Ribonucleotide reductase (RR) is one of the fifty genes reported to be overexpressed in highly malignant tumors with poor prognosis [1
]. RR is essential for DNA synthesis during cell division encoding the rate-limiting enzyme that catalyzes the conversion of ribonucleotides (NTPs) into deoxyribonucleotides (dNTPs) [3
]. The enzyme is composed of two catalytic (RRM1) and two regulatory (RRM2 or p53R2) subunits [9
]. RRM1 expression is detectable throughout the cell cycle in all tissues [10
], while RRM2 and p53R2 are preferentially expressed during cell mitosis and in response to DNA damage, respectively. The reductase activity of RRM2 subunit requires two Fe3+
ions for the formation of the tyrosil radical on Tyr122 that has a key role in the enzyme activity [6
]. Hydroxyurea (HU), gemcitabine, fludarabine and chlorodeoxydenoside are compounds targeting RRM2 activity that showed effects in cancer therapy in vitro, but with some side effects in vivo. In order to improve their efficacy in the inhibition of RRM2 and reduce the side effects, some derivatives of polyhydroxy-substituted benzohydroxamic have been synthesized [3
] and among them there is 3,4 dihydroxybenzohydroxamic acid (didox) in which the amino group of HU is substituted by a catechol group (Figure 1
This compound, targeting RRM2 subunits, was found to trigger cell apoptosis with a different extent depending on the cell types [13
] by increasing the levels of the pro-apoptotic protein Bax and release of cytochrome C from the mitochondria [16
]. Didox revealed good efficacy against multiple myeloma cells [15
], prostate tumor [16
], breast cancer cells [17
] and acute and chronic myeloid leukemia HL-60 and K562 cell lines [14
]. Mouse models have shown that didox significantly caused growth inhibition of breast cancer [17
] and human leukemia [18
]. Furthermore, in phase I/II clinical trials, didox showed minimal toxicity in cancer patients [19
Interestingly, it was also reported that didox forms iron complexes recognized by photometric methods [21
]. The finding that the antiproliferative effect of didox was partially inhibited by iron suggested that iron chelation may be important for its pharmacological activity [21
]. Tumor cells are often characterized by an “iron addiction” status requiring abundant iron to sustain proliferation [22
], thus it has been indicated that iron chelators may improve the chemotherapeutic effects [24
]. The potential clinical impact of the new generation of iron chelators has increased recently due to the improvement of their pharmacokinetic and pharmacodynamic properties [29
]. Iron chelators act to inhibit cell proliferation, by subtracting the iron needed for cellular metabolism, by inducing apoptosis and also by contributing to the generation of reactive oxygen species. This depends largely by the coordination of the chelant, deferoxamine (DFO; Desferal ®
) is a hexadentate iron chelator that blocks the interaction of iron with oxygen, making it inert. Instead, bidentate chelators such as deferiprone (DFP; Ferriprox ®
), or the tridentate deferasirox bind iron in a 3:1 or 2:1 chelator to iron ratio, resulting in a less stable iron complex, thus allowing the formation of potentially toxic free radicals [30
The iron binding properties of didox have been described only by Fritzer-Szekeres [21
] but its pharmacological effects and its impact on iron metabolism have not been further studied. In the present work we studied the effect of didox on the proliferation of the hepatocellular carcinoma HA22T/VGH cell line and we characterized the iron-chelating properties of didox in vitro and in cells, specifically focusing our study to determine the iron binding capacity of didox and its effect on iron related proteins.
