Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chosun University, Gwangju 501-759, Korea.
Korean J Physiol Pharmacol. 2012 Jun;16(3):153-8. doi: 10.4196/kjpp.2012.16.3.153. Epub 2012 Jun 26.
Cellular effects of ethanol in YD-15 tongue carcinoma cells were assessed by MTT assay, caspase activity assay, Western blotting and flow cytometry. Ethanol inhibited the growth and proliferation of YD-15 cells in a dose- and time-dependent manner in an MTT assay. The effects of ethanol on cell cycle control at low percent range of ethanol concentration (0 to 1.5%), the condition not inducing YD-15 cell death, was investigated after exposing cells to alcohol for a certain period of time. Western blotting on the expression of cell cycle inhibitors showed that p21 and p27 was up-regulated as ethanol concentration increases from 0 to 1.5% whilst the cell cycle regulators, cdk1, cdk2, and cdk4 as well as Cyclin A, Cyclin B1 and Cyclin E1, were gradually down-regulated. Flow cytometric analysis of cell cycle distribution revealed that YD-15 cells exposed to 1.5% ethanol for 24 h was mainly arrested at G2/M phase. However, ethanol induced apoptosis in YD-15 cells exposed to 2.5% or higher percent of ethanol. The cleaved PARP, a marker of caspase-3 mediated apoptosis, and the activation of caspase-3 and -7 were detected by caspase activity assay or Western blotting. Our results suggest that ethanol elicits inhibitory effect on the growth and proliferation of YD-15 tongue carcinoma cells by mediating cell cycle arrest at G2/M at low concentration range and ultimately induces apoptosis under the condition of high concentration.
采用 MTT 比色法、半胱天冬酶活性检测、Western blot 及流式细胞术检测乙醇对 YD-15 舌鳞癌细胞的细胞毒性作用。MTT 比色法结果显示乙醇呈时间和剂量依赖性抑制 YD-15 细胞生长和增殖。当乙醇浓度处于低水平（0~1.5%）时，细胞未发生死亡，本研究检测了这一浓度范围内乙醇对细胞周期调控的影响。Western blot 检测细胞周期抑制因子的表达，结果显示随着乙醇浓度从 0%增加至 1.5%，p21 和 p27 逐渐上调，而细胞周期调节因子 CDK1、CDK2、CDK4 以及细胞周期蛋白 A、B1 和 E1 逐渐下调。细胞周期分布流式细胞术分析显示，YD-15 细胞经 1.5%乙醇处理 24 h 后主要停滞在 G2/M 期。然而，乙醇诱导 YD-15 细胞凋亡仅发生在乙醇浓度为 2.5%或更高时。用半胱天冬酶活性检测或 Western blot 检测到 PARP 裂解，即 caspase-3 介导的凋亡的标志物，以及 caspase-3 和 caspase-7 的激活。我们的结果提示，乙醇通过在低浓度时诱导 G2/M 期细胞周期阻滞，在高浓度时诱导细胞凋亡，从而抑制 YD-15 舌鳞癌细胞的生长和增殖。
