In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N⁶-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.
AlkB Homolog 1, Histone H2a Dioxygenase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism* ; Tongue Neoplasms/metabolism*

OBJECTIVE: To examine the correlation of CCBE1 expression in adjacent tissues of tongue squamous cell carcinoma (TSCC) with pericancerous lymphatic vessel proliferation, cervical lymph node metastasis and survival outcomes of the patients. METHODS: Lymphatic vessel density was quantified in pericancerous tissue sections of 44 cases of cT_1-2N₀ TSCC using D2-40 as the lymphatic vessel endothelial marker for calibration and counting of the lymphatic vessels. Of these 44 cases, 22 showed a relatively low lymphatic vessel density (group A) and the other 22 had a high lymphatic vessel density (group B), and the expression levels of CCBE1 in the adjacent tissues determined using immunohistochemistry, immunofluorescence assay and Western blotting were compared between the two groups. The expression level of CCBE1 was also measured in another 90 patients with TSCC using immunohistochemistry, and all the patients were followed up for their survival outcomes. RESULTS: Immunohistochemistry and Western blotting showed a significantly lower rate of high CCBE1 expression in group A than in group B (P < 0.05). Immunofluorescence assay showed co-localization of CCBE1 and D2-40 in the adjacent tissues of TSCC. In the 90 TSCC patients with complete follow-up data, a high expression of CCBE1 was found to correlate with lymph node metastasis and a poor 5-year survival outcomes of the patients (P < 0.05). CONCLUSION A high expression of CCBE1 in the adjacent tissues of TSCC is closely related with pericancerous lymphatic vessel proliferation, cervical lymph node metastasis and a poor 5-year survival of the patients, suggesting the value of CCBE1 as a potential prognostic predictor for TSCC.
Humans ; Tongue Neoplasms/pathology* ; Carcinoma, Squamous Cell/metabolism* ; Lymphatic Metastasis ; Prognosis ; Lymphatic Vessels/pathology* ; Cell Proliferation ; Tongue/pathology* ; Calcium-Binding Proteins/metabolism* ; Tumor Suppressor Proteins/metabolism*

To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbed with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using Cellbed accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.
Carcinoma in Situ ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Culture Techniques ; methods ; Cell Movement ; Cell Proliferation ; Cytoskeletal Proteins ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Neoplasm Invasiveness ; pathology ; Phosphorylation ; Podosomes ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Silicon Dioxide ; Tongue Neoplasms ; metabolism ; pathology ; Tumor Cells, Cultured

OBJECTIVETo investigate the expression of LRG-1 in clinical specimens and Tca8113 cell line of tongue carcinoma and analyze the relationship between LRG-1 expression and the clinicopathological parameters.

METHODSLRG-1 expression was detected in 40 tongue squamous cell carcinoma (TSCC) tissues and paired normal adjacent tissues, 20 atypical hyperplasia tissues of the tongue, and 20 tissues of tongue cancer in situ using immunohistochemical method. The expression of LRG-1 in Tca8113 cell line was detected using flow cytometry. The expression of LRG-1 was also detected in human TSCC tissues and Tca8113 cells with Western blotting. The effect of LRG-1 on the proliferation of HUVECs was determined using MTT assay, and its effect on angiogenesis was evaluated with Matrigel tube formation assays.

RESULTSHuman TSCC tissues had a significantly higher rate of positive expression for LRG-1 (85%, 34/40) than the adjacent tissues (10%, 4/40), invasive tongue cancer (30%, 6/20), and tongue cancer in situ (50%, 10/20) (P<0.05). LRG-1 expression was correlated with the degree of tumor differentiation, clinical stage and lymph node metastasis of the tumor (P<0.05) but not with the patients' age or gender. In the in vitro experiment, LRG-1 promoted HUVEC proliferation and angiogenesis.

CONCLUSIONAbnormal LRG-1 expression is present in the human TSCC tissue and Tca8113 cells. LRG-1 can promote HUVEC proliferation and angiogenesis in vitro, suggesting its possible role in promoting tumor angiogenesis.

