Tongue Squamous Cell Carcinoma Tca8113 Research Articles

Apigenin Inhibits Proliferation, Migration, and Invasion of Human Tongue Carcinoma Tca8113 Cells Through Regulating the MAPK Signaling Pathways.

This study aimed to examine the effects of apigenin (API) on the proliferation, migration, and invasion of human tongue squamous cell carcinoma Tca8113 cells and explore its probable mechanisms. After treating Tca8113 cells with API, the cell proliferation, migration, and invasive capacities were identified by tetrazolium salt colorimetry (MTT) assay, cell scratch test, and Transwell chamber test. Cellular immunofluorescence staining was used to localize mitogen-activated protein kinase 1 (MAPK1) and extracellular regulated protein kinase (ERK) 1/2 proteins. Western blot was used to detect the variations of the related protein expression levels. 1)Through the MTT assay, API significantly inhibited cell proliferation (P<0.01). 2) In the cell scratch test, the distance of lateral migration after the API treatment was significantly shorter compared to the control group (P<0.01). 3) The invasion rate in the lower chamber of the Transwell chamber was lower in the API group (P<0.01). 4) Cellular immunofluorescence staining presented that the total-MEKK1 was localized in the cytoplasm, p-MEKK1 was localized in the nuclear membrane and cytoplasm, and p-ERK1/2 was localized in the cytoplasm and nucleus. ⑤ After API was applied to cells, the expressions of p-MEKK1 and p-ERK1/2 proteins significantly reduced (P<0.01). Apigenin (API) significantly inhibits the proliferation, migration, and invasion of Tca8113 cells and its mechanism may be associated with the MAPK signaling pathway.

Effects of RAB1A on the proliferation, invasion, and metastasis of tongue squamous cell carcinoma cells

This study aimed to investigate the molecular mechanism of RAB1A in the proliferation, invasion, and metastasis of human tongue squamous cell carcinoma. Western blot was used to detect the expression of RAB1A protein in human normal tongue epithelial cells (Hacat) and tongue squamous cell carcinoma Tca8113. The changes in RAB1A after plasmid transfection were also studied. The Tca8113 cells were named SiRAB1A/Tca8113 after RAB1A plasmid transfection. The expression of the epithelial-mesenchymal transition (EMT)-related markers of SiRAB1A/Tca8113 cells was also detected. CCK-8 assay was used to detect the proliferation of SiRAB1A/Tca8113 cells. Transwell and wound healing assays were used to detect the invasive and metastatic abilities of SiRAB1A/Tca8113 cells, respectively. Western blot results showed that the expression of RAB1A in tongue squamous cell carcinoma cells was significantly higher than that in Hacat. RAB1A decreased significantly after SiRAB1A plasmid transfection. CCK-8 proliferation assay showed that the proliferation of SiRAB1A/Tca8113 cells also decreased significantly. Transwell and wound healing assays demonstrated that the invasive and metastatic abilities of SiRAB1A/Tca8113 cells decreased significantly, respectively. In addition, Western blot results demonstrated that RAB1A deletion significantly increased the expression of E-cadherin and inhibited the expression of Vimentin. RAB1A could promote the proliferation, invasion, and metastasis of tongue squamous cell carcinoma cells.

Heavy‑ion beam irradiation inhibits invasion of tongue squamous cell carcinoma Tca8113 cells