Didox has been used for many years as an antitumor agent [14
]. It targets and inhibits RRM2, the enzyme involved in the critical conversion of ribonucleotides to deoxyribonucleotides, essential in DNA replication and one of the most expressed enzymes in tumor cells. Didox is a derivative of HU that is known to inhibit RRM2 activity by quenching the tyrosyl free radical at the active site of the enzyme that is essential for the reductase activity [34
]. The free radical quenching moiety of HU is partially conserved in didox, and it contains an additional catechol group that is known to have iron-chelating properties. In fact, a major class of bacterial siderophores uses catechol as an iron ligand [35
]. The iron-chelating properties of didox were previously studied to define the formation of the iron complex and to show that iron supplementation reduced the cellular toxicity of didox in L1210 leukemia cells [21
]. We used the hepatocellular carcinoma (HCC) cell lines, as a cellular model to study didox antitumor activity since HCC is the most common type of liver cancer. HCC are solid tumors, with a large angiogenic capacity and are often resistant to apoptosis and they are classified on the degree of malignancy and the level of differentiation [36
]. HCC cells derive from liver, which has a high intracellular iron level, and high expression of iron-related proteins, detectable RRM2 levels and normal proliferation rate. The HuH7 cells are highly differentiated while HA22T/VGH cells are poorly differentiated. We found that these cells are similarly sensitive to didox with an IC50
as low as 132 µM in HA22T/VGH and 122 µM in HuH7 after 72 h of incubation. We concluded that the differentiation state did not modify cell sensitivity to the drug, thus we continued the study on the HA22T/VGH cells. We confirmed that didox induced apoptosis also in HA22T/VGH cells with an increase of AnnexinV and mitochondrial ROS production, similar to previous studies reported in multiple myeloma cells [15
]. We compared the antitumor activity and iron binding capacity of didox with those of the two clinically used iron chelators, DFP, which is a catechol bidentate structurally similar to didox, and DFO a hexadentate structurally unrelated to didox. When given to the cells, didox caused modifications of the iron status that were very similar in extent to those caused by the two chelators: changes in the level of TfR1 and ferritins and of the intracellular labile iron. Moreover we found that DFO and DFP had a cytotoxic effect on our cells that was comparable to that of didox, and that was relieved by iron supplementation to the cells at concentrations in the same range of that of the chelators. The cytotoxic potency of HU was lower and, more important, not affected by iron supplementation, indicating a different mechanism of action. Iron is essential for many tumor cells, that require it to proliferate, the so-called “iron addition”, thus the iron chelation activity promoted by didox, DFO and DFP inhibits the growth capacity of tumor cells. For example, sub-lethal concentrations of didox affected the capacity of HCC to close the wound (not shown), showing that iron is important also in this process. Our modeling studies indicate that didox binds iron as a bidentate to form a complex didox-iron of 3:1 and the binding probably occurs through the catechol moiety as it occurs in DFP. Didox seems to combine the free-radical scavenging activity of HU that blocks the tyrosil radical of RRM2 with an iron chelating activity of DFP and DFO, which sequesters iron from many key enzymes, among which RRM2 is central. Thus the observation that didox targets RRM2 with two different mechanisms should make it superior to the iron chelators or HU as antitumor agent.
The published data on the use of didox to treat different tumors and our results on HCC in vitro are promising. In vivo studies on HCC, using didox alone or in combination with different chemotherapeutic drugs, could be an interesting point to be defined in the future to finalize the use of this compound as an antitumor drug.
4. Materials and Methods
4.1. Antibodies and Chemicals
Antibodies used were anti-TfR1 (no. 136800, Thermo Scientific, Waltham, MA, USA) and anti-tubulin (no. T5168, Sigma-Aldrich, Saint Louis, MO, USA). HRP-conjugated secondary antibodies used were anti-mouse (no. sc-516102) and anti-rabbit (no. sc-2357; Santa Cruz Biotechnology, Dallas, TX, USA). The chemicals used in this study were: Didox (3,4 dihydroxybenzohydroxamic acid; no. 10009081, Cayman Chemicals, MI, USA) and hydroxyurea (no. H8627, Sigma-Aldrich, Saint Louis, MO, USA) dissolved in dimethylsulfoxide (DMSO), three different well known iron chelators such as DFO (deferoxamine; no. S0080A, Novartis, Basel, Switzerland), DFP (deferiprone, kind gift of Prof. P. Ponka, University, Montreal, QC, Canada) and BPS (batophenantroline disulfonic acid, no. B1375); FAC (ferric ammonium citrate, no. F5879); FAS (ferrous ammonium sulfate, no. F2262); FeCl3 (ferric chloride, no. 157740) and ascorbic acid (no. A4034; Sigma-Aldrich, Saint Louis, MO, USA) all dissolved in water. Calcein (no. 21030 Sigma-Aldrich, Saint Louis, MO, USA), calcein-AM (no. ALX-610-026 Calcein-acetoxymethyl ester, Enzo Life, Lausen, Switzerland) and MTT (thiazolyl blue tetrazolium bromide, no. M5655 Sigma-Aldrich, Saint Louis, MO, USA) were also used.
4.2. Cell Culture
The human hepatoma cell lines, HuH7 (from IZSLER, Brescia, Italy), were cultured in Dulbecco modified eagle medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% endotoxin-free fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA), 0.04 mg/mL gentamicin (Gibco, Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine (Gibco, Life Technologies, Carlsbad, CA, USA) and 1 mM sodium pyruvate (Carlo Erba, Milan, Italy). The HA22T/VGH cell lines, a kind gift of Dr. A. Salvi and Prof. G. De Petro (University of Brescia, Brescia, Italy), were maintained in RPMI-1640 (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% endotoxin-free fetal bovine serum, Fungizone (Gibco, Life Technologies, Carlsbad, CA, USA), 0.04 mg/mL gentamicin (Gibco, Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine (Life Technologies, Carlsbad, CA, USA) and 1 mM sodium pyruvate (Carlo Erba, Milan, Italy). The cell lines were maintained at 37 °C in a 5% CO2 incubator.