Carcinoma, Squamous Cell ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Glycoproteins ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Lymphatic Metastasis ; Tongue ; metabolism ; pathology ; Tongue Neoplasms ; genetics ; metabolism

OBJECTIVETo study the effects of RhoA down-regulation by RNA interference on the invasion of tongue carcinoma Tca8113 and SCC-4.

METHODSDetermination of the human RhoA sequence as well as the design and constructionof a short specific small interfering RNAs (siRNA) were performed. The siRNA of RhoA gene was transfected into humantongue squamous cell carcinoma Tca8113 and SCC-4 cells line by Lipofectamine 2000. Quantitative real-time polymerasechain reaction was used to examine the mRNA expressionlevels of RhoA. Protein expressions of mRNA, galectin-3,and matrix metalloproteinase (MMP)-9 were evaluated byWestern blot. Transwell invasion assay was performed toassess the invasion ability of tongue carcinoma.

RESULTSRhoA expressions in Tca8113 and SCC-4 cells were reducedsignificantly after transfection of RhoA-siRNA. Protein levels f galectin-3 and MVP-9 were also down-regulated significantly. Invasion ability was inhibited as well.

CONCLUSIONRhoA-siRNA can effectively inhibit RhoA expression in Tca8113 and SCC-4 cells. The invasion ability of tongue carcinoma cells decreased with down-regulation of the protein expressions of galectin-3 and MMP-9, indicating that RhoA-siRNA can inhibit invasion of tongue carcinoma. Results show that RhoA may play an important role in the processes of invasion and metastasis of tongue carcinoma.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Galectin 3 ; metabolism ; Gene Silencing ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; Transfection

OBJECTIVETo investigate the effect of downregulation of N-cadherin expression on cell proliferation, cell cycle, cell apoptosis and cell migration in tongue squamous cell carcinoma cell line Tca8113 cells.

METHODSN-cadherin siRNA was transfected into tongue squamous cell carcinoma cell line Tca8113 cells and Tca8113 cells were divided into three groups: untreated group, control siRNA group and N-cadherin siRNA group. The cells were harvested 48 h after transfection with N-cadherin siRNA. Cell proliferation of Tca8113 cells was examined by cell counting kit (CCK)-8 after transfection with N-cadherin, and the effects of downregulation of N-cadherin on cell cycle and cell apoptosis of Tca8113 cells were investigated by flow cytometry. The effect of downregulation of N-cadherin expression on cell migration of Tca8113 cells was observed by Boyden chamber experiment, and further expression changes of gene-related cell proliferation, cell cycle and cell migration were detected by Western blotting.

RESULTSN-cadherin siRNA downregulated the N-cadherin expression and significantly inhibited cell proliferation of Tca8113 cells (P < 0.05). The results of cell cycle revealed that the percentage of G(0)/G(1) phase in N-cadherin group [(65.41 ± 0.92)%] was significantly higher than that in untreated group [(41.59 ± 1.43)%] or control siRNA group [(43.70 ± 2.08)%], and there was significant difference among the three groups (F = 216.839, P = 0.000). The percentage of cell apoptosis in N-cadherin group [(25.66 ± 1.36)%] was significantly higher than that in untreated group [(2.38 ± 0.14)%] or control siRNA group [(2.81 ± 0.12)%], and there was significant difference among the three groups (F = 850.364, P = 0.000). The cell number migrated into memebrane in N-cadherin group was significantly lower than that in untreated group and control siRNA group, and there was significant difference among the three groups (F = 140.858, P = 0.000). Further, compared with untreated group and control siRNA group, the expressions of matrix metalloproteinase (MMP)-2 and MMP-9 proteins were significantly downregulated and expression of p21 protein was significantly upregulated (P < 0.05).

CONCLUSIONSN-cadherin may play a role in occurrence and development of tongue squamous cell carcinoma.

