Abstract
Aim:
To evaluate the contribution of an autophagic mechanism to the As2O3-induced death of human acute myeloid leukaemia cell line HL60 cells.
Methods:
The growth inhibition of HL60 cells induced by As2O3 was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The activation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As2O3-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms.
Results:
After treatment with As2O3, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutralizing agent NH4Cl 1 h before As2O3 potentiated the As2O3-induced death of HL60 cells. In contrast, 3-MA attenuated As2O3-induced death when administered 30 min after As2O3. 3-MA and NH4Cl also inhibited As2O3-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As2O3, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As2O3 was suggested by the collapse of mitochondrial membrane potential, release of cytochrome c from mitochondria, and the activation of caspase-3. Pretreatment with 3-MA prior to As2O3 amplified these apoptotic signals, while posttreatment with 3-MA 30 min after As2O3 attenuated the apoptotic pathways.
Conclusion:
Autophagy plays complex roles in the As2O3-induced death of HL60 cells; it inhibits As2O3-induced apoptosis in the initiation stage, but amplifies the As2O3-mediated apoptotic program if it is persistently activated.
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References
Yoshimori T . Autophagy: a regulated bulk degradation process inside cells. Biochem Biophys Res Commun 2004; 313: 453–8.
Bursch W, Ellinger A, Gerner C, Schulte-Hermann R . Autophagocytosis and programmed cell death. In: Klionsky DJ, editors. Autophagy. Landes Bioscience, Georgetown, 2004. p 287–303.
Lockshin RA, Osborne B, Zakeri Z . Cell death in the third millennium. Cell Death Differ 2000; 7: 2–7.
Lockshin RA, Zakeri Z . Apoptosis, autophagy and more. Inter J Biochem Cell Biol 2004; 36: 2405–19.
Gozuacik D, Kimchi A . Autophagy as a cell death and tumor suppressor mechanism. Oncogene 2004; 23: 2891–906.
Gorski SM, Chittaranjan S, Pleasance ED, Freeman JD, Anderson CL, Varhol RJ, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Curr Biol 2003; 13: 358–63.
Lee CY, Clough EA, Yellon P, Teslovich TM, Stephan DA, Baehrecke EH . Genome-wide analyses of steroid- and radiation-triggered programmed cell death in Drosophila. Curr Biol 2003; 13: 350–7.
Bauvy C, Gane P, Arico S, Codogno P, Ogier-Denis E . Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res 2001; 268: 139–49.
Knaapen MW, Davies MJ, De-Bie M, Haven AJ, Martinet W, Kockx MM . Apoptotic versus autophagic cell death in heart failure. Cardiovasc Res 2001; 51: 304–12.
Edinger AL, Thompson CB . Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 2004; 16: 663–9.
Lockshin RA, Zakeri Z . Caspase-independent cell deaths. Curr Opin Cell Biol 2002; 14: 727–33.
Knecht E, Hernández-Yago J, GrisolÃa S . Regulation of lysosomal autophagy in transformed and non-transformed mouse fibroblasts under several growth conditions. Exp Cell Res 1984; 154: 224–32.
Kisen GO, Tessitore L, Costelli P, Gordon PB, Schwarze PE, Baccino FM, et al. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells. Carcino-genesis 1993; 14: 2501–5.
Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 1997; 89: 3345–53.
Roboz GJ, Dias S, Lam G, Lane WJ, Soignet SL, Warrell RP Jr, et al. Arsenic trioxide induces dose- and time-dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis. Blood 2000; 96: 1525–30.
Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I . Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 2003; 63: 2103–8.
Kanzawa T, Zhang L, Xiao L, Germano IM, Kondo Y, Kondo S . Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 2005; 24: 980–91.
Qian W, Liu J, Jin J, Ni W, Xu W . Arsenic trioxide induces not only apoptosis but also autophagic cell death in leukemia cell lines via up-regulation of Beclin-1. Leuk Res 2007; 31: 329–39.
