Tongue Squamous Cell Carcinoma Cells Research Articles

Early growth response 1 regulates dual‑specificity protein phosphatase 1 and inhibits cell migration and invasion of tongue squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the head and neck, and among the OSCCs, tongue squamous cell carcinoma (TSCC) is one of the most common types. Although therapy strategies have recently advanced, the prognosis of TSCC has not substantially improved. Metastasis is one of the main causes of patient mortality in TSCC; therefore, it is necessary to elucidate the mechanism by which TSCC metastasis is regulated. In the present study, the early growth response 1 (Egr-1) expression in TSCC was analyzed based on GEO datasets and the effect of Egr-1 in TSCC tumor cell migration and invasion was measured using Transwell assay. By overexpressing dual-specificity protein phosphatase 1 (DUSP1) in cells with Egr-1 knockdown using lentivirus infection, the role of DUSP1 in Egr-1-regulated TSCC cell migration and invasion was determined. By using luciferase and ChIP assays, the mechanism behind how DUSP1 is regulated by Egr-1 was detected. In the present study, it was demonstrated that Egr-1 was downregulated in TSCC and the knockdown of Egr-1 increased TSCC cell migration and invasion. The expression of Egr-1 was also correlated with DUSP1. The overexpression of DUSP1 in Egr-1 knockdown cells, reduced the level of cell migration and invasion. Furthermore, it was demonstrated that knockdown of Egr-1 inhibited the promoter activity of DUSP1 and the site through which Egr-1 regulates DUSP1 transcription was identified. In conclusion, the present study demonstrated that Egr-1 regulates TSCC cell migration and invasion through modulating DUSP1, suggesting the potential of Egr-1 and DUSP1 as therapy targets for TSCC.

Melatonin inhibits tongue squamous cell carcinoma: Interplay of ER stress-induced apoptosis and autophagy with cell migration

Tongue squamous cell carcinoma (TSCC) occupies a high proportion of oral squamous cell carcinoma. TSCC features high lymph node metastasis rates and chemotherapy resistance with a poor prognosis. Therefore, an effective therapy strategy is needed to improve patient prognosis. Melatonin (MT) is a natural indole compound shown to have anti-tumor effects in several cancers. This study focused on the role and mechanism of MT in TSCC cells. The results of the study suggest that MT could inhibit cell proliferation in CRL-1623 cells. Western blot analysis showed the down-regulate of cyclin B1 and the up-regulate P21 protein by MT. MT was also shown to down-regulate the expression of Zeb1, Wnt5A/B, and β-catenin protein and up-regulate E-cadherin to inhibit the migration of CRL-1623 cells. MT also promoted the expression of ATF4, ATF6, Bip, BAP31 and CHOP in CRL-1623 cells leading to endoplasmic reticulum stress, and induced autophagy and apoptosis in CRL-1623 cells. Western blots showed that MT could promote the expression of Bax, LC3, and Beclin1 proteins and inhibit the expression of p62. We screened differentially expressed long non-coding RNAs (lncRNAs) in MT-treated cells and found that the expression of MALAT1 and H19 decreased. Moreover, MT inhibited tumor growth in nude mice inoculated with CRL-1623 cells. These results suggest that MT could induce autophagy, promote apoptosis, and provide a potential natural compound for the treatment of TSCC.

SCUBE3 exerts a tumor-promoting effect in tongue squamous cell carcinoma by promoting CEBPA binding to the CCL2 promoter.

