree, Oroxylum indicum stem bark extract (OIEA) in a 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis model. METHODS: OIEA was prepared by solvent extraction method, and subsequently its in vitro radical scavenging activities were measured. High-performance liquid chromatography (HPLC) analysis of OIEA was done to identify the constituent active compounds. Hemolytic, trypan blue exclusion, and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assays were performed in normal and cancer cells to select an optimum dose of OIEA for antitumor activity study in 4NQO-induced oral cancer in F344 rats. Measurement of tumor volume, weight, and cell count was followed by tumor cell cycle analysis and comet and annexin V/Propidium Iodide (PI) assay. Pro-apoptotic markers were detected by western blot testing. Molecular docking was done to predict the interaction between OIEA active component and EGFR or phosphatidylinositol-3-kinase (PI3K), which was further validated biologically. Finally, hepatic and renal function testing and histopathology were performed. RESULTS: OIEA reduced tumor burden and increased survivability of the tumor-bearing rats significantly as compared to untreated tumor bearers. HPLC revealed oroxylin A as the predominant bioactive component in OIEA. Molecular docking predicted significant binding between oroxylin A and EGFR as well as PI3K, which was confirmed by western blot analysis of in vivo samples. OIEA also ameliorated hepato-, renal- and myelotoxicity induced by 4NQO. CONCLUSION: OIEA reduces 4NQO-induced OSCC by modulating the EGFR/PI3K/AKT signaling cascade and also ameliorated toxicity in tumor bearers.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Ratas , Animales , Neoplasias de la Boca/inducido químicamente , Proteínas Proto-Oncogénicas c-akt/análisis , Fosfatidilinositol 3-Quinasas/análisis , Fosfatidilinositol 3-Quinasa/análisis , Corteza de la Planta/química , Simulación del Acoplamiento Molecular , Ratas Endogámicas F344 , Extractos Vegetales/farmacología , Receptores ErbB/análisis , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Squamous esophageal carcinoma is a common pathological type of esophageal carcinoma around the world. The prognosis of esophageal carcinoma is usually poor and diagnosed at late stages. Recently, research suggested that genomic instability occurred in esophageal cells during the development of esophageal squamous cell carcinoma (ESCC). Identifying prognostic and specific genomic characteristics, especially at the early hyperplasia stage, is critical. Mice were given 4-nitroquinoline 1-oxide (4NQO) with drinking water to induce esophageal cancer. The immortalized human esophageal epithelial cell line (NE2) was also treated with 4NQO. We performed histologic analyses, immunofluorescence, and immunohistochemical staining to detect DNA damage at different time points. Whole-exome sequencing was accomplished on the esophagus tissues at different pathological stages to detect single-nucleotide variants and copy number variation (CNV) in the genome. Our findings indicate that all mice were tumor-forming, and a series of changes from simple hyperplasia (ESSH) to intraepithelial neoplasia (IEN) to esophageal squamous cell carcinoma (ESCC) was seen at different times. The expression of γ-H2AX increased from ESSH to ESCC. In addition, mutations of the Muc4 gene were detected throughout the pathological stages. Furthermore, CNV burden appeared in the esophageal tissues from the beginning of ESSH and accumulated more in cancer with the deepening of the lesions. This study demonstrates that mutations caused by the early appearance of DNA damage may appear in the early stage of malignant tissue before the emergence of atypia. The detection of CNV and mutations of the Muc4 gene may be used as an ultra-early screening indicator for esophageal cancer.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Ratones , Animales , Carcinoma de Células Escamosas de Esófago/inducido químicamente , Carcinoma de Células Escamosas de Esófago/genética , Neoplasias Esofágicas/inducido químicamente , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Variaciones en el Número de Copia de ADN , Hiperplasia , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologíaRESUMEN
OBJECTIVE: This study evaluated the effect of laser photobiomodulation (PBM) on oral leukoplakia and squamous cell carcinomas (OSCC) in a model of oral carcinogenesis. MATERIALS AND METHODS: Forty-one C57Bl/6 female mice were distributed in control group, 4-NQO group, Laser group 1.5 J and Laser group 9 J. Oral cancer was induced on the tongue by nitroquinoline oxide (4-NQO), diluted in the water for 16 weeks. In the 18th and 19th weeks, PBM with a diode laser, 0.028 cm2 spot size, continuous emission mode, 660 nm wavelength was applied on the tongue of animals for seven sessions. Laser group 1.5 J received 30 mW power and 1.5 J energy. In the Laser group 9 J, 100 mW power, and 9 J energy were applied. In the 20th week the animals were euthanized. RESULTS: All animals exposed to carcinogen developed clinical and histological alterations such as leukoplakia and OSCC on the tongue. There was no significant difference among Laser groups 1.5 and 9 J and 4-NQO group (not irradiated) regarding the area of leukoplakia and carcinomas (P > 0.05) or thickness of epithelial tissue and keratin (P > 0.05). There were also no association between PBM and histologic classification of the lesions (P = 0.87), frequency of OSCC (P = 0.57), grade of tumor differentiation (P = 0.88) or depth of invasion (P = 0.45). CONCLUSION: Laser PBM, in both parameters used, does not influence on clinical and histological characteristics of oral leukoplakia and OSCC. CLINICAL RELEVANCE: Results suggest that PBM may be a safe treatment for adverse effects of antineoplastic therapies in patients with leukoplakia and OSCC.
