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1.
Br J Haematol ; 135(1): 52-61, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16925573

ABSTRACT

Ribonucleotide reductase (RR) is the enzyme that catalyses the rate-limiting step in DNA synthesis, the production of deoxynucleotides. RR activity is markedly elevated in tumour tissue and is crucial for cell division. It is therefore an excellent target for cancer chemotherapy. This study examined the anti-myeloma activity of Didox (3,4-Dihydroxybenzohydroxamic acid), a novel RR inhibitor (RRI). Our data showed that Didox induced caspase-dependent multiple myeloma (MM) cell apoptosis. Didox, unlike other RRIs that mainly target the pyrimidine metabolism pathway, targets both purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, a >or=2-fold downregulation of genes in these anabolic pathways was shown as early as 12 h after exposure to Didox. Furthermore, apoptosis was accompanied by downregulation of bcl family proteins including bcl-2, bcl(xl), and XIAP. Importantly, RR M1 component transcript was also downregulated, associated with decreased protein expression. Genes involved in DNA repair mechanisms, specifically RAD 51 homologue, were also downregulated. As Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an agent commonly used in MM, were performed. A strong in vitro synergism was shown, with combination indices of <0.7 as determined by the Chou-Talalay method. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with DNA-damaging agents, to improve patient outcome in MM.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA Repair/drug effects , Hydroxamic Acids/pharmacology , Multiple Myeloma/pathology , Antineoplastic Agents, Alkylating/pharmacology , Caspases/physiology , Cell Cycle/drug effects , Cell Survival/drug effects , DNA Repair/genetics , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Drug Synergism , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melphalan/pharmacology , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Tumor Cells, Cultured
2.
Antivir Chem Chemother ; 13(5): 305-14, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12630679

ABSTRACT

We evaluated the ability of a short course of treatment with the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) and two novel RR inhibitors Trimidox (TX) and Didox (DX) to influence late-stage murine retrovirus-induced lymphoproliferative disease. LPBM5 murine leukaemia virus retrovirus-infected mice were treated daily with HU, TX or DX for 4 weeks, beginning 9 weeks post-infection, after development of immunodeficiency and lymphoproliferative disease. Drug effects on disease progression were determined by evaluating spleen weight and histology. Effects on haematopoiesis were determined by measuring peripheral blood indices (white blood cells and haematocrit) and assay of femur cellularity and femoral and splenic content of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). HU, TX and DX partially reversed late-stage retrovirus-induced disease, resulting in spleen weights significantly below pre-treatment values. Spleen histology was also improved by RR inhibitor treatment (DX>TX>HU). However, as expected, HU was significantly myelosuppressive, inducing a reduction in peripheral indices associated with depletion of femoral CFU-GM and BFU-E. In contrast, although TX and DX were moderately myelosuppressive, both drugs were significantly better tolerated than HU. In summary, short-term treatment in late-stage murine retroviral disease with HU, TX or DX induced dramatic reversal of disease pathophysiology. However, the novel RR inhibitors TX and DX had more effective activity and significantly less bone marrow toxicity than HU.


Subject(s)
Antiviral Agents/adverse effects , Benzamidines/therapeutic use , Bone Marrow/drug effects , Hydroxamic Acids/therapeutic use , Hydroxyurea/adverse effects , Leukemia Virus, Murine/physiology , Lymphoproliferative Disorders/drug therapy , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/therapeutic use , Benzamidines/administration & dosage , Benzamidines/adverse effects , Blood Cell Count , Body Weight/drug effects , Bone Marrow/pathology , Hydroxamic Acids/administration & dosage , Hydroxamic Acids/adverse effects , Hydroxyurea/therapeutic use , Lymphoproliferative Disorders/pathology , Lymphoproliferative Disorders/virology , Mice , Spleen/drug effects , Spleen/pathology , Time Factors
3.
Exp Hematol ; 29(5): 623-32, 2001 May.
Article in English | MEDLINE | ID: mdl-11376876

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.


