|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
RED CELLS
From the Heart Research Institute, The Iron Metabolism
and Chelation Group, Camperdown, Sydney, New South Wales, Australia.
We previously demonstrated that
2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311)
and other aroylhydrazone chelators possess potent antineoplastic
activity because of their ability to bind iron (Fe). From these
studies, we identified structural components of the hydrazones that
provide antineoplastic activity, namely the salicylaldehyde and
2-hydroxy-1-naphthylaldehyde moieties. A related group of chelators
known as the thiosemicarbazones also show pronounced antitumor activity
because of their ability to inhibit ribonucleotide reductase.
Considering this, we designed a new series of "hybrid ligands" by
condensation of the aldehydes described above with a range of
thiosemicarbazides. The parent compound of these ligands is
2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT). Of 8 NT
analogues, 3 chelators, namely NT, N4mT
(2-hydroxy-1-naphthylaldehyde-4-methyl-3-thiosemicarbazone), and
N44mT
(2-hydroxy-1-naphthylaldehyde-4,4-dimethyl-3-thiosemicarbazone), showed high antiproliferative activity against SK-N-MC neuroepithelioma cells (50% inhibitory concentration
[IC50] = 0.5-1.5 µM). Indeed, their activity was
significantly (P < .0001) greater than that of
desferrioxamine (DFO) (IC50 = 22 µM). We demonstrate
that 311, a 311 analogue (311m), and several NT-series chelators have
significantly (P < .001) greater antiproliferative
activity against tumor cells than against a range of normal cell types.
For example, the IC50 values of NT and N4mT in SK-N-MC
neuroepithelioma cells were 0.5 µM, whereas for fibroblasts
the IC50 values were greater than 25 µM. Further, the
effect of one of the most potent chelators (311m) on preventing the
growth of bone marrow stem cell cultures was far less than that of
doxorubicin and similar to that of cisplatin. These studies support the
further development of these chelators as antiproliferative agents.
(Blood. 2002;100:666-676) Iron (Fe) plays a critical role in a variety of
metabolic processes, as Fe-containing proteins catalyze key reactions
involved in energy production and DNA synthesis (eg, ribonucleotide
reductase [RR]).1,2 In the absence of Fe, cells cannot
proceed from the G1 to the S phase of the cell
cycle.1-4 Further, RR is the rate-limiting step in DNA
synthesis, and the fact that Fe is critical for RR activity means it is
an important target for antitumor drugs.5-7
Neuroblastoma (NB) is an aggressive childhood tumor with a poor
prognosis, and new therapies are urgently required.8
Interestingly, NB cells from patients with advanced disease contain
increased amounts of ferritin rich in Fe.9,10 Evidence of
a sensitive relationship between Fe and NB cell growth is suggested by
the observation that the chelator desferrioxamine (DFO) is capable of a
cytotoxic effect on NB cells in vitro while having little effect on
other cells.11,12 Indeed, clinical trials with DFO in
patients with NB and leukemia have shown significant
results.13-17 Despite the link between Fe metabolism and
the antineoplastic effect of DFO and other Fe chelators,2
little is known about the molecular targets of Fe chelators or the
structure-activity relationships involved.
We characterized chelators known as the pyridoxal isonicotinoyl
hydrazone (PIH) analogues.18-24 Some of these (eg, 311)
show much higher antiproliferative activity against tumor cells than DFO.21,24-27 After screening 36 PIH analogues, we
identified structural characteristics of these Fe chelators that result
in antiproliferative activity.21,24 We found that
analogues derived from pyridoxal possess high Fe-chelation activity and
low antiproliferative effects, properties of ligands suitable to treat
Fe overload.21 In contrast, analogues derived from
salicylaldehyde and particularly 2-hydroxy-1-naphthylaldehyde possess
pronounced antiproliferative activity plus high Fe-chelation efficacy,
characteristics of ligands suitable to treat
neoplasia.21,24
Intriguingly, a series of chelators related to PIH known as the
thiosemicarbazones (eg, 3-aminopyridine-2-carboxaldehyde
thiosemicarbazone, Triapine; Vion Pharmaceuticals Inc, New Haven,
CT) have been described as the most effective inhibitors
of RR yet identified.28-31 We showed that the
2-pyridyl component of these latter ligands does not confer
antineoplastic activity on our PIH analogues.32
Considering this, we believe that the other half of the molecule, that
is, the thiosemicarbazide moiety
(NH2-CS-NH-NH2), could mediate
antiproliferative activity. Therefore, condensation of
2-hydroxy-1-naphthylaldehyde with thiosemicarbazide to form
2-hydroxy-1-naphthylaldehyde thiosemicarbazone (NT; Figure
1) may result in a highly active
chelator.
In this study, we designed a novel class of NT analogues that
incorporate features of the thiosemicarbazones into the structural components of the most effective PIH analogues
identified.21,24 Hence, we condensed salicylaldehyde or
2-hydroxy-1-naphthylaldehyde with a range of thiosemicarbazides,
resulting in chelators with a wide range of lipophilicities. It was
important to vary this latter factor because of its relationship to
Fe-chelation efficacy and antiproliferative
activity.19,21,33,34
The characterization of novel ligands is important in terms of
obtaining the patent protection necessary for development by the
pharmaceutical industry. Previous studies with 311 were reported without patent protection,21,24 and this obstructed its
development. Hence, in addition to the NT analogues, we also assessed
the activity of 3 new 311 analogues, known as
2-hydroxy-1-naphthylaldehyde nicotinoyl hydrazone (311m),
2-hydroxy-1-naphthylaldehyde 4,4-diphenylhydrazone (N44pH), and
2-hydroxy-1-naphthylaldehyde octylhydrazone (NoctH) (Figure 1B).
We show that the combination of 2-hydroxy-1-naphthylaldehyde with
various thiosemicarbazides results in novel tridentate "hybrid chelators" known as the NT series, which have significantly greater antiproliferative activity than DFO. Importantly, the antiproliferative activity is relatively selective for tumor cells, the mechanism of
action being due to the chelation of intracellular Fe. Interestingly, the Fe complexes of NT and N4mT appeared to have some redox activity, although this is not as marked as that observed for other
thiosemicarbazones. In summary, we have identified 3 novel hybrid
chelators (NT, N4mT, and N44mT) and a 311 analogue (311m) that warrant
further investigation as suitable candidates for cancer chemotherapy.
