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

Abstract

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GO/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cyclinD3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, CDKls）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-乙醯基半胱氯酸（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GO/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。Carnosic acid (CA) is a phenolic diterpene obtained from rosemary (Rosmarinus officinalis) . Our previous study indicated that CA induced apoptotic cell death through 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 the p38 pathway. IMR-32 cells were treated with 30 μM CA for 12~60 h. A flow cytometric analysis indicated that cells were significantly arrested in the GO/G1 phase when treated with CA for 24 h (p ＜ 0.05). Immunoblotting suggested that CA decreased the levels of cell-cycle regulatory proteins such as cyclin D1, cyclin D3, CDK4, and CDK6 proteins, while increasing the level of p21 protein. Pretreatment with the antioxidant, N-acetylcysteine (NAC), or p38 siRNA inhibited CAinduced p21 expression, whereas it reversed CA-decreased CDK4 expression. In conclusion, CA induced ROS generation and p38 activation, which in turn modulated the protein expressions of p21 and CDK4, and induced GO/G1 cell-cycle arrest in IMR-32 cells. Therefore, CA has the potential to be developed as a therapeutic agent against neuroblastoma.