Tongue squamous cell carcinoma (TSCC) is a common malignant tumor type with aggressive biological characteristics, located in the oral and maxillofacial region. Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) have been implicated in the invasion and metastasis of various malignant tumor types, such as lung cancer and gastric carcinoma. High linear energy transfer (LET) particle irradiation has several advantages over conventional X-rays in suppressing the invasion and metastasis of malignant tumors. The objective of the present study was to investigate the effects of high-LET carbon ions and low-LET X-rays on the expression of VEGF and MMPs, and to identify the associated mechanisms in the Tca8113 TSCC cell line. Tca8113 cells were irradiated with carbon ions or X-rays at doses of 1 and 4 Gy. An immunofluorescence assay indicated that VEGF expression was notably decreased at 24 and 48 h after heavy ion irradiation compared with irradiation with conventional X-rays. The expression of MMP-2 and MMP-9 also decreased in a dose-dependent manner following heavy ion irradiation. These findings indicate that compared with low-LET X-ray irradiation, high-LET carbon ions possess higher biological efficacy in inhibiting the invasive ability of Tca8113 cells via reduction of VEGF, MMP-2 and MMP-9 expression.

Effect of brazilin on apoptosis and autophagy of tongue cancer Tca8113 cells and its molecular mechanism

To investigate the effects of brazilin on the proliferation, apoptosis and autophagy of human tongue squamous cell carcinoma Tca8113 cells in vitro and explore its molecular mechanism. The changes in the proliferation, morphology and apoptosis of Tca8113 cells in response to brazilin treatment were detected using MTT assay, Hoechst33342 staining, and Annexin V/PI double staining, respectively. The expressions of apoptosis-related protein Bax, Bcl-2, cleaved caspase-3 and autophagy-related proteins p-AMPK, p-mTOR, LC3B, and p62 in the treated cells were detected using Western blotting. The effect of treatment with both the AMPK pathway inhibitor and brazilin on the expressions of the pathway-related proteins p-AMPK, p-mTOR, and LC3B was assessed. MTT assay showed that brazilin significantly inhibited the proliferation of Tca8113 cells with an IC50 of 31.17 μmol/L at 24 h. Hoechst33342 staining showed that brazilin induced apoptotic morphological changes in Tca8113 cells in a concentration-dependent manner. Treatment with different concentrations of brazilin resulted in increased apoptosis in the cells. Brazilin obviously inhibited the expression of Bcl-2, p62 and p-mTOR and enhanced the expressions of Bax, cleaved caspase-3, LC3B and p-AMPK. The AMPK pathway inhibitor significantly inhibited the increase in p-AMPK and LC3B expressions and the decrease in p-mTOR expression induced by brazilin. Brazilin can inhibit the proliferation and promote apoptosis in Tca8113 cells and at the same time induces autophagy in the cells through the AMPK/mTOR pathway.

Identification of differentially expressed genes in oral squamous cell carcinoma TCA8113 cells.

Previous studies have demonstrated that cancer cells with increased levels of aldehyde dehydrogenase ‘bright’ activity (ALDHbr) exhibit stem cell properties compared with cells exhibiting decreased ALDH activity (ALDHlow). To screen possible biomarkers of cancer stem cells in tongue squamous cell carcinoma, ALDHbr and ALDHlow cells were isolated from the tongue squamous cell carcinoma TCA8113 cell line, and suppression subtractive hybridization was performed to identify differentially expressed genes in the two subpopulations. A total of 240 positive clones were randomly selected for sequencing and were functionally characterized using bioinformatical tools. The results of the present study identified the differential expression of 104 clones, 62 of which corresponded to known genes and 42 of which corresponded to unknown genes. Cluster analysis revealed that the known genes were involved in the regulation of the cell cycle and cell differentiation. In addition, analysis of 10 signaling pathways revealed that genes were markedly altered in the ALDHbr cell subpopulation. Additional study is required to identify the function that these genes serve in the biomolecular regulatory mechanisms of cancer stem cells and to assist in explaining the biological behavior of oral squamous cell carcinoma.