4.3. Cell Treatments and Cell Viability Analysis
The cells were seeded in a 96-well plate (at a density of 2 × 103 cells for HA22T/VGH; 1.5 × 103 cells for HuH7) and exposed to various concentrations of didox and only HA22T/VGH also to hydroxyurea, DFO or DFP (0, 1, 10, 25, 50, 100, 200 and 500 µM) for 24, 48 and 72 h. In other experiments, HA22T/VGH were seeded in 96-well plates and treated with a single dose of didox, HU, DFO, DFP alone or in combination with increasing doses of FAC (25, 50, 100, 200 and 400 µM) for 48–72 h. In other type of treatment, HA22T/VGH cells were or pre-treated for 16 h with a single dose of didox (200 µM) and then treated in combination with FAC (400–800 µM) or directly in combination didox-FAC for 48–72 h.
Cell viability was evaluated with an MTT assay (Sigma-Aldrich, Saint Louis, MO). After the indicated time points and treatments, the supernatant was removed and 100 µL of the MTT solution (0.5 mg/mL) diluted in the cell medium was added to the wells. After 3.5 h of incubation at 37 °C and 5% CO2, the MTT medium was removed and 75 µL of DMSO was added to each well. Plates were shaken for 15 min at 37 °C until complete dissolution and absorbance was measured at 540 nm emission wavelengths using a Multiskan©EX plate reader (Thermo Scientific, Waltham, MA, USA). Average percentage of cell viability at each concentration was calculated using Microsoft Excel 2016 software.
4.4. Protein Extraction
Cells extracts were prepared using a lysis buffer (200 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM sodium fluoride and 1 mM sodium orthovanadate; Complete Protease Inhibitor Cocktail; Sigma-Aldrich). The protein concentration was quantified using Micro BCATM Protein Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA) and used for different analysis by western blotting and ELISA assays.
4.5. Western Blot Analysis
Western blot was used to analyze protein expression. In brief, after extraction, equal amounts of protein homogenates were boiled at 99 °C for 5 min before separation by SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (GE healthcare, Little Chalfont, UK). Membranes were blocked for 30 min at 37 °C with Tris-buffered saline with 1% Tween-20 (TBS-T) with 2% milk and incubated overnight at 4 °C or 2 h at 37 °C with the primary antibodies (reported in the material and methods paragraph). Following the TBS-T wash, membranes were incubated with HRP-conjugated secondary antibodies for 1 h and 30 min at RT. Membranes were washed again in TBS-T prior to signal visualization using enhanced chemiluminescence (PDS kit, Protein Detection System, GeneSpin, Milan, Italy). The signal was visualized with a Lycor Odyssey instrument and densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD, USA) and normalized against tubulin, as a loading control.
4.6. ELISA Assay
The plates (96 wells) were coated with 0.1 mL of primary antibody against L-ferritin (LFO3) or H-ferritin (RH02; 10 μg/mL diluted in 50 mM carbonate buffer pH 9.6) for 18 h at 4 °C. After three washes with PBS-T (phosphate buffer saline with 0.05% Tween20), the wells were over-coated by adding 0.1 mL of 3% bovine serum albumin (BSA) diluted in PBS for 30 min at 37 °C. After washing with PBS-T, 20 μg of protein extract for L-ferritin and 5 μg of protein extract for the H-ferritin analysis were aliquot in duplicate, diluted in 1% BSA-PBST and incubated at 37 °C for 2 h. A standard curve using recombinant human L- or H-ferritin was added into the plate, as a calibrator. After three washings in PBST, 0.1 mL of anti-L- or H-ferritin antibody HRP labeled (diluted 1:500 in 1% BSA-PBS, respectively) were added and plate incubated for 1 h at 37 °C. After three washings in PBS-T, HRP activity was detected using 1 mg/mL tetramethylbenzene (TMB) dissolved in dimethyl sulfoxide (DMSO) and diluted 1:10 in phosphate-citrate buffer pH 5 with added fresh hydrogen peroxide to a final concentration of 0.006% and the absorbance read at 620 nm by Multiskan©EX plate reader. The reaction was stopped by adding 1 N sulphuric acid and the absorbance was measured at 405 nm. The concentration of ferritins was extrapolated from the calibrator curve and expressed as ng of ferritin/mg of protein extract.
