Rat Tongue Carcinogenesis Research Articles

Serum metabolite profiling of a 4-Nitroquinoline-1-oxide-induced experimental oral carcinogenesis model using gas chromatography-mass spectrometry.

BackgroundOral cancer progresses from hyperplastic epithelial lesions through dysplasia to invasive carcinoma. The critical needs in oral cancer treatment are expanding our knowledge of malignant tumour progression and the development of useful approaches to prevent dysplastic lesions. This study was designed to gain insights into the underlying metabolic transformations that occur during the process of oral carcinogenesis.MethodsWe used gas chromatography-mass spectrometry (GC-MS) in conjunction with multivariate statistical techniques to observe alterations in serum metabolites in a 4-Nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis model. Thirty-eight male rats were randomly divided into two groups, including the 4NQO-induced model group of 30 rats and the healthy control group of five rats. Animals were sacrificed at weeks 9, 13, 20, 24, and 32, post-4NQO treatment. Tissue samples were collected for histopathological examinations and blood samples were collected for metabolomic analysis. Partial least squares discriminate analysis (PLS-DA) models generated from GC-MS metabolic profile data showed robust discrimination from rats with oral premalignant and malignant lesions induced by 4NQO, and normal controls.ResultsThe results found 16 metabolites associated with 4NQO-induced rat tongue carcinogenesis. Dysregulated arachidonic acid, fatty acid, and glycine metabolism, as well as disturbed tricarboxylic acid (TCA) cycle and mitochondrial respiratory chains were observed in the animal model. The PLS-DA models of metabolomic results demonstrated good separations between the 4NQO-induced model group and the normal control group.ConclusionWe found several metabolites modulated by 4NQO and provide a good reference for further study of early diagnosis in oral cancer.

Evaluation of the Chemopreventive Activity of Grape Skin Extract Using Medium-term Oral Carcinogenesis Assay Induced by 4-Nitroquinoline 1-Oxide.

The aim of this study was to evaluate the chemoprotective potential of grape skin extract following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). Male Wistar rats were distributed into four groups (n=5, per group): Control Group: free access to commercial diet and drinking water for 12 weeks; 4NQO Group: received 4NQO diluted in drinking water daily, for 12 weeks; Grape Skin Extract Group: free access to water and received grape skin extract incorporated with diet for 12 weeks; 4NQO + Grape Skin Extract Group: received 4NQO in drinking water daily and grape extract incorporated with diet for 12 weeks. Animals treated with grape skin extract revealed a significant reduction in epithelial dysplasia. Also, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and ki-67 immunoexpression was reduced in animals treated with grape skin extract. Western blot analysis showed a significant decrease of p-NFκB p50 and MyD88 protein expression in the groups treated with grape skin extract. Copper-zinc superoxide dismutase, manganese superoxide dismutase, and catalase gene expression did not present any statistically significant differences (p>0.05). Grape skin extract displayed chemopreventive activity in oral carcinogenesis assays as depicted by its antioxidant, anti-proliferative and anti-inflammatory properties.

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 12weeks; Group 2-received 4NQO at 50ppm dose in drinking water daily and commercial diet for 12weeks; Group 3-free access to water and received diet supplemented with purple carrot extract (0.1g/kg) for 12weeks; and Group 4-received 4NQO at 50ppm dose in drinking water daily and diet supplemented with purple carrot extract (0.1g/kg) for 12weeks. 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.

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

12 4-nitroquinoline 1-oxide誘発ラット舌癌発生過程におけるp21WAF1/Cip1の発現(第511回 大阪歯科学会例会)

12 4-nitroquinoline 1-oxide誘発ラット舌癌発生過程におけるp21WAF1/Cip1の発現(第511回 大阪歯科学会例会)

Usefulness of a fluorescence visualization system for the detection of oral precancerous and early cancerous lesions.

