Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4.
The antiangiogenic, tubulin-binding drug combretastatin A-4 exhibits a selective toxicity for proliferating endothelial cells in vitro and induces vascular shutdown in tumor models in vivo. The mechanism of combretastatin A-4 cytotoxicity has now been investigated with cultured proliferating human umbilical vein endothelial cells by examining various markers of apoptosis. Incubation of cells with 0.1 mM combretastatin A-4 induced the conversion (first detected after 6 h) of the CPP32 proenzyme to active caspase-3, a cysteine protease that plays an important role in apoptosis in many cell types; the drug also increased caspase-3 activity. Another early event observed was the binding of annexin V to 50% of the cells 8 h after drug treatment. Internucleosomal DNA fragmentation, another hallmark of apoptosis, was detected in cells incubated with 0.1 mM combretastatin A-4 for 24 h. Staining with Hoechst 33258 revealed that about 75% of cells exhibited a nuclear morphology characteristic of apoptosis after incubation with drug for 24 h. Incubation of cells for up to 8 h with combretastatin A-4 did not induce the release of lactate dehydrogenase or increase the uptake of propidium iodide, both indicators of membrane integrity. These results indicate that the selective cytotoxic effect of combretastatin A-4 is mediated by the induction of apoptosis rather than by necrosis and may provide an enhanced clinical strategy in cancer chemotherapy with this new agent.
Iyer
S
S
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Chaplin
D J
DJ
Rosenthal
D S
DS
Boulares
A H
AH
Li
L Y
LY
Smulson
M E
ME
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D013267:Stilbenes / Q000494:pharmacology*
D000972:Antineoplastic Agents, Phytogenic; D013267:Stilbenes; D007770:L-Lactate Dehydrogenase; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; C058728:fosbretabulin