Proposal of a secure and efficient protocol for a murine oral carcinogenesis model induced by 4-nitroquinoline-1-oxide (4NQO).

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.

MMP1 Overexpression Promotes Cancer Progression and Associates with Poor Outcome in Head and Neck Carcinoma.

Matrix metalloproteinase-1 (MMP1) has been reported to play key roles in a variety of cancers by degrading the extracellular matrix. However, its carcinogenic roles have not been shown yet in head and neck squamous cell carcinoma (HNSCC). This study aimed to elucidate its expression pattern and functional roles as well as clinical significance in HNSCC. The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and immunohistochemistry (IHC) were utilized to determine the MMP1 expression pattern and the associations between its expression and patients' outcome in HNSCC. Mice tongue squamous cell carcinoma model induced by 4-nitroquinoline 1-oxide (4NQO) and siRNA-mediated cellular assay in vitro were utilized to evaluate the oncogenic role of MMP1. The biological functions and cancer-related pathways involved in MMP1-related genes were found through bioinformatics analysis. Both mRNA and protein abundance of MMP1 were highly increased in HNSCC as compared to its non-tumor counterparts. MMP1 overexpression positively correlated with advanced tumor size, cervical node metastasis, and advanced pathological grade and lower patients' survival. In the 4NQO-induced animal model, MMP1 expression increased along with the progression of the disease. In HNSCC cells, siRNA-mediated knockdown of MMP1 significantly inhibited cell proliferation, migration, and invasion and activated apoptosis and epithelia-mesenchymal transition (EMT). GSEA, GO, and KEGG analyses showed that MMP1 expression was significantly related to cancer-related pathways and cancer-related functions. Together, our results demonstrated MMP1 serves as a novel prognostic biomarker and putative oncogene in HNSCC.

Negative terpinen-4-ol modulate potentially malignant and malignant lingual lesions induced by 4-nitroquinoline-1-oxide in rat model.

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.

Early Detection of Leukoplakic Oral Squamous Cell Carcinoma Using 4NQO-induced Rat Tongue Cancer Model: Study Utilizing Fluorescence Intensity and Histopathological Evaluation.

Early identification of leukoplakic oral squamous cell carcinoma (OSCC) is difficult. The purpose of this study was to determine whether it was possible to detect change from normal epithelium to leukoplakic OSCC using a fluorescence visualization (FV) device in a 4-nitroquinoline 1-oxide (4NQO) -induced rat tongue cancer model. If successful, this would facilitate early detection of OSCC. The rats (3 groups of 5) were administered 50 ppm 4NQO in their drinking water over a period of 10, 15, or 20 weeks. Five non-treated rats were used as a control group. Images of their tongues obtained by FV were analyzed for change in fluorescence intensity (FI) using image analysis software. Immunoreaction for anti-CK13, anti-CK17, and anti-E-cadherin antibodies was also histopathologically evaluated. Receiver operating characteristic (ROC) analysis was used to calculate the cut-off values, sensitivity, specificity, and area under the curve. The most marked change in FI was found between the control and 10-week groups, with an increase observed in its average value and range in the latter. These findings differed from those characteristic of leukoplakia. No significant difference was observed in the positive cell rate for immunoreaction for anti-CK13 or anti-CK17 antibodies between the control and 10-week groups. A significant decrease was observed in the positive pixel ratio of immunoreaction for anti-E-cadherin antibody in the 10-week group in comparison with in the control group (p <0.05). These results showed that disruption of intercellular adhesion could be observed at 10 weeks. In the ROC analysis, the FI cut-off value in the 10-week and control groups was 51.9, sensitivity 95.5%, and specificity 96.9%. This indicated that normal epithelium could be accurately distinguished from low-grade dysplasia with high probability. These results demonstrate that analysis of change in FI as measured by FV could facilitate early detection of leukoplakic OSCC.

Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation.

Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.

The Histopathology of Oral Cancer Pain in a Mouse Model and a Human Cohort.

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.

Can propranolol act as a chemopreventive agent during oral carcinogenesis? An experimental animal study.

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.

Protective and therapeutic effects of pyrrolidine dithiocarbamate in a rat tongue cancer model created experimentally using 4-nitroquinoline 1-oxide.

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.

Single-Cell Analysis of Different Stages of Oral Cancer Carcinogenesis in a Mouse Model.

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

Rhabdomyosarcoma in a child with nephrotic syndrome treated with cyclosporine: a case report with literature review.