Synthesis and characterization of iron chelators and their
preparation for screening in culture
Cell culture
Preparation of 59Fe-transferrin Apotransferrin (Sigma, St Louis, MO) was labeled with 59Fe (Dupont NEN, Boston, MA) to produce 59Fe-transferrin (59Fe-Tf).40,41Effect of chelators on cellular proliferation and [3H]thymidine incorporation The effects of chelators on proliferation and DNA synthesis were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay21 and [3H]thymidine incorporation,24 respectively.Iron uptake and iron efflux experiments The effects of chelators on 59Fe uptake from 59Fe-Tf and 59Fe release from prelabeled cells were studied using standard procedures.21,24,40-42Effect of the chelators at binding 59Fe from 59Fe-transferrin The ability of the chelators to directly remove 59Fe from 59Fe-Tf was analyzed by dialysis studies.19Northern and Western blot analyses Northern and Western blot analyses were performed using established methods in our laboratory.25,27Ascorbate oxidation and benzoate hydroxylation assay These assays were performed using standard procedures (T. Chaston, D.B.L., R. N. Watts, D.R.R., manuscript submitted, Dean and Nicholson,43 and Gutteridge44).Measurement of DNA integrity using the plasmid pGEM-7Zf(±) in the presence of Fe and the chelator Escherichia coli (DH-5 ) was transformed with
pGEM-7Zf(+) (Promega, Madison, WI) and grown in LB medium. Plasmid DNA
was then purified using the Qiagen plasmid purification kit (Qiagen,
Hilden, Germany). Reagents were added in the following order: purified sterile water, chelator (1, 10, and 30 µM), FeSO4 (10 µM), H2O2 (1 mM), and plasmid DNA (10 µg/mL).45 Samples were incubated at room temperature for
30 minutes and then loaded onto a 1% agarose gel.45
For the ascorbate oxidation, benzoate hydroxylation, and plasmid DNA strand-break assays, we used the term "iron-binding equivalents" (IBEs) to express our data. We did this because of the different coordination modes of the ligands to Fe (ie, DFO and EDTA are hexadentate and form 1:1 ligand-Fe complexes, whereas 311, Triapine, and NT and its analogues are tridentate, resulting in 2:1 ligand-Fe complexes). In this study, a range of ligand-Fe IBE ratios were used, namely 0.1, 1, or 3. An IBE of 1 is equivalent to the complete filling of the coordination shell of the Fe atom by the ligand(s). Thus, for a hexadentate chelator (ie, DFO or EDTA), an IBE ratio of 1 represents 1 ligand to 1 Fe atom, whereas for a tridentate chelator (ie, Triapine, NT, or 311), it is equal to 2 ligands to 1 Fe atom. An IBE of 0.1 represents an excess of Fe to chelator (ie, 1 hexadentate chelator or 2 tridentate chelators in the presence of 10 Fe atoms). An IBE of 3 represents an excess of chelator to Fe and is equal to 3 hexadentate chelators or 6 tridentate chelators in the presence of 1 Fe atom. Statistics Data were compared using Student paired t test. Results were considered statistically significant when P < .05.
The effect of the chelators on the proliferation of neoplastic cells Initially, the ability of the ligands to inhibit proliferation was assessed in SK-N-MC neuroepithelioma cells (Figure 2; Table 1). These studies identified 311m, NT, and N4mT as having very high antiproliferative activity (50% inhibitory concentration [IC50] = 0.3-0.5 µM; Table 1). These ligands were significantly (P < .0001) more active than DFO (IC50 = 22 µM) and had efficacy comparable to 311 (IC50 = 0.3 µM; Table 1). Like the Fe complexes of DFO and 311,25,26 the NT-Fe complex was not very active at inhibiting SK-N-MC cell proliferation (IC50 > 12.5 µM; Figure 2; Table 1). This suggests that the metal ion binding activity of this ligand plays a role in its activity. Other chelators with appreciable activity against SK-N-MC cells included N44mT and N4eT, which had IC50 values of 1.5 and 1.6 µM, respectively (Table 1).
To investigate the spectrum of antineoplastic activity, we assessed the most active analogues against SK-N-MC cells (311m, NT, N4mT, and N44mT), K562 erythroleukemia cells, SK-Mel-28 melanoma cells, and MCF-7 breast cancer cell lines (Table 1). These ligands were far more active than DFO at inhibiting proliferation of K562 and MCF-7 cells (Table 1). Against SK-N-MC cells, NT and N4mT were more active (IC50 = 0.5 µM) than N44mT (IC50 = 1.5 µM; Table 1). However, of these chelators, N44mT was most active against K562, SK-Mel-28, and MCF-7 cells (Table 1). Significantly, 311m demonstrated marked activity against all neoplastic cells (Table 1). The chelators have less effect on the proliferation of normal cell types than neoplastic cells Clinically useful antitumor agents have a significant therapeutic index (ie, they have little effect on normal cells while inhibiting neoplastic cell growth). Hence, it was important to compare the antiproliferative effects of the most active chelators (311, 311m, NT, N4mT, and N44mT) between a range of neoplastic and normal cell types (Figure 3; Table 1). In these experiments, N2mT was included as a negative control because the methyl group at the 2-position (Figure 1) hinders electron delocalization and metal binding. These studies showed that in contrast to SK-N-MC cells, in which 311, 311m, NT, N4mT, and N44mT resulted in a potent antiproliferative effect (IC50 = 0.3-1.5 µM), these chelators had relatively little effect on fibroblast proliferation, with the IC50 values being greater than 25 µM (Figure 3; Table 1). The difference in the antitumor effect may be due to the lower rate of proliferation of the MRC-5 fibroblasts (doubling time, 22 hours) compared with the SK-N-MC neuroepithelioma cells (doubling time, 16 hours). For the most active chelators examined (311, 311m, NT, N4mT, and N44mT), their ability to inhibit growth was most pronounced for the neoplastic cells as compared with normal cell types. Of the normal cells examined, MRC-5 fibroblasts were the least sensitive to the action of the 5 chelators, whereas the more rapidly proliferating HUVEC cultures were the most sensitive (Table 1). Despite the similar replicative rates of HUVEC and SK-N-MC cells, the latter tumor cells were more sensitive to chelators. Comparing the 4 neoplastic cell types assessed, we found that SK-N-MC neuroepithelioma cells were the most sensitive and MCF-7 breast cancer cells were least affected.
Further studies examined the effect of one of the most potent
antiproliferative agents identified in this study, namely 311m, on the
proliferation of GM colonies of normal human bone marrow over 14 days.
As relevant controls, the effects of the cytotoxic drugs cisplatin and
doxorubicin were compared with 311m (Figure 4). The ability of 311m to inhibit
proliferation of GM colonies was far less marked than that of
doxorubicin and similar to that of cisplatin (Figure 4).
The relationship between lipophilicity and antiproliferative activity of the NT series To assess the relationship between lipophilicity and antiproliferative activity, we plotted average calculated log P values (n-octanol-water partition coefficients) against IC50 values in SK-N-MC cells (Figure 5). Log P values were calculated (Table 2) by the procedures of Broto et al,46 Ghose and Crippen,47 and Viswanadhan et al48 using Chem Draw Pro (v. 4.5, Cambridge Software, 1997). The means of these values (average calculated log P values; Table 2) showed a strong linear correlation with antiproliferative activity (r = 0.95; Figure 5) if NoctH was excluded. Possible reasons why NoctH did not fit the correlation may relate to structural differences relative to other analogues, namely, only NoctH has a highly lipophilic carbon "tail" (Figure 1B).