Subject(s)
Apoptosis/drug effects , Caspase Inhibitors , Caspases/drug effects , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Oximes/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Annexin A5/metabolism , Caspase 8 , Caspase 9 , DNA Fragmentation , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Gelsolin/metabolism , HL-60 Cells/drug effects , HL-60 Cells/enzymology , Humans , K562 Cells/drug effects , K562 Cells/enzymology , Phosphatidylserines/metabolism , Pilot Projects , Poly(ADP-ribose) Polymerases/metabolism
4.
Exp Hematol ; 28(8): 924-30, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10989193

ABSTRACT

Ribonucleotide reductase (RR) is the rate-limiting enzyme for the de novo synthesis of deoxyribonucleotides. Its activity is significantly increased in tumor cells related to the proliferation rate. Therefore, the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study, we investigated whether the antineoplastic effects of trimidox (3,4, 5-trihydroxybenzamidoxime), a novel inhibitor of RR, were due to induction of apoptosis.HL-60 cells were incubated with various concentrations of trimidox. Consequently, cell morphology, DNA condensation, annexin binding, DNA fragmentation, and signature type cleavage of poly(ADP-ribose)polymerase and gelsolin were determined. We also tested the involvement of CD95 and CD95 ligand in apoptosis induction. Furthermore, we examined the c-myc expression of HL-60 cells after incubation with trimidox in order to elucidate a possible association between c-myc expression and induction of apoptosis in the case of trimidox. Trimidox incubation caused a time-dependent increase of c-myc RNA expression and this was accompanied by the induction of apoptosis. Apoptosis was triggered independently of CD95 by the activation of caspases and PARP cleavage. We conclude that trimidox is able to induce programmed cell death. The induction of apoptosis was demonstrated by various biochemical and morphological methods and seems to be associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and without change of the CD95 and CD95 ligand expression.


Subject(s)
Apoptosis/drug effects , Benzamidines/pharmacology , Caspases/metabolism , Enzyme Inhibitors/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Bisbenzimidazole , DNA Fragmentation , Enzyme Activation/drug effects , Fas Ligand Protein , Fluorescent Dyes , Gelsolin/metabolism , Gene Expression/drug effects , Genes, myc , HL-60 Cells , Humans , Membrane Glycoproteins/analysis , Poly(ADP-ribose) Polymerases/metabolism , Propidium , fas Receptor/analysis
5.
Am J Hematol ; 63(4): 176-83, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10706760

ABSTRACT

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F-cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation. Of the RRIs currently in use, hydroxyurea is the most effective agent for Hb F induction. We have examined the capacity of two novel RRIs, didox (DI) and trimidox (TRI), in combination with streptozotocin (STZ), to induce hemoglobin and F-cell production. The K562 cells were cultured with different concentrations of didox-STZ or trimidox-STZ at a fixed molar ratio of 3:1 and 1:5 for 96 hr, respectively. At pre-determined time intervals, aliquots of cells were obtained and total hemoglobin (benzidine positive) levels, number of F-cells, and Hb F were determined by the differential staining technique, fetal hemoglobin assay kit, and fluorescence cytometry respectively. The effect of combined drug treatment on the growth of K562 cells was examined by isobologram analysis. Our results indicate that a synergistic growth-inhibitory differentiation effect occurred when didox or trimidox was used in combination with STZ on K562 cells. There was an increase in the number of both benzidine-positive normoblasts and F-cells, accompanied by morphologic appearances typical of erythroid maturation. On day 4, the number of benzidine-positive cells showed a 6-9-fold increase and the number of F-cells was between 2.5- and 5.7-fold higher than the respective controls. Based upon these results, treatment with a ribonucleotide reductase inhibitor, such as didox or trimidox, in combination with STZ, might offer an additional promising option in sickle cell disease therapy.