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Carcinoma de Células Escamosas , Neoplasias de la Boca , Femenino , Ratones , Animales , 4-Nitroquinolina-1-Óxido/toxicidad , Neoplasias de la Boca/inducido químicamente , Neoplasias de la Boca/radioterapia , Leucoplasia Bucal , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/radioterapia , Carcinógenos , Láseres de Semiconductores/uso terapéuticoRESUMEN
Background and Objectives: Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy in the world. Transient receptor potential vanilloid 4 (TRPV4) channel has been shown to be involved in angiogenesis in multiple types of tumors. However, not much is known about TRPV4's involvement in OSCC. Thus, in this study, we investigate the effect of administering a TRPV4 agonist on angiogenesis in OSCC. Materials and Methods: Thirty-six Sprague Dawley (SD) rats were used in this study. 4-nitroquinoline 1-oxide (4NQO) was used to induce OSCC. Cisplatin (an anticancer drug), and GSK1016790A (an agonist for TRPV4) was used in this study. Immunohistochemistry was employed to examine the TRPV4 expression. An RT2 Profiler PCR Array was performed for gene expression analysis of TRPV4, vascular growth factors that correspond directly with angiogenesis, such as angiopoietin (Ang-1 and Ang-2), and tyrosine kinase (Tie-1 and Tie-2) receptors. Tumor vessel maturity was assessed by microvessel density and microvessel-pericyte-coverage index. Results: RT2 profiler PCR array showed significant elevated levels of Ang-1 (2.1-fold change; p < 0.05) and Tie-2 (4.5-fold change; p < 0.05) in OSCC following the administration of a combination of GSK1016790A and cisplatin. Additionally, the combination treatment significantly reduced the microvessel density (p < 0.01) and significantly increased the percentage of microvessels covered with pericytes (p < 0.01) in OSCC. Furthermore, tumor size was significantly reduced (p < 0.05) in rats that received cisplatin alone. The combination treatment also greatly reduced the tumor size; however, the data were not statistically significant. Conclusions: The findings suggest that combining a TRPV4 agonist with cisplatin for treatment of OSCC promote vessels normalization via modulation of Ang-1/Tie-2 pathway.
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Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Nitroquinolinas , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Cisplatino/farmacología , Cisplatino/uso terapéutico , Modelos Animales de Enfermedad , Leucina/análogos & derivados , Neoplasias de la Boca/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Óxidos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor TIE-2/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Sulfonamidas , Canales Catiónicos TRPVRESUMEN
Head and neck squamous cell carcinoma (HNSCC) is a common cancer of the oral cavity. Cisplatin (CDDP) is the ideal chemo-radiotherapy used for several tumor types, but resistance to the drug has become a major obstacle in treating patients with HNSCC. 5-methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, reduces inflammation-mediated proliferation and metastasis. This study aimed to assess the anti-oral cancer activity of 5-MTP when used alone or in combination with CDDP. Results showed that CDDP dose dependently reduced the growth of SSC25 cells but not 5-MTP. The combination of CDDP and 5-MTP exerted additional inhibitory effect on the growth of SSC25 cells by attenuating the phosphorylation of STAT3. In the 4-nitroquinoline-1-oxide-induced oral cancer mouse model, 5-MTP sensitized the reduction effect of CDDP on tumorigenesis, which restricted the tongue tissue in hyperkeratotic lesion rather than squamous cell carcinoma. The combination of CDDP and 5-MTP may be a potent therapeutic strategy for HNSCC patients with radiotherapy.