Our investigation identified a significant chelator structure-activity relationship dependent on lipophilicity. This was demonstrated by N4mT and N44mT, which have 1 and 2 methyl groups at N4 (Figure 1) and have IC50 values of 0.5 and 1.5 µM, respectively, in SK-N-MC cells. In addition, N4pT and N44pH (both more lipophilic than N4mT and N44mT; Table 2) have 1 and 2 phenyl groups at N4 and have IC50 values of 3.3 and 5.2 µM, respectively. Hence, for the NT series, antiproliferative activity against SK-N-MC cells decreased when the substituents on the terminal nitrogen atom of the thiosemicarbazide (N4; Figure 1A) became increasingly lipophilic. The effect of the chelators on iron efflux from SK-N-MC neuroepithelioma cells To determine the role of Fe chelation in the antiproliferative effects observed, we examined the ability of the ligands to mobilize 59Fe from SK-N-MC cells. Cells were prelabeled for 3 hours at 37°C with 59Fe-Tf, washed, and then reincubated for 3 hours at 37°C with the chelators (Figure 6A-B). As internal standards, DFO and 311 were used because their activities are well characterized.20,21,24,25
Broadly, the ligands can be grouped into 2 classes depending upon their ability to mobilize 59Fe. The first group has activity similar to DFO and includes ST, NT, and N2mT, which mobilize 3% to 13% of total cellular 59Fe (Figure 6A). The second group includes the remaining NT analogues, which are significantly (P < .0001) more effective than DFO, resulting in the release of 28% to 43% of cellular 59Fe (Figure 6A). Of this latter group, N44mT is the most efficient, having activity comparable to 311 and 311m and resulting in the release of 43% of 59Fe (Figure 6A). The parent analogue, NT, mobilized only 13% of cellular 59Fe and was markedly less efficient than the other NT analogues (Figure 6A). The 59Fe mobilization observed for DFO and 311 correlated with that in our previous studies.21,24 Because NT has high antiproliferative efficacy (Figure 2; Table 1) but low activity at mobilizing 59Fe from cells (Figure 6A), time-course experiments were performed to understand its mechanism of action (Figure 6B). In these studies, N44mT was also assessed because it has high activity at inhibiting proliferation and mobilizing 59Fe. These results with NT and N44mT were compared with those of 311 and DFO. Our experiments showed that DFO and NT behaved similarly, both increasing 59Fe efflux from the SK-N-MC cells as a function of time, being far less effective than N44mT and 311 (Figure 6B). These data suggest that NT and/or its Fe complex may be subject to similar permeability limitations as DFO, which forms a hydrophilic Fe complex that does not exit cells easily.49,50 The effect of the chelators on iron uptake from transferrin by SK-N-MC neuroepithelioma cells To further characterize the effects of the chelators on Fe metabolism, we examined the ability of the ligands to inhibit 59Fe uptake from 59Fe-Tf (0.75 µM) by SK-N-MC cells in the absence or presence of the chelators (25 µM) for 3 hours at 37°C (Figure 7A-B). In agreement with their lower ability to mobilize 59Fe, ST, NT, and N2mT had little effect at preventing 59Fe uptake (Figure 7A). The most efficient chelators, namely 311, 311m, and N44mT, limited 59Fe uptake to 5% to 9% of the control level, their activity being significantly greater (P < .0001) than that of DFO (Figure 7A). These results agree with the ability of these ligands to mobilize cellular 59Fe (Figure 6A). It is significant that all analogues (except ST, NT, and N2mT) showed much greater efficacy than DFO in preventing 59Fe uptake from 59Fe-Tf by SK-N-MC cells (Figure 7A).
We examined whether the ability of the chelators to prevent 59Fe uptake from 59Fe-Tf (Figure 7A) was due to direct removal of 59Fe from the protein. The most effective chelators at inhibiting proliferation, namely 311, 311m, NT, N4mT, and N44mT, were ineffective at removing 59Fe from Tf over the duration of an uptake experiment (3 hours), resulting in the release of 0.1% to 0.3% of the bound 59Fe, whereas DFO removed 0.7% (data not shown). These results demonstrate that the chelators have very little effect on direct 59Fe release from 59Fe-Tf, as found for other aroylhydrazones.19 Further, these data suggest that the ligands inhibit 59Fe uptake only after intracellular delivery from Tf. Considering the limited permeability of NT and/or its Fe complex in kinetic studies assessing 59Fe mobilization (Figure 6B), we conducted time-course experiments examining the effects of NT on 59Fe uptake from 59Fe-Tf. These results were compared with those of the most effective NT analogue, N44mT, and the standards 311 and DFO (Figure 7B). These experiments again showed that NT and/or its Fe complex may be subject to the same permeability limitations as DFO, as both chelators only moderately reduced 59Fe uptake from 59Fe-Tf, even for incubations of 24 hours (Figure 7B). For example, after a 6-hour incubation, NT and DFO reduced 59Fe uptake by SK-N-MC cells to 82% and 85% of the control, respectively. In contrast, after 24 hours, NT and DFO reduced 59Fe uptake to 61% and 48% of the control, respectively (Figure 7B). The relationship between iron-chelation efficacy and antiproliferative activity of the NT series in SK-N-MC neuroepithelioma cells It was of interest to explore the relationship between Fe-chelation efficacy (Figures 6-7) and antiproliferative activity (Table 1; Figure 2). Chelators N4mT and N44mT have high chelation activity (Figures 6-7) and antiproliferative activity (IC50 = 0.5 and 1.5 µM, respectively; Table 1). However, N44pH and NoctH also demonstrate high Fe-chelation efficacy (Figures 6-7), but have much lower antiproliferative activity (IC50 = 5.2 and 10.5 µM, respectively; Table 1). Additionally, NT has marked antiproliferative effects (Table 1) but has low Fe-chelation efficacy (Figures 6-7). The lack of correlation between antiproliferative activity and Fe-chelation efficacy may reflect differences in the Fe pools targeted by these chelators and/or the relative efficacies of the ligand or its Fe complex to permeate membranes.The effect of the chelators on [3H]thymidine incorporation by SK-N-MC neuroepithelioma cells Because chelators can inhibit RR via Fe depletion,2 the effect of the NT analogues on DNA synthesis in SK-N-MC cells was examined (Figure 8; Table 3). Of the NT analogues, the most active inhibitor of DNA synthesis was N44mT (IC50 = 1.5 µM), which had significantly higher activity (P < .0001) than DFO (IC50 = 23 µM) and slightly less effect than 311 (IC50 = 0.6 µM; Figure 8; Table 3). N44mT also showed the highest Fe-chelation efficacy of the NT series in terms of increasing 59Fe mobilization (Figure 6A-B) and inhibiting 59Fe uptake (Figure 7A-B). On the other hand, ST, NT, and N2mT were among the least effective inhibitors of DNA synthesis (Figure 8; Table 3) and had relatively low Fe-chelation efficacy (Figures 6-7). However, overall, there was a very weak relationship between inhibiting DNA synthesis and either preventing 59Fe uptake from 59Fe-Tf (r = 0.48) or increasing 59Fe mobilization from prelabeled cells (r = 0.32). For example, in terms of inhibiting DNA synthesis, chelator NoctH was much less active (IC50 = 17.4 µM; Table 3) than N4pT (IC50 = 3.8 µM; Table 3), even though the ligands have comparable Fe-chelation efficacy (Figures 6-7). When the relationship between antiproliferative activity (Table 1) and [3H]thymidine incorporation (Table 3) was examined, a weak linear relationship was found (r = 0.54).