BACKGROUND: In patients with frequently relapsing nephrotic syndrome, immunosuppressive therapy such as cyclosporine are often required to maintain remission. Cyclosporine has been noted to have tumorgenesis effects. In this case report, we present a child with relapsing nephrotic syndrom developed a rhabdomyosarcoma on her tongue after adout 4 years of continual immunosuppressive therapy. CASE PRESENTATION: A 2-year-old female child had nephrotic syndrome (urine protein-creatinine ratio 749.1 mg/mg; blood urea nitrogen 11 mg/dL; serum creatinine 0.3 mg/dL; and serum albumin 1.8 g/dL.) Proteinuria resolved on treatment with daily prednisolone for 4 weeks at the dose of 45 mg (2.5 mg/kg/day) but recurred with taper from 25 mg/day to 10 mg/day. At least five more episodes of relapse occurred within about a 3-year period. After the third relapse, she was treated with prednisolone and cyclosporine (at initial dose of 50 mg/day [1.7 mg/kg/day]) for immunosuppression. About 4 years after the diagnosis of nephrotic syndrome had been made, an embryonal rhabdomyosarcoma developed on her tongue. The cancer was treated with TPOG-RMS-LR protocol, with vincristine, actinomycin, and cyclophosphamide. Magnetic resonance imaging scan, performed about 3 years after the start of TPOG-RMS-LR therapy, revealed complete remission of the cancer. CONCLUSIONS: Although treatment with cyclosporine cannot be conclusively implicated as the cause the rhabdomyosarcoma in this patient, the association should prompt consideration of its use in the treatment of frequently relapsing nephrotic syndrome in children.

A PIK3CA transgenic mouse model with chemical carcinogen exposure mimics human oral tongue tumorigenesis.

Oral cancer causes significant global mortality and has a five-year survival rate of around 64%. Poor prognosis results from late-stage diagnosis, highlighting an important need to develop better approaches to detect oral premalignant lesions (OPLs) and identify which OPLs are at highest risk of progression to oral squamous cell carcinoma (OSCC). An appropriate animal model that reflects the genetic, histologic, immunologic, molecular and gross visual features of human OSCC would aid in the development and evaluation of early detection and risk assessment strategies. Here, we present an experimental PIK3CA + 4NQO transgenic mouse model of oral carcinogenesis that combines the PIK3CA oncogene mutation with oral exposure to the chemical carcinogen 4NQO, an alternate experimental transgenic mouse model with PIK3CA as well as E6 and E7 mutations, and an existing wild-type mouse model based on oral exposure to 4NQO alone. We compare changes in dorsal and ventral tongue gross visual appearance, histologic features and molecular biomarker expression over a time course of carcinogenesis. Both transgenic models exhibit cytological and architectural features of dysplasia that mimic human disease and exhibit slightly increased staining for Ki-67, a cell proliferation marker. The PIK3CA + 4NQO model additionally exhibits consistent lymphocytic infiltration, presents with prominent dorsal and ventral tongue tumours, and develops cancer quickly relative to the other models. Thus, the PIK3CA + 4NQO model recapitulates the multistep genetic model of human oral carcinogenesis and host immune response in carcinogen-induced tongue cancer, making it a useful resource for future OSCC studies.

Effect of celecoxib on protein expression of FAK and Cx43 in DMBA induced rat tongue carcinoma cells.

OBJECTIVE: The pathogenesis of tongue cancer (TA) has not been fully illustrated. Cyclooxygenase-2 (COX-2) is correlated with the precancerous lesion of oral cavity mucosa and malignant transformation. The focal adhesion kinase (FAK) and gap junction protein connexin 43 (Cx43) are involved in the occurrence and progression of tumors. This study aimed to investigate the effect of celecoxib on the proliferation, malignant transformation, and expression of FAK and Cx43 proteins. MATERIALS AND METHODS: Healthy male Sprague-Dawley (SD) rats (4 months old) were divided into control, model and celecoxib group. 7,12-dimethylbenzanthracene (DMBA) was used to generate tongue mucosal carcinoma, coupled with celecoxib intervention. At 8, 12, 16, and 20 weeks after induction, the rat survival status, the tumor formation rate and the tongue tissue morphology were observed. Meanwhile, the expression of FAK and Cx43 was also evaluated by using immunohistochemistry (IHC). RESULTS: Tumor occurrence rates after induction were 0, 26.67%, 66.67%, and 80% at 8, 12, 16, and 20 weeks, respectively. The celecoxib treatment decreased such rats to 0, 0, 0, and 13.33%, respectively (p<0.05 compared to model group). No significant change was observed in control group, whilst model group had mild to severe hyperplasia and squamous carcinoma with elongated time. Celecoxib treatment significantly improved the tissue morphology (p<0.05). The model group also had elevated FAK and depressed Cx43 protein expression (p<0.05). With elongated time, the FAK expression was further increased whilst Cx43 protein was depressed (p<0.05 compared to model group). CONCLUSIONS: The focal application of celecoxib effectively inhibited the DMBA-induced rat TA, possibly via regulating FAK and CX43 protein expression, and inhibiting oral epidermal hyperplasia.

