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

Abstract

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.

• Authors: Byeong Mo Kim, Yun Jung Choi, Youngsoo Han, Yeon-Sook Yun, Sung Hee Hong
• Journal: Toxicology and applied pharmacology (Toxicol Appl Pharmacol) Vol. 239 Issue 1 Pg. 87-97 (Aug 15 2009) ISSN: 1096-0333 [Electronic] United States
• PMID: 19481559 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• BCL2L1 protein, human
• Caspase Inhibitors
• N,N-dimethylphytosphingosine
• Proto-Oncogene Proteins c-bcl-2
• Reactive Oxygen Species
• bcl-X Protein
• Cytochromes c
• Poly(ADP-ribose) Polymerases
• CASP8 protein, human
• Caspase 3
• Caspase 8
• Sphingosine

Topics

• Apoptosis (drug effects)
• Blotting, Western
• Caspase 3 (metabolism)
• Caspase 8 (physiology)
• Caspase Inhibitors
• Cell Survival (drug effects)
• Cytochromes c (metabolism)
• DNA Fragmentation (drug effects)
• Dose-Response Relationship, Drug
• Down-Regulation
• Flow Cytometry
• HL-60 Cells
• Humans
• Jurkat Cells
• K562 Cells
• Membrane Potential, Mitochondrial (drug effects)
• Poly(ADP-ribose) Polymerases (metabolism)
• Proto-Oncogene Proteins c-bcl-2 (biosynthesis)
• Reactive Oxygen Species (metabolism)
• Sphingosine (analogs & derivatives, pharmacology)
• bcl-X Protein (biosynthesis)