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

与诱导细胞死亡和肿瘤细胞致癌相关的蛋白质的高分辨率分析化学预防剂的开发

基本信息

批准号：
15590068
负责人：
NAKAYA Kazuyasu
金额：
$ 2.3万
依托单位：
Niigata University of Pharmacy and Applied Life Sciences (2004)Showa University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

β-Hydroxyisovalerylshikonin （β-HIVS）是一种atp非竞争性蛋白酪氨酸激酶（如v-Src和EGFR）抑制剂，可诱导多种人类肿瘤细胞凋亡。为了阐明诱导凋亡的机制和开发化学预防药物，我们对β-HIVS最敏感的人肺癌DMS114细胞株进行了β-HIVS处理，并在磷酸化蛋白纯化柱分离磷酸化蛋白后，用2d -聚丙烯酰胺凝胶电泳（2D-PAGE）分析了诱导凋亡的相关蛋白。5 μM β- hiv作用于DMS114细胞后，发现2D凝胶中有一个位点明显减少，质谱鉴定为dUTP核苷酸脱氢酶（dUTP nucleotidehydrolase， dUTP酶）。通过对磷酸化蛋白纯化柱洗脱液进行免疫印迹，证实了β- hiv处理后dUTPase的降低。用siRNA转染DMS114细胞，增强了β- hiv对dUTPase的诱导凋亡作用。β- hiv作用于DMS114细胞30 min后，dUTPase活性立即显著降低。DMS114细胞中dUTPase的降低不受其他凋亡诱导剂如顺铂、喜树碱和依托泊苷（VP16）的影响，提示dUTPase的降低作用是β- hiv的特异性作用。通过β- hiv和胸腺苷酸合成酶特异性抑制剂5-氟尿嘧啶（5-FU）联合处理DMS114细胞，观察其诱导凋亡的累加效应。这些结果提示，毒性较低的β- hiv或其衍生物可能适合用于化学预防。

项目成果

期刊论文数量（55）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Kazuyasu Nakaya et al.: "β-Hydroxyisovalerylshikonin induces apoptosis in human leukemia cells by inhibiting the activity of a polo-like kinase 1"Oncogene. 22(7). 1012-1023 (2003)

Kazuyasu Nakaya 等人：“β-羟基异戊酰紫草素通过抑制 Polo 样激酶 1 的活性诱导人白血病细胞凋亡”Oncogene 1012-1023 (2003)。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

A shikoni derivative, β-hydroxyisovalerylshikonin, is an ATP-non-competitive inhibitor of protein tyrosine kinases.

紫草衍生物 β-羟基异戊酰紫草素是一种 ATP 非竞争性蛋白酪氨酸激酶抑制剂。

DOI：
发表时间：
2003
期刊：
Anti-Cancer Drugs 14・9
影响因子：
0
作者：
Yutaka Masuda;Genryu Shima;Toshihiro Aiuchi;Masayo Horie;Koichi Hori;Shigeo Nakajo;Toshiko Sachiko Kajimotc Shibayama-lmazu;Kazuyasu Nakaya;Kazuyasu Nakaya;Takao Suzuki et al.;Yutaka Masuda et al.;中谷一泰;Yutaka Masuda et al.;Toshiko Shibayama-Imazu et al.;Kazuyasu Nakaya
通讯作者：
Kazuyasu Nakaya

Mechanisms involved in apoptosis of human macrophages induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans in the presence of cycloheximide