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W2004758859 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 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 (ΔΨm) 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 ΔΨm 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 H2O2formation. 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 O2−-derived H2O2 generation, followed by the increase in ΔΨm and subsequent caspase-3 activation, leading to apoptosis. 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 (ΔΨm) 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 ΔΨm 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 H2O2formation. 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 O2−-derived H2O2 generation, followed by the increase in ΔΨm and subsequent caspase-3 activation, leading to apoptosis. mitochondrial permeability transition reactive oxygen species poly(ADP-ribose) polymerase 2′,7′-dichlorofluorescin diacetate 3,3′-dihexyloxacarbocyanine iodide Ac-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin 4-amino-1,8-naphthalimide diphenyleneiodonium chloride 6(5H)-phenanthridinone allopurinol apocynin fetal calf serum phosphate-buffered saline glutathione apoptosis-inducing factor 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 4-morpholinepropanesulfonic acid high performance liquid chromatography A number of anticancer drugs exert their effects by inducing apoptosis (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar). The major apoptotic pathways act either through the induction of death receptors or the loss of mitochondrial membrane integrity (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar); the majority of anticancer drugs induce apoptosis via the mitochondrial pathway (2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar). DNA is the molecular target for many of the drugs that are used in cancer therapeutics (3Hurley L.H. Nat. Rev. Cancer. 2002; 2: 188-200Crossref PubMed Scopus (1167) Google Scholar). DNA damage may cause perturbations in inner mitochondrial membrane permeability (4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar, 5Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z.M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (367) Google Scholar, 6Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun. 1998; 247: 693-696Crossref PubMed Scopus (63) Google Scholar, 7Tada-Oikawa S. Oikawa S. Kawanishi M. Yamada M. Kawanishi S. FEBS Lett. 1999; 442: 65-69Crossref PubMed Scopus (80) Google Scholar). A sudden increase in mitochondrial membrane permeability, so-called mitochondrial permeability transition (MPT),1 is a central coordination event in the apoptotic process (4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar, 8Gottlieb R.A. FEBS Lett. 2000; 482: 6-12Crossref PubMed Scopus (183) Google Scholar, 9Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2754) Google Scholar, 10Costantini P. Jacotot E. Decaudin D. Kroemer G. J. Natl. Cancer Inst. 2000; 92: 1042-1053Crossref PubMed Scopus (492) Google Scholar). Opening MPT pores mediates disruption of mitochondrial membrane potential (ΔΨm) (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). MPT causes the release of cytochrome c from mitochondria; cytochrome c then activates effector caspases to induce DNA ladder formation. Exogenous and endogenous reactive oxygen species (ROS), such as H2O2 and O2− (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar), cause apoptosis through MPT (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 12Hampton M.B. Fadeel B. Orrenius S. Ann. N. Y. Acad. Sci. 1998; 854: 328-335Crossref PubMed Scopus (235) Google Scholar, 13Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1129) Google Scholar); some anticancer drugs induce ROS formation in apoptosis (7Tada-Oikawa S. Oikawa S. Kawanishi M. Yamada M. Kawanishi S. FEBS Lett. 1999; 442: 65-69Crossref PubMed Scopus (80) Google Scholar,14Muller I. Niethammer D. Bruchelt G. Int. J. Mol. Med. 1998; 1: 491-494PubMed Google Scholar, 15Gewirtz D.A. Biochem. Pharmacol. 1999; 57: 727-741Crossref PubMed Scopus (1793) Google Scholar, 16Ikeda K. Kajiwara K. Tanabe E. Tokumaru S. Kishida E. Masuzawa Y. Kojo S. Biochem. Pharmacol. 1999; 57: 1361-1365Crossref PubMed Scopus (76) Google Scholar, 17Kajiwara K. Ikeda K. Kuroi R. Hashimoto R. Tokumaru S. Kojo S. Cell. Mol. Life Sci. 2001; 58: 485-491Crossref PubMed Scopus (29) Google Scholar). However, the mechanism of ROS formation by anticancer drugs remains to be clarified.TAS-103 (Fig. 1), a new quinoline derivative, induces DNA cleavage through the inhibition of DNA topoisomerases I and II (18Utsugi T. Aoyagi K. Asao T. Okazaki S. Aoyagi Y. Sano M. Wierzba K. Yamada Y. Jpn. J. Cancer Res. 1997; 88: 992-1002Crossref PubMed Scopus (126) Google Scholar, 19Byl J.A. Fortune J.M. Burden D.A. Nitiss J.L. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15573-15579Crossref PubMed Scopus (62) Google Scholar, 20Padget K. Stewart A. Charlton P. Tilby M.J. Austin C.A. Biochem. Pharmacol. 2000; 60: 817-821Crossref PubMed Scopus (25) Google Scholar). Recently, TAS-103 is classified as a topoisomerase II-targeted drug (19Byl J.A. Fortune J.M. Burden D.A. Nitiss J.L. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15573-15579Crossref PubMed Scopus (62) Google Scholar). Alteration in the topology of DNA by intercalation of TAS-103 results in inhibition of topoisomerase I-catalyzed DNA relaxation (21Fortune J.M. Velea L. Graves D.E. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15580-15586Crossref PubMed Scopus (84) Google Scholar). The anticancer activity of TAS-103 has a broad spectrum. The cytotoxicity of TAS-103 (IC50: 0.0030–0.23 μm) is much greater against various tumor cell lines than etoposide and comparable with SN-38, an active metabolite of irinotecan (CPT-11) (22Aoyagi Y. Kobunai T. Utsugi T. Oh-hara T. Yamada Y. Jpn. J. Cancer Res. 1999; 90: 578-587Crossref PubMed Scopus (37) Google Scholar). Due to this potent cytotoxicity at low concentrations, TAS-103 must have specific inhibitory effects on topoisomerases. TAS-103 is therefore expected to be a novel and promising anticancer drug, currently under clinical evaluation in the United States (23Ewesuedo R.B. Iyer L. Das S. Koenig A. Mani S. Vogelzang N.J. Schilsky R.L. Brenckman W. Ratain M.J. J. Clin. Oncol. 2001; 19: 2084-2090Crossref PubMed Scopus (36) Google Scholar). Although good evidence exists that TAS-103 induces apoptosis (24Ohyama T., Li, Y. Utsugi T. Irie S. Yamada Y. Sato T. Jpn. J. Cancer Res. 1999; 90: 691-698Crossref PubMed Scopus (13) Google Scholar, 25Kluza J. Lansiaux A. Wattez N. Mahieu C. Osheroff N. Bailly C. Cancer Res. 2000; 60: 4077-4084PubMed Google Scholar), the mechanisms of TAS-103 governing this induction remain unclear.Here, we sought to analyze the mechanism of apoptosis induction by anticancer drugs through the generation of ROS. We investigated apoptosis induction by TAS-103 in HL-60 cells and HP100 cells, H2O2-resistant cells derived from HL-60. Apoptosis was analyzed by examining DNA cleavage, DNA ladder formation, NAD+ depletion, H2O2 generation, mitochondrial membrane potential (ΔΨm) change, and caspase-3 activation. The mechanism of TAS-103-induced H2O2 generation was examined by using inhibitors of poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase. Moreover, to clarify whether H2O2 generation via NAD(P)H oxidase occurs upstream or downstream of the MPT, we examined the effect of the expression of Bcl-2 on TAS-103-induced apoptosis in the human B cell line, BJAB cells.DISCUSSIONTAS-103 induced DNA cleavage in HL-60 cells after a 1-h treatment; the formation of DNA ladders was observed at 4 h. Therefore, DNA cleavage initiates the apoptotic process, subsequently resulting in DNA ladder formation. This study has clarified the mechanism of TAS-103-induced apoptosis from the early events of DNA cleavage to eventual DNA ladder formation (Fig.11). DNA cleavage is initiated by TAS-103-mediated inhibition of topoisomerases (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar, 4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar). Excessive DNA damage causes PARP hyperactivation to repair DNA (35Bertrand R. Solary E. Jenkins J. Pommier Y. Exp. Cell Res. 1993; 207: 388-397Crossref PubMed Scopus (132) Google Scholar, 36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar, 37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). PARP inhibitors, ANI and PHEN, prevented DNA ladder formation following TAS-103 treatment of HL-60 cells, indicating that TAS-103-induced apoptosis requires PARP hyperactivation. PARP hyperactivation accelerates the consumption of NAD+, a PARP substrate (36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar,37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). NAD+ and NADP+ levels decreased in HL-60 cells treated with TAS-103 in less than 3 h. The decrease in NADP+ at 2–3 h appears to be delayed compared with that in NAD+ by ∼30 min, suggesting that NADP+ may be converted to NAD+ by phosphatase for the supplementation of NAD+. Relevantly, the interconversion of pyridine nucleotides in such circumstances has previously been reported (43Stubberfield C.R. Cohen G.M. Biochem. Pharmacol. 