Hsu Yi-Fan, Lee Tong-Sheng, Lin Shyr-Yi, Hsu Sung-Po, Juan Shu-Hui, Hsu Yuan-Hsun, Zhong Wen-Bin, Lee Wen-Sen
Graduate Institute of Cell and Molecular Biology, Medical College, Taipei Medical University, Taipei, Taiwan.
Mol Carcinog. 2007 Apr;46(4):275-83. doi: 10.1002/mc.20274.
Previously, we showed that magnolol induces cell-cycle arrest in cultured colon and liver cancer cells through an upregulation of the p21 protein. The aim of this study was to delineate the molecular mechanism underlying this magnolol-induced increase of p21 protein. Thus our RT-PCR analysis demonstrated that the mRNA levels of p21 were increased at 1 h after magnolol treatment and sustained for at least 24 h. The p21 promoter activity was also increased by magnolol treatment. Western blot analysis demonstrated that treatment of COLO-205 cells with magnolol increased the levels of phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment of the cells with PD98059 abolished the magnolol-induced upregulation of p21 protein, suggesting the involvement of an ERK pathway in the magnolol-induced upregulation of p21 in COLO-205 cells. Ras inhibitor peptide abolished the magnolol-induced increase of phosphorylated ERK protein levels, increase of p21 protein, and decrease of thymidine incorporation. Moreover, treatment of COLO-205 with magnolol increased the phosphorylated Raf-1 protein (the Ras target molecule). Pretreatment of the cells with Raf-1 inhibitor reversed the magnolol-induced decrease in thymidine incorporation. Treatment of the cells with CaM kinase inhibitor, but not protein kinase A (PKA) inhibitor or phosphatidylinosital 3-kinase (PI3K) inhibitor, abolished the magnolol-induced activation of ERK and decrease of thymidine incorporation. Taken together, our results suggest that magnolol activates ERK phosphorylation through a Ras/Raf-1-mediated pathway. Subsequently, p21 expression is increased, and finally thymidine incorporation is decreased.
此前，我们发现厚朴酚通过上调p21蛋白诱导培养的结肠癌细胞和肝癌细胞发生细胞周期阻滞。本研究的目的是阐明厚朴酚诱导p21蛋白增加的分子机制。因此，我们的逆转录聚合酶链反应（RT-PCR）分析表明，厚朴酚处理1小时后p21的mRNA水平升高，并持续至少24小时。厚朴酚处理也增加了p21启动子活性。蛋白质印迹分析表明，用厚朴酚处理COLO-205细胞可增加细胞外信号调节激酶（ERK）的磷酸化水平。用PD98059预处理细胞可消除厚朴酚诱导的p21蛋白上调，提示ERK通路参与了厚朴酚诱导的COLO-205细胞中p21的上调。Ras抑制肽消除了厚朴酚诱导的磷酸化ERK蛋白水平增加、p21蛋白增加和胸苷掺入减少。此外，用厚朴酚处理COLO-205可增加磷酸化Raf-1蛋白（Ras靶分子）。用Raf-1抑制剂预处理细胞可逆转厚朴酚诱导的胸苷掺入减少。用钙调蛋白激酶抑制剂处理细胞可消除厚朴酚诱导的ERK激活和胸苷掺入减少，但蛋白激酶A（PKA）抑制剂或磷脂酰肌醇3激酶（PI3K）抑制剂则不能。综上所述，我们的结果表明，厚朴酚通过Ras/Raf-1介导的途径激活ERK磷酸化。随后，p21表达增加，最终胸苷掺入减少。