The effect of the chelators on the expression of TfR and cell-cycle control molecules Low concentrations of 311 (25 µM) and much higher concentrations of DFO (150 µM) cause SK-N-MC cells to up-regulate TfR and GADD45 mRNA levels.25,27 The TfR plays a crucial role in Fe uptake,1,2 whereas GADD45 plays a role in cell-cycle arrest. Hence, it was of interest to assess whether the most active NT analogues also up-regulate these genes. In line with earlier studies,25,27 DFO (150 µM) and 311 (25 µM) up-regulated TfR and GADD45 mRNA levels (Figure 9). Of the NT series, N44mT (25 µM) markedly up-regulated TfR and GADD45 mRNA levels, whereas NT, N4mT, and particularly N2mT showed less activity. The NT-Fe complex did not increase TfR or GADD45 mRNA levels (Figure 9).
Because Fe deprivation causes G1/S arrest,2 we
assessed changes in the expression of cell-cycle control molecules that play roles in G1/S progression. Hence, we examined the most
cytotoxic NT analogues, namely NT and N4mT, compared with the known
effects of 31127 on the protein levels of key cyclins
(cyclins A, E, D1, D2, and D3) and cyclin-dependent kinases (cdk2 and
cdk4). Hyperphosphorylation of the retinoblastoma gene susceptibility product (pRb) is essential for G1/S progression, and it is
known that D-type cyclins bind cdk4 and/or cdk2 to phosphorylate
pRb.51-54 In line with previous studies,27
DFO (150 µM) and 311 (25 µM) caused a marked decrease in the
expression of cyclins D1, D2, and D3 and also cdk2 in SK-N-MC cells
(Figure 10A-B). Both NT (25 µM) and
N4mT (25 µM) also caused a marked decrease in the expression of these
cell-cycle control molecules, but were slightly less effective
than 311 (Figure 10A-B). Relative to the control, all chelators had
little effect on cdk4 and cyclin-A levels, whereas 311 and NT (25 µM)
increased cyclin E (Figure 10A-B). These latter results may be due to
cell-cycle dysregulation induced by the chelator.27
The effects of the chelators on iron-mediated free-radical damage Doxorubicin and bleomycin partly mediate their antitumor effects by forming metal ion complexes that generate free radicals. We used the ascorbate oxidation, benzoate hydroxylation, and plasmid DNA cleavage assays to assess the redox activity of the Fe complexes of the chelators with the greatest antiproliferative activity in SK-N-MC cells, namely 311m, NT, and N4mT. The redox-active Fe complexes of EDTA and Triapine were used as controls because their effects are well characterized.40,55,56Ascorbate oxidation.
The ability of the Fe complexes of the ligands to reduce Fe(III) as
determined by ascorbate oxidation is shown in Figure
11A. EDTA promoted oxidation of
ascorbate at all ligand-Fe(III) ratios (expressed as IBEs; see
"Materials and methods"). At IBE ratios of 0.1, 1, and 3, EDTA
increased ascorbate oxidation to 145%, 382%, and 376% of the
control, respectively (Figure 11A). In contrast, 311m, NT, and N4mT at
IBE ratios of 1 and 3 were protective against ascorbate oxidation
(Figure 11A). For instance, at an IBE ratio of 3, these chelators
reduced ascorbate oxidation to 19%, 33%, and 77% of the control,
respectively (Figure 11A). Triapine potentiated ascorbate oxidation at
all IBE ratios (Figure 11A).
Benzoate hydroxylation. This assay is based on the ability of hydroxyl radicals to hydroxylate benzoate to fluorescent products (308 nm excitation and 410 nm emission) (T. Chaston, D.B.L., R. N. Watts, D.R.R., manuscript submitted, Dean and Nicholson,43 and Gutteridge44). Previous studies showed that the EDTA-Fe complex increases benzoate hydroxylation.43 At IBE ratios of 0.1, 1, and 3, EDTA increased benzoate hydroxylation to 159%, 341%, and 358% of the control, respectively (Figure 11B). Chelator 311m was mildly protective at an IBE ratio of 3, at which it reduced benzoate hydroxylation to 73% of the control (Figure 11B). NT had little effect on benzoate hydroxylation, whereas N4mT elevated it to 157% of the control (Figure 11B). In contrast, Triapine greatly increased benzoate hydroxylation to 530% and 683% of the control at IBE ratios of 1 and 3, respectively (Figure 11B). Plasmid DNA integrity.
In these studies, we examined the ability of chelators to prevent
Fe-mediated hydroxyl-radical damage to plasmid DNA.45 Untreated plasmid and plasmid treated with H2O2
appeared on gels as a single supercoiled (SC) DNA band (Figure
12). As another control, plasmid
treated with BamHI was included, resulting in a single band
of linearized DNA (Figure 12). When plasmid was treated with Fe(II) and
H2O2, SC DNA was partially converted to the
open circular (OC) form (Figure 12).
Although EDTA was redox active in the ascorbate oxidation (Figure 11A) and benzoate hydroxylation assays (Figure 11B), this chelator was protective of SC DNA at IBE ratios of 1 and 3. This agrees with the well-characterized protective effects of EDTA against Fe-mediated DNA damage. However, 311m was not protective of SC DNA in the presence of Fe(II) and H2O2, resulting in the formation of OC DNA and also linearized DNA, particularly at IBE ratios of 1 and 3 (Figure 12). These findings were observed even though 311m was redox inactive in the ascorbate oxidation and benzoate hydroxylation assays (Figure 11A-B). The reason for this inconsistency may be that the PIH class of chelators has low affinity for Fe(II).57 Additionally, differences in the interactions of 311m and/or Fe(II) with benzoate and DNA may contribute to this discrepancy. The Fe complexes of NT and N4mT at an IBE of 0.1 showed effects on SC DNA that were similar to those of the Fe complex of 311m (Figure 12). However, at IBE ratios of 1 and 3, N4mT and particularly NT caused plasmid degradation. This latter effect was pronounced for NT at an IBE of 3 (Figure 12). Triapine was more aggressive than NT, completely degrading plasmid DNA at IBE ratios of 1 and 3 (Figure 12).
