Shyu Kou-gea, Huang Sheng-tung, Kuo Hsien-shou, Cheng Wen-pin, Lin Yuh-ling
Division of Cardiac, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 106, Taiwan, China.
Acta Pharmacol Sin. 2007 Apr;28(4):559-66. doi: 10.1111/j.1745-7254.2007.00508.x.
The cytotoxic activities of a series of bis-aziridinylnaphthoquinone, AZ1 to AZ4, on human lung carcinoma cell lines, H460, and normal lung cells fibroblast cell line, MRC-5, and the mechanisms of H460 cells induced by AZ4 were investigated.
The MTT assay was used to determine the cell proliferation. Cell cycle was analysed by FACS. The activity of caspase 3, 8 and 9 was determined by cell-permeable fluorogenic detection system. Western blot assay was used to evaluate the regulation of cyclin B, Cdc-2, p53, p21, and the Bcl-2 protein.
AZ1 to AZ4 displayed various cytotoxicity activities against H460 and MRC-5 cells. Compared to those compounds, AZ4 was with the most effective agent among the 5 tested analogues at reducing H460 cell viability with an IC(50) value of 1.23 micromol/L; it also exhibited weak cytotoxicity against MRC-5 cells with an IC(50) value of 12.7 micromol/L. The results show that growth arrest on the G2-M phase of H460 cells induced by AZ4 for 24 h was discovered, and this might be altered with the reduced Cdc-2 protein expression of 47% at 2.0 micromol/L AZ4, but not with cyclin B protein expression. The AZ4 treated cells were then led to apoptosis after 48 h. This was associated with the activation of apoptotic enzyme caspase 3 and mediated by caspase 8, but not caspase 9 at various concentrations of AZ4 after being cultured for 48 h and 30 h, respectively. The anti-apoptotic protein (Bcl-2) expression in H460 cells altered by 39% with downregulation, and the p53 protein by 25% with upregulation after being cultured with 2.0 micromol/L AZ4 for 48 h. In a time-dependent manner, the expression of the p53 and p21 proteins were increased to the maximum at 24 h, and then decreased at 48.
AZ4 represents a novel antitumor aziridinylnaphthoquinone with therapeutic potential against the non-small cell lung cancer cells.
研究一系列双氮丙啶基萘醌（AZ1至AZ4）对人肺癌细胞系H460和正常肺成纤维细胞系MRC-5的细胞毒性作用，以及AZ4诱导H460细胞的作用机制。
采用MTT法检测细胞增殖。通过流式细胞术分析细胞周期。采用细胞可渗透荧光检测系统测定半胱天冬酶3、8和9的活性。用蛋白质免疫印迹法评估细胞周期蛋白B、细胞分裂周期蛋白2（Cdc-2）、p53、p21和Bcl-2蛋白的调控情况。
AZ1至AZ4对H460和MRC-5细胞表现出不同的细胞毒性活性。与这些化合物相比，AZ4是所测试的5种类似物中对降低H460细胞活力最有效的药物，其半数抑制浓度（IC50）值为1.23 μmol/L；它对MRC-5细胞也表现出较弱的细胞毒性，IC50值为12.7 μmol/L。结果表明，发现AZ4诱导H460细胞在G2-M期生长停滞24小时，这可能与在2.0 μmol/L AZ4作用下细胞分裂周期蛋白2（Cdc-2）蛋白表达降低47%有关，但与细胞周期蛋白B蛋白表达无关。AZ4处理的细胞在48小时后发生凋亡。这与凋亡酶半胱天冬酶3的激活有关，并由半胱天冬酶8介导，但在分别培养48小时和30小时后，不同浓度的AZ4作用下与半胱天冬酶9无关。在2.0 μmol/L AZ4作用下培养48小时后，H460细胞中抗凋亡蛋白（Bcl-2）表达下调39%，p53蛋白表达上调25%。p53和p21蛋白的表达呈时间依赖性，在24小时时增加到最大值，然后在48小时时下降。
AZ4是一种新型的具有治疗非小细胞肺癌细胞潜力的氮丙啶基萘醌类抗肿瘤药物。
