Effects of 6,8-Diprenylgenistein on VEGF-A-Induced Lymphangiogenesis and Lymph Node Metastasis in an Oral Cancer Sentinel Lymph Node Animal Model

Abstract

Background: The major determining factor of prognosis of oral squamous cell carcinoma is cervical lymph node metastasis. 6,8-Diprenylgenistein (6,8-DG), an isoflavonoid isolated from Cudrania tricuspidata has been reported to have anti-microbial and anti-obesity activities. However, its effects on lymphangiogenesis and lymph node metastasis in oral cancer have not yet been reported.

Methods: To investigate the in vitro inhibitory effects of 6,8-DG on VEGF-A-induced lymphangiogenesis, we performed the proliferation, tube formation, and migration assay using human lymphatic microvascular endothelial cells (HLMECs). RT-PCR, Western blot, immunoprecipitation, ELISA and co-immunoprecipitation assays were used to investigate the expression levels of proteins, and mechanism of 6,8-DG. The in vivo inhibitory effects of 6,8-DG were investigated using an oral cancer sentinel lymph node (OCSLN) animal model.

Results: 6,8-DG inhibited the proliferation, migration and tube formation of rhVEGF-A treated HLMECs. In addition, the in vivo lymphatic vessel formation stimulated by rhVEGF-A was significantly reduced by 6,8-DG. 6,8-DG inhibited the expression of VEGF-A rather than other lymphangiogenic factors in CoCl₂-treated SCCVII cells. 6,8-DG inhibited the expression and activation of VEGFR-2 stimulated by rhVEGF-A in HLMECs. Also, 6,8-DG inhibited the activation of the lymphangiogenesis-related downstream signaling factors such as FAK, PI3K, AKT, p38, and ERK in rhVEGF-A-treated HLMECs. Additionally, 6,8-DG inhibited the expression of the hypoxia-inducible factor (HIF-1α), which is involved in the expression of VEGF-A in CoCl₂-treated SCCVII cells, and 6,8-DG inhibited VEGF-A signaling via interruption of the binding of VEGF-A and VEGFR-2 in HLMECs. In the VEGF-A-induced OCSLN animal model, we confirmed that 6,8-DG suppressed tumor-induced lymphangiogenesis and SLN metastasis.

Conclusion: These data suggest that 6,8-DG inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in vitro and in vivo. Furthermore, the inhibitory effects of 6,8-DG are probably mediated by inhibition of VEGF-A expression in cancer cells and suppression of the VEGF-A/VEGFR-2 signaling pathway in HLMEC. Thus, 6,8-DG could be novel and valuable therapeutic agents for metastasis prevention and treatment of oral cancer.

Keywords: 6,8-diprenylgenistein; VEGF-A-induced lymphangiogenesis; oral cancer; sentinel lymph node metastasis.

Figure 1

Structure of 6,8-diprenylgenistein (6,8-DG) and effects of 6,8-DG on VEGF-A-induced human lymphatic microvascular endothelial cells (HLMEC) proliferation. (A) Structure of 6,8-DG. (B) Proliferation in HLMECs stimulated rhVEGF-A. Cells were detached and counted using a hemocytometer. Data are presented as a mean ± S.D. of three independent experiments (^## p < 0.001, *** p < 0.001).

Figure 2

Effects of 6,8-DG on VEGF-A-induced HLMEC tube formation and migration. (A,C) Tube formation in HLMECs stimulated rhVEGF-A. Cells were imaged under an inverted phase contrast microscope using a digital single-lens reflex camera. Total tube lengths of a unit area were calculated using the Image J program. (B,D) The migrated cells to the underside of membranes were fixed with methanol, stained with hematoxylin solution, and then imaged under an inverted phase contrast microscope using a digital camera. Five digital images per well for (B) were obtained, and the numbers of migrated HLMECs were counted. Each sample was assayed in duplicate. Numbers of migrated HLMECs present in 320 mm² are presented as a bar diagram. Data are presented as a mean ± S.D. of three independent experiments (^## p < 0.001, ** p < 0.01, *** p < 0.001).

Figure 3

Effects of 6,8-DG on VEGF-A-induced lymphangiogenesis in an in vivo Matrigel plug. (A) Matrigel plugs were excised and photographed using a digital camera. The lymphatic vessel density values in Matrigel plug sections were measured using the immunohistochemical analysis with anti-LYVE-1 antibody. All Matrigel sections were digitalized and microscopic images were captured under 200× objective magnification. Scale bar = 200 μm. (B) Immunohistochemical intensity values (LYVE-1) from captured images were analyzed by the Image J program and represented as a bar diagram. Data are presented as a mean ± S.D. (** p < 0.01).