Apoptosis ; Cadherins ; genetics ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Down-Regulation ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Tongue Neoplasms ; metabolism ; pathology ; Transfection

OBJECTIVE: To determine the expression levels of EphA7 and MTDH and detect their clinicopathological significance in the peritumoral normal tissues and the squamous cell cancer of the tongue. METHODS: Envision immunohistochemistry was used to assay the expression levels of EphA7 and MTDH in the conventional paraffin-embedded sections from specimens of squamous cell cancer (n=45) and peritumoral normal tissues (n=10). RESULTS: The positIVe rates of EphA7 and MTDH were significantly higher in the squamous cell cancer than those in the peritumoral normal tissues ( χ(2)(EphA7)=4.14; χ(2)(MTDH)= 5.25; P < 0.05). The positIVe rates of EphA7 and MTDH expression were significantly lower in the cases of histological grade I-II,clinical stage I-II, and no-metastasis of neck lymph node than those in the histological grade III-IV, clinical stage III-IV, and metastasis of neck lymph node (P <0.05 or P <0.01). CONCLUSION The expression levels of EphA7 and/or MTDH might have important effect on the carcinogenesis and progression of tongue cancer. Overexpression of EphA7 and/or MTDH might have poor prognosis.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Adhesion Molecules ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Receptor, EphA7 ; metabolism ; Tongue Neoplasms ; metabolism ; pathology

OBJECTIVETo investigate the relationship between the extracellular matrix (ECM) and neoplastic progression in hamster with tongue cancer.

METHODSForty-eight specimens of hamster tongue cancer were divided into control group (n=6) and experimental group (n=42). The pathological grade of the specimens was assessed (including 3 stages, namely atypical hyperplasia, carcinoma in situ and early invasive carcinoma). The sections of the tongue were stained with Masson and aldehyde-fuchsin (AF) staining for microscopic observation of the elastic fiber and collagen fiber changes.

RESULTSWithin the connective tissue cores (CTC) of the papillae in the control group was a framework of numerous and fine Gomrori's aldehyde fuchsin-positive elastic fibers. But in the stages of dysplasia and carcinoma in situ, these elastic fibers decreased and further diminished in the CTC in early invasive carcinoma. In dysplasia and carcinoma in situ stages, most of the elastic fibers collapsed with scattered elastic fibers, and the elastic fibers decreased significantly in early invasive carcinoma. The control group showed a significantly greater number of elastic fibers in the experimental group. The collagen fiber was obviously increased and irregularly arranged in dysplasia and carcinoma in situ stage; in early invasive carcinoma, the collagen fibers became thicker with deposition in the lamina propria.

CONCLUSIONAn excessive deposition of collagen fiber and reduction of the elastic fibers is an important factor contributing to the development of tongue carcinoma in hamsters.

Animals ; Carcinoma ; pathology ; Collagen ; metabolism ; Connective Tissue ; pathology ; Cricetinae ; Elastic Tissue ; pathology ; Extracellular Matrix ; pathology ; Neoplasms, Experimental ; pathology ; Tongue Neoplasms ; pathology

Glossectomy ; methods ; Humans ; In Situ Hybridization, Fluorescence ; Keratins ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Sarcoma, Synovial ; genetics ; metabolism ; pathology ; surgery ; Tongue Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Translocation, Genetic ; Vimentin ; metabolism

OBJECTIVETo investigate the effect of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib on prostaglandin E(2) (PGE2) release and vascular endothelial growth factor C (VEGF-C) and COX-2 mRNA expression in Tca8113 cell lines.

METHODSMTT assay was used to analyze the proliferation of Tca8113 cells. The PGE2 level was detected with enzyme-linked immunosorbent assay (ELISA), and the expressions of COX-2 and VEGF-C mRNA were examined with RT-PCR.

RESULTSCelecoxib could induce inhibitory effects on the growth and PGE2 release in Tca8113 cells. The RT-PCR results showed that celecoxib significantly down-regulated the expression of VEGF-C mRNA, but produced a weak effect on COX-2 mRNA expression.

CONCLUSIONThe inhibitory effect of celecoxib on Tca8113 cell growth and the expressions of VEGF-C and COX-2 may be related to the release of PGE2.

Carcinoma, Squamous Cell ; blood supply ; metabolism ; pathology ; Celecoxib ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Dinoprostone ; metabolism ; Humans ; Pyrazoles ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Sulfonamides ; pharmacology ; Tongue Neoplasms ; blood supply ; metabolism ; pathology ; Vascular Endothelial Growth Factor C ; genetics ; metabolism