Doxorubicin induces apoptosis in Jurkat cells by mitochondria-dependent and mitochondria-independent mechanisms under normoxic and hypoxic conditions.
In this study, we investigated the molecular mechanism of doxorubicin (dxr)-induced cytotoxicity on Jurkat cells - a model cell of human acute lymphoblastic leukemia - under normoxic (20% O2) and hypoxic (5% O2) conditions. Using in-cell western analysis, immunofluorescence, flow cytometry analysis, and biochemical inhibitors, we evaluated several oxidative stress (OS) and cell death markers. It was found that dxr (5-100 μmol/l) induced apoptosis by OS mechanisms involving DNA fragmentation (8-48%), loss of mitochondrial membrane potential (ΔΨm, 33-92%), and H2O2 production (15-42%) under normoxia. In addition, dxr (10 μmol/l) induced activation and/or nuclei translocation of NF-κB (6.6, 1.6-fold increase), p53 (4.3, 3.1 f), c-Jun (9.5, 5.0 f), apoptosis-inducing factor (AIF) (1.9, 3.9 f), caspase-3 (3.7, 1.9 f), overexpression of Parkin (2.1, 1.2 f)/PINK-1 (2.1 f) proteins, and reduced DJ-1 levels by half compared with untreated cells under normoxia, according to immunofluorescence and in-cell western analysis, respectively. In contrast, dxr (10 μmol/l) could not induce apoptosis in Jurkat cells under hypoxia. Effectively, dxr significantly reduced DNA fragmentation (6%), expression levels of cell death (e.g. p53, c-Jun, caspase-3, AIF), and OS (e.g. Parkin) markers, whereas it increased ΔΨm, hypoxia-inducible factor 1-α (HIF-1α, 3.1, 2.3 f), NF-κB (6.8, 2.0 f), and DJ-1 (1.3, 1.0 f) levels. This investigation suggests that dxr might efficiently eliminate acute lymphoblastic leukemia cells by OS-induced apoptosis under normoxic conditions through a minimal completeness of cell death signaling (i.e. mitochondria-caspase-3/AIF-dependent pathways) and through a direct DNA damage process. However, hypoxic conditions may reduce the effectiveness of dxr toxicity.
Neuroscience Research Group, Medical Research Institute, Faculty of Medicine, University of Antioquia (UdeA), Medellin, Colombia.
Miguel
M
Mendivil-Perez
Carlos
C
Velez-Pardo
Marlene
M
Jimenez-Del-Rio
D000111:Acetylcysteine / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D004317:Doxorubicin / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D000071617:Protein Deglycase DJ-1; D011494:Protein Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000099202:PTEN-Induced Putative Kinase
D000111:Acetylcysteine; D000903:Antibiotics, Antineoplastic; D051033:Apoptosis Inducing Factor; D053148:Caspase 3; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D015513:Oncogene Proteins; D000071617:Protein Deglycase DJ-1; D011494:Protein Kinases; D016755:Proto-Oncogene Proteins c-jun; D016159:Tumor Suppressor Protein p53; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C494829:AIFM1 protein, human; C497442:HIF1A protein, human; C111567:parkin protein; C105131:PARK7 protein, human