Molecular mechanism of P75NTR gene-induced apoptosis in tongue squamous cell carcinoma cell

Objective To investigate the molecular mechanism of P75NTR gene-induced apoptosis in tongue squamous cell carcinoma Tca8113 cell lineage. Methods P75NTR specific siRNA was transferred into P75NTR positive tongue squamous cell carcinoma Tca8113 cells. P75NTR positive Tca8113 cells were divided into 4 groups: blank group (without transfection), negative control group (transfected with negative control siRNA), experiment group-776 (transfected with siRNA-P75NTR-776) and experiment group-1234 (transfected with siRNA-P75NTR-1234). Transfection efficiency and cell apoptosis were detected by flow cytometry. The interference effect of P75NTR mRNA expression was detected by fluorescence quantitative PCR. 3-(4, 5-dimethyl-2-thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide assay was applied in measuring cell prolife-ration. The protein changes of P75NTR were detected by Western blotting. The distributions of nuclear factor-κB(NF-κB) of cells were observed by cell immunofluorescence labeling method. Results The transfection efficiency was 30%. The apoptosis rate of experiment group-776, experiment group-1234 and negative control group was (20.35±0.18) %, (12.32±1.51)% and (2.63±0.10)% respectively. Compared with the negative control group, the differences of the former two group had statistical significance (t=177.20, P<0.005; t=37.12, P<0.005). The P75NTR gene interference was successful. The inhibition rate of P75NTR protein reached 31% in experiment group-776. The cell viability of Tca8113 cells after P75NTR-siRNA interference was 70.02%, 78.01% and 95.81% in experiment group-776, experiment group-1234 and negative control group. And there were significant differences between experiment group-776 and negative control group(χ2= 235.3, P<0.010), and between experiment group-1234 and negative control group (χ2= 117.5, P<0.005). NF-κB distribution was increased in cell cytoplasm in the interference group than that in control group. Conclusion P75NTR may promote the proliferation or inhibit the apoptosis of tongue squamous cell carcinoma, and the molecular mechanism may be correlated with hindering the transportion of NF-κB into cell nuclear. Key words: Apoptosis; Nerver growth factor; NF-Kappa B

HMME combined with green light-emitting diode irradiation results in efficient apoptosis on human tongue squamous cell carcinoma.

Hematoporphyrin monomethyl ether (HMME) is a novel and promising porphyrin-related photosensitizer for photodynamic therapy (PDT). This study aimed to investigate the efficacy and potential mechanism of HMME-PDT under irradiation of green light-emitting diode (LED) with wavelength of 530 ± 20nm in treating human tongue squamous cell carcinoma Tca8113 cells in vitro. The HMME concentrations were 1.25, 2.5, and 5μg/ml while the energy densities were 0.6, 1.2, 1.8, 2.4, and 3.0J/cm(2). MTT assay demonstrated that HMME-PDT significantly inhibited the proliferation of Tca8113 cells, and the cytotoxicity was improved with increased HMME concentration and light intensity. The amount of cells decreased significantly and the morphology of cells changed drastically after HMME-PDT. Flow cytometry analysis revealed that HMME-PDT induced both apoptosis and necrosis, but apoptosis was the main form of cell death. Apoptotic morphology was confirmed by Hoechst 33342 staining. Laser scanning confocal microscopy observation showed that HMME was mainly localized in mitochondria. The production of intracellular reactive oxygen species increased remarkably after PDT treatment, and both sodium azide (the singlet oxygen quencher) and D-mannitol (the hydroxyl radical scavenger) could protect Tca8113 cells from death induced by HMME-PDT. Additionally, the activity of caspase-3 also increased markedly in treated groups, and the cell death could be rescued by a reversible inhibitor (Ac-DEVD-CHO) of caspase-3. These results demonstrated that HMME combined with green LED significantly induced apoptosis of Tca8113 cells, suggesting that HMME-PDT using green LED might be a potential therapeutic strategy for human tongue squamous cell carcinoma.

Down-Regulating Receptor Interacting Protein Kinase 1 (RIP1) Promotes Oxaliplatin-Induced Tca8113 Cell Apoptosis.