Figure 4

Effects of 6,8-DG on the expression of VEGF-A and VEGF-C in SCCVII cells treated with CoCl₂. (A) cDNAs were generated from total RNAs treated with DNase I, and PCR reaction was performed with specific primers of VEGF-A and -C and β-actin. (B) PCR products from three independent experiments (A) were quantified and represented as a bar diagram. The levels of the VEGF-A and -C transcripts in the control (6,8-DG- and CoCl₂-untreated cells) were estimated as 100%. (C) The protein levels of VEGF-A and -C in the intracellular fraction were determined using Western blot with anti-VEGF-A and anti-VEGF-C antibodies. (D) The protein level of VEGF-A in medium fractions was determined using ELISA assay. Data are presented as a mean ± S.D. of three independent experiments (^# p < 0.01, * p < 0.05, ** p < 0.01).

Figure 5

Effects of 6,8-DG on activation of VEGFR-1, VEGFR-2 and lymphangiogenesis-related downstream signaling factors in rhVEGF-A-treated HLMECs. (A,C) Cell lysates were immunoprecipitated with anti-phospho-Tyr (anti-p-Tyr). The levels of phosphorylated VEGFR-1 and -2 in immunoprecipitates were detected using Western blot analysis with anti-VEGFR-1 and anti-VEGFR-2 antibodies. (B,D) HLMECs were serum starved for 6 h, then were treated with different concentrations of 6,8-DG (0,1, 2.5, 5 μM) in the presence of rhVEGF-A (20 ng/mL) for 30 min. The phosphorylation levels of FAK, PI3K, AKT, JNK, ERK1/2, and p38 were determined using Western blot analysis with anti-p-FAK, anti-p-PI3K, anti-p-AKT, anti-p-JNK, anti-p-ERK1/2, and anti-p-p38 antibodies. Data are presented as a mean ± S.D. of three independent experiments (^# p < 0.01, * p < 0.05, ** p < 0.01).

Figure 6

Effects of 6,8-DG Effect of 6,8-DG in oral cancer related lymphangiogenesis. (A) SCCVII cells were treated with different concentrations of 6,8-DG (0, 1, 2.5, 5 μM) in the presence or absence of 100 μM of CoCl₂ for 24 h. cDNAs were generated from total RNAs treated with DNase I, and PCR reaction was performed with specific primers of HIF-1α and β-actin. (B) PCR products from three independent experiments (A) were quantified and represented as a bar diagram. The levels of the VEGF-A and -C transcripts in the control (6,8-DG- and CoCl₂-untreated cells) were estimated as 100%. (C,D) The cells were lysed and samples subjected to SDS-PAGE. Western blot was performed with anti-HIF-1α and anti-β-actin antibodies. (E) Total protein extracts of HLMECs were collected and analyzed by co-immunoprecipitation with rhVEGF-A and anti-VEGF-A antibody. The protein levels of VEGFR-1, VEGFR-2, and VEGF-A were detected by Western blot using anti-VEGFR-1, anti-VEGFR-2, and anti-VEGF-A antibodies. (F) Schematic illustration of the mechanism of action of 6,8-DG in oral cancer related lymphangiogenesis. Data are presented as a mean ± S.D. of three independent experiments (^# p < 0.01, ^## p < 0.001).

Figure 7

Effects of 6,8-DG on lymphangiogenesis and lymph node metastasis in a VEGF-A-induced OCSLN animal model. (A) Sentinel lymph nodes sections of mice of each group were analyzed using hematoxylin and eosin staining, and immunohistochemical analysis with anti-cytokeratin antibody. (B) Sentinel lymph nodes volumes were measured using a caliper. (C) Lymphatic vessel density values in sentinel lymph nodes sections were measured by immunohistochemical analysis using anti-LYVE-1 antibody. All sections were digitalized and images were captured under 200× objective magnification. Scale bar = 200 μm. (D) Immunohistochemical intensity values of LYVE-1 from captured images of tumors and sentinel lymph nodes were analyzed via the Image J program and represented as a bar diagram. Data are presented as a mean ± S.D. (^# p < 0.01, ^## p < 0.001, ** p < 0.01).