Early detection of precancerous and early cancerous lesions could greatly reduce both the mortality and morbidity of oral cancer. The objective of this study was to analyze a fluorescence visualization(FV) system for the detection of precancerous and early cancerous lesions in rat tongue carcinogenesis and human oral cancerous lesions using for the first time a 4NQO rat model and human tissue. Based on the results from the rat tongue carcinogenesis model, under direct FV, the normal oral mucosa emitted various shades of pale green autofluorescence. In the precancerous and early cancerous cases, the lesion appeared as an irregular dark area. Histological examination of the lesions showed that the VELscope system had a sensitivity of 95% and specificity of 100% in discriminating normal mucosa from dysplasia/carcinoma insitu(CIS) or invasive carcinoma. The proliferating cell nuclear antigen(PCNA) protein level was gradually increased with progression of carcinogenic transformation. Furthermore, the results of PCNA and FV loss (FVL) were correlated. Next, results from 17 patients were also presented. Histological examination of the lesions showed that the VELscope system had a sensitivity of 95% and specificity of 100% in discriminating normal mucosa from severe dysplasia/CIS or invasive carcinoma. There were no normal epithelium cells in any of the FVL regions. Furthermore, to clarify the usefulness of FV compared to vital staining with iodine, we investigated the surgical margins of early oral squamous cell carcinoma(OSCC) tissues and compared the FVL and iodine unstained area(IU). The percentage of various types of dysplasia were almost equal when comparing the FVL and IU. These results suggest that this direct FV device has the potential for simple, cost-effective screening, detection and margin determination of oral precancerous and early cancerous lesions.

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis

Antioxidant activity of apple extract protects against rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

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

HnRNP K Is A New Prospective Marker of Early Detection for Tongue Carcinogenesis

Previously, we have reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) was associated with a pre-malignant state during 4-nitroquinoline 1-oxide induced rat tongue carcinogenesis.

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

One of the theories regarding oral carcinogenesis is that the tumor growth is initiated from cancer stem cells (CSCs) that self-renew and give rise to differentiated tumor cells, like stem cells do in normal tissues. The most common methods of CSC identification are based on CSC marker expression in carcinogenesis. This study examined the expression of CD133 and CD44, the most commonly used CSC biomarkers in oral squamous cell sarcoma (SCC), with the goal of identifying molecular biomarkers whose expression is associated with the multistep oral carcinogenesis. The expression of CD133, CD44, proliferating cell nuclear antigen (PCNA), and Cytokeratin (CK) was examined by Western blot analysis and confirmed by immunohistochemistry in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model. Also, the expression of aldehyde dehydrogenase 1 (ALDH1), OCT-4 and Nanog were investigated for alteration of cancer cell stemness by Western blot. Along with the progress of multistep carcinogenesis, there were slight increases of CD133 and CD44 expression in the dysplasia group compared with normal rats. However, CD133 protein level was significantly overexpressed in SCC. The expression of PCNA and CK were low in normal group, but sequentially increased in SCC. ALDH1, Nanog and OCT-4 expression were significantly increased according to SCC grade during carcinogenesis. The findings indicate that CD133 is useful in identifying oral CSCs, which suggests that CD133 may serve as a predictor to identify CSCs with a high risk of oral cancer development.

Chemopreventive activity of apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline-1-oxide

ObjectiveThe aim of this study was to evaluate the chemopreventive activity of an apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline-1-oxide (4NQO). MethodsA total of 30 male Wistar rats were distributed into five groups as follows (n=6 per group): Group 1, negative control group (non-treated group); Group 2, received 4NQO during 8 weeks in drinking water and treated with apple extract at 1% by gavage between the first and fourth weeks daily (initiation phase); Group 3, received 4NQO for 8 weeks in drinking water and treated with apple extract by gavage at 1% between the fifth and eighth weeks daily (promotion phase); Group 4, received apple extract at 1% by gavage for 8 consecutive weeks only; and Group 5, received 4NQO for 8 weeks in drinking water daily. ResultsHistopathological analysis revealed decreased hyperplasic lesions in Group 2 when compared with Group 5. Likewise, decreased dysplastic lesions in Group 3 were observed when compared with Group 5. In Groups 2 and 3, decreased COX-2 and TNF-alpha gene expressions were observed when compared with Group 5. Cytochrome c and caspase 3 levels increased in Groups 2 and 3 when compared with Group 5. ConclusionIn conclusion, our results demonstrate that apple extract suppresses rat tongue carcinogenesis as a result of anti-inflammatory activity and apoptosis through the intrinsic mitochondrial pathway.

