Bcl-2Overexpression Induces Head And Neck Squamous Carcinoma Cell A Partial Epithelial To Mesenchymal Transition And Mechanism Investigating Of Promoting Its Invasion And Metastasis

Head and Neck Squamous Cell Carcinoma (HNSCC) is serious threat to human life as a malignant epidermis cancer, which has become one of six common cancer in the world. Diagnosis and treatment of HNSCC has been one of the challenges we face since its special anatomy and complex function.60%patients are diagnosed at late stage (Ⅲ,Ⅳ) and the rate of survive of five year is about10%-20%. However, in the last50years, it has not significantly increased. One of important causes is that HNSCC is often easy to lymph node metastasis, which also resultes in clinically difficult to detect owing to high-frequency micrometastais. Therefore, it is particularly important to reveal the potential mechanism of invasion and metastasis.Bcl-2gene is an important anti-apoptotic gene, which playes an important role in many biological processes, such as placental development, cell homing, multidrug resistance, cell cycle regulation, angiogenesis, and gene regulation. Research shows that50%of the tumors have high Bcl-2expression and HNSCC is no exception. Elevated expression of Bcl-2in some tumors is often associated with shorter survival time, generally poorer clinical outcomes, even invasion and metastasis. However, the mechanism of overexpression of Bcl-2promoting invasion and metastasis of HNSCC remains unclear. In this paper, we want to detect the mechanism of invasion and metastasis of Bcl-2overexpression in HNSCC.Firstly, we want to study high expression of Bcl-2effecting on tumor cell invasion, metastasis. After the establishment of high expression of Bcl-2stable transfection of HNSCC cell lines, the subcutaneous inoculation of tumor cells into BALB/c mice was to oberve the impact of high expression of Bcl-2on tumorigenicity in vivo. BALB/c mice were injected by tumor cells via tail vein to study lung metastasis by high expression of Bcl-2tumor cell. Cell invasion assay, migration assay and motility assay were used to test ability of cell invasion and migration. FACS was carried out to examine the changes of cell surface adhesion molecules (Integrin a2, Integrin α3, Integrin av, Integrin α5, Integrin a6, Integrin β1and Integrin (34) owing to the overexpression of Bcl-2. The results showed that the overexpression of Bcl-2enhanced the tumorigenicity and lung metastasis in vivo, invasion, migration and motility in vitro, decreased the ability of cell adhesion and the expression of Integrina2and Integrinβ1.In order to reveal the mechanism of improving their invasion and metastasis in the Bcl-2cell, Western blotting, FACS, Immunostaining, and real time PCR were carried out to examine the changes of epithelial cell marker protein (E-cadherin, Cytokeratin, ZO1and Dsg3, etc.) and mesenchymal cell marker protein (N-cadherin, Fibronectin and Vimentin) expression by high expression of Bcl-2. The Dual-Luciferase Reporter Assay System was applied to analysis Bcl-2on N-cadherin promoter activity. Gelatin zymography was used to study high expression of Bcl-2on the metal matrix. The expression of N-cadherin was knoched down by siRNA N-cadherin to investigate the effect on mestastasis and invasion. ERK, FGFR, EGFR inhibitor U0126, PD174073and AG1478, respectively were applied to block its biological function to observe the relevant signaling pathway on promoting HNSCC cell invasion and metastasis in Bcl-2. The results showed that Bcl-2induced EMT-like cell phenotype, decreased epithelial cell marker proteins (E-cadherin, Cytokeratin, ZO1and Dsg3, etc.) and up-regulated mesenchymal cell marker protein (N-cadherin, Fibronectin and Vimentin). Bcl-2could activate N-cadherin promoter activity and increased production of MMP-9. Bcl-2improved cell invasion and metastasis through N-cadherin/FGFR/ERK signaling pathway. To sum up,(1) The overexpression of Bcl-2promoted HNSCC invasion and migratory;(2) The overexpression of Bcl-2induced EMT-like cell phenotype;(3) The overexpression of Bcl-2induced the expression of MMP-9by through N-cadherin/FGFR/ERK signaling pathway. Our results for the first time reported that Bcl-2has a function of regulating HNSCC invasion and metastasis.