1989; 38: 2631-2637Crossref PubMed Scopus (48) Google Scholar). Thus, PARP hyperactivation results in the depletion of both NAD+ and NADP+.FIG. 11Suggested mechanism of TAS-103-induced apoptosis mediated by hydrogen peroxide generation.View Large Image Figure ViewerDownload (PPT)TAS-103 induced apoptosis in both HL-60 and the H2O2-resistant clone, HP100. DNA cleavage was induced in these cells at 1 h to a similar extent by TAS-103, suggesting that the cleavage is caused by the inhibition of topoisomerases without H2O2 generation. DNA ladder formation in HP100 cells, however, was delayed to 6 h from 4 h in HL-60 parental cells. This delay can be explained by the involvement of H2O2 generation in the apoptotic pathway. In HP100 cells, H2O2 generation, increases in ΔΨm and caspase-3 activation were suppressed relative to the levels observed in HL-60 cells. H2O2formation precedes increases in ΔΨm and caspase-3 activation in TAS-103-induced apoptosis; in HL-60 cells, H2O2generation occurred at 3 h, followed by increases in ΔΨm and caspase-3 activation at 4 h (Fig. 11), whereas in HP100 cells, H2O2 generation occurred at 3–4 h, with increases in ΔΨm at 5 h and caspase-3 activation at 5–6 h. Therefore, intracellular H2O2 generation is critical in these TAS-103-induced apoptotic events. PARP inhibitors inhibited both H2O2 generation at 3 h and changes in ΔΨm at 4–5 h in HL-60 cells, indicating that TAS-103-induced PARP hyperactivation is important for H2O2 generation.Here, the question has been raised how TAS-103 induces generation of H2O2. On the basis of our data, we propose the possible mechanism by which TAS-103 induces the generation of intracellular H2O2 as follows: the PARP-catalyzed depletion of both NAD+ and NADP+in cells treated with TAS-103 may result in the activation of NAD(P)H oxidase to maintain the cellular redox balance by converting NAD(P)H to NAD(P)+ (44Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). NAD(P)H oxidase catalyzes the following reaction: NAD(P)H + 2O2 → 2O2− + NAD(P)+ + H+. O2− is then rapidly converted to H2O2 (45Jones R.D. Hancock J.T. Morice A.H. Free Radic. Biol. Med. 2000; 29: 416-424Crossref PubMed Scopus (148) Google Scholar). This possibility is supported by our observations that inhibitors of NAD(P)H oxidase inhibited TAS-103-induced DNA ladder formation in HL-60 cells. On the other hand, rotenone, which can inhibit mitochondrial ROS production (46Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (397) Google Scholar), did not inhibit TAS-103-induced DNA ladder formation (data not shown). This result excludes the possibility that ROS are released from mitochondria. The decreases in intracellular GSH may also induce ROS generation (13Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1129) Google Scholar, 47Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (643) Google Scholar). Significant GSH decreases were not observed until 6 h, suggesting this GSH decrease does not mediate early generation of H2O2. Therefore, NAD(P)H oxidase is the primary enzyme mediating H2O2formation in cells treated with TAS-103. In addition, p53activation by DNA damage induces apoptosis by the transcriptional induction of redox-related genes and the formation of ROS (48Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2226) Google Scholar, 49Johnson T.M., Yu, Z.X. Ferrans V.J. Lowenstein R.A. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (521) Google Scholar). This possibility is also excluded as HL-60 cells lack functionalp53 (50Dou Q.P., An, B. Will P.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9019-9023Crossref PubMed Scopus (155) Google Scholar).The activation of NAD(P)H oxidase has been reported to either precede (51Chen Y.C. Lin-Shiau S.Y. Lin J.K. J. Cell. Physiol. 1998; 177: 324-333Crossref PubMed Scopus (424) Google Scholar, 52Hiraoka W. Vazquez N. Nieves-Neira W. Chanock S.J. Pommier Y. J. Clin. Invest. 1998; 102: 1961-1968Crossref PubMed Scopus (96) Google Scholar) or follow (53Simizu S. Takada M. Umezawa K. Imoto M. J. Biol. Chem. 1998; 273: 26900-26907Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) caspase-3 activation. TAS-103-induced H2O2 generation was not inhibited by a caspase-3 inhibitor (data not shown), confirming that activation of NAD(P)H oxidase occurs upstream of caspase-3 activation. Our results revealed that H2O2 generation via NAD(P)H oxidase activation precedes the ΔΨm increase in TAS-103-induced apoptosis. Bcl-2 binds to the voltage-dependent anion channel to regulate the mitochondrial membrane potential, and controls the release of mitochondrial apoptogenic factors, cytochromec and apoptosis-inducing factor (AIF), a mitochondrial flavoprotein (54Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1910) Google Scholar, 55Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (629) Google Scholar). Our experiment using BJAB/Bcl-2 cells confirmed that Bcl-2 prevented TAS-103-induced apoptosis by inhibiting MPT mediated by H2O2 generation. AIF has an NADH oxidase activity, but DPI, an NAD(P)H oxidase inhibitor, does not affect the apoptogenic activity of AIF (56Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Our data that DPI prevented TAS-103-induced apoptosis exclude the possibility that the NAD(P)H oxidase activity of AIF is involved in apoptosis. It has been reported that the activity of NAD(P)H oxidase in microsomes is linked to cytosolic NAD(H) redox and appears to be a key source of O2− production (57Mohazzab K.M. Kaminsky P.M. Wollin M.S. Circulation. 1997; 96: 614-620Crossref PubMed Scopus (137) Google Scholar). Taken together, the activation of microsomal NAD(P)H oxidase may participate in TAS-103-induced H2O2 generation. In conclusion, H2O2 generation precedes MPT, leading to subsequent apoptotic events, including cytochrome c release and caspase-3 activation in TAS-103-induced apoptosis. A number of anticancer drugs exert their effects by inducing apoptosis (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar). The major apoptotic pathways act either through the induction of death receptors or the loss of mitochondrial membrane integrity (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar); the majority of anticancer drugs induce apoptosis via the mitochondrial pathway (2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar). DNA is the molecular target for many of the drugs that are used in cancer therapeutics (3Hurley L.H. Nat. Rev. Cancer. 2002; 2: 188-200Crossref PubMed Scopus (1167) Google Scholar). DNA damage may cause perturbations in inner mitochondrial membrane permeability (4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar, 5Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z.M. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (367) Google Scholar, 6Tada-Oikawa S. Oikawa S. Kawanishi S. Biochem. Biophys. Res. Commun. 1998; 247: 693-696Crossref PubMed Scopus (63) Google Scholar, 7Tada-Oikawa S. Oikawa S. Kawanishi M. Yamada M. Kawanishi S. FEBS Lett. 1999; 442: 65-69Crossref PubMed Scopus (80) Google Scholar). A sudden increase in mitochondrial membrane permeability, so-called mitochondrial permeability transition (MPT),1 is a central coordination event in the apoptotic process (4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar, 8Gottlieb R.A. FEBS Lett. 2000; 482: 6-12Crossref PubMed Scopus (183) Google Scholar, 9Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2754) Google Scholar, 10Costantini P. Jacotot E. Decaudin D. Kroemer G. J. Natl. Cancer Inst. 2000; 92: 1042-1053Crossref PubMed Scopus (492) Google Scholar). Opening MPT pores mediates disruption of mitochondrial membrane potential (ΔΨm) (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). MPT causes the release of cytochrome c from mitochondria; cytochrome c then activates effector caspases to induce DNA ladder formation. Exogenous and endogenous reactive oxygen species (ROS), such as H2O2 and O2− (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar), cause apoptosis through MPT (11Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 12Hampton M.B. Fadeel B. Orrenius S. Ann. N. Y. Acad. Sci. 1998; 854: 328-335Crossref PubMed Scopus (235) Google Scholar, 13Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1129) Google Scholar); some anticancer drugs induce ROS formation in apoptosis (7Tada-Oikawa S. Oikawa S. Kawanishi M. Yamada M. Kawanishi S. FEBS Lett. 1999; 442: 65-69Crossref PubMed Scopus (80) Google Scholar,14Muller I. Niethammer D. Bruchelt G. Int. J. Mol. Med. 1998; 1: 491-494PubMed Google Scholar, 15Gewirtz D.A. Biochem. Pharmacol. 1999; 57: 727-741Crossref PubMed Scopus (1793) Google Scholar, 16Ikeda K. Kajiwara K. Tanabe E. Tokumaru S. Kishida E. Masuzawa Y. Kojo S. Biochem. Pharmacol. 1999; 57: 1361-1365Crossref PubMed Scopus (76) Google Scholar, 17Kajiwara K. Ikeda K. Kuroi R. Hashimoto R. Tokumaru S. Kojo S. Cell. Mol. Life Sci. 2001; 58: 485-491Crossref PubMed Scopus (29) Google Scholar). However, the mechanism of ROS formation by anticancer drugs remains to be clarified. TAS-103 (Fig. 1), a new quinoline derivative, induces DNA cleavage through the inhibition of DNA topoisomerases I and II (18Utsugi T. Aoyagi K. Asao T. Okazaki S. Aoyagi Y. Sano M. Wierzba K. Yamada Y. Jpn. J. Cancer Res. 1997; 88: 992-1002Crossref PubMed Scopus (126) Google Scholar, 19Byl J.A. Fortune J.M. Burden D.A. Nitiss J.L. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15573-15579Crossref PubMed Scopus (62) Google Scholar, 20Padget K. Stewart A. Charlton P. Tilby M.J. Austin C.A. Biochem. Pharmacol. 2000; 60: 817-821Crossref PubMed Scopus (25) Google Scholar). Recently, TAS-103 is classified as a topoisomerase II-targeted drug (19Byl J.A. Fortune J.M. Burden D.A. Nitiss J.L. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15573-15579Crossref PubMed Scopus (62) Google Scholar). Alteration in the topology of DNA by intercalation of TAS-103 results in inhibition of topoisomerase I-catalyzed DNA relaxation (21Fortune J.M. Velea L. Graves D.E. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15580-15586Crossref PubMed Scopus (84) Google Scholar). The anticancer activity of TAS-103 has a broad spectrum. The cytotoxicity of TAS-103 (IC50: 0.0030–0.23 μm) is much greater against various tumor cell lines than etoposide and comparable with SN-38, an active metabolite of irinotecan (CPT-11) (22Aoyagi Y. Kobunai T. Utsugi T. Oh-hara T. Yamada Y. Jpn. J. Cancer Res. 1999; 90: 578-587Crossref PubMed Scopus (37) Google Scholar). Due to this potent cytotoxicity at low concentrations, TAS-103 must have specific inhibitory effects on topoisomerases. TAS-103 is therefore expected to be a novel and promising anticancer drug, currently under clinical evaluation in the United States (23Ewesuedo R.B. Iyer L. Das S. Koenig A. Mani S. Vogelzang N.J. Schilsky R.L. Brenckman W. Ratain M.J. J. Clin. Oncol. 2001; 19: 2084-2090Crossref PubMed Scopus (36) Google Scholar). Although good evidence exists that TAS-103 induces apoptosis (24Ohyama T., Li, Y. Utsugi T. Irie S. Yamada Y. Sato T. Jpn. J. Cancer Res. 1999; 90: 691-698Crossref PubMed Scopus (13) Google Scholar, 25Kluza J. Lansiaux A. Wattez N. Mahieu C. Osheroff N. Bailly C. Cancer Res. 2000; 60: 4077-4084PubMed Google Scholar), the mechanisms of TAS-103 governing this induction remain unclear. Here, we sought to analyze the mechanism of apoptosis induction by anticancer drugs through the generation of ROS. We investigated apoptosis induction by TAS-103 in HL-60 cells and HP100 cells, H2O2-resistant cells derived from HL-60. Apoptosis was analyzed by examining DNA cleavage, DNA ladder formation, NAD+ depletion, H2O2 generation, mitochondrial membrane potential (ΔΨm) change, and caspase-3 activation. The mechanism of TAS-103-induced H2O2 generation was examined by using inhibitors of poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase. Moreover, to clarify whether H2O2 generation via NAD(P)H oxidase occurs upstream or downstream of the MPT, we examined the effect of the expression of Bcl-2 on TAS-103-induced apoptosis in the human B cell line, BJAB cells. DISCUSSIONTAS-103 induced DNA cleavage in HL-60 cells after a 1-h treatment; the formation of DNA ladders was observed at 4 h. Therefore, DNA cleavage initiates the apoptotic process, subsequently resulting in DNA ladder formation. This study has clarified the mechanism of TAS-103-induced apoptosis from the early events of DNA cleavage to eventual DNA ladder formation (Fig.11). DNA cleavage is initiated by TAS-103-mediated inhibition of topoisomerases (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar, 4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar). Excessive DNA damage causes PARP hyperactivation to repair DNA (35Bertrand R. Solary E. Jenkins J. Pommier Y. Exp. Cell Res. 1993; 207: 388-397Crossref PubMed Scopus (132) Google Scholar, 36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar, 37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). PARP inhibitors, ANI and PHEN, prevented DNA ladder formation following TAS-103 treatment of HL-60 cells, indicating that TAS-103-induced apoptosis requires PARP hyperactivation. PARP hyperactivation accelerates the consumption of NAD+, a PARP substrate (36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar,37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). NAD+ and NADP+ levels decreased in HL-60 cells treated with TAS-103 in less than 3 h. The decrease in NADP+ at 2–3 h appears to be delayed compared with that in NAD+ by ∼30 min, suggesting that NADP+ may be converted to NAD+ by phosphatase for the supplementation of NAD+. Relevantly, the interconversion of pyridine nucleotides in such circumstances has previously been reported (43Stubberfield C.R. Cohen G.M. Biochem. Pharmacol. 