Department of Food and Biotechnology, Chungju National University, Chungju, Chungbuk 380-702, South Korea.
Eur J Pharmacol. 2012 Apr 15;681(1-3):15-22. doi: 10.1016/j.ejphar.2012.02.003. Epub 2012 Feb 17.
Cordycepin, the main constituent of Cordyceps militaris, demonstrated an anti-atherogenic effect in experimental animals. However, the effects of cordycepin on cell-cycle regulation and the signaling pathway in vascular smooth muscle cells (VSMC) remain largely unknown; therefore, unexpected roles of cordycepin-induced inhibition in VSMC growth were investigated. Mechanisms in cordycepin-treated VSMC were examined via an MTT assay, a thymidine uptake experiment, FACS analysis, immunoblot analysis, kinase assay, immunoprecipitation assay, and transient transfection assays. Cordycepin inhibited cell growth, induced G1-phase cell-cycle arrest, down-regulated cyclins and cyclin-dependent kinase (CDK) expression, and up-regulated p27KIP1 expression in VSMC. Cordycepin induced activation of JNK, p38MAPK and ERK1/2. Blocking of the ERK function using either ERK1/2-specific inhibitor U0126 or a small interfering RNA (si-ERK1) reversed p27KIP1 expression, inhibition of cell growth, and decreased cell-cycle proteins in cordycepin-treated VSMC. Ras activation was increased by cordycepin. Transfection of cells with dominant negative Ras (RasN17) mutant genes rescued cordycepin-induced ERK1/2 activity, increased p27KIP1 expression, inhibited cell proliferation, and reduced cell cycle proteins. In conclusion, our findings indicate that Ras/ERK1 pathways participate in p27KIP1-mediated G1-phase cell-cycle arrest induced by cordycepin via a decrease in cyclin/CDK complexes in VSMC.
蛹虫草中的主要成分蛹虫草素有抗动脉粥样硬化作用。然而，蛹虫草素对血管平滑肌细胞（VSMC）细胞周期调控和信号通路的影响在很大程度上尚不清楚；因此，研究了蛹虫草素诱导的 VSMC 生长抑制的意想不到的作用。通过 MTT 测定、胸苷摄取实验、FACS 分析、免疫印迹分析、激酶测定、免疫沉淀测定和瞬时转染实验，研究了蛹虫草素处理的 VSMC 中的机制。蛹虫草素抑制细胞生长，诱导 G1 期细胞周期停滞，下调细胞周期蛋白和细胞周期蛋白依赖性激酶（CDK）的表达，并上调 p27KIP1 的表达。蛹虫草素诱导 JNK、p38MAPK 和 ERK1/2 的激活。使用 ERK1/2 特异性抑制剂 U0126 或小干扰 RNA（si-ERK1）阻断 ERK 功能，逆转了 p27KIP1 的表达、细胞生长抑制和细胞周期蛋白减少，这表明蛹虫草素在 VSMC 中诱导 ERK1/2 激活。蛹虫草素增加 Ras 激活。用显性负 Ras（RasN17）突变基因转染细胞可挽救蛹虫草素诱导的 ERK1/2 活性，增加 p27KIP1 的表达，抑制细胞增殖，并降低细胞周期蛋白。综上所述，我们的研究结果表明，Ras/ERK1 途径参与了蛹虫草素通过降低 VSMC 中环素/CDK 复合物诱导的 p27KIP1 介导的 G1 期细胞周期阻滞。