Subject(s)
Benzamidines/pharmacology , Enzyme Inhibitors/pharmacology , Fetal Hemoglobin/biosynthesis , Hemoglobin, Sickle/biosynthesis , Hydroxamic Acids/pharmacology , K562 Cells/cytology , Streptozocin/therapeutic use , Antineoplastic Agents/therapeutic use , Benzamidines/therapeutic use , Cell Differentiation/drug effects , Cell Division/drug effects , Drug Therapy, Combination , Humans , Hydroxamic Acids/therapeutic use , K562 Cells/metabolism , Ribonucleotide Reductases/antagonists & inhibitors
6.
Life Sci ; 67(26): 3131-42, 2000 Nov 17.
Article in English | MEDLINE | ID: mdl-11191620

ABSTRACT

Trimidox (3,4,5-trihydroxybenzohydroxamidoxime), a recently synthesized inhibitor of ribonucleotide reductase (RR), was shown to exert anti-proliferative activities in HL-60 and K562 human leukemia cell lines and to prolong the life span of mice inoculated with L1210 mouse leukemia cells. Here we test whether trimidox also exhibits anti-neoplastic properties in ovarian carcinoma cells. Since the mode of action of trimidox on cell fate has not been investigated so far, we addressed this unresolved item and find that this polyhydroxybenzoic acid derivative induces apoptosis of N.1 human ovarian carcinoma cells when tested in growth factor deprived medium. Utilizing an improved analysis, based on Hoechst 33258/propidium iodide double staining, apoptosis is quantified and discriminated from necrosis. Trimidox induces c-myc expression, which is indispensible for apoptosis of N.1 cells, and expression of plasminogen activator/urokinase type (upa), which supports the apoptotic process under more physiological conditions. Surprisingly, trimidox does not block dNTP synthesis in N.1 cells at the concentrations tested and, therefore, trimidox induces apoptosis independent of RR-inhibition. Like TNFalpha or benzamide riboside, which are also inducers of apoptosis of N.1 cells, trimidox also down-regulates the G1 cell cycle phosphatase cdc25A, whereas cyclin D1 becomes up-regulated. This report shows that trimidox destroys human ovarian carcinoma cells by inducing them to undergo apoptosis as well as corroborating previous investigations which demonstrated that apoptosis of these cells depends on c-myc over-expression when survival factors are withdrawn.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Benzamidines/pharmacology , Enzyme Inhibitors/pharmacology , Genes, myc , Ovarian Neoplasms/drug therapy , Apoptosis/genetics , Cyclin D1/biosynthesis , Deoxyribonucleotides/metabolism , Drug Screening Assays, Antitumor , Female , Gene Expression/drug effects , Genes, cdc/drug effects , HL-60 Cells , Humans , Ribonucleotide Reductases/antagonists & inhibitors , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/biosynthesis , cdc25 Phosphatases/biosynthesis
8.
Stem Cells ; 17(6): 345-56, 1999.
Article in English | MEDLINE | ID: mdl-10606163

ABSTRACT

Inhibitors of the cellular enzyme ribonucleotide reductase (hydroxyurea, [HU]) have been proposed as a new therapeutic strategy for the treatment of HIV type-1 (HIV-1) infection. However, HU use may be limited by the frequent development of hematopoietic toxicity. We report here short-term hematopoietic toxicity in mice receiving HU when compared to either of two more potent enzyme inhibitors, didox (DX) and trimidox (TX). High dose HU, DX, and TX monotherapy (500, 460, and 220 mg/kg/day respectively) was administered by daily i.p. injection (Monday-Friday) to C57BL/6 mice for 10 weeks. Effects on hematopoiesis were established by quantitating peripheral blood indices (hematocrit, hemoglobin, mean corpuscular volume, mean cell hemoglobin, mean corpuscular hemoglobin concentration, RBC, and WBC) and numbers of colony-forming units-granulocyte-macrophage (CFU-GM) and BFU-E from bone marrow and spleen. HU produced rapid induction of a macrocytic hypochromic anemia and altered white blood cell kinetics associated with myelosuppression defined as reduced marrow organ cellularity and induction of splenic extramedullary hematopoiesis. Compared to HU, TX and DX induced fewer changes in peripheral blood indices and CFU-GM and BFU-E per hematopoietic organ. In vitro human and murine marrow CFU-GM and BFU-E colony formations were assayed in the presence of dose escalation HU, DX, or TX (0, 1, 10, 50, 100, and 200 microM). HU inhibited colony formation more than either DX or TX. These in vivo and in vitro studies suggest that novel ribonucleotide reductase inhibitors TX and DX may provide an effective alternative to HU in HIV-1 therapy because they demonstrate reduced hematopoietic toxicity.