Yan CH, Liang ZQ, Gu ZL, Yang YP, Reid P, Qin ZH . Contributions of autophagic and apoptotic mechanisms to CrTX-induced death of K562 cells. Toxicon 2006; 47: 521–30.
Yan CH, Qin ZH, Yang YP, Gu ZL, Liang ZQ . Involvement of autophagy and caspase-independent apoptosis in crotoxin-induced death of MCF-7 cells. Acta Pharmacol Sin 2007; 28: 540–8.
Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A, Boyd MR . Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. Cancer Res 1991; 51: 2515–20.
Biederbick A, Kern HF, Elsasser HP . Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol 1995; 66: 3–14.
Munafo DB, Colombo MI . Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24. Traffic 2002; 3: 472–82.
Thomas CE, Mayle DA . NMDA-sensitive neurons profoundly influence delayed staurosporine-induced apoptosis in rat mixed cortical neuronal cultures. Brain Res 2000; 884: 163–73.
Gottron FJ, Ying HS, Choi DW . Caspase inhibition selectively reduces the apoptotic component of oxygen-glucose deprivation-induced cortical neuronal cell death. Mol Cell Neurosci 1997; 9: 159–69.
Hong QT, Song YT, Tang YP, Liu CM . Determination and application of leakage rate of lactate dehydrogenase in the cultured medium of cells. Chin J Cell Biol 2004; 26: 89–92.
Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A . JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess DY changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 1997; 411: 77–82.
Gravance CG, Garner DL, Baumber J, Ball BA . Assessment of equine sperm mitochondrial function using JC-1. Theriogenology 2000; 53: 1691–703.
Qin ZH, Wang YM, Kikly KK, Sapp E, Kegel KB, Aronin N, et al. Pro-caspase-8 is predominantly localized in mitochondia and released into cytoplasm upon apoptotic stimulation. J Biol Chem 2001; 276: 8079–86.
Akao Y, Nakagawa Y, Akiyama K . Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro. FEBS Lett 1999; 455: 59–62.
Chow SK, Chan JY, Fung KP . Inhibition of cell proliferation and the action mechanisms of arsenic trioxide (As2O3) on human breast cancer cells. J Cell Biochem 2004; 93: 173–87.
Yi J, Gao F, Shi G, Li H, Wang Z, Shi X, et al. The inherent cellular level of reactive oxygen species: One of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide. Apoptosis 2002; 7: 209–15.
Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood 1997; 89: 3354–60.
Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti L, et al. Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med 1998; 339: 1341–8.
Park JW, Choi YJ, Jang MA, Baek SH, Lim JH, Passaniti T, et al. Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in prominute socytic U937 cells. Biochem Biophys Res Commun 2001; 286: 726–34.
Zhu Q, Zhang JW, Zhu HQ, Shen YL, Flexor M, Jia PM, et al. Synergic effects of arsenic trioxide and Camp during acute promyelocytic leukemia cell maturation subtends a novel signaling cross-talk. Blood 2002; 99: 1014–22.
Jiang XH, Wong BC, Yuen ST, Jiang SH, Cho CH, Lai KC, et al. Arsenic trioxide induce s apoptosis in human ga stric cancer cells through up-regulation of p53 and activation of caspase-3. Int J Cancer 2001; 91: 173–9.
Kelekar A . Autophagy. Ann N Y Acad Sci 2005; 1066: 259–71.
Meijer AJ, Codogno P . Signalling and autophagy regulation in health, aging and disease. Mol Aspects Med 2006; 27: 411–25.
Shintani T, Klionsky DJ . Autophagy in health and disease: a double-edged sword. Science 2004; 306: 990–5.
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000; 19: 5720–8.
Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P . Distinct classes of phosphatidylinositol 3′ -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 2000; 275: 992–8.
Seglen PO, Gordon PB . 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA 1982; 79: 1889–92.
