1-磷酸钙-尿嘧啶对卵巢上皮癌腹膜粘附作用的抑制及其相关机制研究

Ovarian cancer is the fifth leading cause of cancer-related deaths for women in the world. Intraperitoneal metastases can cause peritoneal organ adhesion and malfunction, massive ascites, and/or pleural effusions, leading to mortality.1-calcium phosphate-uracil (1-CP-U) is a synthetic uracil derivative, which has been suggested to demonstrate a wide range of highly selective functions. The previous study demonstrated the function of 1-CP-U in tumor proliferation, apoptosis and invasion with specific effects against cancer cells in vitro, suggesting 1-CP-U as a potential anticancer agent. More importantly, 1-CP-U has been shown to inhibit adhesion of ovarian cancer cells to peritoneal mesothelium though the effects remains undefined. Hence, in order to fulfill this niche we designed the in vitro experiments in which three lines of ovarian cancer cells SKOV3, CAOV3 and OVCAR3 were subjected to Res, and then cell adhesion assay, stable transfections with luciferase, ex vivo co-culturing of mouse omenta with luciferase-marked human EOC cells and in vivo tumor inoculation and bioluminescence imaging were assayed.To address this issue mechanistically, the expression of major mediators of ovarian cancer cell adhesion to peritoneal mesothelial, including integrin, FAK, Paxillin, Src were evaluated by PCR, western blotting analysis, co-Immunoprecipitation and immunohistochemical. These results are supposed to support the continued evaluation of 1-CP-U as a promising clinical treatment for ovarian cancer.

卵巢癌易发生腹膜腔浸润和转移是造成高病死率的原因之一，离巢肿瘤细胞对腹膜的粘附作用在这个过程中起到关键作用。我们前期发现，新型嘧啶类衍生物1-磷酸钙-尿嘧啶（1-calcium phosphate-uracil，1-CP-U）能够较为有效地在一定程度上特异性地抑制多种肿瘤细胞生长，诱导其发生凋亡，降低其侵袭能力。更重要的是，1-CP-U能够抑制多种卵巢癌细胞株发生腹膜粘附，但相关机制尚不清楚。申请人拟通过细胞粘附实验，构建稳定表达荧光素酶的肿瘤细胞，建立体内外裸鼠腹膜种植模型等方法检测1-CP-U对卵巢癌细胞腹膜粘附的影响，再通过荧光定量PCR、Western Blot、免疫共沉淀、免疫组化、基因干扰等方法检测药物干预后Integrin β1/Fak通路粘附因子的表达，阐明1-CP-U所作用的信号通路及粘附分子，为其作用于临床治疗奠定理论基础。

背景：卵巢癌是女性生殖器官恶性肿瘤中死亡率最高的疾病。卵巢癌易发生腹膜腔浸润和转移是造成高病死率的原因之一，离巢肿瘤细胞对腹膜的粘附作用在这个过程中起到关键作用。如果能够成功抑制肿瘤细胞对腹膜的粘附，则可以切断肿瘤细胞的侵袭与转移途径，并可以促进离巢肿瘤细胞的死亡，抑制恶性肿瘤的发展。1-磷酸钙-尿嘧啶（1-calcium phosphate-uracil，1-CP-U）是人工合成的嘧啶类衍生物，对多种肿瘤细胞株具有显著的生长抑制作用，并且能够诱导肿瘤细胞发生凋亡，同时抑制肿瘤细胞的侵袭能力，且1-CP-U在较低药物浓度水平上，抑制肿瘤细胞侵袭的能力更为显著。本项目初步数据显示1-CP-U干预后，卵巢癌细胞株粘附作用降低，粘附分子Fak、p-Fak及Integrin β1的表达逐渐减弱。.研究内容：本研究以1-CP-U为对象，深入研究其对卵巢癌细胞腹膜粘附的抑制作用，探讨1-CP-U所作用的粘附分子及信号通路。.重要结果及关键数据：在体外实验中，发现1-CP-U抑制多种EOC细胞发生粘附，迁移以及侵袭，呈现时间和浓度依赖性；在体内实验中，对5-6周大小的裸鼠腹腔注射SKOV3-Luc细胞，建立卵巢癌细胞腹腔转移模型。选择腹腔注射1-CP-U 200 mg/kg作为处理剂量，并且在裸鼠腹腔荷瘤的第一天及第五天进行药物处理，处死裸鼠后进行大网膜取材，用荧光素酶试剂盒检测大网膜上转移病灶，发现处理组病灶荧光强度低于对照组；随后通过体内成像仪连续检测裸鼠腹腔镜内荧光素活性，发现在治疗21天后，1-CP-U组荧光素活性显著降低，表明1-CP-U发挥抑制转移效应需要2-3周时间。对照组裸鼠腹腔肿瘤转移病灶累及器官更多，且肿瘤个数及体积均明显增多。上述体内试验中裸鼠腹腔转移瘤行免疫荧光以及免疫印迹检查，对照组肿瘤细胞 FAK及其相关通路蛋白呈强阳性表达，而处理组中表达减弱。.科学意义：表明1-CP-U可以抑制卵巢癌发生腹腔转移，且可能通过抑制FAK相关信号通路从而发挥抑制卵巢癌腹腔转移作用，为其作用于临床治疗奠定理论基础。