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.

中文翻译：

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6,8-二异戊二烯基染料木黄酮对口腔癌前哨淋巴结动物模型中VEGF-A诱导的淋巴管生成和淋巴结转移的影响

背景：口腔鳞状细胞癌预后的主要决定因素是颈部淋巴结转移。6,8-Diprenylgenistein (6,8-DG) 是一种从柘木中分离的异黄酮类化合物，据报道具有抗微生物和抗肥胖活性。然而，其对口腔癌淋巴管生成和淋巴结转移的影响尚未见报道。方法：为了研究 6,8-DG 对 VEGF-A 诱导的淋巴管生成的体外抑制作用，我们使用人淋巴微血管内皮细胞 (HLMEC) 进行了增殖、管形成和迁移试验。RT-PCR、Western印迹、免疫沉淀、ELISA和免疫共沉淀试验用于研究蛋白质的表达水平和6,8-DG的机制。6、体内抑制作用 使用口腔癌前哨淋巴结 (OCSLN) 动物模型研究了 8-DG。结果：6,8-DG抑制rhVEGF-A处理的HLMECs的增殖、迁移和管形成。此外，6,8-DG 显着减少了由 rhVEGF-A 刺激的体内淋巴管形成。在 CoCl2 处理的 SCCVII 细胞中，6,8-DG 抑制 VEGF-A 而非其他淋巴管生成因子的表达。6,8-DG 抑制 HLMECs 中由 rhVEGF-A 刺激的 VEGFR-2 的表达和激活。此外，6,8-DG 抑制了 rhVEGF-A 处理的 HLMEC 中淋巴管生成相关的下游信号因子如 FAK、PI3K、AKT、p38 和 ERK 的激活。此外，6,8-DG 抑制缺氧诱导因子 (HIF-1α) 的表达，该因子参与 CoCl2 处理的 SCCVII 细胞中 VEGF-A 的表达，并且 6, 8-DG 通过中断 HLMECs 中 VEGF-A 和 VEGFR-2 的结合来抑制 VEGF-A 信号传导。在 VEGF-A 诱导的 OCSLN 动物模型中，我们证实 6,8-DG 抑制肿瘤诱导的淋巴管生成和 SLN 转移。结论：这些数据表明 6,8-DG 在体外和体内抑制 VEGF-A 诱导的淋巴管生成和淋巴结转移。此外，6,8-DG 的抑制作用可能是通过抑制癌细胞中 VEGF-A 的表达和抑制 HLMEC 中的 VEGF-A/VEGFR-2 信号通路介导的。因此，6,8-DG 可能成为预防和治疗口腔癌转移的新型且有价值的治疗剂。8-DG 抑制肿瘤诱导的淋巴管生成和 SLN 转移。结论：这些数据表明 6,8-DG 在体外和体内抑制 VEGF-A 诱导的淋巴管生成和淋巴结转移。此外，6,8-DG 的抑制作用可能是通过抑制癌细胞中 VEGF-A 的表达和抑制 HLMEC 中的 VEGF-A/VEGFR-2 信号通路介导的。因此，6,8-DG 可能成为预防和治疗口腔癌转移的新型且有价值的治疗剂。8-DG 抑制肿瘤诱导的淋巴管生成和 SLN 转移。结论：这些数据表明 6,8-DG 在体外和体内抑制 VEGF-A 诱导的淋巴管生成和淋巴结转移。此外，6,8-DG 的抑制作用可能是通过抑制癌细胞中 VEGF-A 的表达和抑制 HLMEC 中的 VEGF-A/VEGFR-2 信号通路来介导的。因此，6,8-DG 可能成为预防和治疗口腔癌转移的新型且有价值的治疗剂。