Subject(s)
Anti-HIV Agents/toxicity , Benzamidines/toxicity , Enzyme Inhibitors/toxicity , Hematopoietic Stem Cells/drug effects , Hydroxamic Acids/toxicity , Hydroxyurea/toxicity , Ribonucleotide Reductases/antagonists & inhibitors , Acquired Immunodeficiency Syndrome/drug therapy , Anemia/chemically induced , Animals , Cells, Cultured , Colony-Forming Units Assay , Female , Femur , Hematopoiesis/drug effects , Humans , In Vitro Techniques , Lymphocytes/cytology , Lymphocytes/drug effects , Macrophages/cytology , Macrophages/drug effects , Mice , Mice, Inbred C57BL , Neutrophils/cytology , Neutrophils/drug effects , Organ Size , Spleen/cytology
9.
Blood Cells Mol Dis ; 25(3-4): 255-69, 1999.
Article in English | MEDLINE | ID: mdl-10575551

ABSTRACT

Pharmacologic agents such as hydroxyurea (HU), N, 3-4 trihydroxybenzamide (didox), and isobutyramide (ISB) can elevate gamma-globin as a potential treatment for the beta-hemoglobinopathies. In these experiments, transgenic mice with 5'HS2 from the human beta-globin locus control region, the fetal (A gamma), and adult (beta s) globin genes were used. Mice were treated with HU, didox, or ISB individually, or with combinations of HU or didox with ISB. The aim was to determine whether these drugs have synergistic effects on the induction of fetal hemoglobin (HbF) and whether the combination regimens are more hematotoxic. In the combination regimens, injections of HU or didox for five weeks were concomitant with ISB treatment every other day for the final three weeks of treatment. The combination of HU + ISB was more hematotoxic than the individual drugs based on significantly increased percentages of reticulocytes and reduced hemoglobin, indicating that caution should be taken in treatments involving combinations of these types of drugs. The didox + ISB combination was not more hematotoxic than the individual drugs. HbF was not induced in the groups treated with the combinations of HU or didox with ISB compared to the individual agents. There was a negligible effect on the percentage of HbF and an unexpected negative effect on the percentage of F cells. The results also have implications for future testing of HbF-inducing drugs in mouse models. In control mice that were phlebotomized but not treated with any drugs, increased percentages of F cells were observed, indicating that blood sampling can cause this effect. In addition, increases in the percentage of F cells did not correlate with increases in the percentage of HbF, indicating that monitoring F cells alone is not a sufficient measure of HbF induction.


Subject(s)
Amides/toxicity , Amides/therapeutic use , Fetal Hemoglobin/metabolism , Hydroxyurea/toxicity , Hydroxyurea/therapeutic use , Animals , Disease Models, Animal , Drug Therapy, Combination , Fetal Hemoglobin/analysis , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/drug effects , Hemoglobinopathies/drug therapy , Humans , Mice , Mice, Transgenic , RNA, Messenger/analysis , Reticulocytes/chemistry , Reticulocytes/metabolism , gamma-Globulins/drug effects , gamma-Globulins/genetics
10.
Antimicrob Agents Chemother ; 42(9): 2456-8, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9736585

ABSTRACT

Several known mammalian ribonucleotide reductase inhibitors featuring a polyhydroxyphenyl and/or hydroxamate moiety as the active group were screened for potency in inhibiting growth of the malaria parasite Plasmodium falciparum. Compounds containing a 2,3- or 3,4-dihydroxyphenyl group as well as benzohydroxamate appear to be the most effective inhibitors of the malaria parasite.