BackgroundOxaliplatin is a crucial chemotherapy drug that plays an important role in colorectal cancer and oral cancer treatment. However, the molecular mechanism of oxaliplatin in killing tongue squamous cell cancer cells is still unknown. This paper investigates the mechanism of by which oxaliplatin regulates tongue squamous cell carcinoma Tca8113 cell survival and death.Material/MethodsTca8113 was treated with 1 μmol/L oxaliplatin for 24 h. Tca8113 cell proliferation and apoptosis were determined by MTT method and flow cytometry, respectively. Western blot was applied to detect receptor-interacting protein kinase 1 (RIP1) level. Tca8113 was transfected with siRNA RIP1 and then treated with 1 μmol/L oxaliplatin, and the cell apoptosis was detected.ResultsWe found that 1 μmol/L oxaliplatin could inhibit Tca8113 cell growth (cell survival rate was 19.3%), reduce mitochondrial membrane potential (reduce 82.3%) and phosphatidylserine eversion (positive rate was 62.7%), and activate caspase-3 (increased 2.6 times). We also found that 1 μmol/L oxaliplatin treatment could increase RIP1 expression in Tca8113 cells. Cell apoptosis rate increased after siRNA RIP1 and 1 μmol/L oxaliplatin treatment (apoptosis rate was 90.2%).ConclusionsDown-regulating RIP1 promotes oxaliplatin induced Tca8113 cells apoptosis.

Subcellular location and photodynamic therapeutic effect of chlorin e6 in the human tongue squamous cell cancer Tca8113 cell line

The present study aimed to investigate the distribution and photodynamic therapeutic effect of chlorin e6 (Ce6) in the human tongue squamous cell carcinoma Tca8113 cell line in vitro. The distribution of Ce6 in the Tca8113 cells was observed in situ combined with mitochondrial and lysosomal fluorescent probes. Next, 630-nm semiconductor laser irradiation was performed. The MTS colorimetric method was used to determine cell survival. Annexin V fluorescein isothiocyanate/propidium iodide (PI) double staining was used to detect early apoptosis following photodynamic therapy (PDT). The flow cytometer was used to analyze the DNA content subsequent to PI-staining. It was observed that Ce6 could combine with the cellular membrane following 30 min of incubation with the Tca8113 cells. As the length of incubation increased, Ce6 gradually entered the cells in a particular distribution and reached saturation by 3 h. Co-localization analysis demonstrated that Ce6 was more likely to be present in the mitochondria than in the lysosomes. The cells incubated with 5 μg/ml Ce6 for 24 h exhibited a low toxicity of 5%, however, following light irradiation, Ce6-PDT was able to kill the Tca8113 cells in vitro. The cell toxicity was positively correlated with Ce6 concentration and light dose, therefore, the effect of Ce6 was concentration/dose-dependent (P<0.01). The lower Ce6 concentrations and light doses could significantly induce apoptosis in the Tca8113 cells, while higher doses increased necrosis/percentage of dead cells. In summary, Ce6 saturated the Tca8113 cells following 3 h of incubation. Furthermore, Ce6-PDT effectively killed the cultured Tca8113 cells in vitro at a safe concentration. At a low concentration and light dose, Ce6 is more likely to induce cell apoptosis via the mitochondria than the lysosomes.

Effects of RhoA siRNA on the proliferation, adhesion, migration and invasion of tongue squamous cell carcinoma Tca8113 cells in vitro

