Liu Tsan-Zon, Chen Chung-Yi, Yiin Shuenn-Jiun, Chen Ching-Hsein, Cheng Jiin-Tsuey, Shih Ming-Kuei, Wang Yu-Shan, Chern Chi-Liang
Center for Gerontological Research and Graduate Institute of Medical Biotechnology, Chang-Gang University, Kwei-Shan, Taoyuan, Taiwan, ROC.
Food Chem Toxicol. 2006 Feb;44(2):227-35. doi: 10.1016/j.fct.2005.07.003. Epub 2005 Aug 19.
Reports elsewhere demonstrated that Epimedin C, a constituent isolated from the leaves of Epimedium sagittatum, possessed anti-tumor activity. However, its mechanism of action remains unresolved. Using SK-Hep-1 cells, a poorly-differentiated hepatoma subline, as an experimental model, we present evidence here that the anti-tumor activity of Epimedin C may involve cell cycle blockage. Immunoblotting analyses demonstrated that Epimedin C caused a decreased expression of hyperphosphorylated retinoblastoma (Rb) protein, cyclin D1, c-Myc, and c-Fos. In parallel, we measured the kinase activities and found that CDK2 and CDK4 were suppressed with commensurate increased levels of CDK inhibitors, p21(Cip1) and p27(Kip1). These data suggested that Epimedin C arrested the proliferation of these cells at G0/G1 phase through inhibition of CDK2 and CDK4 activities via an increased induction of p21(Cip1) and p27(Kip1). Alternatively, we investigated whether the anti-proliferative effect of Epimedin C on these cells might involve MAP kinase cascade. Using western blotting technique, we demonstrated that Epimedin C also selectively decreased ERK1/2 phosphorylation. Among the downstream effectors of ERK examined, we found that Epimedin C selectively decreased the expression of c-Fos, but not c-Jun. By EMSA assay, we further demonstrated that decreased c-Fos resulted in the downregulation of AP-1/DNA binding activity. Taken together, the molecular mechanisms of anti-tumor activity of Epimedin C may be proceeded by the combined effects of the cell cycle blockage via either the inhibition of CDK2 and CDK4 activities, with commensurate increase in their inhibitors, p21(Cip1) and p27(Kip1) or negatively modulates the ERK/c-Fos/AP-1 signaling pathway.
其他地方的报告表明，从箭叶淫羊藿叶片中分离出的成分淫羊藿苷C具有抗肿瘤活性。然而，其作用机制仍未明确。我们使用低分化肝癌亚系SK-Hep-1细胞作为实验模型，在此提供证据表明淫羊藿苷C的抗肿瘤活性可能涉及细胞周期阻滞。免疫印迹分析表明，淫羊藿苷C导致高磷酸化视网膜母细胞瘤（Rb）蛋白、细胞周期蛋白D1、c-Myc和c-Fos的表达降低。同时，我们测量了激酶活性，发现CDK2和CDK4受到抑制，同时CDK抑制剂p21（Cip1）和p27（Kip1）水平相应增加。这些数据表明，淫羊藿苷C通过增加p21（Cip1）和p27（Kip1）的诱导来抑制CDK2和CDK4活性，从而使这些细胞在G0/G1期停止增殖。另外，我们研究了淫羊藿苷C对这些细胞的抗增殖作用是否可能涉及丝裂原活化蛋白激酶（MAP）级联反应。使用蛋白质印迹技术，我们证明淫羊藿苷C还选择性地降低了ERK1/2的磷酸化。在所检测的ERK下游效应物中，我们发现淫羊藿苷C选择性地降低了c-Fos的表达，但不影响c-Jun的表达。通过电泳迁移率变动分析（EMSA）测定，我们进一步证明c-Fos的减少导致AP-1/DNA结合活性的下调。综上所述，淫羊藿苷C抗肿瘤活性的分子机制可能是通过抑制CDK2和CDK4活性并相应增加其抑制剂p21（Cip1）和p27（Kip1）来阻断细胞周期，或者负向调节ERK/c-Fos/AP-1信号通路的联合作用来实现的。
