| Project/Area Number |
15590068
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Niigata University of Pharmacy and Applied Life Sciences (2004)
Showa University (2003) |
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Principal Investigator |
NAKAYA Kazuyasu Niigata University of Pharmacy and Applied Life Sciences, Professor, 応用生命科学部, 教授 (40053855)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Chemopreventive agent / dUTPase / Apoptosis / DNA synthesis / Shikonin derivative / Tyrosine kinase / Molecular target / Cell death / チロシンキナーゼ阻害剤 / 質量分析装置 / 癌細胞 |
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| Research Abstract |
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.
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