4.7. Labile Iron Pool Assay and Calcein-AM Assay
The cellular LIP was measured as described elsewhere, with minor modification [37
]. Briefly, HA22T/VGH cells (3 × 104
) were seeded on 96-well plates and treated with 100 µM DFO, DFP or didox for 4 h. The cells were incubated with 0.25 µM calcein-AM in MEM with 1 mg/mL BSA for 30 min at 37 °C. After washing with 1X phosphate-buffered saline (PBS), 100 μL of 1X Hank’s Balanced Salt Solution (HBSS) was added to the cells and the fluorescence was monitored at an excitation of 488 nm and an emission of 517 nm using an EnSight Multimode plate reader (Perkin Elmer). Cells were then fixed in 4% PFA, stained with crystal violet solution (0.1% crystal violet, 20% methanol) for 15 min. After washings, 100 μL of 10% acetic acid was added and absorbance was detected at 540 nm using a Multiskan©
EX plate reader (Thermo Scientific, Waltham, MA, USA). The data were expressed as fold change over the not treated cells (ratio of fluorescence of calcein-AM/absorbance at 540 of crystal violet). The quenching of calcein-AM is inversely proportional to the concentration of intracellular iron.
4.8. MitoSOX™ Red Mitochondrial Superoxide Indicator Assay
HA22T/VGH cells were seeded in six-well plates and 24 h after seeding, treated with 200 µM didox, respectively. After 24, 48 and 72 h, cells were collected and labeled with 5 µM MitoSOX™ (Molecular Probes) diluted in medium and incubated in the dark for 20 min at 37 °C. Cells were then washed and suspended in medium and fluorescence detected by a citofluorimeter instrument (MACSQuant Analyzer, Miltenyi Biotec, Germany).
4.9. AnnexinV/Propidium Iodide Assay
HA22T/VGH cells were seeded in six-well plates (250 × 105 cells/well). Twenty-four hours after the seeding, cells were untreated or treated with 200 µM didox and the apoptotic cell death analyzed after 24, 48 and 72 h using the commercial kit AnnexinV-FITC Apoptosis Detection (Immunostep) and following the manufacturer’s instructions. Briefly, cells were harvested, washed with 1X PBS and resuspended in 1X Annexin-binding buffer followed by the addition of 5 μL of AnnexinV-FITC and 5 μL of propidium iodide (PI). The cells were then incubated at RT for 15 min in darkness. After incubation, 400 μL of 1X Annexin-binding buffer was added and cells analyzed by flow cytometry within one hour, using the MACSQuant Analyzer (Miltenyi Biotec). Analysis of apoptosis was performed by counting cells stained simultaneously with AnnexinV–FITC and PI, to discriminate intact cells (AnnexinV-FITC and PI negative) from cells in the early apoptotic state (AnnexinV-FITC positive and PI negative) and late apoptotic state (AnnexinV-FITC and PI positive). The percentage of cells positive to each dye was represented in the plot, whereas the histogram showed the cells positive to AnnexinV-FITC (the sum of the percentage of cells in early and late apoptosis).
4.10. Dequencing of Calcein Fluorescence In Vitro
Calcein fluorescence (excitation 488 nm; emission 517 nm) was detected by an EnSight Multimode plate reader (Perkin Elmer), following the protocol from Breuer W. et al. (1995) [32
]. Briefly, 1 μM of calcein (Sigma-Aldrich, Saint Louis, MO, USA) was incubated with an equimolar concentration of iron (II), as 1 μM FAS (ferrous ammonium sulfate) and allowed to equilibrate for 8 min before analyzing the quenching of fluorescence. Iron (III) chelators such as DFO and DFP, or iron (II) chelator such as BPS, or didox or HU were subsequentially added at the final concentration of 100 μM. The dequenching of calcein fluorescence was analyzed after 1, 2, 3, 4, 5, 15, 25, 35, 45, 55 and 65 min.
4.11. UV-Vis Spectroscopy
The absorption spectra of didox combined with iron (III) were measured with a Jasco 815SE instrument. Conditions were 2-nm resolution, 200 nm/min scan speed and 1 cm quartz cuvette. A solution of 300 µM of ferric chloride was prepared in the Tris-HCl buffer, 25 mM, pH 7.2. Increasing amounts of didox (from 0 to a final concentration of 900 µM) were added to the solution. After each addition of didox, the solution was left for 30 min at 25 °C, then UV-vis spectra were recorded.
4.12. Computational Method
Geometry optimizations and UV-vis spectra calculations were performed with the use of the Gaussian16 program (Gaussian 16, Revision B.01, M. J. Frisch 2016), with CAM-B3LYP functional and 6 − 31 + G** basis set and MDF10 pseudopotential in the IEF-PCM approximation for the solvent. The 0.2 eV broad Gaussian band-shape was used.
4.13. Statistical Analysis
Data are presented as mean ± standard error of mean (SD). Statistical significance was assessed by a two-way ANOVA, unless otherwise indicated in the figure legends, and performed by GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). p-values < 0.05 were considered significant.