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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 αßT 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.
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Antineoplásicos/metabolismo , Carcinógenos/toxicidad , Células de Langerhans/metabolismo , 4-Nitroquinolina-1-Óxido/toxicidad , Línea Celular Tumoral , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Células Epiteliales/metabolismo , Epitelio/efectos de los fármacos , Epitelio/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/patología , Histonas/metabolismo , Humanos , Inmunidad/efectos de los fármacos , Células de Langerhans/efectos de los fármacos , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Fagocitos/patología , Quinolonas/toxicidad , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Lengua/patología , Transcriptoma/genéticaRESUMEN
OBJECTIVES: To investigate the role of hepatocyte growth factor (HGF)/c-Met signaling in oral malignant transformation. METHODS: We used immunohistochemistry to investigate HGF and c-Met expression in 53 oral squamous cell carcinoma (OSCC) specimens and 21 adjacent nontumor specimens and evaluated the associations between HGF and c-Met expression and clinicopathological parameters. Additionally, HGF-overexpression transgenic (HGF-Tg) and wild-type (Wt) mice were treated with 4-nitroquinoline-1-oxide (4NQO) to induce oral carcinogenesis for 16 weeks. At 16, 20, and 24 weeks, tongue lesions were collected for clinical observation; estimation of HGF, c-Met, and PCNA expression; apoptosis (TUNEL) assays; and RNA sequencing (RNA-seq). RESULTS: HGF and c-Met were positively expressed in 92.5% and 64% of OSCC samples, respectively. High HGF expression was significantly associated with smaller tumor size (p = 0.006) and inferior TNM stage (p = 0.032). No correlation between HGF and c-Met levels and other clinical parameters or prognosis was noted. In addition, HGF and c-Met expression was elevated in 4NQO-induced lesions of Wt mice. Compared with Wt mice, HGF-Tg mice have lower tumor incidence, number, volume, and lesion grade. In addition, the percentage of PCNA-positive cells in Wt mice was significantly higher than that in HGF-Tg mice at different time points. At 16 weeks, HGF-Tg mice exhibited less apoptotic cells compared with Wt mice (p < 0.000), and these levels gradually increased until the levels were greater than that of Wt mice at 24 weeks (p < 0.000). RNA-seq data revealed that 140 genes were upregulated and 137 genes were downregulated in HGF-Tg mice. KEGG enrichment analysis showed that upregulated differentially expressed genes (DEGs) are highly correlated with oxidative and metabolic signaling and that downregulated DEGs are related to MAPK and PI3K-AKT signaling. CONCLUSIONS: HGF and c-Met expression is upregulated in OSCC tissues and is associated with the occurrence and development of OSCC. HGF overexpression in normal oral epithelial tissue can inhibit 4NQO-induced tumorigenesis potentially through inhibiting proliferation and accelerating apoptosis via MAPK and PI3K-AKT signaling.
