Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox.
OBJECTIVE
Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy.


METHODS
HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis.


RESULTS
Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels. The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed.


CONCLUSIONS
Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.
Grusch
M
M
Institute of Clinical Pathology, University of Vienna, Vienna, Austria.
Fritzer-Szekeres
M
M
Fuhrmann
G
G
Rosenberger
G
G
Luxbacher
C
C
Elford
H L
HL
Smid
K
K
Peters
G J
GJ
Szekeres
T
T
Krupitza
G
G
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000187:drug effects*; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018260:Gelsolin / Q000378:metabolism; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D020014:K562 Cells / Q000187:drug effects / Q000201:enzymology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors*; D010091:Oximes / Q000494:pharmacology*; D010718:Phosphatidylserines / Q000378:metabolism; D010865:Pilot Projects; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*
D017304:Annexin A5; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D018260:Gelsolin; D006877:Hydroxamic Acids; D009363:Neoplasm Proteins; D010091:Oximes; D010718:Phosphatidylserines; C070491:Amidox; D012264:Ribonucleotide Reductases; D011065:Poly(ADP-ribose) Polymerases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C035419:3,4-dihydroxybenzohydroxamic acid