Tongue squamous cell carcinoma (TSCC) is the main pathological subtype of oral cancer, and the current therapeutic effect is far from satisfactory. The signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) has been shown to be a tumor-promoting factor in several malignancies. However, little is known about the role of SCUBE3 in TSCC. In this study, we identified that SCUBE3 was highly expressed in TSCC. Clinically, high expression of SCUBE3 was positively associated with tumor stage and T stage of TSCC. Functionally, SCUBE3 silence remarkably restrained cell proliferation, migration and invasion, induced apoptosis as well as cell cycle arrest in G2 phase, and weakened the tumorigenicity of TSCC cells in vivo. Mechanistically, SCUBE3 promoted the direct binding of CCAAT enhancer binding protein alpha (CEBPA) to C-C motif chemokine ligand 2 (CCL2) promoter in TSCC cells. Interestingly, CCL2 overexpression partially reversed the inhibitory effect of SCUBE3 deficiency on TSCC cell viability and migration. Moreover, signal transducer and activator of transcription 3 (STAT3) signaling contributed to CCL2-mediated phenotypes in TSCC cells. Implications: our data revealed a tumor-promoting role for SCUBE3 in TSCC via the CEBPA/ CCL2/STAT3 axis, which provided new insight into novel potential therapeutic target for TSCC.

Novel Prognostic Model Construction of Tongue Squamous Cell Carcinoma Based on Apigenin-Associated Genes.

Clinical indexes are often selected as relevant factors for constructing prognostic models of tongue squamous cell carcinoma (TSCC) patients, while factors related to therapeutic targets are less frequently included. As Apigenin (API) shows anti-tumor properties in many tumors, in this study, we construct a novel prognostic model for TSCC patients based on Apigenin-associated genes through transcriptomic analysis. The effect of Apigenin (API) on the cell characteristics of TSCC cells was measured by several phenotype experiments. RNA-seq was executed to ensure differentially expressed genes (DEGs) in squamous cell carcinoma-9 (SCC-9) cells after API treatment. Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were performed to verify the expression of API-related genes. Then, combined with the gene expression data and relevant individual information of TSCC samples acquired from The Cancer Genome Atlas (TCGA), an API-related model was built through Lasso regression and multivariate Cox regression. A receiver operating characteristic (ROC) curve and a nomogram and calibration curve were created to forecast patient outcomes to improve the clinical suitability of the API-related signature. The relationships between the two risk groups and function enrichment, immune infiltration characteristics, and drug susceptibility were analyzed. We demonstrated that API could inhibit the malignant behavior of TSCC cells. Among API-related genes, TSCC cells treated with API, compared to the control group, have higher levels of transmembrane protein 213 (TMEM213) and G protein-coupled receptor 158 (GPR158), and lower levels of caspase 14 (CASP14) and integrin subunit alpha 5 (ITGA5). An 7 API-associated gene model was built through Lasso regression and multivariate Cox regression that could direct TSCC prognostic status and tumor immune cell infiltration. In addition, we acquired 6 potential therapeutic agents for TSCC based on the prognostic model. Our research suggested the inhibition effect of API on TSCC cells and provided a novel prognostic model combined with therapeutic factors that can guide the prognosis of TSCC and clinical decision-making in TSCC.

Integrated bioinformatics analysis of SEMA3C in tongue squamous cell carcinoma using machine-learning strategies

Tongue squamous cell carcinoma (TSCC) is an aggressive oral cancer with a high incidence of metastasis and poor prognosis. We aim to identify and verify potential biomarkers for TSCC using bioinformatics analysis. To begin with, we examined clinical and RNA expression information of individuals with TSCC from the Gene Expression Omnibus (GEO) database. Differential expression analysis and functional analysis were conducted. Multiple machine-learning strategies were next employed to screen and determine the hub gene, and receiver operating characteristic (ROC) analysis was used to assess diagnostic value. Semaphorin3C (SEMA3C) was identified as a critical biomarker, presenting high diagnostic accuracy for TSCC. In the validation cohorts, SEMA3C exhibited high expression levels in TSCC. The high expression of SEMA3C was a poor prognostic factor in TSCC by the Kaplan–Meier curve. Based on the Gene Ontology (GO) analysis, SEMA3C was mapped in terms related to cell adhesion, positive regulation of JAK-STAT, positive regulation of stem cell maintenance, and positive regulation of NF-κB activity. Single-cell RNA sequencing (ScRNA-seq) analysis showed cells expressing SEMA3C were predominantly tumor cells. Then, we further verified that SEMA3C had high expression in TSCC clinical samples. In addition, the knockdown of SEMA3C suppressed the proliferation, migration, and invasion of TSCC cells in vitro. This study is the first to report the involvement of SEMA3C in TSCC, suggesting that upregulated SEMA3C could be a novel and critical potential biomarker for future predictive diagnostics, prevention, prognostic assessment, and personalized medical services in TSCC.

