Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox.

Abstract

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.

MATERIALS AND 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.

• Authors: M Grusch, M Fritzer-Szekeres, G Fuhrmann, G Rosenberger, C Luxbacher, H L Elford, K Smid, G J Peters, T Szekeres, G Krupitza
• Journal: Experimental hematology (Exp Hematol) Vol. 29 Issue 5 Pg. 623-32 (May 2001) ISSN: 0301-472X [Print] Netherlands
• PMID: 11376876 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Annexin A5
• Caspase Inhibitors
• Enzyme Inhibitors
• Gelsolin
• Hydroxamic Acids
• Neoplasm Proteins
• Oximes
• Phosphatidylserines
• Amidox
• Ribonucleotide Reductases
• Poly(ADP-ribose) Polymerases
• CASP8 protein, human
• CASP9 protein, human
• Caspase 8
• Caspase 9
• Caspases
• 3,4-dihydroxybenzohydroxamic acid

Topics

• Annexin A5 (metabolism)
• Apoptosis (drug effects)
• Caspase 8
• Caspase 9
• Caspase Inhibitors
• Caspases (drug effects)
• DNA Fragmentation
• Dose-Response Relationship, Drug
• Enzyme Activation (drug effects)
• Enzyme Inhibitors (pharmacology)
• Gelsolin (metabolism)
• HL-60 Cells (drug effects, enzymology)
• Humans
• Hydroxamic Acids (pharmacology)
• K562 Cells (drug effects, enzymology)
• Neoplasm Proteins (antagonists & inhibitors)
• Oximes (pharmacology)
• Phosphatidylserines (metabolism)
• Pilot Projects
• Poly(ADP-ribose) Polymerases (metabolism)
• Ribonucleotide Reductases (antagonists & inhibitors)