New strategies for selectively inhibiting cancer cell growth are vital, especially considering that some tumors become resistant to conventional chemotherapy. Considering this, many studies using cell culture, animal models, and clinical trials have shown that tumors are sensitive to Fe-chelation therapy using DFO.2,11-17 However, DFO has serious disadvantages, including its requirement for long infusions, its short plasma half-life, and its poor permeability, which leads to poor antitumor activity.58,59 Hence, it is essential to examine new chelators with greater activity. We have designed novel "hybrid" Fe chelators known as the NT series. These ligands incorporate structural elements of the highly active thiosemicarbazones28-31 with the features of the most effective PIH analogues.21,24 Our current studies have identified new ligands that show selective antitumor activity that is much greater than that of DFO. These results confirm our design strategy and provide insights into the structural requirements for optimal antitumor activity. In contrast to previous chelators of the PIH class, our new ligands are the subject of a patent application. This is important in terms of characterizing these agents as antitumor drugs because pharmaceutical development will not proceed without patent protection. One problem with all cytotoxic agents for cancer treatment is the lack of selectivity against tumor cells as opposed to normal cells. The use of Fe as a target is a relatively new therapeutic strategy that has shown promising results, even using DFO, which is not highly membrane permeable.11-17 More permeable lipophilic chelators that rapidly inhibit RR show more promise. Indeed, Triapine is already in phase II clinical trials and demonstrates high activity and appreciable selectivity.30,31 Some of our ligands are more effective chelators than Triapine,41 and we have shown that 311 is a potent RR inhibitor that can overcome hydroxyurea resistance in vitro.60 Significantly, the most cytotoxic NT analogues against SK-N-MC neuroepithelioma cells, namely NT and N4mT, showed selective antitumor activity, having significantly less effect on a range of normal cells (Table 1). However, although these compounds showed high activity against neuroepithelioma cells, less efficacy was observed in other tumor cell types (Table 1). These results indicate that NT and N4mT may have more potential in the management of neuroepithelioma and related tumors. Although N44mT showed slightly less antiproliferative activity against SK-N-MC cells than NT or N4mT, this ligand displayed high efficacy against K562 leukemia and SK-Mel-28 melanoma cells. In addition to these "hybrid" ligands, we also identified the 311 analogue 311m, which shows high antitumor activity against neoplastic cells and significantly less activity against normal cells (Table 1). The basis of selectivity for the chelators is that rapidly
growing tumor cells have a higher Fe requirement for DNA synthesis and
metabolism than more slowly growing normal cells. Indeed, the more
slowly growing fibroblasts (doubling time, 22 hours) were far less
affected than the more rapidly growing SK-N-MC neuroepithelioma cells (doubling time, 16 hours) (Table 1). Considering the effect on
normal cells, it was significant that one of the most potent Fe
chelators studied, 311m, showed similar activity to cisplatin and far
less effect than doxorubicin at inhibiting the growth of bone marrow
stem cells. Furthermore, injection of 311 intraperitoneally (300 mg · kg Considering the role of Fe in proliferation1,2 and the fact that tumors are sensitive to Fe depletion,2 it was vital to determine the Fe-chelation efficacy of the ligands. Our studies showed that the Fe-chelation efficacy of most NT ligands was higher than that of DFO. However, NT demonstrated Fe-chelation efficacy comparable to DFO. In light of its high antiproliferative activity, it is possible that the lower chelation efficacy of NT may be due to the formation of an Fe complex that is less lipophilic and subject to impaired permeability. It is relevant to discuss that chelator N2mT showed no appreciable Fe-chelation activity. These data are in agreement with its low antiproliferative efficacy (Table 1) and reflect its difficulty in coordinating to Fe. In this case, the methyl group at the 2-position of N2mT (Figure 1) hinders the electron delocalization that stabilizes Fe binding. Nonetheless, this result, together with the fact that the NT-Fe complex shows far less antiproliferative activity than NT and no ability to induce TfR or GADD45 mRNA expression (Figure 9), suggests that the antiproliferative activity of the NT series is due to their ability to bind Fe. In the present study, NT was one of the most effective ligands in terms of its ability to inhibit proliferation, despite its lower Fe-chelation efficacy than other NT analogues or 311. The fact that NT does not markedly deplete cells of Fe could be an advantage over 311 and 311m, which are the most effective chelators ever assessed in our laboratories. One possible problem with 311 and 311m is their potential to markedly deplete Fe levels within the organism. However, the use of NT, which does not possess such high Fe-chelation efficacy, could be more appropriate. Obviously, detailed dose-response studies in animals should be conducted, and these are under way. Indeed, the high antiproliferative activity of these chelators (Table 1) and marked Fe-chelation efficacy (eg, 311m; Figures 6-7) could necessitate very low doses, preventing profound Fe depletion but maintaining antitumor activity. Because thiosemicarbazones are RR inhibitors,63,64 we assessed the effects of our chelators on DNA synthesis. For 311, 311m, and N44mT, inhibition of DNA synthesis (Table 3) was associated with high Fe-chelation efficacy (Figures 6A,7A) and antiproliferative activity (Table 1). However, for some chelators there was less correlation. For instance, although NT markedly inhibited proliferation (Table 1), it only moderately prevented DNA synthesis (Table 3), perhaps reflecting its lower Fe-chelation efficacy. The modest prevention of DNA synthesis by NT indicates that it has other modes of action besides inhibition of RR. Indeed, we showed that these chelators also affect the expression of cell-cycle control molecules (Figure 10). It should also be noted that our previous studies20,21 showed that there was a lack of correlation between Fe-chelation efficacy and antiproliferative activity. This probably reflects the different cellular Fe pools (which have divergent functions) that are targeted by the chelators. Our studies of the aroylhydrazone chelators with the same Fe-binding
site as 311, namely 311m, N44pH, and NoctH, demonstrate that these
ligands show high affinity and selectivity for Fe(III) and much lower
affinity for Mg(II), Ca(II), and Zn(II).57,65 Further,
thiosemicarbazones are well-known chelators with high avidity for
Fe.66-68 Formation-constant calculations show that all our
chelators (apart from N2mT) have similar and high Fe-binding affinity
and selectivity for this metal (D.R.R., in preparation). In addition,
there was no defined correlation between Fe-binding affinity and
biologic activity. Indeed, our previous studies showed that for
aroylhydrazone ligands, one of the most important effects on activity
appears to be lipophilicity.19 Considering the affinity and selectivity of one of our most active chelators, 311m, at pH 7.4 using a ligand concentration of 10 Another mode of activity exerted by NT analogues may be the formation of Fe complexes that generate toxic free radicals. Thiosemicarbazone-Fe complexes have antitumor activity and inhibit RR,63,64 and in fact, Triapine30 forms an Fe complex with marked antiproliferative and redox activity (Figure 11; T. Chaston, D.B.L., R. N. Watts, D.R.R., manuscript submitted). In contrast, the NT-Fe complex was relatively inactive in terms of antitumor activity (Table 1), and both the Fe complexes of NT and N4mT failed to oxidize ascorbate or hydroxylate benzoate (Figure 11). However, NT did degrade DNA at an IBE ratio of 3, although its effect was far less than that of Triapine (Figure 12). Thus, the redox activity of NT and N4mT was less than that of Triapine and may play a lesser role in their biologic effects. In summary, the combination of 2-hydroxy-1-naphthylaldehyde with various thiosemicarbazides results in novel hybrid chelators with significantly greater antiproliferative activity than DFO. We demonstrated that this activity was selective for tumor cells, as the most efficient analogues were less effective at inhibiting the growth of normal cells. Because of these properties, NT, N4mT, and N44mT, as well as the 311 analogue 311m, warrant further investigation for cancer chemotherapy.
