Overexpression of angiopoietin 2 promotes the formation of oral squamous cell carcinoma by increasing epithelial-mesenchymal transition-induced angiogenesis

Abstract

Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck and is associated with a high rate of lymph node metastasis. The initial step in the metastasis and transition of tumors is epithelial-mesenchymal transition (EMT)-induced angiogenesis, which can be mediated by angiopoietin 2 (ANG2), a key regulatory factor in angiogenesis. In the present study, immunohistochemistry and real-time quantitative reverse transcriptase (qRT-PCR) were used to measure the expression of ANG2 in OSCC tissues. Plasmids encoding ANG2 mRNA were used for increased ANG2 expression in the OSCC cell line TCA8113. The short interfering RNA (siRNA)-targeting ANG2 mRNA sequences were used to inhibit ANG2 expression in TCA8113 cells. Subsequently, transwell assays were performed to examine the effects of ANG2 on TCA8113 cell migration and invasion. Furthermore, in vivo assays were performed to assess the effect of ANG2 on tumor growth. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemistry were used to examine cell apoptosis and angiogenesis in tumor tissues, respectively. Finally, western blot analysis was performed to evaluate tumor formation-related proteins in OSCC tissues. We found that protein expression of ANG2 was remarkably upregulated in OSCC tissues. Overexpression of ANG2 increased the migration and invasion of TCA8113 cells by regulating EMT. Further investigations showed that overexpression of ANG2 increased tumor growth in nude mice, and angiogenesis of OSCC tissues increased in the presence of ANG2 overexpression. Overexpression of ANG2 also reduced cell apoptosis in tumor tissue cells. Finally, we found that overexpression of ANG2 resulted in changes in the expression of tumor formation-related proteins including vimentin, E-cadherin, Bim, PUMA, Bcl-2, Bax, Cyclin D1, PCNA and CD31. Our findings show that ANG2 has an important role in the migration and invasion of OSCC. More importantly, further investigations suggested that overexpression of ANG2 might increase OSCC metastasis by promoting angiogenesis in nude mice. This stimulatory effect could be achieved by inducing abnormal EMT and by reducing apoptosis and increasing proliferation of cells.

Figure 1

Angiopoietin 2 (ANG2) was upregulated in oral squamous cell carcinoma (OSCC) tissues. (a) Immunohistochemical and quantification analysis of ANG2 protein expression in normal liver tissue (up) and OSCC (bottom) samples using specific anti-ANG2 antibody. Representative photographs were obtained at × 200 magnification. (b) ANG2 mRNA expression levels in OSCC tissue samples and normal tissue samples (12 cases) were examined by real-time quantitative reverse transcriptase (qRT-PCR), respectively. (c) The expression of ANG2 in OSSC cell lines (TCA8113, cal27, SCC4, SCC15 and SCC25). GAPDH was used as an internal quantitative control. Three independent experiments were performed, and the data represent the means±s.d. *P<0.05.

Figure 2

Effects of angiopoietin 2 (ANG2) on apoptosis and the cell cycle in oral squamous cell carcinoma (OSCC) cells. (a) Proliferation rates were determined by the MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay on days 1–3 in TCA8113 mock cells or cells transfected with ANG2-encoded plasmids, ANG-targeted short interfering RNA (siRNA) or nonspecific (NC) siRNA. Before performing the MTS assays, cells were transfected with the indicated plasmids or siRNA for 24 h. Three independent experiments were performed, and the data represent the means±s.d. Treated and control groups of TCA8113 cells were used in subsequent experiments. (b) Representative dot plots showing fluorescence channel analysis of the treated and control groups of TCA8113 cells after dual staining with Annexin V and propidium iodide (PI) and analysis by flow cytometry. Columns represent the mean of three individual experiments; Bars, s.d. (c) Cell cycle distributions of the treated and control groups of TCA8113 cells were analyzed by flow cytometry. Data represent the mean±s.d. of three independent experiments.

Figure 3

Overexpression of angiopoietin 2 (ANG2) increased the migration and invasion of TCA8113 cells by regulating epithelial–mesenchymal transition (EMT). (a) Cell migration and invasion were observed using a transwell chamber. (b) Western blot analysis of the cell proliferation-related proteins vimentin, Snail, Twist and E-cadherin in treated and control groups of TCA8113 cells. GAPDH served as a loading control. (c) Enzyme-linked immunosorbent assay (ELISA) was performed to analyze VEGF concentrations in cell-free supernatants. *Compared to Crtl or NC group. VEGF, vascular endothelial growth factor.

Figure 4

Overexpression of angiopoietin 2 (ANG2) promoted tumorigenicity, increased angiogenesis and reduced apoptosis in nude mice. Subcutaneous injection of treated and control groups of TCA8113 cells into nude mice. (a, b) Tumor volume was examined every week. Three independent experiments were performed, and the data represent the means±s.d. *P<0.05. (c) Tumor tissues from nude mice 4 weeks after injection were subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Green TUNEL-positive cells are apoptotic cells with fragmented DNA, whereas blue Hoechst-positive cells represent all cells in this assay. Magnification is × 200. (d) Immunohistochemical and quantification analysis of CD31 protein expression in the treated and control groups of oral squamous cell carcinoma (OSCC) tissues with specific anti-CD31 antibody. Representative photographs were obtained at × 200 magnification. (e) Western blot analysis of tumor formation-related proteins including Bim, PUMA, Bcl-2, Bax, Cyclin D1 and PCNA in the treated and control groups of OSCC tissue samples. GAPDH served as a loading control.