Levine B, Yuan J . Autophagy in cell death: an innocent convict? J Clin Invest 2005; 115: 2679–88.
Rubinsztein DC, DiFiglia M, Heintz N, Nixon RA, Qin ZH, Ravikumar B, et al. Autophagy and its possible roles in nervous system diseases, damage and repair. Autophagy 2005; 1: 11–22.
Ogier-Denis E, Codogno P . Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta 2003; 1603: 113–28.
Kroemer G, Reed JC . Mitochondrial control of cell death. Nat Med 2000; 6: 513–9.
Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA . Apoptosis definition, mechanism, and relevance to disease. Am J Med 1999; 107: 489–506.
Budihardjo I, Oliver H, Lutter M, Luo X, Wang X . Biochemical pathways of caspase activation during apoptosis. Annau Rev Cell Dev Biol 1999; 15: 269–90.
Mijaljica D, Prescott M, Devenish RJ . Different fates of mitochondria: alternative ways for degradation? Autophagy 2007; 3: 4–9.
Elmore SP, Qian T, Grissom SF, Lemasters JJ . The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J 2001; 15: 2286–7.
Cardenas ME, Cutler NS, Lorenz MC, Di-Como CJ, Heitman J . The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev 1999; 13: 3271–9.
Levine B, Klionsky DJ . Development by Self-Digestion:Molecular Mechanisms and Biological Functions of Autophagy. Dev Cell 2004; 6: 463–77.
Lemasters JJ . Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res 2005; 8: 3–5.
Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 2005; 169: 425–34.
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, et al. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1998; 1366: 177–96.
Daugas E, Susin SA, Zamzami N, Ferri KF, Irinopoulou T, Larochette N, et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J 2000; 14: 729–39.
Guicciardi ME, Leist M, Gores GJ . Lysosomes in cell death. Oncogene 2004; 23: 2881–90.
Barrett AJ, Kirschke H . Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol 1981; 80: 535–61.
Takahashi T, Tang J . Cathepsin D from porcine and bovine spleen. Methods Enzymol 1981; 80: 565–81.
Nikawa T, Towatari T, Katunuma N . Purification and characterization of cathepsin J from rat liver. Eur J Biochem 1992; 204: 381–93.
Nakamura Y, Takeda M, Suzuki H, Morita H, Tada K, Hariguchi S, et al. Lysosome instability in aged rat brain. Neurosci Lett 1989; 97: 215–20.
Bi X, Yong AP, Zhou J, Gall CM, Lynch G . Regionally selective changes in brain lysosomes occur in the transition from young adulthood to middle age in rats. Neuroscience 2000; 97: 395–404.
Bechet D, Tassa A, Taillandier D, Combaret L, Attaix D . Lysosomal proteolysis in skeletal muscle. Int J Biochem Cell Biol 2005; 37: 2098–114.
Jaattela M . Multiple cell death pathways as regulators of tumour initiation and progression. Oncogene 2004; 23: 2746–56.
Cirman T, Oresic K, Mazovec GD, Turk V, Reed JC, Myers RM, et al. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 2004; 279: 3578–87.
Furuno K, Ishikawa T, Kato K . Appearance of autolysosomes in rat liver after leupeptin treatment. J Biochem 1982; 91: 1485–94.
Kominami E, Hashida S, Khairallah EA, Katunuma N . Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin. J Biol Chem 1983; 258: 6093–100.
Ohshita T, Kominami E, Ii K, Katunuma N . Effect of starvation and refeeding on autophagy and heterophagy in rat liver. J Biochem 1986; 100: 623–32.
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Yang, Yp., Liang, Zq., Gao, B. et al. Dynamic effects of autophagy on arsenic trioxide-induced death of human leukemia cell line HL60 cells. Acta Pharmacol Sin 29, 123–134 (2008). https://doi.org/10.1111/j.1745-7254.2008.00732.x
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DOI: https://doi.org/10.1111/j.1745-7254.2008.00732.x
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