Subject(s)
Antimalarials/pharmacology , Hydroxamic Acids/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Animals , Hydroxyurea/pharmacology , Plasmodium falciparum/drug effects , Structure-Activity Relationship
11.
Cancer Lett ; 129(2): 199-204, 1998 Jul 17.
Article in English | MEDLINE | ID: mdl-9719462

ABSTRACT

Ribonucleotide reductase, a key enzyme in deoxyribonucleotide synthesis, is an important target for cancer chemotherapy. Drugs that inhibit its individual components may act synergistically to block DNA synthesis. Prior work has established that gallium inhibits the R2 subunit of ribonucleotide reductase. We show that gallium acts synergistically with the ribonucleotide reductase inhibitors gemcitabine and hydroxyurea to inhibit the proliferation of CCRF-CEM cells. In contrast, combinations of gallium with the ribonucleotide reductase inhibitors amidox, didox, or trimidox produced antagonistic effects on cell growth. Spectroscopy analysis revealed that as a result of their metal-binding properties, amidox, didox and trimidox formed complexes with gallium, thus negating potential synergistic actions. Our results have important implications in the design of clinical trials using these ribonucleotide reductase inhibitors in combination.


Subject(s)
Gallium/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Ribonucleotide Reductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Benzamidines/pharmacology , Cell Division/drug effects , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Synergism , Enzyme Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Hydroxyurea/pharmacology , Oximes/pharmacology , Tumor Cells, Cultured , Gemcitabine
12.
Life Sci ; 63(7): 545-52, 1998.
Article in English | MEDLINE | ID: mdl-9718080

ABSTRACT

Ribonucleotide reductase is the rate limiting enzyme of de novo DNA synthesis; its activity is significantly increased in tumor cells related to the proliferation rate. Therefore the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study we tested the in vitro and in vivo antitumor effects of a drug combination using trimidox (3,4,5-trihydroxybenzamidoxime), a novel inhibitor of ribonucleotide reductase with adriamycin, a widely used anticancer drug. This combination was selected because adriamycin generates free radicals being responsible for cardiotoxic side effects; trimidox has been shown to be a good free radical scavenger. The in vitro cytotoxic effect of the drug combination was examined in L1210 mouse leukemia cells employing a MTT chemosensitivity assay. Incubation of these cells with adriamycin and trimidox together yielded less than additive cytotoxic effects compared to either drug alone. These effects were not caused by the involvement of p-glycoprotein mediated drug efflux. However, when the effect of trimidox and adriamycin in combination was examined in L1210 leukemia bearing mice antitumor effects of adriamycin could be enhanced by the presence of trimidox. Our data indicate, that the in vivo combination of adriamycin together with trimidox might be beneficial for the treatment of malignancies.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Benzamidines/therapeutic use , Doxorubicin/therapeutic use , Enzyme Inhibitors/therapeutic use , Leukemia L1210/drug therapy , Ribonucleotide Reductases/antagonists & inhibitors , Animals , Mice , Mice, Inbred DBA , Neoplasm Transplantation
13.
Biochem Biophys Res Commun ; 247(3): 759-64, 1998 Jun 29.
Article in English | MEDLINE | ID: mdl-9647767

