www.nature.com/scientificreports
OPEN
received: 20 April 2016 accepted: 08 November 2016 Published: 07 December 2016
Iron chelators target both proliferating and quiescent cancer cells Mårten Fryknäs1, Xiaonan Zhang2,3, Ulf Bremberg4, Wojciech Senkowski1, Maria Hägg Olofsson3, Peter Brandt5, Ingmar Persson6, Padraig D’Arcy2, Joachim Gullbo1,7, Peter Nygren7, Leoni Kunz Schughart8, Stig Linder2,3,* & Rolf Larsson1,* Poorly vascularized areas of solid tumors contain quiescent cell populations that are resistant to cell cycle-active cancer drugs. The compound VLX600 was recently identified to target quiescent tumor cells and to inhibit mitochondrial respiration. We here performed gene expression analysis in order to characterize the cellular response to VLX600. The compound-specific signature of VLX600 revealed a striking similarity to signatures generated by compounds known to chelate iron. Validation experiments including addition of ferrous and ferric iron in excess, EXAFS measurements, and structure activity relationship analyses showed that VLX600 chelates iron and supported the hypothesis that the biological effects of this compound is due to iron chelation. Compounds that chelate iron possess anti-cancer activity, an effect largely attributed to inhibition of ribonucleotide reductase in proliferating cells. Here we show that iron chelators decrease mitochondrial energy production, an effect poorly tolerated by metabolically stressed tumor cells. These pleiotropic features make iron chelators an attractive option for the treatment of solid tumors containing heterogeneous populations of proliferating and quiescent cells. Iron is an essential nutrient that enables a plethora of biological processes including DNA replication and mitochondrial respiration. Cancer cells display increased rate of iron uptake and usage1. Thus, iron may have an even more fundamental role in tumor cell hemostasis than is generally appreciated. Ferrous iron is present in a cytoplasmic pool of soluble and chelatable iron, i.e. the labile iron pool1. Increases in the size of the labile iron pool has been reported to lead to increased tumor cell proliferation2. Iron is a necessary component of haem and iron-sulfur clusters, present in enzymes involved in oxidative phosphorylation (OXPHOS) and the Krebs cycle3. Iron is also required for the enzymatic activity of ribonucleotide reductase (RR), catalyzing the conversion of ribonucleotides to deoxyribonucleotides4. Indeed, several iron chelators have been shown to possess anti-cancer activity1,5–9. We recently identified the small molecule VLX600 (Fig. 1A) as a candidate drug that preferentially targets quiescent cells in colon cancer 3-D multicellular tumor spheroids (MCTS)10. Similar to other compounds targeting quiescent cells in MCTS11,12, VLX600 affects mitochondrial function. The anti-cancer activity of VLX600 is attributed to the limited metabolic plasticity of cancer cells in hypoxic and nutritionally compromised environments, in which cells are unable to compensate for decreased mitochondrial OXPHOS by other means of energy production. This ultimately leads to a bioenergetic catastrophe and tumor cell death13. In contrast to other agents that decrease the viability of MCTS such as nitazoxanide11, VLX600 also inhibits the proliferation of tumor cells in 2-D monolayer culture10. This observation prompted us to investigate the 1
Department of Medical Sciences, Division of Cancer Pharmacology and Computational Medicine, Uppsala University, SE-751 85 Uppsala, Sweden. 2Department of Medical and Health Sciences, Linköping University, SE-58183 Linköping, Sweden. 3Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska Institute, SE-171 76 Stockholm, Sweden. 4Beactica, SE-754 50 Uppsala, Sweden. 5Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden. 6Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, P.O. Box 7015, SE-756 51 Uppsala, Sweden. 7Department of Immunology, Genetics and Pathology, Section of Oncology, Uppsala University, SE-75185, Uppsala, Sweden. 8OncoRay-National Center for Radiation Research in Oncology, TU Dresden, D-01307 Dresden, Germany. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.F. (email:
[email protected]) Scientific Reports | 6:38343 | DOI: 10.1038/srep38343
1
www.nature.com/scientificreports/ A
B
VLX600
MCF7 Rank
N
HC
N
HN
N
Agent
Dose
Agent
Dose
1
Ciclopirox
15 μM MCF7
1
1
Ciclopirox
15 μM MCF7
1
2
5109870
25 μM MCF7
1
2
Ciclopirox
15 μM MCF7
.936
N
HN
Cell
Score
Ciclopirox
Rank
15 μM MCF7
.973
3
Ciclopirox
4
Deferoxamine 100 μM MCF7
.920
4
5109870
5
Deferoxamine 100 μM MCF7
.795
5
3
N
HCT116
15 μM
Cell
Score
PC3
.922
25 μM MCF7
.921
Deferoxamine 100 μM MCF7
.854
HC
D Co2+ Fe 3+ Fe 2+
VLX600 Ni 2+ Zn 2+ Cu2+ Al 3+
0.1
0 300
150
Survival Index (%)
Absorbance
0.2
E
132
100
VLX600 + FeCl VLX600 + FeCl VLX600
50
400
450
500
96
Control VLX600 VLX600 + FeCl
59 22
0 350
FCCP
169
OCR (%)
C
10
20
Concentration (μmol/L)
-15
0
83
165
248
331
Time (min)
Wavelength(nm)
Figure 1. VLX600 is an iron chelator. (A) Molecular formula for VLX600. (B) Drug-specific query signatures based on the 30 most up and down regulated genes in MCF-7 cells (monolayer culture) or HCT116 cells (multicellular spheroid culture) exposed to VLX600 were uploaded to the CMAP data base to identify other compounds with similar mechanism of action. (C) Analysis of metal binding by VLX600 using spectrophotometry as described16. Note the reduction in A340 after addition of Fe2+, Fe3+ and Co2+, whereas Cu2+ and other metal ions do not affect A340. Representative of three independent experiments (D) Antiproliferative activity of VLX600 on HCT116 cells is abrogated by addition of iron chloride (FeCl2 and FeCl3). Cells were grown for 72 h in the presence or absence of VLX600 and iron chloride and viability was assessed by MTT assay. Mean ± S.D. (n = 4), representative repeated experiments. (E) The reduction of oxygen consumption by VLX600 in HCT116 cells is reversed by the addition of iron. Mean ± S.D. (n = 4), representative of two independent experiments. molecular mechanism of action of VLX600. We here report that VLX600 binds iron and that this property is the underlying mechanism of the ability of VLX600 to reduce cell proliferation and to decrease mitochondrial OXPHOS. We show that also other iron chelators have the ability to affect the viability of MCTS, albeit with lower potency than VLX600. The ability of iron chelators to reduce mitochondrial energy production adds to the evidence of this class of compounds as having attractive anti-neoplastic activities.
Results
VLX600 is an iron chelator. The molecular structure of VLX600 is shown in Fig. 1A. The precise molecular
mechanism of action of VLX600 was unknown and we therefore performed a Connectivity Map-based mechanistic exploration by examining the gene expression profile of drug-treated tumor cells14. We employed two different cellular models; the breast cancer cell line MCF-7 and colon carcinoma cell line HCT116, grown as 2-D monolayer and 3-D MCTS, respectively. MCF-7 cells were chosen since it is the most frequently used cell model in the Connectivity Map database. We selected MCTS HCT116 to investigate if the response is the similar when cells were grown in 3-D cell culture. The gene expression signature induced by VLX600 was most similar to that of ciclopirox olamine (CPX; 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone 2-aminoethanol), the ChemBridge compound 5109870 (2-hydroxy-3-methoxybenzaldehyde 2-pyridinylhydrazone), and deferoxamine (Fig. 1B). All of these compounds were previously described as iron chelators15–17, suggesting that the anticancer activity of VLX600 could be attributed to iron chelation and sequestering. Complex formation between VLX600 and different metals was examined using spectrophotometry (Fig. 1C). VLX600 was indeed found to form complexes with ferrous and ferric iron as well as with Co2+, whereas only minor changes in the UV/Vis spectra were observed with Cu2+, Zn2+, Ni2+ or Al3+ (Fig. 1C). Addition of extracellular iron (Fe2+ and Fe3+) abolished the cytotoxic activity of the compound (Fig. 1D), supporting the hypothesis of iron chelation being instrumental to biological activity of VLX600. As previously described, VLX600 is able to reduce mitochondrial OXPHOS, particularly uncoupled respiration10. We found that addition of iron chloride restored OXPHOS capacity in cells exposed to VLX600 (Fig. 1E).