to investigate the effect of RhoA on the metastasis of tongue squamous cell carcinoma Tca8113 cells in vitro. a group of RhoA specific small interfering RNAs (siRNA) was constructed and confined by sequencing analysis. The siRNA of RhoA gene was transfected into human tongue squamous cell carcinoma Tca8113 cells line by Lipofectamine(TM) 2000. The protein transient expression of RhoA in the transfectants was examined 48 h after transfection. The cell line with stable expression of siRNA of RhoA was obtained by blasticidin screening and colony culture. Cell growth rate was determined with a cell counting. Millicell chambermodel and wound healing assay were used to examine the abilities of migration and invasion, respectively in vitro. RhoA was overexpressed in tongue squamous cell carcinoma Tca8113 cell line. Silencing of endogenous RhoA gene expression in Tca8113 cells resulted in inhibition of the proliferation, adhesion, chemotaxis and invasion of Tca8113 cells in vitro. RhoA siRNA inhibits the proliferation, adhesion, chemotaxis and invasion of oral squamous cell carcinoma Tca8113 cell lines. siRNA of RhoA is a potential factor controlling the proliferation and metastasis of Tca8113 cells. RhoA may play an important role in metastasis of oral squamous cell carcinoma.

Hyperthermia-induced apoptosis in Tca8113 cells is inhibited by heat shock protein 27 through blocking phospholipid scramblase 3 phosphorylation

Purpose: Hyperthermia induces tumour cell apoptosis through the mitochondrial apoptotic pathway; however, the signal transduction mechanism underlying this process still needs to be fully elucidated. Phospholipid scramblase 3 (PLS3), a target of protein kinase C-δ (PKC-δ), resides in mitochondria and plays pivotal roles in regulating apoptotic response. Activated PLS3 facilitates cardiolipin (CL) translocation from the mitochondrial inner membrane to the outer leaflet of the mitochondrial outer membrane and triggers apoptosis.Materials and methods: The tongue squamous cell carcinoma Tca8113 cells were transfected or co-transfected using Lipofectamine 2000 with plasmids pCMV-6×His-PLS3, pCMV-6×His-PLS3 (T21A), pHA-PKC-δ, pHA-PKC-δ-KD (K376R), pHA-Hsp27, and empty control plasmid pcDNA3.1. The transfected cells were heated in water bath at 43°C for 20 min, 40 min and 60 min. Assessments of apoptosis and redistribution of mitochondrial cardiolipin were performed by flow cytometry. PLS3, PKC-δ, Hsp27, phosphorylation of PLS3 and PLS3/PKC-δ interaction were detected by western blotting.Results: In our study the results show that elevated levels of the wild-type PLS3, but not the PLS3 (T21A) mutant, is able to increase hyperthermia-induced CL translocation and apoptosis. Wild-type PKC-δ facilitates PLS3 phosphorylation, PKC-δ/PLS3 interaction, and CL translocation, which consequently promote apoptosis. In contrast, heat shock protein 27 (Hsp27) blocks PKC-δ-induced PLS3 phosphorylation, suppresses PKC-δ/PLS3 interaction and CL translocation, and inhibits apoptosis.Conclusions: Our findings suggest that phosphorylation of PLS3 by PKC-δ is involved in the hyperthermia-induced apoptotic signal transduction pathway in Tca8113 cells, and that Hsp27 blocks this pathway to suppress hyperthermia-induced apoptosis.

Apoptosis induces by exogenous nitric oxide in Tca8113 cells

To observe the apoptosis induced by exogenous NO in Tca8113 cells and to investigate the possible mechanism. SNP as NO donor was used to treat the tongue squamous cell carcinoma Tca8113 cells. Cytotoxic and apoptotic effects of NO on Tca8113 cells were examined by using MTT assay, acridine orange (AO) staining, Wright-Giemsa staining, agarose gel electrophoresis and flow cytometry. Western blot was performed for investigating the apoptotic mechanism. NO had a remarkable proliferation inhibiting effect on Tca8113 cells. After being exposed to exogenous NO, Tca8113 cells showed series of apoptotic morphological changes such as cell shrinkage, nuclear condensation; and also showed DNA fragmentation, G2/M phase arrest as well as upregulation of the tumor suppressor P53 protein. Exogenous NO has a proliferation inhibition and apoptosis induction effect on Tca8113 cells in a concentration and time-dependent manner, P53 protein may be involved in the apoptosis induced by NO.