Analysis on the mechanism of growth inhibition by vitamin K2 in hepatoma cells

（目的）为了阐明维生素K2对肝癌细胞生长抑制的作用机制，我们研究了维生素K2对凋亡和细胞周期相关因子的影响。（方法）以人肝癌细胞系HepG2、Hep 3B和Huh7为研究对象，检测1)细胞凋亡和细胞增殖的诱导，2)细胞周期，3)细胞周期蛋白依赖性激酶（CDK）抑制剂（p16、p21、p27）的表达，4)细胞周期蛋白D1的表达，5)细胞周期蛋白D1启动子的激活，6)转录因子（AP-1、NF-κB）与细胞周期蛋白D1启动子的结合活性，7)抗癌药物（CDDP、5- fu、VP-16、Docetaxel）的加性作用，8)基质金属蛋白酶（MMP-1、MMP-3、MMP-7）的表达。通过补充维生素K2（结果）1)维生素K2抑制细胞生长，但不诱导细胞凋亡，2)维生素K2诱导G1阻滞，3)维生素K2增加CDK抑制剂的表达，4)维生素K2降低cyclin D1的表达，5)维生素K2降低cyclin D1启动子活性，6)维生素K2降低AP-1和NF-κB与cyclin D1启动子区的结合活性，7)维生素K2抵消CDDP的作用，但增强了对5- fu、VP-16和Docetaxel细胞生长的抑制作用。8)维生素K2抑制MMP-1、MMP-3和MMP-7的表达。（结论）维生素K2可通过抑制转录因子、NF-κB与cyclinD1启动子的结合以及增强CDK抑制剂的表达，从而抑制cyclinD1的表达，从而诱导肝癌细胞G1阻滞。此外，这些结果提示了维生素K2在抗癌药物联合治疗和抑制肿瘤侵袭方面的临床应用的可能性。

(Purpose) To elucidate the mechanism of growth inhibition by vitamin K2 in hepatoma cells, we examined how apoptosis- and cell cycle-related factors were influenced by vitamin K2.(Methods) Using human hepatoma cell lines, HepG2, Hep 3B and Huh7, we tested about 1)induction of apoptosis and cell proliferation, 2)cell cycle, 3)expression of cyclin dependent kinase (CDK) inhibitors (p16, p21, p27), 4)expression of cyclin D1, 5)activation of cyclin D1 promoter, 6)binding activity of transcription factors (AP-1, NF-κB) to cyclin D1 promoter, 7)additive effect of anti-cancer agents (CDDP, 5-FU, VP-16, Docetaxel), 8)expression of matrix metalloproteinase (MMP-1, MMP-3, MMP-7), by addition of vitamin K2.(Results) 1)Vitamin K2 suppressed cell growth, but could not induce apoptosis, 2)vitamin K2 induced G1 arrest, 3)vitamin K2 increase the expression of CDK inhibitors, 4)vitamin K2 decrease the expression of cyclin D1, 5)vitamin K2 decrease promoter activity of cyclin D1, 6)vitamin K2 decrease binding activity of AP-1 and NF-κB to promoter region of cyclin D1, 7)vitamin K2 counteracts the action of CDDP, but enhances the suppressive effect on cell growth of 5-FU, VP-16 and Docetaxel, 8)vitamin K2 inhibits the expression of MMP-1, MMP-3 and MMP-7.(Conclusion) Vitamin K2 can induce G1 arrest in hepatoma cells by suppression of cyclin D1 expression through inhibition of transcription factor, NF-κB binding to cyclinD1 promoter as well as enhancement of CDK inhibitors expression. Furthermore, these results suggest the possibility on clinical application of vitamin K2 in the field of concomitant treatment with anti-cancer agents and suppression of tumor invasion.