The high incidence of lymphatic metastasis is closely related to poor prognosis and mortality in cancers. Potent inhibitors to prevent pathological lymphangiogenesis and lymphatic spread are urgently needed. The VEGF-C-VEGFR3 pathway plays a vital role in driving lymphangiogenesis and lymph node metastasis. In addition, COX2 in tumor cells and tumor-associated macrophages (TAMs) facilitates lymphangiogenesis. We recently reported that aiphanol, a natural stilbenolignan, attenuates tumor angiogenesis by repressing VEGFR2 and COX2. In this study, we evaluated the antilymphangiogenic and antimetastatic potency of aiphanol using in vitro, ex vivo and in vivo systems. We first demonstrated that aiphanol directly bound to VEGFR3 and blocked its kinase activity with an half-maximal inhibitory concentration (IC₅₀) value of 0.29 μM in an in vitro ADP-Glo^TM kinase assay. Furthermore, we showed that aiphanol (7.5−30 μM) dose-dependently counteracted VEGF-C-induced proliferation, migration and tubular formation of lymphatic endothelial cells (LECs), which was further verified in vivo. VEGFR3 knockdown markedly mitigated the inhibitory potency of aiphanol on lymphangiogenesis. In 4T1-luc breast tumor-bearing mice, oral administration of aiphanol (5 and 30 mg· kg⁻¹ ·d⁻¹) dose-dependently decreased lymphatic metastasis and prolonged survival time, which was associated with impaired lymphangiogenesis, angiogenesis and, interestingly, macrophage infiltration. In addition, we found that aiphanol decreased the COX2-dependent secretion of PGE2 and VEGF-C from tumor cells and macrophages. These results demonstrate that aiphanol is an appealing agent for preventing lymphangiogenesis and lymphatic dissemination by synergistically targeting VEGFR3 and inhibiting the COX2-PGE2-VEGF-C signaling axis.

淋巴转移的高发生率与癌症的不良预后和死亡率密切相关。迫切需要有效的抑制剂来防止病理性淋巴管生成和淋巴扩散。 VEGF-C-VEGFR3 通路在驱动淋巴管生成和淋巴结转移中发挥着至关重要的作用。此外，肿瘤细胞和肿瘤相关巨噬细胞（TAM）中的COX2促进淋巴管生成。我们最近报道了 aiphanol，一种天然的二苯乙烯木脂素，通过抑制 VEGFR2 和 COX2 来减弱肿瘤血管生成。在这项研究中，我们使用体外、离体和体内系统评估了 aiphanol 的抗淋巴管生成和抗转移效力。我们首先在体外 ADP-Glo^{​​ TM}激酶测定中证明，aiphanol 直接与 VEGFR3 结合并阻断其激酶活性，半数抑制浓度 (IC ₅₀ ) 值为 0.29 μM。此外，我们发现，aiphanol (7.5−30 μM) 剂量依赖性地抵消 VEGF-C 诱导的淋巴内皮细胞 (LEC) 的增殖、迁移和管状形成，这在体内得到了进一步验证。 VEGFR3 敲低显着减轻了 aiphanol 对淋巴管生成的抑制效力。在4T1-luc乳腺肿瘤小鼠中，口服aiphanol（5和30 mg·kg ^-1 ·d ^-1 ）剂量依赖性地减少淋巴转移并延长生存时间，这与受损的淋巴管生成、血管生成有关，有趣的是，巨噬细胞浸润。此外，我们发现 aiphanol 减少了肿瘤细胞和巨噬细胞中 COX2 依赖性的 PGE2 和 VEGF-C 分泌。 这些结果表明，aiphanol 是一种通过协同靶向 VEGFR3 和抑制 COX2-PGE2-VEGF-C 信号轴来预防淋巴管生成和淋巴传播的有吸引力的药物。