鼠尾草酸對人類神經母細胞瘤imr-32細胞株細胞週期之影響.docVIP

鼠尾草酸對人類神經母細胞瘤IMR-32細胞株細胞週期之影響 林家媛1 蔡佳文1* Effect of Carnosic Acid on the Induction of Cell Cycle Arrest in Human Neuroblastoma IMR-32 Cells Chia-Yuan Lin1, Chia-Wen Tsai1* 1 Department of Nutrition, China Medical University, Taiwan, ROC *Corresponding author: Chia-Wen Tsai, Department of Nutrition, China Medical University, Taiwan, ROC. Tel: 886-4ext.7521; Fax: 886-4 E-mail: cwtsai@mail.cmu.edu.tw Running title: carnosic acid induces cell cycle arrest in neuroblastoma cells Figure: 6 摘要 鼠尾草酸(carnosic acid, CA)為迷迭香中的二萜類(diterpene)，由於我們先前的研究發現CA會透過增加活性氧化物質(reactive oxygen species, ROS)而活化p38蛋白，以促進人類神經母細胞瘤(neuroblastoma) IMR-32細胞發生凋亡。因此，本研究欲探討CA減少IMR-32細胞生長是否與ROS和p38路徑影響其細胞週期有關。IMR-32細胞以30 μM的CA處理12-60小時，流式細胞儀結果顯示在CA處理24小時後，細胞滯留在G0/G1期的比例顯著地增加，而且CA隨處理濃度的增加而降低週期素(cyclins)中的cyclin D1和cyclin D3以及週期素激酶(cyclin-dependent kinases, CDKs)中的CDK4和CDK6蛋白表現，然而CA對於細胞週期抑制性蛋白(cyclin dependent kinase inhibitors, CDKIs)中p21蛋白的表現卻隨著濃度的增加而增加。另外，細胞若先處理抗氧化劑N-乙醯基半胱氨酸(N-acetylcysteine, NAC)或進行p38 siRNA實驗，則會抑制CA增加p21蛋白的能力，並回復CA抑制CDK4蛋白的能力。結論：CA可能透過調節p21和CDK4蛋白表現，使IMR-32細胞停滯在G0/G1期，且此路徑可能與ROS和p38路徑有關，因此，CA 未來可能可發展為抵抗神經母細胞瘤的化學預防治療策略。 關鍵字：鼠尾草酸、活性氧化物質、細胞週期、p38激酶、人類神經母細胞瘤IMR-32細胞株 *蔡佳文 通訊地址：中國醫藥大學營養系，台中市學士路91 號 ABSTRACT Carnosic acid (CA) is a phenolic diterpene obtained from rosemary (Rosmarinus officinalis). Our previous study indicated that CA induced apoptotic cell death though reactive oxygen species (ROS)-mediated phosphorylation of p38 in human neuroblastoma IMR-32 cells. In this study, we explored whether CA reduced cell viability was associated with cell cycle arrest through ROS generation and p38 pathway. IMR-32 cells were treated with 30 μM CA for 12-60 h. In flow cytometric analysis indicated that cells were significantly arrested in G0/G1 phase when the cells were treated with CA for 24 h (p0.05). Immunoblotting suggested that CA decreased the levels of cell cycle regulatory proteins such as cyclin D1, cyclin D3, CDK4, and CDK6 proteins, while increased the le