Rac1/Cdc42的活化在内皮抑素联合CA4P阻断肿瘤血管网中的作用机制研究

Combretastatin A4 phosphate (CA4P) is the quintessential Vascular Disrupting Agent (VDA), which induces rapid vascular shutdown in the center of tumors and causes tumor necrosis. The protein Endostatin, a 20-kD proteolytic fragment of type ⅩⅧ collagen, is one of the most active natural inhibitors of angiogenesis. Our previous study shows that Endostatin enhances the antineoplastic effects of CA4P in an osteosarcoma xenograft because Endostatin and CA4P respectively induce breakdown of the peripheral and central tumor vessel. However, the in vitro study of HUVEC (Human Umbilical Vein Endothelial Cells) shows that over-activation of Rac1/Cdc42, the members of RhoGTPases which control the polymerization of microfilaments, restrict the synergistic anti-tumor effect of Endostatin and CA4P. The mechanisms remain elusive. In this study, Rac1 inhibitor NSC23766 and Cdc42 inhibitor ML141 will be used to interfere HUVEC. The role of RhoGTPases in interact of Endostatin and CA4P will be clarified by Real-time immunofluorescence, Pull-down Assays Western blot, Transwell assay and tube formation researches. On this basis, we will explore the feasibility of enhancing the in vivo synergistic anti-tumor effect of Endostatin and CA4P by inhibit the activation of Rac1/Cdc42. The study will provide scientific basis of rational combination of Endostatin and CA4P and promote understanding to tumor vascular-targeted therapy.

CA4P是最具代表性的肿瘤血管阻断剂，靶向阻断肿瘤中心血管；内皮抑素是最广谱的内源性血管形成抑制因子，抑制肿瘤周边新生血管形成。项目组前期动物实验证明，CA4P联合内皮抑素可同时阻断肿瘤中心和周边血管，产生协同抗肿瘤作用。两药对人脐静脉内皮细胞HUVEC体外作用的结果提示，细胞内RhoGTPases激酶成员Rac1/Cdc42活性的过度激活会限制两药协同作用，但机制不明。本项目拟借助HUVEC细胞体外血管模型，以Rac1抑制剂NSC23766和Cdc42抑制剂ML141进行干预，通过实时细胞免疫荧光和基于Pull-down Assays的Western blot等技术，阐明RhoGTPases激酶影响内皮抑素和CA4P相互作用机制。进而探索抑制Rac1/Cdc42活性以增强两药协同抗肿瘤作用的可行性，并以动物实验验证，为CA4P和内皮抑素这两大肿瘤血管靶向药物的合理联合使用提供理论依据。

CA4P是最具代表性的肿瘤血管阻断剂，靶向阻断肿瘤中心血管；内皮抑素是最广谱的内源性血管形成抑制因子，抑制肿瘤周边新生血管形成。项目组前期动物实验证明，CA4P联合内皮抑素可同时阻断肿瘤中心和周边血管，产生协同抗肿瘤作用。两药对人脐静脉内皮细胞HUVEC体外作用的结果提示，细胞内RhoGTPases激酶成员Rac1/Cdc42活性的过度激活会限制两药协同作用，但机制不明。本项目通过体内和体外实验观察了联合使用CA4P和内皮抑素对HUVEC细胞及MNNG-HOS细胞的影响。结果发现：1）在人脐静脉内皮细胞上短时间内联合使用内皮抑素和CA4P，MTT法检测细胞活力发现较低浓度的内皮抑素（0.05-50μg/ml）和CA4P（0.1-100nM）均无明显细胞毒作用。划痕实验和Transwell实验发现内皮抑素（10μg/ml）和CA4P（10nM）均能抑制内皮细胞迁移，间隔3h联合使用则迁移能力强于单药组；管状结构形成实验也是类似结果。2）用共聚焦显微镜观察内皮抑素和CA4P作用后内皮细胞F-actin的生成，结果提示两药单药均可增加F-actin生成，间隔3h联合使用F-actin生成减少。用pull-down assays联合western blot的方法检测了药物作用后RhoGTPases家族的活化RhoA、Rac1及Cdc42的相对表达量，发现内皮抑素可能通过活化Rac1/Cdc42，抑制了RhoA的活化，进而干扰了CA4P的生物学效应。3）延长药物作用时间间隔，在划痕实验和Transwell实验中发现间隔12h联合内皮抑素和CA4P，两者干扰作用基本消失。随后我们构建了HUVEC细胞和MNNG/HOS骨肉瘤细胞裸鼠皮下移植瘤模型，当内皮抑素和CA4P间隔24h给药时，两者联合发挥最强的抗肿瘤效果，且肿瘤的微血管密度最低。本研究为CA4P和内皮抑素这两大肿瘤血管靶向药物的合理联合使用提供理论依据。