Characterization of iron chelation. By means of density functional theory (DFT) calculations on Fe(II) (VLX600)2, the N2 nitrogen in the 1,2,4-triazine moiety was found to preferentially coordinate iron over the N4 Scientific Reports | 6:38343 | DOI: 10.1038/srep38343
2
www.nature.com/scientificreports/
A
B VLX600
VLX641
VLX642
N N
HC
HC
N
HN
N
N
HN
N
N
N
N HC
N
HN
N
N
N
HN
HN
HN
HC
HC
HC
N N
D
C
150
75
VLX600 VLX641 VLX642
50
OCR (%)
Survival Index (%)
100 100
Control VLX600 VLX641 VLX642
50
25 0 0.1
1
10
Concentration (μmol/L)
100
0
0
100
200
Time (min)
300
400
Figure 2. Iron binding by VLX600. (A) Model for binding of VLX600 to iron generated by DFT and EXAFS. (B) Molecular structures of VLX600, VLX641 and VLX642. The nitrogen atom in the pyridine moiety (in red) is positioned in ortho- meta- and para-position, respectively. (C) Cytotoxicity was completely abrogated (HCT116 colon cancer cell line, FMCA, 72 hours) when the nitrogen atom in the pyridine is placed in meta- or para position (VLX641 and VLX642). Data is based on four independent experiments, were each concentration is tested in quadruplicates (mean ± S.E.M). (D) The ability of VLX600 to reduce oxygen consumption is dependent on the position of the nitrogen in the pyridine ring. Mean ± S.D. (n = 4), representative of three independent experiments.
nitrogen by as much as 18 kcal/mol (Fig. 2A). Using this coordination mode, the geometries were calculated for both oxidation states and spin states of Fe(VLX600)2 (Supplementary Table 1). Extended X-Ray Absorption Fine Structure (EXAFS) confirmed that iron(II) binds two VLX600 ligands to form a bis-complex where iron binds six nitrogen atoms in a pseudo-octahedral configuration at very short Fe-N bond distances, 1.90 and 1.915 Å for iron(II) and iron(III), respectively. This is in good agreement with reported iron(II)-terpyridine complexes, Supplementary Table 3 and the DFT calculated distances. Furthermore, Fe-C/N distances at ~2.80, 3.35 and 4.05 Å were identified and fit with distances observed in the iron(II)-terpyridine complexes and those in the calculated model presented in Fig. 2A. The very short Fe-N bond distances are consistent with both iron(II) and iron(III) being present as low-spin complexes, and with significant back-bonding for iron(II) making the bond distances even shorter and the complex being significantly stabilized. The refined structure parameters are given in Supplementary Table 2, and the model fits of the experimental EXAFS and corresponding Fourier transforms (Supplementary Figures 1 and 2, respectively). The positions of the absorption edges clearly show that the VLX600 complexes of both iron(II) and iron(III) in principle remain in their oxidation states after 48 hours of storage, see Figure S3. The iron(III) complex seems to be completely stable, while there is maybe a sign that the iron(II) complex is partly oxidized as seen in the slightly longer mean Fe-N bond distance (Supplementary Table 2), and the somewhat different shape of the absorption edge, see Figure S3. The pre-edge at 7111–7114 eV is a strong indicator of the oxidation state of iron18. However, in these cases the pre-peak is observed at 7112 eV for both complexes, likely due to that they are low-spin complexes and with significant back-bonding in the iron(II) VLX600 complex, Figure S3. The chemical structure of VLX600 was modified by altering the position of the nitrogen in the pyridine moiety (VLX641 and VLX642; Fig. 2B). According to the model describing the coordination mode (Fig. 2A), this modification would result in loss of tridentate iron binding and, thus, biological activity of the molecules. Indeed, the VLX600 analogues showed no cytotoxic activity (Fig. 2C), suggesting limited off-target effects of the molecular scaffold in this biological system. Furthermore, the chemical alterations abolished the effect on mitochondrial respiration (Fig. 2D).