ABSTRACT

Trimidox (3,4,5-trihdroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase with accompanied growth inhibition and differentiation of mammalian cells. Hydroxyurea (HU) is the only ribonucleotide reductase inhibitor in clinical use for the treatment and management of sickle cell anemia, since this compound increases fetal hemoglobin (Hb F) production: a potent inhibitor of sickle hemoglobin (Hb SS) polymerization. However, the main limitations of HU is its lack of potency, myelosuppression and short half life. These studies investigated the effects of trimidox on the induction of hemoglobin and F-cells production in K562 erythroleukemia cells. Our study reveals that trimidox exhibits concentration dependent inhibitory effect on K562 cells with increase in benzidine positive normoblasts and F-cells production as well as morphological changes typical of erythroid differentiation. These findings provide the first evidence that the growth inhibitory differentiation of cells induced by trimidox enhance hemoglobin and F-cells production.


Subject(s)
Benzamidines/pharmacology , Cell Differentiation/drug effects , Ribonucleotide Reductases/antagonists & inhibitors , Cell Division/drug effects , Enzyme Inhibitors , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/drug effects , Fetal Hemoglobin/metabolism , Histocytochemistry , Humans , Leukemia, Erythroblastic, Acute/metabolism , Tumor Cells, Cultured
14.
Adv Exp Med Biol ; 431: 599-604, 1998.
Article in English | MEDLINE | ID: mdl-9598136

ABSTRACT

Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Didox and amidox are newly synthesized compounds, which inhibit this enzyme and have in vitro and in vivo antitumor activity. We have now investigated the capability of didox and amidox to interfere with the iron metabolism. We show by photometric and polarographic methods, that didox and amidox are capable of forming an iron complex. However, their cytotoxic action cannot be circumvented by addition of Fe-ammoniumcitrate, indicating the iron complexing capacity not to be responsible for the mechanism of action of these compounds. When L1210 leukemia cells were incubated with the didox-iron or amidox-iron complex itself, only slight changes of the 50% growth inhibitory capacity of the complex in comparison with didox or amidox alone could be shown. We conclude, that didox and amidox are capable of forming an iron complex, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Further studies will have to elucidate the molecular mechanism of action of these new and promising anticancer agents.


Subject(s)
Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , Hydroxamic Acids/chemistry , Iron/chemistry , Oximes/chemistry , Ribonucleotide Reductases/antagonists & inhibitors , Animals , Antineoplastic Agents/toxicity , Cell Division/drug effects , Enzyme Inhibitors/toxicity , Humans , Hydroxamic Acids/toxicity , Iron/pharmacology , Leukemia L1210 , Mice , Oximes/toxicity , Polarography , Tumor Cells, Cultured
15.
Anticancer Res ; 17(5A): 3437-40, 1997.
Article in English | MEDLINE | ID: mdl-9413183

ABSTRACT

The DNA-protective activity of hydroxyurea (HU) and novel ribonucleotide reductase (RR) inhibitors amidox (AX), didox (DX) and trimidox (TX) was examined using hydrogen peroxide as the DNA-damaging agent. The exposure of superspiralized plasmid DNA molecules (pBR 322) to H2O2 under precisely defined in vitro conditions initiates a change in DNA topology (DNA from I relaxes to DNA form II). This electrophoretically monitored change in the plasmid DNA topology is related to the induction of ss-DNA breaks and corresponds with DNA exposition to free radicals. The inhibition of DNA relaxation (the prevention of DNA damage induced by hydrogen peroxide) depended on the free radical scavenging capacity of the drugs investigated. HU exerted DNA protective activity at a concentration of 4 mM, AX at concentration of 1 microM, TX at a concentration of 5 microM and DX at a concentration of 25 microM (the free radical scavenging activity increases from HU to AX in following manner: HU << DX < TX < AX). It can be concluded that the new synthetic RR-inhibitor AX which is being investigated at the preclinical level as a potential anti-cancer drug possess the highest capacity for scavenging of free radicals.