1989; 38: 2631-2637Crossref PubMed Scopus (48) Google Scholar). Thus, PARP hyperactivation results in the depletion of both NAD+ and NADP+.TAS-103 induced apoptosis in both HL-60 and the H2O2-resistant clone, HP100. DNA cleavage was induced in these cells at 1 h to a similar extent by TAS-103, suggesting that the cleavage is caused by the inhibition of topoisomerases without H2O2 generation. DNA ladder formation in HP100 cells, however, was delayed to 6 h from 4 h in HL-60 parental cells. This delay can be explained by the involvement of H2O2 generation in the apoptotic pathway. In HP100 cells, H2O2 generation, increases in ΔΨm and caspase-3 activation were suppressed relative to the levels observed in HL-60 cells. H2O2formation precedes increases in ΔΨm and caspase-3 activation in TAS-103-induced apoptosis; in HL-60 cells, H2O2generation occurred at 3 h, followed by increases in ΔΨm and caspase-3 activation at 4 h (Fig. 11), whereas in HP100 cells, H2O2 generation occurred at 3–4 h, with increases in ΔΨm at 5 h and caspase-3 activation at 5–6 h. Therefore, intracellular H2O2 generation is critical in these TAS-103-induced apoptotic events. PARP inhibitors inhibited both H2O2 generation at 3 h and changes in ΔΨm at 4–5 h in HL-60 cells, indicating that TAS-103-induced PARP hyperactivation is important for H2O2 generation.Here, the question has been raised how TAS-103 induces generation of H2O2. On the basis of our data, we propose the possible mechanism by which TAS-103 induces the generation of intracellular H2O2 as follows: the PARP-catalyzed depletion of both NAD+ and NADP+in cells treated with TAS-103 may result in the activation of NAD(P)H oxidase to maintain the cellular redox balance by converting NAD(P)H to NAD(P)+ (44Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). NAD(P)H oxidase catalyzes the following reaction: NAD(P)H + 2O2 → 2O2− + NAD(P)+ + H+. O2− is then rapidly converted to H2O2 (45Jones R.D. Hancock J.T. Morice A.H. Free Radic. Biol. Med. 2000; 29: 416-424Crossref PubMed Scopus (148) Google Scholar). This possibility is supported by our observations that inhibitors of NAD(P)H oxidase inhibited TAS-103-induced DNA ladder formation in HL-60 cells. On the other hand, rotenone, which can inhibit mitochondrial ROS production (46Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (397) Google Scholar), did not inhibit TAS-103-induced DNA ladder formation (data not shown). This result excludes the possibility that ROS are released from mitochondria. The decreases in intracellular GSH may also induce ROS generation (13Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1129) Google Scholar, 47Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (643) Google Scholar). Significant GSH decreases were not observed until 6 h, suggesting this GSH decrease does not mediate early generation of H2O2. Therefore, NAD(P)H oxidase is the primary enzyme mediating H2O2formation in cells treated with TAS-103. In addition, p53activation by DNA damage induces apoptosis by the transcriptional induction of redox-related genes and the formation of ROS (48Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2226) Google Scholar, 49Johnson T.M., Yu, Z.X. Ferrans V.J. Lowenstein R.A. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (521) Google Scholar). This possibility is also excluded as HL-60 cells lack functionalp53 (50Dou Q.P., An, B. Will P.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9019-9023Crossref PubMed Scopus (155) Google Scholar).The activation of NAD(P)H oxidase has been reported to either precede (51Chen Y.C. Lin-Shiau S.Y. Lin J.K. J. Cell. Physiol. 1998; 177: 324-333Crossref PubMed Scopus (424) Google Scholar, 52Hiraoka W. Vazquez N. Nieves-Neira W. Chanock S.J. Pommier Y. J. Clin. Invest. 1998; 102: 1961-1968Crossref PubMed Scopus (96) Google Scholar) or follow (53Simizu S. Takada M. Umezawa K. Imoto M. J. Biol. Chem. 1998; 273: 26900-26907Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) caspase-3 activation. TAS-103-induced H2O2 generation was not inhibited by a caspase-3 inhibitor (data not shown), confirming that activation of NAD(P)H oxidase occurs upstream of caspase-3 activation. Our results revealed that H2O2 generation via NAD(P)H oxidase activation precedes the ΔΨm increase in TAS-103-induced apoptosis. Bcl-2 binds to the voltage-dependent anion channel to regulate the mitochondrial membrane potential, and controls the release of mitochondrial apoptogenic factors, cytochromec and apoptosis-inducing factor (AIF), a mitochondrial flavoprotein (54Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1910) Google Scholar, 55Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (629) Google Scholar). Our experiment using BJAB/Bcl-2 cells confirmed that Bcl-2 prevented TAS-103-induced apoptosis by inhibiting MPT mediated by H2O2 generation. AIF has an NADH oxidase activity, but DPI, an NAD(P)H oxidase inhibitor, does not affect the apoptogenic activity of AIF (56Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Our data that DPI prevented TAS-103-induced apoptosis exclude the possibility that the NAD(P)H oxidase activity of AIF is involved in apoptosis. It has been reported that the activity of NAD(P)H oxidase in microsomes is linked to cytosolic NAD(H) redox and appears to be a key source of O2− production (57Mohazzab K.M. Kaminsky P.M. Wollin M.S. Circulation. 