Anti-cancer effects of green synthesized gold nanoparticles using leaf extract of Annona muricata. L against squamous cell carcinoma cell line 15 through apoptotic pathway

Background: Oral cancer remains one of the most dreadful diseases in developing nations. Currently, there has been a rise in the prevalence of tongue squamous cell carcinoma (SCC), with a poor prognosis. The use of standard treatment approaches against oral cancer patients brings about several side effects. In recent years, nanomedicine has provided a versatile platform for developing new targeted therapeutic modalities. However, safety remains a concern in the synthesis of nanoparticles (NPs). Therefore, the present study aims to synthesize safer phytoconstituent-mediated gold NPs (AuNPs) utilizing leaf extracts of Annona muricata, where the biochemical components of the plant leaf act as the reducing and capping agents in the synthesis of NPs, and to evaluate its anti-cancer activity against SCC. Materials and Methods: In this in vitro experimental study, AuNPs were synthesized through an effective, simple, and ecologically sound green synthesis method. After characterization of these synthesized AuNPs, in vitro assays such as 3-(4, 5-dimethylthiazole2-yl)-2, 5-biphenyl tetrazolium bromide, wound healing, and clonogenic assays were carried out to investigate the anti-cancer potential of green synthesized AuNPs in the human tongue SCC cell line (SCC-15), and the possible mechanism of action was evaluated through gene and protein expression analysis of Bax, Bcl-2, and p53 genes. The results were expressed as mean ± standard deviation using Statistical Package for Social Sciences (SPSS) 20.0 software and Student's t-test was performed for experimental data. P ≤0.05 were considered statistically significant. Results: The in vitro assays demonstrated that the synthesized AuNPs are exhibiting anti-cancer activity by apoptosis of SCC-15 cells in a dose-dependent manner. Further, it also revealed a highly significant decrease in anti-apoptotic Bcl-2 gene expression, whereas pro-apoptotic genes p53 and Bax revealed a highly significant increase, which is statistically significant compared to the control (P < 0.05). Conclusion: Our findings demonstrated that the AuNPs synthesized from A. muricata leaf extract could act as a novel anticancer agent, particularly against SCC, after further scrutiny.

Regulating Lipid Metabolism via Mitochondrial Dynamics in Tongue Squamous Cell Carcinoma Cancer Stem Cells.

Cancer stem cells (CSCs) are a sub-population of cancer cells present in many kinds of malignant tumors that have the potential for self-proliferation and differentiation. These cells have been demonstrated as the main cause of tumor recurrence and metastasis. Strong evidence indicates that CSCs prefer reprogrammed fatty acid β-oxidation over oxidative phosphorylation for sustaining energy supply. Although mitochondrial dynamics participate in the regulation of cancer stemness, the correlation between the inhibition of mitochondrial fission and the regulation of lipid metabolism in CSCs remains poorly understood. The human tongue squamous cell carcinoma (TSCC) cell lines CAL27 and SAS were used to obtain the CSCs by 3D Spheroid Culture. Then,western blot methods, RT-PCR and flow cytometry analysis were used to identify the TSCC CSCs. Next, Immunofluorescence method, transmission electron microscopy detection and western blot methods were used to evaluate the mitochondrial morphology and the quantity of lipid droplets (LDs). Lastly, lipidomic analysis was applied to explored the lipidomic alterations of TSCC CSCs with different mitochondrial morphology. Here, we show that the quantity of lipid droplets containing intracellular triglyceride (TG) can be decreased by regulating mitochondrial morphology. Lipidomic analysis using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) also compared alterations in lipid metabolites in tongue squamous cell carcinoma (TSCC) CSCs, TSCC cells (non-CSCs), and CSCs with different mitochondrial morphology. Discriminant lipids of statistical significance were successfully annotated, including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), sphingomyelins (SMs), triacylglycerols (TGs), phosphatidylglycerols (PGs), phosphatidylserines (PSs), lysophosphatidylcholines (LPCs), and lysophosphatidylethanolamines (LPEs). This study provides a deeper insight into the alterations of lipid metabolism associated with TSCC CSCs, non-CSCs and CSCs regulated by mitochondrial dynamics and thus serves as a guide toward novel targeted therapies.

