Vincristine induced apoptosis in acute lymphoblastic leukaemia cells: a mitochondrial controlled pathway regulated by reactive oxygen species?
Vincristine (VCR), a microtubule interfering anti-cancer agent, plays a key role in the treatment of childhood acute lymphoblastic leukaemia (ALL). The route of VCR induced apoptosis in ALL cells is not well defined. In this study we demonstrated caspase-9 and -3 activation in vivo in bone marrow leukaemic cells of a child with newly diagnosed ALL, after treatment with a single dose of VCR. We hypothesized that VCR induced apoptosis in ALL cells proceeds by a mitochondrial controlled pathway. We further studied the route of VCR induced apoptosis in Jurkat acute lymphoblastic leukaemia cells. First we showed that VCR induces activation of caspase-9 and -3 in Jurkat cells. With the caspase-9 inhibitor Z-LEHD-FMK we proved that caspase-9 was activated prior to caspase-3. Loss of mitochondrial transmembrane potential was independent of caspase-9 activation. To confirm the mitochondrial role in VCR induced apoptosis, the effect of blocking the mitochondrial route upstream of caspase-9 activation was investigated at two different levels: reactive oxygen species (ROS) scavenging and Bcl-2 overexpression. Generation of ROS was detected early in Jurkat cells during VCR exposure. Ascorbic acid, a ROS scavenger, inhibited ROS generation as well as caspase-9 and -3 activation and cell death induced by VCR. Furthermore, in Bcl-2 overexpressing Jurkat cells mitochondrial membrane potential changes, caspase-9 and -3 activation and cell death upon VCR exposure were decreased, in comparison to parental Jurkat cells. However, generation of ROS was not decreased in Jurkat cells with Bcl-2 overexpression. We concluded that ROS play a regulatory role in the initial phase of a mitochondrial controlled pathway of VCR induced apoptosis in Jurkat cells.
Groninger
E
E
Division of Paediatric Oncology Haematology, Beatrix Children's Hospital Groningen, Groningen, The Netherlands.
Meeuwsen-De Boer
G J
GJ
De Graaf
S S N
SS
Kamps
W A
WA
De Bont
E S J M
ES
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001205:Ascorbic Acid / Q000494:pharmacology; D015153:Blotting, Western; D001854:Bone Marrow Cells; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014750:Vincristine / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D000972:Antineoplastic Agents, Phytogenic; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D014750:Vincristine; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D001205:Ascorbic Acid