Subject(s)
Ribonucleotide Reductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Benzamidines/pharmacology , DNA Damage/drug effects , DNA, Superhelical/drug effects , Enzyme Inhibitors/pharmacology , Free Radicals , Hydrogen Peroxide , Hydroxamic Acids/pharmacology , Hydroxyurea/pharmacology , Oximes/pharmacology , Plasmids
16.
J Pharm Biomed Anal ; 15(7): 951-6, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9160261

ABSTRACT

Amidox (AX), didox (DX) and trimidox (TX), compounds synthetized as new ribonucleotide reductase inhibitors, have been investigated by ultraviolet (UV) spectrophotometry, polarography and high performance liquid chromatography (HPLC). The experiments have been performed at various pH values. The changes in UV absorption of the compounds studied were recorded and it was demonstrated that these changes are related to the pH and to structural features of the investigated molecules. From the compounds included in our series of experiments, only amidox and trimidox are reduced during polarographic experiments in Britton-Robinson buffer. The reduction of both compounds proceeded in two one-electron steps in acidic pH. One two-electron diffuse irreversible wave was observed at basic pH. The values of the half-wave potential became more negative in accordance with the increasing pH. HPLC assay also showed changes in the retention of compounds investigated, particularly when the pH of the mobile phase was close to the dissociation constant of the particular drug. The changes of physico-chemical properties detected by the all used methods are related to different chemical structures (the most significant changes were observed in alkaline pH).


Subject(s)
Benzamidines/chemistry , Enzyme Inhibitors/chemistry , Hydroxamic Acids/chemistry , Oximes/chemistry , Ribonucleotide Reductases/antagonists & inhibitors , Benzamidines/pharmacology , Chromatography, High Pressure Liquid , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Hydroxamic Acids/pharmacology , Oximes/pharmacology , Polarography , Spectrophotometry, Ultraviolet
17.
Life Sci ; 61(22): 2231-7, 1997.
Article in English | MEDLINE | ID: mdl-9393942

ABSTRACT

Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Didox and amidox are newly synthesized compounds, which inhibit this enzyme and have in vitro and in vivo antitumor activity. We have now investigated the capability of didox and amidox to interfere with the iron metabolism. We show by photometric and polarographic methods, that didox and amidox are capable of forming an iron complex. However, their cytotoxic action cannot be completely circumvented by addition of Fe-ammoniumcitrate, indicating that the iron complexing capacity may not be responsible for the mechanism of action of these compounds. When L1210 leukemia cells were incubated with the didox-iron or amidox-iron complex itself, changes of the 50% growth inhibitory capacity of the complex in comparison with didox or amidox alone could be shown. We conclude, that didox and amidox are capable of forming iron complexes, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Future studies will have to elucidate the molecular mechanism of action of these new and promising anticancer agents.


Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/metabolism , Hydroxamic Acids/pharmacology , Iron Compounds/metabolism , Oximes/metabolism , Oximes/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Animals , Antineoplastic Agents/toxicity , Enzyme Inhibitors/toxicity , Humans , Hydroxamic Acids/toxicity , Leukemia L1210/drug therapy , Leukemia, Erythroblastic, Acute/drug therapy , Mice , Oxidation-Reduction , Oximes/toxicity , Polarography , Tumor Cells, Cultured
18.
Crit Rev Clin Lab Sci ; 34(6): 503-28, 1997.
Article in English | MEDLINE | ID: mdl-9439883

ABSTRACT

Ribonucleotide reductase is the rate-limiting enzyme of DNA synthesis, and it has been shown to be linked with malignant transformation and tumor cell proliferation. It was therefore considered as an excellent target for cancer chemotherapy. This article reviews the in vitro and in vivo effects of hydroxyurea the first inhibitor of the enzyme, which is currently used in general clinical practice. In addition, we summarize the results obtained with other inhibitors of the enzyme; for instance, polyhydroxy-substituted benzohydroxamic acid derivatives, a promising group of inhibitors of ribonucleotide reductase that was synthesized by Bart van'T Riet and investigated by our group. In vitro as well as animal data and pharmacokinetic results are reviewed and possible implications for an improvement in the management of various patient groups are outlined.