We thank Ms Juliana Kwok for her suggestions concerning the manuscript prior to submission. We gratefully acknowledge Mr Tim Chaston for his expertise in assisting with redox activity studies and Ms Vicky Antonenas, Department of Hematology, Institute of Clinical Pathology and Medical Research, Sydney, for help with bone marrow stem cell studies.
Submitted October 10, 2001; accepted February 28, 2002.
Supported by grants from the National Health and Medical Research Council and an Australian Research Council Large Grant. The Heart Research Institute also provided financial support.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Des R. Richardson, The Heart Research Institute, 145 Missenden Rd, Camperdown, Sydney, New South Wales, 2050 Australia; e-mail: d.richardson{at}hri.org.au.
1.
Andrews NC.
Disorders of iron metabolism.
N Engl J Med.
1999;341:1986-1995 2. Hershko C. Control of disease by selective iron depletion: a novel therapeutic strategy utilizing iron chelators. Baillières Clin Haematol. 1994;7:965-1000[Medline] [Order article via Infotrieve]. 3. Chitamber CR, Seligman PA. Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells: mechanisms responsible for the specific cytotoxicity of transferrin-gallium. J Clin Invest. 1986;78:1538-1546[Medline] [Order article via Infotrieve].
4.
Trowbridge IS, Lopez F.
Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits tumor cell growth in vitro.
Proc Natl Acad Sci U S A.
1982;79:1175-1179 5. Witt L, Yap T, Blakely RL. Regulation of ribonucleotide reductase activity and its possible exploitation in chemotherapy. Adv Enzyme Regul. 1979;17:157-171[CrossRef]. 6. Takeda E, Weber G. Role of ribonucleotide reductase in expression in the neoplastic program. Life Sci. 1981;28:1007-1014[CrossRef][Medline] [Order article via Infotrieve]. 7. Thelander L, Reichard P. The reduction of ribonucleotides. Annu Rev Biochem. 1979;48:133-158[CrossRef][Medline] [Order article via Infotrieve]. 8. Voute PA. Neuroblastoma. In: Sutow WW,Fernbach DJ,Vietta TJ, eds. Clinical Pediatric Oncology. St Louis, MO: Mosby; 1984:559. 9. Iancu TC, Shiloh H, Kedar A. Neuroblastomas contain iron-rich ferritin. Cancer. 1988;61:2497-2502[CrossRef][Medline] [Order article via Infotrieve].
10.
Munro HN, Linder MC.
Ferritin: structure, biosynthesis and role in iron metabolism.
Physiol Rev.
1978;58:317-396
11.
Blatt J, Stitely S.
Antineuroblastoma activity of desferrioxamine in human cell lines.
Cancer Res.
1987;47:1749-1750
12.
Becton DL, Bryles P.
Deferoxamine inhibition of human neuroblastoma viability and proliferation.
Cancer Res.
1988;48:7189-7192
13.
Donfrancesco A, Deb G, Dominici C, et al.
Effects of a single course of deferoxamine in neuroblastoma patients.
Cancer Res.
1990;50:4929-4930 14. Donfrancesco A, Deb G, Dominici C, et al. Deferoxamine, cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECat): a new cytoreductive chelation-chemotherapy regimen in patients with advanced neuroblastoma. Am J Clin Oncol. 1992;15:319-322[Medline] [Order article via Infotrieve]. 15. Donfrancesco A, De Bernardi B, Carli M, et al. Deferoxamine (D) followed by cytoxan (C), etoposide (E), carboplatin (Ca), thio-TEPA (T), induction regimen in advanced neuroblastoma. Eur J Cancer. 1995;31A:612-615[CrossRef][Medline] [Order article via Infotrieve].
16.
Estrov Z, Tawa A, Wang X-H, et al.
In vivo and in vitro effects of desferrioxamine in neonatal acute leukemia.
Blood.
1987;69:757-761 17. Dezza L, Cazzola M, Danova M, et al. Effects of desferrioxamine on normal and leukemic human hematopoietic cell growth: in vitro and in vivo studies. Leukemia. 1989;3:104-107[Medline] [Order article via Infotrieve]. 18. Ponka P, Richardson D, Baker E, Schulman HM, Edward JT. Effect of pyridoxal isonicotinoyl hydrazone (PIH) and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes. Biochim Biophys Acta. 1988;967:122-129[Medline] [Order article via Infotrieve]. 19. Baker E, Richardson DR, Gross S, Ponka P. Evaluation of the iron chelation potential of hydrazones of pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde using the hepatocyte in culture. Hepatology. 1992;15:492-501[Medline] [Order article via Infotrieve]. 20. Richardson DR, Ponka P. The iron metabolism of the human neuroblastoma cell: lack of relationship between the efficacy of iron chelation and the inhibition of DNA synthesis. J Lab Clin Med. 1994;124:580-671.
21.
Richardson DR, Tran E, Ponka P.
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents.
Blood.
1995;86:4295-4306 22. Richardson DR. Cytotoxic analogues of the iron(III) chelator pyridoxal isonicotinoyl hydrazone: effect of complexation with copper(II), gallium(III), and iron(III) on their anti-proliferative activity. Antimicrob Agents Chemother. 1997;41:2061-2063[Abstract]. 23. Richardson DR. Mobilization of iron from neoplastic cells by some iron chelators is an energy-dependent process. Biochim Biophys Acta. 1997;1320:45-57[Medline] [Order article via Infotrieve].
24.
Richardson DR, Milnes K.
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective anti-proliferative agents, II: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone.
Blood.
1997;89:3025-3038
25.
Darnell G, Richardson DR.
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, III: the effect of the ligands on molecular targets involved in proliferation.