1997; 96: 614-620Crossref PubMed Scopus (137) Google Scholar). Taken together, the activation of microsomal NAD(P)H oxidase may participate in TAS-103-induced H2O2 generation. In conclusion, H2O2 generation precedes MPT, leading to subsequent apoptotic events, including cytochrome c release and caspase-3 activation in TAS-103-induced apoptosis. TAS-103 induced DNA cleavage in HL-60 cells after a 1-h treatment; the formation of DNA ladders was observed at 4 h. Therefore, DNA cleavage initiates the apoptotic process, subsequently resulting in DNA ladder formation. This study has clarified the mechanism of TAS-103-induced apoptosis from the early events of DNA cleavage to eventual DNA ladder formation (Fig.11). DNA cleavage is initiated by TAS-103-mediated inhibition of topoisomerases (1Sellers W.R. Fisher D.E. J. Clin. Invest. 1999; 104: 1655-1661Crossref PubMed Scopus (194) Google Scholar, 2Kaufmann S.H. Earnshaw W.C. Exp. Cell Res. 2000; 256: 42-49Crossref PubMed Scopus (1052) Google Scholar, 4Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (703) Google Scholar). Excessive DNA damage causes PARP hyperactivation to repair DNA (35Bertrand R. Solary E. Jenkins J. Pommier Y. Exp. Cell Res. 1993; 207: 388-397Crossref PubMed Scopus (132) Google Scholar, 36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar, 37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). PARP inhibitors, ANI and PHEN, prevented DNA ladder formation following TAS-103 treatment of HL-60 cells, indicating that TAS-103-induced apoptosis requires PARP hyperactivation. PARP hyperactivation accelerates the consumption of NAD+, a PARP substrate (36Scovassi A.I. Poirier G.G. Mol. Cell. Biochem. 1999; 199: 125-137Crossref PubMed Scopus (131) Google Scholar,37Martin D.S. Bertino J.R. Koutcher J.A. Cancer Res. 2000; 60: 6776-6783PubMed Google Scholar, 42Chiarugi A. Trends Pharmacol. Sci. 2002; 23: 122-129Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). NAD+ and NADP+ levels decreased in HL-60 cells treated with TAS-103 in less than 3 h. The decrease in NADP+ at 2–3 h appears to be delayed compared with that in NAD+ by ∼30 min, suggesting that NADP+ may be converted to NAD+ by phosphatase for the supplementation of NAD+. Relevantly, the interconversion of pyridine nucleotides in such circumstances has previously been reported (43Stubberfield C.R. Cohen G.M. Biochem. Pharmacol. 1989; 38: 2631-2637Crossref PubMed Scopus (48) Google Scholar). Thus, PARP hyperactivation results in the depletion of both NAD+ and NADP+. TAS-103 induced apoptosis in both HL-60 and the H2O2-resistant clone, HP100. DNA cleavage was induced in these cells at 1 h to a similar extent by TAS-103, suggesting that the cleavage is caused by the inhibition of topoisomerases without H2O2 generation. DNA ladder formation in HP100 cells, however, was delayed to 6 h from 4 h in HL-60 parental cells. This delay can be explained by the involvement of H2O2 generation in the apoptotic pathway. In HP100 cells, H2O2 generation, increases in ΔΨm and caspase-3 activation were suppressed relative to the levels observed in HL-60 cells. H2O2formation precedes increases in ΔΨm and caspase-3 activation in TAS-103-induced apoptosis; in HL-60 cells, H2O2generation occurred at 3 h, followed by increases in ΔΨm and caspase-3 activation at 4 h (Fig. 11), whereas in HP100 cells, H2O2 generation occurred at 3–4 h, with increases in ΔΨm at 5 h and caspase-3 activation at 5–6 h. Therefore, intracellular H2O2 generation is critical in these TAS-103-induced apoptotic events. PARP inhibitors inhibited both H2O2 generation at 3 h and changes in ΔΨm at 4–5 h in HL-60 cells, indicating that TAS-103-induced PARP hyperactivation is important for H2O2 generation. Here, the question has been raised how TAS-103 induces generation of H2O2. On the basis of our data, we propose the possible mechanism by which TAS-103 induces the generation of intracellular H2O2 as follows: the PARP-catalyzed depletion of both NAD+ and NADP+in cells treated with TAS-103 may result in the activation of NAD(P)H oxidase to maintain the cellular redox balance by converting NAD(P)H to NAD(P)+ (44Toomey D. Mayhew S.G. Eur. J. Biochem. 