Effect of methotrexate and 5-fluorodeoxyuridine on ribonucleotide reductase activity in mammalian cells.

A number of studies in bacteria have indicated that deoxythymidine 5'-triphosphate may be a repressor or corepressor of ribonucleotide reductase. For determination of whether a similar regulating mechanism exists in mammalian cells, HeLa cells and partially hepatectomized rats were treated with either methotrexate, 5-fluorouracil, or 5-fluorodeoxyuridine in order to block thymidylate synthesis and consequently lower the intracellular pools of deoxythymidine 5'-triphosphate. In HeLa cells there was a significant (360 to 400 percent) increase in reductase activity in both the methotrexate and 5-fluorodeoxyuridine-treated cells. The administration of methotrexate to partially hepatectomized rats resulted in a 2.7-fold enhancement of the hepatectomy-induced increase in reductase activity, and the 5-fluorouracil treatment yielded a 60 percent increment in the increase of ribonucleotide reductase activity after partial hepatectomy. Cycloheximide prevented the increase in reductase activity after the exposure of HeLa cells to methotrexate and 5-fluorodeoxyuridine, indicating that the stimulation of ribonucleotide reductase activity was the result of enhancement of de novo enzyme synthesis rather than of enzyme activation. The data support the thesis that deoxythymidine 5'-triphosphate or a thymidylate metabolite may be involved in the regulation of ribonucleotide reductase levels in mammalian cells.

Trimidox, an inhibitor of ribonucleotide reductase, induces apoptosis and activates caspases in HL-60 promyelocytic leukemia cells.

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.

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. 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.

Synthesis of hydroxy- and amino-substituted benzohydroxamic acids: inhibition of ribonucleotide reductase and antitumor activity.

Benzohydroxamic acids inhibit mammalian ribonucleotide reductase and exhibit antineoplastic activity in L1210 leukemic mice. Five new hydroxy- and amino-substituted benzohydroxamic acids (3,4- and 3,5-OH, 3,4-NH2, 2,3,4- and, 3,4,5-OH) were prepared and tested along with 12 previously reported benzohydroxamic acids (BHA) for enzyme inhibition and antitumor activity. The most potent enzyme inhibitor in this series was 2,3,4-OH-BHA (ID50 = 3.5 microM), which is 140 times more potent than hydroxyurea, but its toxicity limited the antitumor activity to a 30% increase in life span of L1210 bearing mice at 125 (mg/kg)/day ip for 8 days. The most effective antitumor agent in this series was 3,4-OH-BHA which prolonged the life span of L1210 bearing mice 103% at 600 (mg/kg)/day ip for 8 days.

Interaction of gallium nitrate with other inhibitors of ribonucleotide reductase: effects on the proliferation of human leukemic cells.

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.

Biochemical and antitumor activity of trimidox, a new inhibitor of ribonucleotide reductase.

Trimidox (3,4,5-trihydroxybenzamidoxime), a newly synthesized analog of didox (N,3,4-trihydroxybenzamide) reduced the activity of ribonucleotide reductase (EC 1.17.4.1) in extracts of L1210 cells by 50% (50% growth-inhibitory concentration, IC50) at 5 microM, whereas hydroxyurea, the only ribonucleotide reductase inhibitor in clinical use, exhibited an IC50 of 500 microM. Ribonucleotide reductase activity was also measured in situ by incubating L1210 cells for 24 h with trimidox at 7.5 microM, a concentration that inhibits cell proliferation by 50% (IC50) or at 100 microM for 2 h; these concentrations resulted in a decrease in enzyme activity to 22% and 50% of the control value, respectively. Trimidox and hydroxyurea were cytotoxic to L1210 cells with IC50 values of 7.5 and 50 microM, respectively. Versus ribonucleotide reductase, trimidox and hydroxyurea yielded IC50 values of 12 and 87 microM, respectively. A dose-dependent increase in life span was observed in mice bearing intraperitoneally transplanted L1210 tumors. Trimidox treatment (200 mg/kg; q1dx9) significantly increased the life span of mice bearing L1210 leukemia (by 82% in male mice and 112% in female mice). The anti-tumor activity appeared more pronounced in female mice than in male mice. Viewed in concert, these findings suggest that trimidox is a new and potent inhibitor of ribonucleotide reductase and that it is a promising candidate for the chemotherapy of cancer in humans.

Studies on the mechanisms of inhibition of L1210 cell growth by 3,4-dihydroxybenzohydroxamic acid and 3,4-dihydroxybenzamidoxime.

Didox and Amidox inhibit L1210 cell growth in culture. At least one of the mechanisms in the mode(s) of action of the compounds is directed at the ribonucleotide reductase site. Partially purified preparations of ribonucleotide reductase activity are inhibited by Amidox and Didox. The formation of deoxycytidine nucleotides from [14C]cytidine in intact L1210 cells is also blocked. Didox and Amidox cause the decrease in the intracellular pools of the four dNTPs. Hydroxyurea-resistant L1210 cells are not cross-resistant to either Didox or Amidox. These data suggest that Didox and Amidox are not inhibiting ribonucleotide reductase through a mechanism similar to hydroxyurea.

Short-term treatment with novel ribonucleotide reductase inhibitors Trimidox and Didox reverses late-stage murine retrovirus-induced lymphoproliferative disease with less bone marrow toxicity than hydroxyurea.

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.

Iron binding capacity of didox (3,4-dihydroxybenzohydroxamic acid) and amidox (3,4-dihydroxybenzamidoxime) new inhibitors of the enzyme ribonucleotide reductase.

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.

The ribonucleotide reductase inhibitor trimidox induces c-myc and apoptosis of human ovarian carcinoma cells.

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.

Enhanced effects of adriamycin by combination with a new ribonucleotide reductase inhibitor, trimidox, in murine leukemia.

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.

DNA-protective activity of new ribonucleotide reductase inhibitors.

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.

Structure-activity relationships of benzohydroxamic acid inhibitors of ribonucleotide reductase.

A structure-activity relationship study of 28 substituted benzohydroxamic acids that inhibit ribonucleotide reductase was undertaken to discern the structural features of the molecule contributing to the inhibitory potency of these compounds. An equation containing three molecular connectivity indexes, but not including Hammett sigma values, was developed which gives close correlation with observed values for ribonucleotide reductase inhibition. It is postulated that the inhibitory potency involves two parts of the benzohydroxamic acid molecule. One is the hydroxamic portion, which complexes with the metal component of the enzyme, providing a qualitative effect. The other is an interaction involving the benzene ring and its substituents and may provide the quantitative aspect of the observed inhibition values.

The new inhibitors of ribonucleotide reductase--comparison of some physico-chemical properties.

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).

Iron binding capacity of didox (3,4 dihydroxybenzohydroxamic acid) and amidox (3,4 dihydroxybenzamidoxime) two inhibitors of the enzyme ribonucleotide reductase.

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.