N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells.
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.
Kim
Byeong Mo
BM
Korea Institute of Radiological and Medical Sciences, Gongneung-Dong, Nowon-Gu, Seoul, Republic of Korea.
Choi
Yun Jung
YJ
Han
Youngsoo
Y
Yun
Yeon-Sook
YS
Hong
Sung Hee
SH
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000502:physiology*; D061945:Caspase Inhibitors; D002470:Cell Survival / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051020:bcl-X Protein / Q000096:biosynthesis
C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; C542681:N,N-dimethylphytosphingosine; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051020:bcl-X Protein; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D013110:Sphingosine