Apoptotic response of HL-60 human leukemia cells to the antitumor drug NB-506, a glycosylated indolocarbazole inhibitor of topoisomerase 1.
The antitumor drug NB-506 is a glycosylated indolocarbazole derivative targeting topoisomerase I. This DNA-intercalating agent, which is currently undergoing phase I/II clinical trials, was shown to induce apoptosis in HL-60 human leukemia cells. We compared the cellular dysfunctions induced by NB-506 and the reference topoisomerase I poison camptothecin (CPT) at the nuclear, mitochondrial, and cytoplasmic levels. The two drugs NB-506 and CPT were almost equally toxic to HL-60 cells and produced similar cell cycle changes with a considerable increase in the fraction of cells with DNA content less than G1. The sub-G1 fraction, which can be considered as the apoptotic cell population, appeared more rapidly with CPT than with NB-506 but in both cases, the cell cycle perturbation was accompanied by a marked decrease in the mitochondrial transmembrane potential and the intracellular pH. In contrast, no change in the intracellular calcium concentration was detected. Treatment of HL-60 cells with NB-506 resulted in an increase in the activity of the intracellular protease caspase-3, as determined by a DEVD-based colorimetric assay and direct monitoring of poly(ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. The initiator caspase-8 was also stimulated by NB-506 but, as for caspase-3, the extent of the caspase activation was weaker with NB-506 compared to CPT. With both drugs, the protease activation resulted in DNA degradation, as independently confirmed via the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and characterization of internucleosomal DNA fragmentation. Collectively, these findings identify some of the molecular events leading to NB-506-induced apoptosis and as such, provide important mechanistic insights into the mode of action of topoisomerase I-targeted indolocarbazole antitumor drugs.
Facompre
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INSERM U-524, Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun, F-59045, Lille, France.
Goossens
J F
JF
Bailly
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C
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation; D005960:Glucosides / Q000494:pharmacology*; D006031:Glycosylation; D018922:HL-60 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D059004:Topoisomerase I Inhibitors
D000970:Antineoplastic Agents; D002227:Carbazoles; D005960:Glucosides; C092476:NB 506; D059004:Topoisomerase I Inhibitors; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002118:Calcium