Actin filament-associated protein 1-antisense RNA1 promotes the development and invasion of tongue squamous cell carcinoma via the AFAP1-AS1/miR-133a-5p/ZIC2 axis.

The present study aimed to explore the biological role and underlying mechanism of the long non-coding RNA actin filament-associated protein 1-antisense RNA1 (lncRNA AFAP1-AS1) in the progression of tongue squamous cell carcinoma (TSCC). A quantitative reverse transcriptase-PCR (RT-qPCR) was conducted to assess relative levels of the miR-133a-5p, lncRNAs AFAP1-AS1 and zinc finger family member 2 (ZIC2) in TSCC cell lines and specimens, whereas ZIC2 protein levels were measured using western blotting. After modifying the levels of expression of lncRNA AFP1-AS1, miR-133a-5p and ZIC2 using lentivirus or plasmid transfection, we examined AKT/epithelial-mesenchymal transition signaling pathway alterations, in vivo carcinogenesis of TSCC in nude mice and in vitro malignant phenotypes. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between ZIC2 and miR-133a-5p, as well as between miR-133a-5p and lncRNA AFAP1-AS1. Based on The Cancer Genome Atlas (TCGA) database, we additionally validated AFP1-AS1. The potential biological pathway for AFP1-AS1 was investigated using gene set enrichment analysis (GSEA). We also evaluated the clinical diagnostic capacities of AFP1-AS1 and clustered the most potential biomarkers with the Mfuzz expression pattern. Finally, we also made relevant drug predictions for AFP1-AS1. In TSCC cell lines and specimens, lncRNA AFAP1-AS1 was upregulated. ZIC2 was upregulated in TSCC cells as a result of lncRNA AFAP1-AS1 overexpression, which also promoted TSCC cell migration, invasion, viability, and proliferation. Via the microRNA sponge effect, it was found that lncRNA AFAP1-AS1 could upregulate ZIC2 by competitively inhibiting miR-133a-5p. Interestingly, knockdown of ZIC2 reversed the biological roles of lncRNA AFAP1-AS1 with respect to inducing malignant phenotypes in TSCC cells. In addition, in vivo overexpression of lncRNA AFAP1-AS1 triggered subcutaneous tumor growth in nude mice implanted with TSCC cells and upregulated ZIC2 in the tumors. The TCGA database findings revealed that AFAP1-AS1 was significantly upregulated in TSCC specimens and had good clinical diagnostic value. The results of GSEA showed that peroxisome proliferator-activated receptor signaling pathway was significantly correlated with low expression of AFP1-AS1. Finally, the results of drug prediction indicated that the group with high AFAP1-AS1 expression was more sensitive to docetaxel, AZD4547, AZD7762 and nilotinib. The upregulation of lncRNA AFAP1-AS1, which increases TSCC cell viability, migration, proliferation and invasion via the AFAP1-AS1/miR-133a-5p/ZIC2 axis, aids in the progression of TSCC.

Investigating whether exosomal miR-205-5p derived from tongue squamous cell carcinoma cells stimulates the angiogenic activity of HUVECs by targeting AMOT.

Although exosomal microRNAs (exo-miRNAs) regulate angiogenesis, they are not sufficient for the development of anti-vascular drugs for tongue squamous cell carcinoma (TSCC). miR-205-5p is an exo-miRNA that is highly expressed in the saliva of patients with oral SCC. We aimed to clarify the role and molecular mechanism of exosomal miR-205-5p in regulating TSCC angiogenesis. Effect of exosomes derived from TSCC cells on human umbilical vein endothelial cell (HUVEC) function was determined using the CCK-8, Transwell, Transwell-Matrigel, and Matrigel-based tube formation assays. Protein levels were detected by western blot. The binding between miR-205-5p and the 3'UTR of AMOT was verified using a luciferase reporter assay. Exosomal miR-205-5p (exo-miR-205-5p) promoted the proliferation, migration, and invasion of HUVECs, increased the number of tubes formed by HUVECs, and increased the vascular endothelial growth factor receptor 2 levels in HUVECs. Exo-miR-205-5p downregulated the AMOT level in HUVECs. Results of the luciferase reporter assay showed that miR-205-5p could bind to the 3'UTR of AMOT. AMOT overexpression blocked the effect of exo-miR-205-5p on HUVEC functions. Exo-miR-205-5p derived from TSCC regulates the angiogenic activity of HUVECs by targeting AMOT and might be a new molecular target for the development of anti-vascular drugs for TSCC.

Silencing of heat shock factor 1 (HSF1) inhibits proliferation, invasion, and epithelial-mesenchymal transition in oral squamous cell carcinoma.

Oral squamous cell carcinoma is characterized by high rates of morbidity and mortality. Evidence obtained for different types of cancer shows that tumor initiation, progression, and therapeutic resistance are regulated by heat shock factor 1. This research aimed to analyze the effects of heat shock factor 1 on the biological behavior of oral squamous cell carcinoma. Clinicopathological and immunoexpression study of heat shock factor 1 in 70 cases of oral tongue SCC and functional assays by gene silencing of this factor in an oral tongue SCC cell line. Heat shock factor 1 was overexpressed in oral tongue SCC specimens compared to normal oral mucosa (p < 0.0001) and in the SCC15 line compared to immortalized keratinocytes (p < 0.005). No significant associations were observed between overexpression of heat shock factor 1 and clinicopathological parameters or survival rates of the oral tongue SCC cases in the present sample. In vitro experiments showed that heat shock factor 1 silencing inhibited cell proliferation (p < 0.005) and cell cycle progression, with the accumulation of cells in the G0/G1 phase (p < 0.01). In addition, heat shock factor 1 silencing reduced cell invasion capacity (p < 0.05) and epithelial-mesenchymal transition, characterized by a decrease in vimentin expression (p < 0.05) and an increase in E-cadherin expression (p < 0.001). Heat shock factor 1 may exert several functions that help maintain cell stability under the stressful conditions of the tumor microenvironment. Thus, strategies targeting the regulation of this protein may in the future be a useful therapeutic tool to control the progression of oral squamous cell carcinoma.

Brucea javanica oil inhibits tongue squamous cell invasion and metastasis by regulating miR-138-EZH2 pathway

Tongue squamous cell carcinoma (TSCC) is one of the most common malignant tumors of head and neck. Its incidence is on the rise, and the proportion of young patients is gradually increasing, which is prone to tumor recurrence and metastasis. At present, there is no effective method to completely treat TSCC. Studies have shown that brucea javanica oil (BJO) has good antitumor activity against lung cancer and gastrointestinal tumors, but its therapeutic effect on TSCC is not clear. We have previously confirmed that oleic acid, the main component of BJO, can induce apoptosis of TSCC and reduce its invasion and metastasis ability. However, the anticancer effect and mechanism of BJO in TSCC remain unclear. In order to further explore the effects of BJO on the biological characteristics of TSCC cells, we studied the effects of different concentrations of BJO on the migration, invasion ability and epithelial mesenchymal transition (EMT) progression of TSCC cells and the possible mechanisms through in vitro experiments. We found that BJO could inhibit the invasion and metastasis of TSCC and up-regulate miR-138. After BJO treatment, the expression of E-cad was significantly increased, while the expression of EZH2, Slug, p-ERK1/2 and Vimentin was significantly decreased. EZH2 is a miR-138 target gene involved in TSCC. BJO inhibits TSCC invasion and metastasis by regulating the miR-138-EZH2 pathway. In vivo experiments have also well demonstrated the targeting effect of this pathway. This study provides a new therapeutic strategy for the treatment of TSCC.