Method for diagnosing neoplastic lesions by quantitative fluorescence value.

Fluorescence visualization devices (FVs) are useful for detecting malignant lesions because of their simple and noninvasive application. However, their quantitative application has been challenging. This study aimed to quantitatively and statistically evaluate the change in fluorescence intensity (FI) during the progression from normal epithelium to squamous cell carcinoma using a reproducible animal tongue carcinogenesis model. To establish this model, rats were treated with 50 ppm 4-Nitroquinoline 1-oxide (4NQO) in their drinking water for 10, 15, and 20 weeks. After 4NQO administration, each rat tongue was evaluated by gross observation, histology, and FI measurements. Fluorescence images were captured by FV, and ImageJ was used to measure FI, which was analyzed quantitatively and statistically. The establishment of a reproducible tumor progression model was confirmed, showing precancerous lesions (low-grade dysplasia [LGD]), early cancers (high-grade dysplasia/carcinoma in situ [HGD/CIS]), and advanced cancers (Cancer). This carcinogenesis model was quantitatively evaluated by FI. The FI of LGD stage was 54.6, which was highest intensity of all groups. Subsequently, the HGD/CIS and Cancer stages showed decreased FI (HGD/CIS: 46.1, Cancer: 49.1) and manifested as dark spots. This result indicates that FI had more variation and a wider range with increasing tumor progression. We demonstrated that FI migration and an uneven distribution are consistent with tumor progression. Since each step of tumor progression occurs reproducibly in this animal model, statistical evaluation was possible. In addition, tumor progression can be monitored by this new FI analysis method in humans.

Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.

BACKGROUND: The survival of OSCC patient needs to be further improved. miR-211 is oncogenic in OSCC and its upregulation is associated with tumor progression and poor patient survival. K14-EGFP-miR-211 transgenic mice also exhibit augmented potential for OSCC induction. METHODS: Four murine OSCC cell lines, designated MOC-L1 to MOC-L4, are established from tongue tumors induced by 4-nitroquinoline 1-oxide using the K14-EGFP-miR-211 transgenic mouse model. The genetic disruption, in vitro oncogenicity, and the eligibilities of tumorigenesis and metastasis of the cell lines are analyzed. RESULTS: All cell lines show green fluorescence and express a range of epithelial markers. The MOC-L1, MOC-L2 and MOC-L3 cells carry missense mutations in the DNA binding domain of the p53 gene. MOC-L1 exhibits a high level of epithelial-mesenchymal transition and has the aggressive characteristics associated with this. MOC-L1 and MOC-L2 are clonogenic in vitro as well as being tumorigenic when implanted into the dermis or tongue of syngeneic recipients. Nonetheless, only MOC-L1 exhibits immense potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts is drastically decreased on cisplatin treatment, it would seem that targeting of miR-196b might facilitate tumor abrogation. CONCLUSIONS: As cell lines established in this study originated from the C57BL/6 mouse, the strain most suitable for transgenic engineering, exploring the interplay of these OSCC cells with other genetically modified cells in immune-competent mice would provide important insights into OSCC pathogenesis.

No difference in 4-nitroquinoline induced tumorigenesis between germ-free and colonized mice.

Variations in oral bacterial communities have been linked to oral cancer suggesting that the oral microbiome is an etiological factor that can influence oral cancer development. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine oral and esophageal cancer model is frequently used to assess the effects of preventive and/or therapeutic agents. We used this model to assess the impact of the microbiome on tumorigenesis using axenic (germ-free) and conventionally housed mice. Increased toxicity was observed in germ-free mice, however, no difference in tumor incidence, multiplicity, and size was observed. Transcriptional profiling of liver tissue from germ-free and conventionally housed mice identified 254 differentially expressed genes including ten cytochrome p450 enzymes, the largest family of phase-1 drug metabolizing enzymes in the liver. Gene ontology revealed that differentially expressed genes were enriched for liver steatosis, inflammation, and oxidative stress in livers of germ-free mice. Our observations emphasize the importance of the microbiome in mediating chemical toxicity at least in part by altering host gene expression. Studies on the role of the microbiome in chemical-induced cancer using germ-free animal models should consider the potential difference in dose due to the microbiome-mediated changes in host metabolizing capacity, which might influence the ability to draw conclusions especially for tumor induction models that are dose dependent.

