Abstract
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 H(2)O(2)-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 H(2)O(2). 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 H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) 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 DeltaPsim 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 H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) 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(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.
Highlights
TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II
DNA Cleavage and DNA Ladder Formation in HL-60 and HP100 Cells Treated with TAS-103—We analyzed DNA strand breaks in cells treated with 0.2 M TAS-103 using pulsed field gel electrophoresis
DNA fragments of 1–2 Mb appeared in both HL-60 and HP100 cells treated following a 1-h treatment with TAS-103; smaller fragments appeared at 3 h in HL-60 cells (Fig. 2A), suggesting that the 1–2-Mb fragments were further digested to generate 50-kb fragments
Summary
Materials—TAS-103 (6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy7H-indeno[2,1-c] quinolin-7-one dihydrochloride) was provided by Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). Measurement of Caspase-3 Activity—To analyze caspase-3 activity, TAS-103-treated cells (1 ϫ 106 cells) were washed twice with PBS and resuspended in a 100 l solution containing 0.1 M HEPES (pH 7.4), 2 mM dithiothreitol, 0.1% CHAPS, and 1% sucrose. Measurement of Intracellular NADϩ and NADPϩ Levels—After treatment with TAS-103, cells (2 ϫ 106 cells) were washed twice in PBS and resuspended in 50 l PBS. Following centrifugation at 3000 ϫ g for 10 min, NADϩ and NADPϩ levels were analyzed by HPLC with a Shimadzu photodiode array UV detector (SPD-M10A, Kyoto, Japan) as described by Litt et al [32]. Cells (1 ϫ 106 cells) were resuspended in 1 ml cytoplasm extraction buffer, followed by treatment with trifluoroacetic acid according to the method as described previously [33]. The GSH level was analyzed by HPLC with an Eicom electrochemical detector (ECD-300, Kyoto, Japan) with a gold disk electrode that can detect GSH [33, 34]
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