N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

• Published in: Toxicology and applied pharmacology 2009-08, Vol.239 (1), p.87-97
• Main Authors: Kim, Byeong Mo, Choi, Yun Jung, Han, Youngsoo, Yun, Yeon-Sook, Hong, Sung Hee
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; ADP; APOPTOSIS; Apoptosis - drug effects; bcl-X Protein - biosynthesis; Biological and medical sciences; Blotting, Western; Caspase 3 - metabolism; Caspase 8 - physiology; Caspase Inhibitors; Caspase-8; Cell death mechanisms; Cell Survival - drug effects; Cytochromes c - secretion; DEPOLARIZATION; DNA; DNA Fragmentation - drug effects; Dose-Response Relationship, Drug; Down-Regulation; ENZYME INHIBITORS; ENZYMES; Flow Cytometry; FRAGMENTATION; HL-60 Cells; HUMAN POPULATIONS; Humans; Inhibitors; Jurkat Cells; K562 Cells; LEUKEMIA; Medical sciences; Membrane Potential, Mitochondrial - drug effects; MITOCHONDRIA; MORPHOLOGICAL CHANGES; N,N-dimethyl phytosphingosine (DMPS); PHOSPHATES; Poly(ADP-ribose) Polymerases - metabolism; Proto-Oncogene Proteins c-bcl-2 - biosynthesis; QUERCETIN; Reactive oxygen species (ROS); Reactive Oxygen Species - metabolism; RNA; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; TIRON; TOXICITY; Toxicology
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1016/j.taap.2009.05.020

N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of the bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8–Bid–Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.