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Tobacco exposure and human papillomavirus (HPV) infection are among the main risk factors for the development of head and neck squamous cell carcinoma (HNSCC). Interestingly, recent studies show that tumors from HPV positive (HPV+) smokers and non-smokers have similar mutational profiles, which suggests that HPV could prevent mutation induction or accumulation in the intermediate risk group composed of HPV+ smokers. Hence, we tested this observation by analyzing the effects of 4-Nitroquinoline N-oxide (4NQO), a mutagen and smoking mimetic, in NOK (normal oral keratinocytes), NOKE6.E7 (NOK cells transfected with E6.E7 oncogenes of HPV), HPV+ and HPV negative (HPV-) HNSCC cells. Oxidative DNA damage, γH2AX foci formation, DNA repair protein activation, cell cycle phase analysis, apoptotic cell death, cell viability and clonogenic cell survival were analyzed after 4NQO treatment in NOK, NOKE6.E7, HPV+ and HPV- HNSCC cells. 4NQO increased oxidative base damage and γH2AX foci formation in NOKE6.E7, HPV+ and HPV- HNSCC cells. Phosphorylation of homologous recombination (HR) repair proteins was higher in NOKE6.E7 and HPV+ HNSCC cells compared to NOK and HPV- HNSCC cells respectively. HPV+ and HPV- HNSCC cells showed differential activation of cell cycle regulatory proteins, increased apoptosis, and decreased cell viability upon 4NQO-induced DNA damage. Taken together, 4NQO (a smoking mimetic), induced higher activation of HR repair in HPV+ HNSCC cells compared to HPV- HNSCC cells. This may allow for increased mutational resistance and help explain why HPV+ smokers have a worse prognosis than HPV+ non-smokers.
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Reparación del ADN , Neoplasias de Cabeza y Cuello , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Carcinoma de Células Escamosas de Cabeza y Cuello , 4-Nitroquinolina-1-Óxido , Células Cultivadas , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/virología , Humanos , Queratinocitos/efectos de los fármacos , Proteínas E7 de Papillomavirus/genética , Fumar , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/virologíaRESUMEN
We investigated the role of PI3Kγ in oral carcinogenesis by using a murine model of oral squamous carcinoma generated by exposure to 4-nitroquinoline 1-oxide (4NQO) and the continuous human cancer cell line HSC-2 and Cal-27. PI3Kγ knockout (not expressing PI3Kγ), PI3Kγ kinase-dead (carrying a mutation in the PI3Kγ gene causing loss of kinase activity) and wild-type (WT) C57Bl/6 mice were administered 4NQO via drinking water to induce oral carcinomas. At sacrifice, lesions were histologically examined and stained for prognostic tumoral markers (EGFR, Neu, cKit, Ki67) and inflammatory infiltrate (CD3, CD4, CD8, CD19 and CD68). Prevalence and incidence of preneoplastic and exophytic lesions were significantly and similarly delayed in both transgenic mice versus the control. The expression of prognostic markers, as well as CD19+ and CD68+ cells, was higher in WT, while T lymphocytes were more abundant in tongues isolated from transgenic mice. HSC-2 and Cal-27 cells were cultured in the presence of the specific PI3Kγ-inhibitor (IPI-549) which significantly impaired cell vitality in a dose-dependent manner, as shown by the MTT test. Here, we highlighted two different mechanisms, namely the modulation of the tumor-infiltrating cells and the direct inhibition of cancer-cell proliferation, which might impair oral cancerogenesis in the absence/inhibition of PI3Kγ.
RESUMEN
Understanding the dynamics of the immune microenvironment is critical to the development of immuno-based strategies for the prevention of oral potentially malignant disorders transformation to oral squamous cell carcinoma (OSCC). We used laser capture microdissection and RNA-sequencing to profile the expression of 13 matched pairs of epithelial versus stromal compartments from normal mucosa, hyperplasia, dysplasia, and invasive tumors in the 4-nitroquinolein (4-NQO) murine model of oral carcinogenesis. Genes differentially expressed at each step of transformation were defined. Immune cell deconvolution and enrichment scores of various biological processes including immune-related ones were computed. Immunohistochemistry was also performed to characterize the immune infiltrates by T-cells (T-cells CD3+, helper CD4+, cytotoxic CD8+, regulatory FoxP3+), B-cells (B220+), and macrophages (M1 iNOS+, M2 CD163+) at each histological step. Enrichment of three independent M2 macrophages signatures were computed in 86 oral leukoplakia with available clinical outcome. Most gene expression changes were observed in the stromal compartment and related to immune biological processes. Immune cell deconvolution identified infiltration by the macrophage population as the most important quantitatively especially at the stage of dysplasia. In 86 patients with oral leukoplakia, three M2 macrophages signatures were independently associated with improved oral cancer-free survival. This study provides a better understanding of the dynamics of the immune microenvironment during oral carcinogenesis and highlights an unexpected association of M2 macrophages gene expression signatures with oral cancer free survival in patients with oral leukoplakia.