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J. Biol. Chem., Vol. 277, Issue 34, 30684-30689, August 23, 2002

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Mechanism of Apoptosis Induced by a New Topoisomerase Inhibitor through the Generation of Hydrogen Peroxide^*

Hideki Mizutani^§, Saeko Tada-Oikawa, Yusuke Hiraku, Shinji Oikawa, Michio Kojima^§, and Shosuke Kawanishi^¶

From the  Department of Environmental and Molecular Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie 514-8507, Japan and the ^§ Department of Pharmacy, Mie University Hospital, Edobashi, Tsu, Mie 514-8507, Japan

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H₂O₂-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H₂O₂. TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H₂O₂-mediated pathways in apoptosis. Flow cytometry revealed that H₂O₂ formation preceded increases in mitochondrial membrane potential (m) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H₂O₂ generation and DNA ladder formation. The levels of NAD⁺, a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD⁺ levels preceded both increases in m and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H₂O₂ formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H₂O₂ generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD⁺ depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O-derived H₂O₂ generation, followed by the increase in m and subsequent caspase-3 activation, leading to apoptosis.

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