Yang Yang-Ming, Jhanwar-Uniyal Meena, Schwartz Joel, Conaway C Clifford, Halicka H Dorota, Traganos Frank, Chung Fung-Lung
Division of Carcinogenesis, American Health Foundation Cancer Center, Institute for Cancer Prevention, Valhalla, New York, USA.
Cancer Res. 2005 Sep 15;65(18):8538-47. doi: 10.1158/0008-5472.CAN-05-0236.
We previously showed that dietary treatment with the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) inhibited benzo(a)pyrene-induced lung tumorigenesis in A/J mice, and that tumor inhibition was associated with induction of activator protein-1 (AP-1) activity and stimulation of apoptosis in the lungs of mice. In the present study, we show that PEITC-NAC also induces apoptosis and AP-1 activity in human lung adenocarcinoma A549 cells, and that activation of AP-1 is important in PEITC-NAC induced apoptosis in these cells. PEITC-NAC induced AP-1 binding activity in A549 cells in a dose- and time-dependent manner; peak activity appeared at 10 micromol/L after 24 hours. At that time, flow cytometric analysis showed a sub-G1 peak, indicating that approximately 4.5% of the cells had undergone apoptosis. When wild-type c-jun cDNA was transfected into A549 cells, PEITC-NAC-mediated apoptosis was greatly increased in the c-jun-transfected cells compared with the control vector-transfected cells, based on cell morphology and analysis of DNA fragmentation. Furthermore, cells that were pretreated with 100 nmol/L 12-O-tetradecanoyl phorbol-13-acetate, and then treated with 25 micromol/L PEITC-NAC, underwent enhanced apoptosis compared with cells that were treated with PEITC-NAC alone; cells treated with 12-O-tetradecanoyl phorbol-13-acetate alone showed active cell growth without apoptosis. Bivariate flow cytometric analysis of DNA strand breaks versus DNA content showed that apoptosis induced by PEITC-NAC occurred predominantly in the G2-M phase. These findings suggest that growth-stimulated cells with an elevated basal AP-1 activity, i.e., A549 cells transfected with wild-type c-jun or treated with a tumor promoter, were more sensitive to PEITC-NAC-mediated apoptosis. The observation that PEITC-NAC induces apoptosis predominantly in growth-promoted cells, such as neoplastic cells, suggests a selective mechanism by which PEITC-NAC inhibits lung carcinogenesis.
我们之前的研究表明，用异硫氰酸苯乙酯的N - 乙酰半胱氨酸共轭物（PEITC - NAC）进行饮食治疗可抑制A/J小鼠中苯并(a)芘诱导的肺癌发生，并且肿瘤抑制与激活蛋白-1（AP - 1）活性的诱导以及小鼠肺部细胞凋亡的刺激有关。在本研究中，我们发现PEITC - NAC还可诱导人肺腺癌A549细胞发生凋亡并激活AP - 1活性，并且AP - 1的激活在PEITC - NAC诱导这些细胞凋亡过程中起重要作用。PEITC - NAC以剂量和时间依赖性方式诱导A549细胞中的AP - 1结合活性；24小时后，在10微摩尔/升时出现活性峰值。此时，流式细胞术分析显示出现亚G1峰，表明约4.5%的细胞发生了凋亡。当将野生型c - jun cDNA转染到A549细胞中时，基于细胞形态和DNA片段化分析，与对照载体转染细胞相比，PEITC - NAC介导的c - jun转染细胞凋亡显著增加。此外，用100纳摩尔/升12 - O - 十四烷酰佛波醇-13 - 乙酸酯预处理，然后用25微摩尔/升PEITC - NAC处理的细胞，与单独用PEITC - NAC处理的细胞相比，凋亡增强；单独用12 - O - 十四烷酰佛波醇-13 - 乙酸酯处理的细胞显示活跃的细胞生长且无凋亡。DNA链断裂与DNA含量的双变量流式细胞术分析表明，PEITC - NAC诱导的凋亡主要发生在G2 - M期。这些发现表明，基础AP - 1活性升高的生长刺激细胞，即转染了野生型c - jun或用肿瘤启动子处理的A549细胞，对PEITC - NAC介导的凋亡更敏感。PEITC - NAC主要在生长促进细胞（如肿瘤细胞）中诱导凋亡的观察结果提示了一种PEITC - NAC抑制肺癌发生的选择性机制。