Scientific Reports | 6:38343 | DOI: 10.1038/srep38343
3
www.nature.com/scientificreports/
0.1
1
10
100
Concentration (μmol/L)
D
RKO 100
1000
Ciclopirox Deferoxamine VLX50 VLX600
50
0 0.01
0.1
1
10
100
HCT8 100
50
0 0.01
0.1
10
100
DLD
50
0.1
1
10
100
Concentration (μmol/L)
C
HT-29
Ciclopirox Deferoxamine VLX50 VLX600
100
50
0 0.01
1000
Ciclopirox Deferoxamine VLX50 VLX600
100
0 0.01
Concentration (μmol/L)
1
Concentration (μmol/L)
E
1000
Ciclopirox Deferoxamine VLX50 VLX600
Survival index (%)
50
B
0.1
1
10
100
Concentration (μmol/L)
F Survival index (%)
100
0 0.01
Survival index (%)
Ciclopirox Deferoxamine Triapine VLX50 VLX600
Survival index (%)
HCT116
Survival index (%)
Survival index (%)
A
SW620
1000
Ciclopirox Deferoxamine VLX50 VLX600
100
50
0 0.01
1000
0.1
1
10
100
1000
Concentration (μmol/L)
G IC50 concentrations in monolayer cell cultures (μM)
H
HCT116 28.2 144.9 24 24.2 1.9
HCT8 16.2 137.2 NA 24.4 1.4
HT-29 69.9 195.7 NA 63.3 3.7
RKO 31 153.1 NA 43.2 2.1
DLD 40.4 >200 NA 45.7 3.3
I Control
SW620 48.5 201.2 NA 51.1 3
J EdU incorporation
VLX600
200
VLX600 (μM)
0 0.4 0.8 1.6 3
400
300
600 Activity (CPM)
Ciclopirox Deferoxamine Triapine VLX50 VLX600
200
100
μM 10 00
VL X6
Co nt ro
l
6 12.5
Figure 3. VLX600 is more potent compared to other iron chelators in reducing tumor cell proliferation. (A–F) Six different colon cancer cell lines (HCT116, HCT8, HT-29, RKO, DLD and SW620) were tested exposed to the indicated concentrations of VLX600 and viability was determined by FMCA at 72 hours. All concentrations tested in quadruplicate, experiments repeated three times (except HT-29, repeated twice), graphs indicate mean ± S.E.M. (G) Summary of IC50 concentrations from the data in (A–F). (H) VLX600 reduces DNA synthesis in HCT116 cells as evidenced by reduced EdU (5-ethynyl-2′-deoxyuridine) incorporation after 24-hour treatment, mean + S.D., (n = 3) representative of repeated experiment. (C) VLX600 inhibits ribonucleotide reductase activity in vitro. Results are shown as mean + S.D. (difference significant at the level of p 264 64.5 3.4
>32 >264 3.5 0.75
0.1 1 10 100 Concentration (μmol/L)
Figure 4. Iron chelators impact oxidative phosphorylation and cell viability. (A) Iron chelators diminish respiratory reserve capacity in monolayer cell cultures. Shown are Seahorse recordings of oxygen consumption of HCT116 cells exposed to the different compounds shown. Samples were run in quadruplicates, all 3 iron chelators tested induced a lower level of uncoupled respiration (p