ARNTL inhibits the malignant behaviors of oral cancer by regulating autophagy in an AKT/mTOR pathway-dependent manner.

Current studies have shown that ARNTL, an important clock gene, plays an anticancer role and is downregulated in certain types of cancer. However, the biological functions and mechanisms of ARNTL in tumors remain largely unknown. This study aimed to elucidate how ARNTL-induced autophagy impacts the biological properties of tongue squamous cell carcinoma (TSCC) cells and the mechanisms by which ARNTL expression activates autophagy. ARNTL was stably overexpressed in TSCC cells to investigate its functions in vitro and in vivo. We found that activation of autophagy induced by ARNTL decreases cell proliferation, enhances cell death, and hinders the migratory ability of TSCC cells. Moreover, ARNTL antagonizes the AKT/mTOR pathway, which potentiates autophagy induction. The manipulation of Akt activation cancels the effects of ARNTL overexpression on the biological behaviors of TSCC cells. Furthermore, after the addition of SC79, the upregulated BAX expression due to ARNTL overexpression and downregulated expressions of BCL-2 and MMP2 were remarkably rescued. In vivo tumorigenicity assays and immunohistochemistry also confirmed that ARNTL overexpression suppresses tumor development. Our study found for the first time that ARNTL inhibits the malignant behaviors of oral cancer cells by regulating autophagy in an AKT/mTOR pathway-dependent manner, which provides a novel theoretical basis for the potential future application of ARNTL to treat patients with oral cancer.

KLK4 affects the biological behavior of oral squamous cell carcinoma through Wnt/β catenin and PI3K/Akt signaling pathways

Purpose: To investigate the effect of kallikrein 4 (KLK4) on oral squamous cell carcinoma (OSCC), and its mechanism of action.&#x0D; Method: Human tongue SCC cell line TCA-8113 was cultured, and the interference fragment and irrelevant sequence fragment of KLK4 gene were designed and synthesized. These fragments were constructed into pgcsi-h1 plasmid. Then, the TCA-8113 cells were cultured for 24 h, followed by transfection, after which TCA-8113-NC and TCA-8113-siKLK4 cells were obtained. The mRNA expression of KLK4 was determined using quantitative real-time-polymerase chain reaction (PCR). Proliferative potential was assessed using MTT method, while apoptosis was determined by flow cytometry. Furthermore, cell migration was evaluated by scratch test, and Transwell cell invasion experiment was used to determine cell invasion. Immunoblot assay was applied to determine the expressions of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt.&#x0D; Result: Compared with TCA-8113-nc, the expression of KLK4 in TCA-8113-siKLK4 cells was significantly down-regulated, while, relative to TCA-8113-NC, TCA-8113-siKLK4 cells had significantly reduced cell proliferation, invasion and migration, and enhanced apoptosis (p &lt; 0.05). Moreover, there was significant up-regulation of E- and N-cadherins, vimentin, uPA, Wnt1, β-Catenin, p-pi3k and p-Akt in TCA-8113-siKLK4 group, relative to TCA-8113-NC (p &lt; 0.05).&#x0D; Conclusion: KLK4 enhances the multiplication, metastasis and invasive potential of OSCC cells, but suppresses apoptotic changes via activation of Wnt/β-catenin/PI3K/Akt signal route. The potential of Kallikrein-4 as biomarker for OSCC cells should be determined in suitable animal models.

