Inhibition of ribonucleotide reductase by a new class of isoindole derivatives: drug synergism with cytarabine (Ara-C) and induction of cellular apoptosis.
The hydroxyisoindole dione derivatives ISID and MISID are new compounds with structures resembling purines and possessing a hydroxamic acid moiety which is the pharmacophore of hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RR). ISID and MISID exhibited 100- to 500-fold higher cytotoxicity as compared to HU against cell lines sensitive (CEM/0) or resistant to ara-C (CEM/ara-C/7A; CEM/dCk[-]). Both MISID and ISID showed significant inhibitory activity of ribonucleotide reductase (RR). Treatment of CEM/0 cells with 10 microM ISID showed a linear decrease in all the dNTPs leading to a complete depletion by 4 hours with no recovery of enzymatic activity of RR up to 48 hours in the presence of the drug, suggesting an irreversible inhibition of this enzyme. However, 10 microM MISID caused a significant time dependent, but reversible inhibition of RR in a whole cell assay in CEM/0 cells. Pretreatment of CEM/0 cells with 10 microM MISID for 1 hour increased cellular ara-CTP concentrations approximately 2-fold as compared to untreated controls. However, a reduction in intracellular ara-CTP concentration was observed following a commensurate depletion of ATP in these cells after 4 hrs of ISID pretreatment. Similarly, the ara-CTP concentration was augmented by 1.6-fold following pretreatment of CEM/0 cells with 10 microM MISID for 4 hours. Significant apoptotic cell death was detected in CEM/0 cells treated with ara-C, ISID or MISID alone or in combination. Ara-C treatment induced HMW (high molecular weight) DNA fragmentation at earlier times which subsequently led to oligonucleosomal DNA fragmentation by 48 hrs. The sequential treatment of CEM/0 cells with MISID followed by ara-C resulted in increased DNA fragmentation in the 2.0 to 4.0 Kb range in comparison to those cells treated with either ara-C or MISID alone. The increased apoptotic cell death explained the synergistic cytotoxicity of the combination of ara-C and MISID against CEM/0 cells observed earlier. We conclude that the inhibition of RR by these agents induces leukemic cell apoptosis, a mechanism which is further potentiated when these RR inhibitors are combined with ara-C. Since new compounds do not require activation, as do other clinically useful RR inhibitors, further studies for their potential use against leukemias and solid tumors are warranted.
Nandy
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Department of Pediatrics, USC School of Medicine, Childrens Hospital Los Angeles 90027, USA.
Lien
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EJ
Avramis
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001085:Arabinofuranosylcytosine Triphosphate / Q000032:analysis; D003561:Cytarabine / Q000494:pharmacology*; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis; D003854:Deoxyribonucleotides / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D016521:Electrophoresis, Gel, Pulsed-Field; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology*; D054833:Isoindoles; D007938:Leukemia / Q000473:pathology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
C121189:2-hydroxy-1(H)-isoindole-1,3-dione; C121190:6-methyl-2-isopropylsulfonyloxy-1(H)-isoindole-1,3-dione; D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D003854:Deoxyribonucleotides; D004791:Enzyme Inhibitors; D007211:Indoles; D054833:Isoindoles; D009363:Neoplasm Proteins; D003561:Cytarabine; D001085:Arabinofuranosylcytosine Triphosphate; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea