| Part ⅠTWIST expression in hypopharyngeal cancer and the mechanism of TWIST-induced promotion of metastasisObjective To explore the functions of TWIST in hypopharyngeal cancer, we investigated if overexpression of TWIST has an effect on FaDu cell morphology, and if alteration of TWIST has an effect on E-cadherin and N-cadherin, as well as in cell migration, and the invasion ability of FaDu cells. Moreover, we also studied the relationship between TWIST overexpression and clinicopathological characteristics in hypopharyngeal cancer tissue samples.Methods Morphological changes of FaDu cells were observed by reserved microscope. Cell migration and invasion were determined by Transwell chamber assays. RT-PCR and Western blot was performed in order to examine the expression of TWIST, E-cadherin, N-cadherin expression. Immunohistochemical assays was used to examine the TWIST expression in hypopharyngeal cancer tissue samples.Results TWIST expression increase in the transfected pcDNA3.1-TWIST vector FaDu cells and decrease in the transfected miR-TWIST vector FaDu cells. The results showed that morphology of FaDu cells, after transfecting the pcDNA3.1-TWIST vector, was changed from well organized cell-cell adhesion and cell polarity to loss of cell-cell contacts and cell scattering. TWIST overexpression also promoted the EMT in the transfected pcDNA3.1-TWIST vector FaDu cells. In the migration assay, the number of invaded cells in the pcDN A3.1-TWIST group was 1.8-fold, compared with the number of invaded cells in the control group (P<0.05). Similar results were obtained in the invasion assay. The number of invaded cells in pcDNA3.1-TWIST group was 1.6-fold higher compared with the number of invaded cells in the control group (P<0.05). TWIST overexpression had notably inhibited activation of E-cadherin in the FaDu TWIST cell lines compared to the controls at the mRNA and protein levels, respectively. Examination of N-cadherin revealed that the TWIST-overexpressing cell line increased the N-cadherin expression at the mRNA and protein levels, respectively. By contrast, use of the microRNA to knockdown TWIST expression, showed that down-regulation of TWIST expression strikingly increased the activation of E-cadherin and inhibited N-cadherin expression. The expression of TWIST in hypopharyngeal cancer tissues was higher than in the adjacent non-tumor tissue. Immunohistochemical assays showed that TWIST was expressed in both the nucleus and cytoplasm of tumor cells, but the expression of TWIST in the cytoplasm was more prominent than in the nucleus. TWIST expression was negative in the adjacent non-tumor tissue. The results indicated that TWIST expression is related to tumor differentiation (P=0.038), tumor size (P=0.048), and lymph node metastasis (P=0.044). In contrast, no correlation was observed between TWIST expression and gender or age (P>0.050).Conclusions Alteration in TWIST expression induces morphologic changes in FaDu cells. Alteration in the TWIST expression affects the migration and invasion of FaDu cells. Alteration in the TWIST expression leads to an alteration in E-cadherin and N-cadherin expression in the FaDu cell line. TWIST expression is related to tumor differentiation, tumor size, and lymph node metastasis in hypopharyngeal tumors. The data presented reveal that overexpression of TWIST plays a significant role in the metastasis of hypopharyngeal tumors, and alteration of TWIST has an effect on the EMT in FaDu cells.PartⅡTumor necrosis factor alpha induces epithelial-mesenchymal transition via NF-κB signaling pathway mediated TWIST expression in the hypopharyngeal cancerObjective Epithelial-mesenchymal transition (EMT) is an important mechanism in cancer metastasis. TNFα can induce cancer invasion and metastasis associated with EMT. However, the underlying mechanisms are not entirely clear. So we investigated TNFα had the an effect on EMT and invasion and metastasis in FaDu cells, and further explored its potential mechanism.Methods Morphological changes of FaDu cells that were treated by TNFα were observed by reserved microscope. Scratch-wound assay was used to examine cell motion. Cell migration and invasion were determined by Transwell chamber assays. Immunofluorescence was performed in order to examine the expression of TWIST, p65, E-cadherin and N-cadherin. SiRNA-p65 and BAY 11-7082 were used to inhibit p65 expression. Western blot was performed to examine the protein expression of p65, TWIST, p-Ikk, p-IκBα, E-cadherin and vimentin.Results After 24 h following exposure to TNFα, the speed of motility of the FaDu cells was found to be more rapid than that of the control group; the former was closer to the center of the wound area than the control group. The numbers of cells that had migrated in the control and TNFα treatment groups were 124±15 and 276±38, respectively (P<0.05). In the in vitro invasion assay, the number of invasive cells in the TNFα treatment group was 95±3, significantly more than the number in the control group (63±6, P<0.05). Morphology of the FaDu cells following treatment with TNFa was altered from well organized cell-cell adhesion and cell polarity to loss of cell-cell contacts and cell scattering. Cells underwent a significant change in morphology from a cobblestone morphology to exhibiting mesenchymal spindle-like features.Expression of the epithelial marker E-cadherin was downregulated, whereas the mesenchymal markers, vimentin and N-cadherin, were significantly upregulated in the FaDu cells. TWIST expression was increased in a time-dependent manner in the FaDu cells following exposure to TNFa. P65 expression following exposure to TNFa was increased in a time-dependent manner. This suggests that P65 was upregulated together with TNFa-induced TWIST expression in the FaDu cells. p-IKK and p-IicBa expression was increased in the TNFa-activated p65-expressing cells, respectively. Compared to the control group, we observed that the levels of nuclear p65 and TWIST were increased in the TNFa treatment group. TNFa-induced nuclear expression of p65 and TWIST was significantly increased by western blotting. Both BAY11-7082 (inhibitor of NF-κB) and siRNA-p65 inhibited p65 expression and blocked TNFa-induced TWIST expression.At the same time, TWIST expression was also decreased in the siRNA-p65 group and BAY 11-7082 group. These results showed that silencing of p65 expression also attenuated TNFa-induced TWIST expression.Conclusions TNFa increases cancer cells motion, migration and invasion abilities. TNFa induces the Epithelial-mesenchymal transition. TNFa induces TWIST expression in the FaDu cells. P65 expression is up-regulated and translocated into the nucleus in TNFα-induced TWIST expression. Downregulation of p65 expression inhibits TNFα-induced TWIST expression. TNFa induces EMT and promotes metastasis via NF-κB signaling pathway mediated TWIST expression in the hypopharyngeal cancer. |
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