1998; 251: 935-945Crossref PubMed Scopus (24) Google Scholar). NAD(P)H oxidase catalyzes the following reaction: NAD(P)H + 2O2 → 2O2− + NAD(P)+ + H+. O2− is then rapidly converted to H2O2 (45Jones R.D. Hancock J.T. Morice A.H. Free Radic. Biol. Med. 2000; 29: 416-424Crossref PubMed Scopus (148) Google Scholar). This possibility is supported by our observations that inhibitors of NAD(P)H oxidase inhibited TAS-103-induced DNA ladder formation in HL-60 cells. On the other hand, rotenone, which can inhibit mitochondrial ROS production (46Li Y. Trush M.A. Biochem. Biophys. Res. Commun. 1998; 253: 295-299Crossref PubMed Scopus (397) Google Scholar), did not inhibit TAS-103-induced DNA ladder formation (data not shown). This result excludes the possibility that ROS are released from mitochondria. The decreases in intracellular GSH may also induce ROS generation (13Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1129) Google Scholar, 47Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (643) Google Scholar). Significant GSH decreases were not observed until 6 h, suggesting this GSH decrease does not mediate early generation of H2O2. Therefore, NAD(P)H oxidase is the primary enzyme mediating H2O2formation in cells treated with TAS-103. In addition, p53activation by DNA damage induces apoptosis by the transcriptional induction of redox-related genes and the formation of ROS (48Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2226) Google Scholar, 49Johnson T.M., Yu, Z.X. Ferrans V.J. Lowenstein R.A. Finkel T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11848-11852Crossref PubMed Scopus (521) Google Scholar). This possibility is also excluded as HL-60 cells lack functionalp53 (50Dou Q.P., An, B. Will P.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9019-9023Crossref PubMed Scopus (155) Google Scholar). The activation of NAD(P)H oxidase has been reported to either precede (51Chen Y.C. Lin-Shiau S.Y. Lin J.K. J. Cell. Physiol. 1998; 177: 324-333Crossref PubMed Scopus (424) Google Scholar, 52Hiraoka W. Vazquez N. Nieves-Neira W. Chanock S.J. Pommier Y. J. Clin. Invest. 1998; 102: 1961-1968Crossref PubMed Scopus (96) Google Scholar) or follow (53Simizu S. Takada M. Umezawa K. Imoto M. J. Biol. Chem. 1998; 273: 26900-26907Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) caspase-3 activation. TAS-103-induced H2O2 generation was not inhibited by a caspase-3 inhibitor (data not shown), confirming that activation of NAD(P)H oxidase occurs upstream of caspase-3 activation. Our results revealed that H2O2 generation via NAD(P)H oxidase activation precedes the ΔΨm increase in TAS-103-induced apoptosis. Bcl-2 binds to the voltage-dependent anion channel to regulate the mitochondrial membrane potential, and controls the release of mitochondrial apoptogenic factors, cytochromec and apoptosis-inducing factor (AIF), a mitochondrial flavoprotein (54Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1910) Google Scholar, 55Tsujimoto Y. Shimizu S. FEBS Lett. 2000; 466: 6-10Crossref PubMed Scopus (629) Google Scholar). Our experiment using BJAB/Bcl-2 cells confirmed that Bcl-2 prevented TAS-103-induced apoptosis by inhibiting MPT mediated by H2O2 generation. AIF has an NADH oxidase activity, but DPI, an NAD(P)H oxidase inhibitor, does not affect the apoptogenic activity of AIF (56Miramar M.D. Costantini P. Ravagnan L. Saraiva L.M. Haouzi D. Brothers G. Penninger J.M. Peleato M.L. Kroemer G. Susin S.A. J. Biol. Chem. 2001; 276: 16391-16398Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Our data that DPI prevented TAS-103-induced apoptosis exclude the possibility that the NAD(P)H oxidase activity of AIF is involved in apoptosis. It has been reported that the activity of NAD(P)H oxidase in microsomes is linked to cytosolic NAD(H) redox and appears to be a key source of O2− production (57Mohazzab K.M. Kaminsky P.M. Wollin M.S. Circulation. 1997; 96: 614-620Crossref PubMed Scopus (137) Google Scholar). Taken together, the activation of microsomal NAD(P)H oxidase may participate in TAS-103-induced H2O2 generation. In conclusion, H2O2 generation precedes MPT, leading to subsequent apoptotic events, including cytochrome c release and caspase-3 activation in TAS-103-induced apoptosis." @default.
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W2004758859 title "Mechanism of Apoptosis Induced by a New Topoisomerase Inhibitor through the Generation of Hydrogen Peroxide" @default.
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