Analysis of the mechanism of cancer invasion and metastasis due to heparanase

将大鼠肝细胞癌（HCC）组织移植到另一个同基因大鼠肝脏，所有动物均死于肺转移，平均生存期为116.7天。如果用他克莫司治疗的同种异体肝移植代替肝癌，大鼠的生存时间不会因肿瘤复发而明显延长。他克莫司联合ROCK抑制剂治疗显著延长了肝移植大鼠的生存期，平均生存期为303.1天。未使用ROCK抑制剂治疗的大鼠在肺部显示多发性转移结节，而使用ROCK抑制剂治疗的大鼠未发现转移性肺结节。这是一种在免疫充足动物体内模拟肿瘤复发和转移的高贵模型系统。我们现在正在研究肝素酶诱导的HCC细胞是否可以改变肿瘤的复发状态。我们在体外研究表明，他克莫司激活Rho/ROCK信号通路可增强大鼠肝癌细胞的细胞迁移，ROCK抑制剂可减少他克莫司诱导的大鼠肝癌细胞的迁移。他克莫司诱导的细胞迁移与Rho/ROCK信号下游效应物MLC的磷酸化状态有关。ROCK抑制剂的加入显著抑制了磷酸化MLC的活性。另一方面，通过MTT试验，他克莫司不会增加肝癌细胞的增殖。我们还开发了肝素酶诱导细胞系，在体外产生肝素酶。我们研究了肝素酶诱导的HCC细胞是否增加了迁移能力。我们的研究结果表明，他克莫司激活Rho/ROCK信号通路，刺激大鼠HCC细胞的细胞运动，增强大鼠HCC细胞的侵袭性。此外，我们还发现ROCK抑制剂在大鼠HCC模型中抑制肝移植后肿瘤复发。

If the rat hepatocellular carcinoma (HCC) tissue was implanted into another syngeneic rat liver, all animals died of pulmonary metastasis with a mean survival period of 116.7 days. If the HCC bearing liver was replaced by the allogeneic liver graft with tacrolimus administration, survival of rats did not significantly prolonged due to tumor recurrence. Tacrolimus plus ROCK inhibitor-treatment, however, significantly prolonged the survival of liver grafted rats with a mean survival period 303.1 days. Rats that had not been treated with the ROCK inhibitor showed multiple metastatic nodules in the lungs, whereas no metastatic pulmonary nodules were found in rats treated with the ROCK inhibitor. This is a noble model system that mimics the tumor recurrence and metastasis in the immune-sufficient animals. We are now investigating whether heparanase induced HCC cells can alter the tumor recurrence status.We have shown that tacrolimus activated Rho/ROCK signal pathway to enhance cell migration of rat HCC cells and the ROCK inhibitor reduced tacrolimus-induced migration of rat hepatocellular carcinoma cells in vitro. Tacrolimus-induced cell migration was associated with phosphorylation state of MLC, a downstream effector of Rho/ROCK signaling. The activity of phosphorylated MLC was significantly suppressed by the addition of ROCK inhibitor. On the other hand, tacrolimus does not increase the proliferation of hepatocellular carcinoma cells by MTT assays. We also developed the heparanase induced cell lines that produced heparanase in vitro. We investigated whether heparanase induced HCC cells increased the capacity of migration. Our findings indicate that tacrolimus activates the Rho/ROCK signal pathway to stimulate cell motility of rat HCC cells and enhances the invasiveness of rat HCC cells. Further, we have also shown that the ROCK inhibitor suppressed tumor recurrence after liver transplantation in a rat HCC model.