Chemopreventive effect of green tea catechin on rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Treatments for oral cancer may have significant adverse effect on the patient’s activities of daily living (ADL) and quality of life (QOL). However, foods may offer potential for chemoprevention. In recent years, attention has been focused on the anti-tumor and carcinogenesis-inhibiting actions of green tea. (−)Epigallocatechin-3-gallate (EGCG), the main component of green tea catechins, has been shown to exert a particularly pronounced carcinogenesis-inhibiting effect even among catechins. However, many issues concerning its mechanism remain unclear. Nuclear factor κB (NF-κB) p65 is a protein complex Chemopreventive effect of green tea catechin on rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Anti-tumor activity of grape juice concentrate in the rat tongue two-stage initiation–promotion protocol induced by 4-nitroquinoline 1-oxide

The aim of this study was to evaluate the anti-tumor activity of grape juice concentrate following medium-term oral carcinogenesis assay induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were distributed into five groups, as follows (n = 6 per group): Group 1 – negative control group (non-treated group); Group 2 – received grape juice concentrate at 1% dose by gavage for eight consecutive weeks; Group 3 – received 4NQO for 8 weeks at 20 ppm dose in drinking water daily; Group 4 – received 4NQO at 20 ppm dose during 8 weeks in drinking water and treated with grape juice concentrate at 1% dose orally by gavage for first 4 weeks after 4-NQO administration; Group 5 – received 4NQO at 20 ppm dose for 8 weeks in drinking water and treated with grape juice concentrate at 1% dose orally by gavage between the 5th and 8th weeks daily. Histopathological analysis revealed a decrease in hyperplasic and dysplastic lesions in Group 4. Groups 4 and 5 showed decreased COX-2 and TNF-alpha and eNOS gene expression. Grape juice concentrate also increased SOD Cu/Zn and catalase expression. However, Ki-67 immunoexpression was reduced at the promotion step of oral carcinogenesis (G5). Taken together, our results demonstrate that grape juice concentrate modulates rat tongue carcinogenesis as a result of anti-inflammatory activity, antioxidant activity and down-regulation of oral cells proliferation.

Quantitative proteomic analysis of different stages of rat lingual carcinogenesis

Background: In India, oral squamous cell carcinoma (OSCC) is the single largest group of malignancies in males. Early diagnosis of cancer is difficult because of the lack of specific symptoms and/or biomarkers for early disease. Animal models provide an opportunity to study development and progression of cancers. Materials and Methods: In this study, we have explored the 4-nitroquinoline 1-oxide (4NQO)-induced tongue cancer model in Sprague Dawley rats. We compared the protein expression profiles of normal tissues with different stages of rat tongue cancer using isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics strategy. We validated some known and novel proteins by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR). Results: We observed hyperplasia, papillomas, and carcinomas after 120, 160, and 200 days treatment of 4NQO, respectively. LC-MS/MS analysis resulted in identification of 2223 proteins. Of these, 415 proteins were found to be differentially expressed in tumors, 333 proteins in papilloma and 109 proteins in hyperplasia. We have found alterations in several previously reported as well as novel proteins during rat tongue carcinogenesis. We validated known molecules such as vimentin, fascin, periostin, transglutaminase 3 by IHC and cornulin by real-time PCR on rat tissues. We also validated tenascin N, a novel protein by IHC on rat as well as in human tongue tissues. Conclusion: To the best of our knowledge, this is the first in-depth differential proteomics study carried out using an experimental rat model of OSCC. Proteomic alterations observed in this study provide insights into carcinogenesis process and may serve as a valuable resource for oral cancer biomarker discovery.

Chemopreventive activity of apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline 1-oxide

The aim of this study was to evaluate the chemopreventive activity of an apple extract following medium-term oral carcinogenesis assay induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were distributed into five groups, as follows (n=6 per group): Group 1, negative control group (non-treated group); Group 2, received 4NQO during 8 weeks in drinking water and treated with apple extract at 1% by gavage between the 1st and 4th weeks daily (initiation phase); Group 3, received 4NQO for 8 weeks in drinking water and treated with apple extract by gavage at 1% between the 5th and 8th weeks daily (promotion phase); Group 4, received apple extract at 1% by gavage for eight consecutive weeks only; and Group 5, received 4NQO for 8 weeks in drinking water daily. Histopathological analysis revealed decreased hyperplasic lesions in Group 2 when compared to Group 5. Likewise, decreased dysplastic lesions in Group 3 were observed when compared to Group 5. Groups 2 and 3 decreased COX-2 and TNF-alpha gene expression when compared to Group 5. Cytochrome c and caspase 3 levels increased in Groups 2 and 3 when compared to Group 5 as well. In conclusion, our results demonstrate that apple extract suppresses rat tongue carcinogenesis as a result of anti-inflammatory activity and apoptosis through intrinsic mitochondrial pathway.