Blood.
1999;94:781-792 26. Richardson DR, Bernhardt PV. Crystal and molecular structure of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron(III) complex: an iron chelator with anti-tumor activity. J Biol Inorg Chem. 1999;4:266-273[CrossRef][Medline] [Order article via Infotrieve].
27.
Gao J, Richardson DR.
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective anti-proliferative agents, IV: the mechanisms involved in inhibiting cell cycle progression.
Blood.
2001;98:842-850
28.
Agrawal KC, Sartorelli AC.
The chemistry and biological activity of
29.
Liu M-C, Lin T-S, Sartorelli AC.
Chemical and biological properties of cytotoxic 30. Finch RA, Liu M-C, Grill SP, et al. Triapine (3-aminopyridine-2-carboxaldehyde-thiosemicarbazone): a potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity. Biochem Pharmacol. 2000;59:983-991[CrossRef][Medline] [Order article via Infotrieve]. 31. Finch RA, Liu M-C, Cory AH, Cory JG, Sartorelli AC. Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): an inhibitor of ribonucleotide reductase with antineoplastic activity. Adv Enzyme Regul. 1999;39:3-12[CrossRef][Medline] [Order article via Infotrieve]. 32. Becker E, Richardson DR. Development of novel aroylhydrazone ligands for iron chelation therapy: the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) analogues. J Lab Clin Med. 1999;134:510-521[CrossRef][Medline] [Order article via Infotrieve]. 33. Ponka P, Richardson DR, Edward JT, Chubb FL. Iron chelators of the pyridoxal isonicotinoyl hydrazone class: relationship of the lipophilicity of the apochelator to its ability to mobilise iron from reticulocytes in vitro. Can J Physiol Pharmacol. 1994;72:659-666[Medline] [Order article via Infotrieve].
34.
Porter JB, Gyparaki M, Burke LC, et al.
Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant.
Blood.
1988;72:1497-1503 35. Johnson DK, Murphy TB, Rose NJ, Goodwin WH, Pickart L. Cytotoxic chelators and chelates I. Inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazones and by a copper(II) complex of salicylaldehyde benzoyl hydrazone. Inorg Chim Acta. 1982;67:159-162[CrossRef]. 36. Lovejoy D, Richardson DR, Bernhardt PV. X-ray crystal structure of the chelator 2-hydroxy-1-naphthylaldehyde-2-methyl-3-thiosemicarbazone. Acta Crystallogr C. 2000;C56:341-342.
37.
McCrohon JA, Jessup W, Handelsman DJ, Celermeyer DS.
Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular cell adhesion molecule-1.
Circulation.
1999;99:2317-2322
38.
Brown AJ, Watts GF, Burnett JR, Dean RT, Jessup W.
Sterol 27-hydroxylase acts on 7-ketocholestrol in human atherosclerotic lesions and macrophages in culture.
J Biol Chem.
2000;275:27627-27633 39. Eaves CJ. Assays of hemopoietic progenitor cells. In: Beutler E,Lichtman MA,Coller BS,Kipps TJ, eds. Williams Hematology. 5th ed. New York, NY: McGraw-Hill; 1995. 40. Richardson DR, Baker E. The uptake of iron and transferrin by the human melanoma cell. Biochim Biophys Acta. 1990;1053:1-12[Medline] [Order article via Infotrieve].
41.
Richardson DR, Baker E.
Two mechanisms of iron uptake from transferrin by melanoma cells: the effect of desferrioxamine and ferric ammonium citrate.
J Biol Chem.
1992;267:13972-13979 42. Baker E, Baker SM, Morgan EH. Characterisation of non-transferrin-bound iron (ferric citrate) uptake by rat hepatocytes in culture. Biochim Biophys Acta. 1998;1380:21-30[Medline] [Order article via Infotrieve]. 43. Dean RT, Nicholson P. The action of nine chelators on iron-dependent radical damage. Free Radic Res. 1994;20:83-101[Medline] [Order article via Infotrieve]. 44. Gutteridge JMC. Superoxide-dependent formation of hydroxyl radicals from ferric complexes and hydrogen peroxide: an evaluation of fourteen iron chelators. Free Radic Res Commun. 1990;9:119-125[Medline] [Order article via Infotrieve]. 45. Hermes-Lima M, Nagy E, Ponka P, Schulman HM. The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against .OH-mediated strand breaks. Free Radic Biol Med. 1998;25:875-880[CrossRef][Medline] [Order article via Infotrieve]. 46. Broto P, Moreau G, Vandycke C. Molecular structures: perception, autocorrelation descriptor and sar studies: system of atomic contributions for the calculation of the n-octanol/water partition coefficients. Eur J Med Chem Chim Theor. 1984;19:71-78. 47. Ghose AK, Crippen GM. Atomic physiochemical parameters for 3-dimensional-structure directed quantitative structure-activity-relationships 2. Modelling dispersive and hydrophobic interactions. J Chem Inf Comput Sci. 1987;27:21-35[CrossRef][Medline] [Order article via Infotrieve]. 48. Viswanadhan VN, Chose AK, Revankar GR, Robins RK. Atomic physiochemical parameters for 3-dimensional-structure directed quantitative structure-activity-relationships 4. Additional parameters for hydrophobic and dispersive interactions and their application for an automated superposition of certain naturally-occurring nucleoside antibiotics. J Chem Inf Comput Sci. 1987;29:163-172.
49.
Bottomley SS, Wolfe LC, Bridges KR.
Iron metabolism in K562 erythroleukemia cells.
J Biol Chem.
1985;260:6811-6815
50.
Richardson DR, Ponka P, Baker E.
The effect of the iron(III) chelator, desferrioxamine, on iron and transferrin uptake by the human malignant melanoma cell.
Cancer Res.
1994;54:685-689 51. Reed SI. Control of the G1/S transition. Cancer Surv. 1997;29:7-23[Medline] [Order article via Infotrieve]. 52. Sherr CJ. G1 phase progression: Cycling on cue. Cell. 1994;79:551-555[CrossRef][Medline] [Order article via Infotrieve]. 53. Zetterberg A, Larsson O, Wiman KG. What is the restriction point? Curr Opin Cell Biol. 1995;7:835-842[CrossRef][Medline] [Order article via Infotrieve]. 54. Kulp KS, Green SL, Vulliet R. Iron deprivation inhibits cyclin-dependent kinase activity and decreases cyclin D/CDK protein levels in asynchronous MDA-MB-453 human breast cancer cells. Exp Cell Res. 1996;229:60-68[CrossRef][Medline] [Order article via Infotrieve]. 55. Gutteridge JMC. Ferrous-salt promoted damage to deoxyribose and benzoate. Biochem J. 1987;243:709-714[Medline] [Order article via Infotrieve]. 56. Singh S, Hider RC. Colorimetric detection of the hydroxyl radical: comparison of the hydroxyl-radical generating ability of various iron chelators. Anal Biochem. 1988;171:47-54[CrossRef][Medline] [Order article via Infotrieve]. 57. Richardson DR, Hefter GT, May PM, Webb J, Baker E. Iron chelators of the pyridoxal isonicotinoyl hydrazone class. III. Formation constants with calcium(II), magnesium(II) and zinc(II). Biol Met. 1989;2:161-167[CrossRef][Medline] [Order article via Infotrieve].
