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

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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.