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

如果将大鼠肝细胞癌组织移植到另一只同基因大鼠肝脏，所有动物均死于肺转移，平均生存期为116.7天。如果用他克莫司给药的同种异体肝移植来替代承载肝癌的肝脏，大鼠的生存时间不会因为肿瘤复发而显著延长。然而，他克莫司加ROCK抑制剂治疗显著延长了肝移植大鼠的存活时间，平均存活时间为303.1天。没有接受ROCK抑制剂治疗的大鼠显示肺部有多个转移性结节，而接受ROCK抑制剂治疗的大鼠没有发现转移性肺结节。这是一个高贵的模型系统，模拟了免疫充足动物的肿瘤复发和转移。我们正在研究乙酰肝素酶诱导的肝癌细胞是否可以改变肿瘤复发状态。我们已经证明，他克莫司激活Rho/ROCK信号通路促进大鼠肝癌细胞的迁移，而ROCK抑制剂在体外抑制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信号的下游效应分子MLC的磷酸化状态有关。加入ROCK抑制剂后，磷酸化MLC的活性明显受到抑制。另一方面，通过四甲基偶氮唑盐比色法检测，他克莫司不能促进肝癌细胞的增殖。我们还建立了肝素酶诱导的体外产生肝素酶的细胞系。我们研究了乙酰肝素酶诱导的肝癌细胞是否增加了迁移能力。我们的发现表明，他克莫司激活Rho/ROCK信号通路，刺激大鼠肝癌细胞的细胞运动，增强大鼠肝癌细胞的侵袭性。此外，我们还表明，ROCK抑制剂抑制了大鼠肝细胞癌模型肝移植后的肿瘤复发。

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.