Expression and effect of heterogeneous nuclear ribonucleoprotein A2/B1 in tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.

Mena as a key enhancer factor of EMT to promote metastasis of human tongue squamous cell carcinoma.

The aim of this study was to investigate the effect of mammalian-enabled (Mena) on tongue squamous cell carcinoma (TSCC) metastasis and its mechanism. Immunochemistry was performed to investigate the Mena and tumor-related markers expression, and its clinicopathological characteristics in 46 TSCC specimens. TSCC cell SCC9 and Cal27 untransfected or stable transfected with Mena overexpression and small interfering RNA were used to determine the role of Mena in cell proliferation, cell migration, invasion and metastasis, and EMT-related markers in vitro, and the effect of Mena on TSCC growth and metastasis through tumor-bearing and tumor metastasis immunodeficient mice models in vivo. Immunochemistry showed that the expression of Mena was significantly correlated with lymphatic metastasis and TNM stage, E-cadherin, Vimentin, and MMP2. Mena did not affect cell proliferation and colony formation in vitro, and tumor growth in vivo. However, it promoted cell migration and invasion in vitro, and TSCC metastasis in vivo. Mena expression is associated with lymphatic metastasis and tumor stage and promotes TSCC invasion and metastasis by inducing the EMT process. Thus, Mena may be a biomarker for prognosis and targeted therapy in TSCC patients.

The Clinical and Biological Effects of Receptor Expression-Enhancing Protein 6 in Tongue Squamous Cell Carcinoma.

There are currently no effective biomarkers for the diagnosis and treatment of tongue squamous cell carcinoma (TSCC), which causes a poor 5-year overall survival rate. Thus, it is crucial to identify more effective diagnostic/prognostic biomarkers and therapeutic targets for TSCC patients. The receptor expression-enhancing protein 6 (REEP6), a transmembrane endoplasmic reticulum resident protein, controls the expression or transport of a subset of proteins or receptors. Although it was reported that REEP6 plays a role in lung and colon cancers, its clinical impact and biological role in TSCC are still unknown. The present study aimed to identify a novel effective biomarker and therapeutic target for TSCC patients. Expression levels of REEP6 in specimens from TSCC patients were determined with immunohistochemistry. Gene knockdown was used to evaluate the effects of REEP6 in cancer malignancy (colony/tumorsphere formation, cell cycle regulation, migration, drug resistance and cancer stemness) of TSCC cells. The clinical impact of REEP6 expression and gene co-expression on prognosis were analyzed in oral cancer patients including TSCC patients from The Cancer Genome Atlas database. Tumor tissues had higher levels of REEP6 compared to normal tissues in TSCC patients. Higher REEP6 expression was related to shorter disease-free survival (DFS) in oral cancer patients with poorly differentiated tumor cells. REEP6-knocked-down TSCC cells showed diminished colony/tumorsphere formation, and they also caused G1 arrest and decreased migration, drug resistance and cancer stemness. A high co-expression of REEP6/epithelial-mesenchymal transition or cancer stemness markers also resulted in poor DFS in oral cancer patients. Thus, REEP6 is involved in the malignancy of TSCC and might serve as a potential diagnostic/prognostic biomarker and therapeutic target for TSCC patients.

Rhaponticin suppresses the hypoxia-induced factor-1 alpha-mediated aggressive phenotype of tongue squamous cell carcinoma