Increased frequency of CD4+ CD25+ FOXP3+ cells correlates with the progression of 4-nitroquinoline1-oxide-induced rat tongue carcinogenesis

ObjectivesCD4+ CD25+ FoxP3+ T cells (Tregs) play an essential role in sustaining self-tolerance by negatively regulating immune responses. Increased frequencies of Tregs have been reported in a variety of human cancers. The aim of this study was to evaluate the prevalence of Tregs infiltration in the peripheral blood and regional lymph nodes during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO).Materials and methodsForty-eight Sprague–Dawley rats were divided into the control (n = 16) and experimental groups (n = 32) to which 4NQO in drinking water was administered. Flow cytometry was used to analyze the prevalence of Tregs in lymphocytes of peripheral blood and regional lymph nodes during 4NQO-induced rat tongue carcinogenesis. CD4+ CD25+ FoxP3+ cells were expressed as a percentage of the total CD4+ cells.ResultsThe frequency of Tregs in peripheral blood from squamous cell carcinoma rats was significantly higher than controls (3.82 ± 0.62 versus 1.40 ± 0.31 %, P < 0.001). The proportion of Tregs was sequentially increased from moderate dysplasia to severe dysplasia and SCC (1.94 ± 0.72, 2.29 ± 0.82, and 3.82 ± 0.62 %, respectively). The frequency of Tregs in regional lymph nodes from squamous cell carcinoma rats was also significantly higher than normal rat mucosa (14.67 ± 3.09 versus 5.53 ± 2.07 %, P < 0.001). The percentage of Tregs was gradually increased in moderate dysplasia, severe dysplasia, and SCC groups (8.93 ± 1.74, 10.15 ± 0.86, 14.67 ± 3.09 %, respectively) as compared to control group (5.53 ± 2.07 %).Conclusion and clinical relevanceTregs in peripheral blood and lymph nodes were associated with disease progression during 4NQO-induced rat tongue carcinogenesis. This study indicated that the upregulation of Tregs might play important role during oral mucosa malignant transformation.

The influence of sleep deprivation on expression of apoptosis regulatory proteins p53, bcl-2 and bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to evaluate whether paradoxical sleep deprivation could affects the mechanisms and pathways essentials for cancer cells in tongue cancer induced by 4-nitroquinole 1-oxide in Wistar rats. For this purpose, the animals were distributed into 4 groups of 5 animals each treated with 50 ppm 4 nitroquinoline 1 oxide (4 NQO) solution through their drinking water for 4 and 12 weeks. The animals were submitted to paradoxical sleep deprivation (PSD) for 72 h using the modified multiple platform method, which consisted of placing 5 mice in a cage (41 × 34 × 16 cm) containing 10 circular platforms (3.5 cm in diameter) with water 1 cm below the upper surface. The investigations were conducted using immunohistochemistry of p53, Bax and Bcl-2 proteins related to apoptosis and its pathways. Statistical analysis was performed by Kruskal-Wallis non-parametric test followed by the Dunn's test using SPSS software pack (version 1.0). P value < 0.05 was considered for statistic significance. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre-neoplasic lesions. Data analysis revealed statistically significant differences (P < 0.05) in 4 weeks group for p53 and for bcl-2 and for all immunomarkers after 12 weeks of 4NQO administration. Our results reveal that sleep deprivation exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis.

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析

4-nitroquinoline 1-oxide誘発ラット舌癌発生過程における蛍光光学機器解析

Analysis of fluorescence visualization in rat tongue carcinogenesis by 4-nitroquinoline 1-oxide

Analysis of fluorescence visualization in rat tongue carcinogenesis by 4-nitroquinoline 1-oxide

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

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