Subject(s)
Antineoplastic Agents , Antiviral Agents , Enzyme Inhibitors/therapeutic use , HIV/drug effects , Ribonucleotide Reductases/antagonists & inhibitors , HIV/growth & development , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Hydroxamic Acids/therapeutic use , Hydroxyurea/therapeutic use , Virus Replication/drug effects
19.
Cell Mol Biol (Noisy-le-grand) ; 43(7): 1019-29, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9449534

ABSTRACT

Ribonucleotide reductase inhibitors (RRIs) have been recently shown to inhibit retroviral replication. We examined a new series of RRIs, 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5-trihydroxybenzohydroxamidoxime (Trimidox) for their ability to alter disease progression in murine acquired immunodeficiency syndrome (MAIDS), both alone and in combination with 2',3'-dideoxyinosine (ddI). MAIDS disease was induced by inoculation of female C57BL/6 mice with the LP-BM5 murine leukemia virus (MuLV) and disease progression characterized by extensive peripheral lymphadenopathy and splenomegaly. Efficacy of treatment with these drugs was based upon their ability to influence survival and disease pathophysiology by monitoring the development of splenomegaly. Toxicity was determined by changes in body weight, total peripheral white blood cell count and hematocrit. Didox or trimidox monotherapy was associated with increased survival and decreased disease pathophysiology, with no apparent toxicity. Combined with ddI, their ability to reduce development of viral induced splenomegaly was enhanced compared to trimidox, didox or ddI alone. These results demonstrate RRIs have potent activity in reversing the disease manifestations characteristic of MAIDS. Further studies are warranted to determine human clinical efficacy.


Subject(s)
Antiviral Agents/pharmacology , Murine Acquired Immunodeficiency Syndrome/drug therapy , Ribonucleotide Reductases/antagonists & inhibitors , Ribonucleotide Reductases/pharmacology , Animals , Benzamidines/pharmacology , Biomarkers , Didanosine/pharmacology , Disease Progression , Drug Therapy, Combination , Female , Hydroxamic Acids/pharmacology , Leukemia Virus, Murine/drug effects , Mice , Mice, Inbred C57BL , Murine Acquired Immunodeficiency Syndrome/mortality , Murine Acquired Immunodeficiency Syndrome/physiopathology , Murine Acquired Immunodeficiency Syndrome/virology , Survival Rate
20.
Eur J Clin Chem Clin Biochem ; 33(11): 785-9, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8620054

ABSTRACT

Ribonucleotide reductase is the rate limiting enzyme of deoxynucleoside triphosphate synthesis and is considered to be an excellent target of cancer chemotherapy. Trimidox, a newly synthesized compound, inhibits this enzyme and has in vitro and in vivo antitumour activity. As trimidox was able to upregulate the expression of the transferrin receptor in HL-60 human promyelocytic leukaemia cells, we have now investigated the capability of trimidox to interfere with iron metabolism. We show by photometric and polarographic methods that trimidox is able for form an iron complex. However, its cytotoxic action cannot be circumvented by addition of iron-saturated transferrin or iron-ammonium citrate, indicating that the iron complexing capacity is not responsible for the mechanism of action of this compound. When HL-60, K562 or L1210 leukaemia cells were incubated with the trimidox-iron complex itself, we could observe increases of the 50% growth inhibitory capacity of the complex in comparison with trimidox alone. We conclude that trimidox is able to form an iron complex, but in contrast to other agents, the anticancer activity cannot be contributed to this effect alone. Further studies will have to elucidate the molecular mechanism of action of this new and promising anticancer agent.


Subject(s)
Benzamidines/chemistry , Benzamidines/toxicity , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/toxicity , Iron , Ribonucleotide Reductases/antagonists & inhibitors , Cell Division/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , HL-60 Cells , Humans , Iron/pharmacology , Kinetics , Oxidation-Reduction , Polarography
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