BackgroundEmerging evidence suggests that rhaponticin, a stilbene monomeric compound isolated from North China rhubarb, has been shown to exhibit significant biological activity against tumors. However, the anticancer effects and mechanisms of rhaponticin in tongue squamous cell carcinoma (TSCC) remain elusive.ObjectiveWe investigated the changes of migration and invasion abilities and EMT progression of TSCC cells treated with different concentrations of rhaponticin under hypoxia, as well as the possible mechanisms, in order to initially explore the effects of rhaponticin on the biological characteristics of TSCC cells under hypoxia.ResultsThe number of cell migration and invasion was prominently increased, E-cadherin protein was down-regulated, and N-cadherin and HIF-1α protein expression was elevated under hypoxia. Rhaponticin intervention strikingly prevented the increased abilities of migration and invasion and EMT of TSCC cells under hypoxia. This was followed by further validation finding that rhaponticin indeed leads to reduced HIF-1α post-transcriptional activity. Mechanistically, rhaponticin may bind to aryl-hydrocarbon nuclear translocator (ARNT) domain of HIF-1α.ConclusionsRhaponticin repressed the invasion and migration abilities and EMT process of TSCC cells under a hypoxic environment in vitro by targeted suppression of HIF-1α.

Plumbagin Enhances the Anticancer Effects of PF Chemotherapy via Downregulation of the PI3K/AKT/mTOR/p70S6K Pathway in Human Tongue Squamous Cell Carcinoma.

Cisplatin plus 5-fluorouracil (PF) is used as the standard neoadjuvant chemotherapy (also called preoperative chemotherapy) in the treatment of tongue squamous cell carcinoma (TSCC). Although PF chemotherapy reduces the distant metastasis of TSCC, the five-year survival rate has not significantly improved. In recent years, components considered in traditional Chinese medicine have been researched as adjuvant drugs for radiotherapy and chemotherapy. Plumbagin (PB) is a quinone component isolated from Plumbago zeylanica L. Notably, PB demonstrates numerous anticancer properties. In order to examine the chemosensitization effect of PB on PF and its associated mechanisms, in vitro experiments using TSCC Cal27 and cisplatin (CDDP)-resistant Cal27/CDDP cells were carried out in the present study, and the results were subsequently verified using nude mice xenografts. Results of the present study demonstrated that PB enhanced the anticancer effects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/CDDP cells. Cell cycle assays demonstrated that both Cal27 and Cal27/CDDP cells were arrested in the S phase following the combined treatment of PF and PB. Moreover, the PF and PB combination group induced higher levels of apoptosis in Cal27 and Cal27/CDDP cells compared with the group treated with PF alone. In addition, the results of the present study demonstrated that combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. Moreover, the weight and volumes of tumors in nude mice were reduced following treatment with a combination of PF and PB. Results of the present study also demonstrated that the expression levels of Ki67 were markedly reduced in the combined treatment group compared with the group treated with PF alone. In summary, the results of the present study demonstrated that PB enhanced the PF sensitivity of TSCC through induction of S-phase arrest and apoptosis via the PI3K/AKT/mTOR/p70S6K pathway.

Oral-specific microenvironments regulate cell behavior and anticancer drug sensitivity of tongue squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most major malignant tumor of the tongue. The tongue exists at the air-liquid interface and is covered with saliva. In addition, the tongue constituent cells and tongue cancer are present under fluid flow stimulation due to the abundant capillary network and contraction of muscle tissue. Therefore, replicating both cell-cell interactions (the cellular microenvironment) and the aforementioned physical microenvironment is very important for understanding the kinetics of tongue SCC. To elucidate the effects of the cellular and physical microenvironment on tongue SCC and to investigate the relationships between these factors, we developed a collagen cell disc, with double dish under a rotational culture method to generate cancer-stroma interactions and to create fluid flow stimulation. Mesenchymal cells, NIH-3T3 cells and tongue-derived fibroblasts influenced the proliferative potential. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by cellular interactions and fluid flow stimulation, depending on the SCC cell type. The cell-cell interactions and fluid flow stimulation independently, synergistically or contradictorily affected the behavior of tongue SCC. Fluid flow stimulation synergistically enhanced the antiproliferative effect of cis-diamminedichloroplatinum on tongue SCC cells, but mesenchymal cells abolished the synergistic antiproliferative effect related to fluid flow stimulation. In conclusion, a reconstructed model was established to investigate the cellular and physical microenvironments of tongue SCC in vitro. The newly established system is a promising model for the development of further regimes to treat general oral cancer.