Mutational signatures and the genomic landscape of betel quid chewing-associated tongue carcinoma.

Our study presents the genetic landscape betel quid chewing-associated tongue carcinomas (BQ-TCs). We compared the genetic landscape and mutational signatures of 15 BQ-TCs, five nonbetel quid chewing-associated tongue carcinomas (nBQ-TCs), and 82 tongue carcinomas in general population from the TCGA (TCGA-TCs) project. The highlights of this research mainly include: (a) The genetic landscape of BQ-TC was characterized with frequent mutations in RASA1 gene and in CpG islands throughout the genome. (b) The BQ-TC had a distinct mutational signature from that of nBQ-TC and tongue carcinomas in the general population, and this signature was associated with the mutations in RASA1 and in CpG islands. (c) Our study indicates that betel quid (BQ) chewing classifies a distinct group of tongue carcinoma. The BQ chewing might not contribute to the tumorigenesis of tongue carcinomas as a mutagen.

Loss of Notch1 predisposes oro-esophageal epithelium to tumorigenesis.

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

Protective effects of purple carrot extract (Daucus carota) against rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

The aim of this study was to evaluate the chemopreventive potential of purple carrot extract following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). For this purpose, histopathological analysis, proliferative status, antioxidant activity and inflammatory status were investigated in this setting. A total of 20 male rats were distributed into four groups as follows (n = 5 per group): Group 1-free access to water and commercial diet for 12 weeks; Group 2-received 4NQO at 50 ppm dose in drinking water daily and commercial diet for 12 weeks; Group 3-free access to water and received diet supplemented with purple carrot extract (0.1 g/kg) for 12 weeks; and Group 4-received 4NQO at 50 ppm dose in drinking water daily and diet supplemented with purple carrot extract (0.1 g/kg) for 12 weeks. Histopathological analysis revealed that animals treated with purple carrot extract reduced the oral lesions such as dysplasia and squamous cell carcinoma. Animals with oral pre-neoplastic lesions and treated with purple carrot extract decreased ki-67 and 8-OHdG immunoexpression. Moreover, pNFκBp50 and MyD88 protein expressions were decreased after purple carrot treatment associated or not with 4NQO exposure. SOD-Mn mRNA levels increased with treatment with purple carrot extract as well. In conclusion, our results demonstrated that purple carrot extract was able to protect oral lesions induced by 4NQO in Wistar rats as a result of antioxidant activity, anti-inflammatory potential and antiproliferative and antimutagenic actions.

Effects of low-intensity ultrasound combined with low-dose carboplatin in an orthotopic hamster model of tongue cancer: A preclinical study.

Low-intensity ultrasound (LIUS) combined with chemotherapy is an innovative modality for cancer treatment, but its effect on orthotopic carcinoma remains unknown. Our previous study revealed that LIUS enhanced the growth inhibitory effects of several chemotherapeutic drugs in nude mice with transplanted tumors. In the present study, we used 7,12-dimethylbenz(alpha)anthracene to induce orthotopic tongue carcinogenesis in hamsters. We used the first-line chemotherapy drug for tongue cancer, carboplatin (CBP) in combination with LIUS to investigate the synergistic effect. The results revealed that LIUS combined with low-dose CBP enhanced the inhibitory effects of CBP on tumor growth, prolonged survival, and did not increase the incidence of side-effects. It also enhanced the inherent DNA damage caused by CBP, suppressed the expression of the DNA repair proteins O6-methylguanine DNA methyltransferase (MGMT) and Chk1, and increased the expression of DNA damage-inducible protein GADD45α. Furthermore, compared with CBP alone, LIUS combined with CBP reduced the expression of cyclin D1 and cyclin B1, induced the expression of caspase-3, cleaved caspase-3, caspase-8, Bax, and Bak, and inhibited the expression of Bcl-2. Examination of clinical samples revealed that MGMT, Chk1, and Gadd45α were higher in OTSCC than in adjacent normal tissue. Hence, our results indicated that LIUS enhanced the ability of low-dose CBP to damage DNA in an orthotopic hamster model of tongue cancer, induced apoptosis, inhibited tumor growth and progression, while it did not increase the toxic side-effects of the drug, suggesting additional clinical benefits for patients treated with the combination of CBP with LIUS.