58.
Selig RA, White L, Gramacho C, Sterlinglevis K, Fraser IW, Naidoo D.
Failure of iron chelators to reduce tumor growth in human neuroblastoma xenografts.
Cancer Res.
1998;58:473-478 59. Blatt J. Deferoxamine in children with recurrent neuroblastoma. Anticancer Res. 1994;14:2109-2112[Medline] [Order article via Infotrieve].
60.
Green DA, Antholine WE, Richardson DR, Chitambar CR.
Ribonucleotide reductase as a preferential target for the inhibition of leukemic cell growth by 311, a novel iron chelator of the pyridoxal isonicotinoyl hydrazone class.
Clin Cancer Res.
2001;7:3574-3579 61. Sah P. Nicotinoyl and isonicotinoyl hydrazones of pyridoxal. J Am Chem Soc. 1954;76:300. 62. Pickart L, Goodwin WH, Burgua W, Murphy TB, Johnson DK. Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Biochem Pharmacol. 1983;32:3868-3871[CrossRef][Medline] [Order article via Infotrieve].
63.
Sartorelli AC, Agrawal KC, Tsiftsoglou AS, Moore EC.
Characterization of the biochemical mechanism of action of
64.
Thelander L, Gräslund A.
Mechanism of inhibition of mammalian ribonucleotide reductase by the iron chelate of 1-formylisoquinoline thiosemicarbazone. Destruction of the tyrosine free radical of the enzyme in an oxygen-requiring reaction.
J Biol Chem.
1983;258:4063-4066 65. Vitolo LMW, Hefter GT, Clare BW, Webb J. Iron chelators of the pyridoxal isonicotinoyl hydrazone class. Part 2. Formation constants with iron(III) and iron(II). Inorg Chim Acta. 1990;170:171-174[CrossRef]. 66. Krakoff IH, Etcubanas E, Tan C, Mayer K, Bethune V, Burchenal JH. Clinical trial of 5-hydroxypicolinaldehyde thiosemicarbazone (5-HP; NSC 107392), with special reference to its iron chelating properties. Cancer Chemother Rep. 1974;58:207-212[Medline] [Order article via Infotrieve].
67.
Antholine W, Knight J, Whelan H, Petering DH.
Studies of the reaction of 2-formylpyridine thiosemicarbazone and its iron and copper complexes with biological systems.
Mol Pharmacol.
1977;13:89-98
68.
Agrawal KC, Sartorelli AC.
The chemistry and biological activity of
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S. J. Assinder, Q. Dong, H. Mangs, and D. R. Richardson Pharmacological Targeting of the Integrated Protein Kinase B, Phosphatase and Tensin Homolog Deleted on Chromosome 10, and Transforming Growth Factor-{beta} Pathways in Prostate Cancer Mol. Pharmacol., March 1, 2009; 75(3): 429 - 436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Rao, S. R. Klein, K. K. Agama, E. Toyoda, N. Adachi, Y. Pommier, and E. B. Shacter The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase II{alpha} in Breast Cancer Cells Cancer Res., February 1, 2009; 69(3): 948 - 957. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Sen, S. Mukhopadhyay, M. Ray, and T. Biswas Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity J. Antimicrob. Chemother., May 1, 2008; 61(5): 1066 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nurtjahja-Tjendraputra, D. Fu, J. M. Phang, and D. R. Richardson Iron chelation regulates cyclin D1 expression via the proteasome: a link to iron deficiency-mediated growth suppression Blood, May 1, 2007; 109(9): 4045 - 4054. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yu, J. Wong, D. B. Lovejoy, D. S. Kalinowski, and D. R. Richardson Chelators at the Cancer Coalface: Desferrioxamine to Triapine and Beyond Clin. Cancer Res., December 1, 2006; 12(23): 6876 - 6883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Kalinowski and D. R. Richardson The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer Pharmacol. Rev., December 1, 2005; 57(4): 547 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-H. Hou and A. H.-J. Wang Mithramycin forms a stable dimeric complex by chelating with Fe(II): DNA-interacting characteristics, cellular permeation and cytotoxicity Nucleic Acids Res., March 1, 2005; 33(4): 1352 - 1361. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. B. Chaston, R. N. Watts, J. Yuan, and D. R. Richardson Potent Antitumor Activity of Novel Iron Chelators Derived from Di-2-Pyridylketone Isonicotinoyl Hydrazone Involves Fenton-Derived Free Radical Generation Clin. Cancer Res., November 1, 2004; 10(21): 7365 - 7374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. T.V. Le and D. R. Richardson Iron chelators with high antiproliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a link between iron metabolism and proliferation Blood, November 1, 2004; 104(9): 2967 - 2975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Yuan, D. B. Lovejoy, and D. R. Richardson Novel di-2-pyridyl-derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment Blood, September 1, 2004; 104(5): 1450 - 1458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.X. Liang and D.R. Richardson The effect of potent iron chelators on the regulation of p53: examination of the expression, localization and DNA-binding activity of p53 and the transactivation of WAF1 Carcinogenesis, October 1, 2003; 24(10): 1601 - 1614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. T.V. Le and D. R. Richardson Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21CIP1/WAF1, but paradoxically inhibit its translation: a potential mechanism of cell cycle dysregulation Carcinogenesis, June 1, 2003; 24(6): 1045 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. B. Chaston, D. B. Lovejoy, R. N. Watts, and D. R. Richardson Examination of the Antiproliferative Activity of Iron Chelators: Multiple Cellular Targets and the Different Mechanism of Action of Triapine Compared with Desferrioxamine and the Potent Pyridoxal Isonicotinoyl Hydrazone Analogue 311 Clin. Cancer Res., January 1, 2003; 9(1): 402 - 414. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-actin (loading
control) in SK-N-MC neuroepithelioma cells.
Total RNA was extracted from cells after a 20-hour incubation with
medium alone (control) or medium containing DFO (150 µM) or the other
chelators (25 µM). The isolated RNA was subjected to electrophoresis
on a 1.2% agarose-formaldehyde gel, transferred to a hybridization
membrane, and probed under high-stringency conditions (see "Materials
and methods"). The result illustrated is a typical experiment from 3 experiments performed.
1 · d
log of the free metal
concentration (pM) for Fe(III) was 28. In contrast, the affinity of
this ligand for Ca(II) and Mg(II) was negligible (pM = 6). As
observed for DFO and PIH,