High resolution analysis of proteins associated with the induction of cell death and carcinogenesis of tumor cells development of chemopreventive agents

β-羟基异戊基紫草素(β-HIVS)是一种对v-Src和EGFR等蛋白酪氨酸激酶的非竞争性抑制物，可诱导多种肿瘤细胞系的凋亡。为了阐明诱导细胞凋亡的机制和开发化学预防药物，对对β敏感的人肺癌DMS114细胞株进行了β-HIVS处理，并用磷酸蛋白纯化柱分离磷酸化蛋白后，用2D-PAGE分析了诱导细胞凋亡的蛋白质。当5μMβ-HIVS作用于DMS114细胞时，我们发现2D凝胶中有一个斑点明显减少，并经质谱仪鉴定为dUTP核苷酸脱氢酶(DUTPase)。免疫印迹证实β-HIVS处理后dUTP酶活性降低。将抗dUTPase的小干扰RNA导入DMS114细胞，可增强β-HIVS诱导的细胞凋亡。β-HIVS处理DMS114细胞30min后，dUTP酶活性显著降低。顺铂、喜树碱和足叶乙甙等其他诱导细胞凋亡的药物不能引起dUTPase的降低，提示dUTPase的降低作用是β-HIVs所特有的。β-HIV与胸苷合成酶特异性抑制剂5-氟尿嘧啶(5-FU)联合作用于DMS114细胞，观察其诱导细胞凋亡的相加作用。这些结果提示，毒性较低的β-HIV或其衍生物可能适合于化学预防。

β-Hydroxyisovalerylshikonin (β-HIVS) is an ATP-noncompetitive inhibitor for protein tyrosine kinases such as v-Src and EGFR and induces apoptosis in various lines of human tumor cells. For elucidating of the mechanism of the induction of apoptosis and developing chemopreventive agents, a human lung cancer DMS114 cell line, which is one of the most sensitive to β-HIVS, was treated with β-HIVS and proteins responsible for the induction of apoptosis was analyzed by 2D-polyacrylamide gel electrophoresis (2D-PAGE) after separation of phosphoproteins using phosphoprotein purification column. When DMS114 cells were treated with 5 μM β-HIVS, we found that one spot in 2D gel was decreased markedly and identified this by mass spectrometry as dUTP nucleotidehydrolase (dUTPase). The decrease of dUTPase by the treatment with β-HIVS was confirmed by immunoblotting of the eluate fraction from the phosphoprotein purification column. Transfection of DMS114 cells with siRNA against dUTPase enhanced the induction of apoptosis by treatment with β-HIVS. The activity of dUTPase was markedly decreased immediately 30 min after treatment of DMS114 cells with β-HIVS. The reduction of dUTPase in DMS114 cells were not caused by other inducers of apoptosis such as cisplatin, camptothecin, and etoposide (VP16), suggesting that the dUTPase-decreasing effect is specific to the action of β-HIVS. Additive effects on the induction of apoptosis was observed by combined treatment of DMS114 cells with β-HIVS and 5-fluorouracil (5-FU), which is a specific inhibitor of thymidylate synthase. These results suggest that β-HIVS or its derivative with lower toxicity may be suitable for chemopreventive.