Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species
© Fatfat et al.; licensee BioMed Central Ltd. 2014
Received: 21 October 2013
Accepted: 15 July 2014
Published: 21 July 2014
Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action.
Multiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN–metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo.
We show that metal chelation using TPEN (5μM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts.
Our data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells.
KeywordsColon cancer Metal chelation TPEN Redox cycling Copper Reactive oxygen species
Cancer is one of the leading causes of mortality and represents a tremendous burden on patients and societies. Colorectal cancers are associated with one of the highest morbidity and mortality rates in both men and women (Globocan 2008, IARC 2010). Although the etiology of cancer varies greatly between different types of neoplasms, a hallmark finding is a defect in cell cycle regulation [1, 2]. Such cell cycle disruption is associated with genomic instability, due to mutations and chromosomal aberrations that disrupt critical housekeeping functions, including DNA repair and cell cycle checkpoints. Because these homeostatic mechanisms are essential for both normal and cancerous cells, treatments targeted at selectively destroying cancer cells become challenging.
The G2-M transition marks the entry of cells into the division phase of the cell cycle. Interestingly, metal chelation using the acyclic amino metal chelator N, N, N’, N’-tetrakis-[2-pyridylmethyl]-ethylenediamine (TPEN) prevents frog oocytes from entering meiosis . TPEN, an uncharged polydentate ligand with nitrogens as donor atoms, has remarkably high affinity for a broad spectrum of metal ions, including copper, iron and zinc . TPEN-induced meiotic arrest is due to the lack of activation of Cdc25C, a dual specificity phosphatase in oocytes that represents the rate limiting step in activating the master regulator of the G2-M transition cyclin-dependent kinase 1 (Cdk1) . This is because Cdc25C is a Zn2+-binding protein and removal of Zn2+ inhibits its ability to interact with and dephosphorylate Cdk1 . TPEN treatment also results in meiosis arrest in mouse oocytes .
TPEN-dependent metal chelation has noticeably distinct effects during the mitotic cell cycle as compared to meiosis. TPEN induces apoptosis in different cell types, including lymphocytes and splenocytes [7, 8], epithelial cells , hepatocytes , breast cancer , HT-29 colorectal cancer , ovarian cancer , pancreatic cancer , and prostate cancer . However the mechanisms of actions proposed for the TPEN-dependent cell killing are quite varied: TPEN was proposed to decrease the levels of the apoptosis inhibitor XIAP due to Zn2+ chelation . Zn2+ depletion has also been implicated in mitochondrial injury, activation of caspases (primarily caspase 3) and apoptosis [8, 9, 14]. Furthermore, TPEN treatment results in depletion of glutathione causing increased redox stress . Collectively these findings argue that TPEN induces cell killing through varied mechanisms, which may be expected given the diverse roles that metals chelated by TPEN play in physiological processes. Indeed TPEN has been used as a selective Zn2+ chelator despite the fact that it has a significantly higher affinity for Cu2+ (Stability constant for Zn 15.5 and for Cu 20.5) . There is evidence at least in the hippocampus that TPEN in vivo chelates Zn2+ with better efficiency as compared to Cu2+.
Metal homeostasis is important for biological function and needs to be tightly regulated since either metal deficiencies or metal excesses tend to be toxic. Metals have played important roles in cancer treatment since ancient times with the use of arsenic trioxide to treat different cancers including leukemia in the 18th and 19th century . More recently platinum based compounds such as cysplatin and carboplatin have become the chemotherapeutic agents of choice for many cancers . Interestingly cancer cells are addicted to high iron levels and accumulate the metal through transferrin-dependent uptake [20, 21]. Furthermore cancer cells concentrate high levels of copper, which is presumed to be important for both angiogenesis and metastasis . Therefore, transition metals are likely to play important roles in the development and growth and neoplasms.
Here we show that TPEN-mediated metal chelation results in selective killing of HCT116 colon cancer cells without affecting normal cells. TPEN cytotoxicity is due to the generation of ROS as it is reversed by antioxidants. Interestingly, HCT116 colon cancer cells accumulate 7-fold higher levels of copper compared to normal cells. The TPEN-copper complex undergoes redox cycling reactions. These results suggest that TPEN chelates accumulated copper in HCT116 cells making it available for redox cycling leading to cell toxicity and death. We further show that TPEN effectively inhibits colon cancer tumor growth in human colon cancer xenografts in mice. Therefore metal chelation provides a promising selective approach to target colon cancer.
Human colorectal cancer cells, SW480, HT-29 and LOVO were kindly provided by the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Sigma-Aldrich, UK) with 20mM HEPES and L-Glutamine at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Media was supplemented with 1% Penicillin-Streptomycin (100 U/ml) and 10% heat-inactivated FBS (Sigma-Aldrich, Germany). Unless otherwise mentioned, cells were seeded at 1.2 ×105 cells/ml and treated with TPEN (Sigma-Aldrich) at 50% confluence. TPEN was prepared in DMSO and the final DMSO concentration used on cells <0.3%.
Cell viability assays & antibodies
Human HCT116 p53+/+ colon cancer cells were cultured as previously described . Cell viability was measured using the MTT-based Cell Titer 96 non-radioactive cell proliferation kit (Promega Corp, Madison, Wisconsin, USA). Cell cycle analyses were performed on propidium iodide stained cells using flow cytometry (Becton Dickinson, Research Triangle, NC). The TUNEL assay used the In Situ Cell Death Detection Kit according to the manufacture instructions (Roche Diagnostics Corporation, Mannheim, Germany). For Annexin V staining cells were incubated in Annexin-V-Fluos labeling solution [20 μl Annexin reagent and 20 μl PI (50 μg/ml) in 1000 μl incubation buffer pH 7.4 (10 mM Hepes/NaOH, 140 mM NaCl, 5 mM CaCl2), then analyzed by flow cytometry. Caspase 3, 8 and 9 activities were assessed using Colorimetric Assay kits according to manufacturer insutructions (R & D Systems-BF4100). Primary antibody used for Western blots: XIAP #2042S; Caspase 3 #9665S; Caspase 9 #9502S; Bax #2772; PARP #9542S, from Cell Signaling. Cytochrome C sc-13560 from Santa-Cruz and GAPDH #5476 from Abnova.
Cells were treated with TPEN for 10, 20, 30 and 45 min. In experiments which involved addition of the antioxidant N-acetyl-L-cysteine (NAC), cells were treated with 5 mM NAC for 2 h before TPEN after which 10 μM of the CM-H2DCFDA dye was added for 20 min. Cells were washed, harvested by centrifugation and the pellet washed and re-suspended in 500 μl PBS followed by flow cytometery.
Mitochondrial membrane potential
Cells were washed, pelleted and incubated in 500 μl of rhodamine buffer [5 μm rhodamine 123, 130 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 1 mM CaCl2, 1 mM MgCl2, and 25 mM Hepes (pH7.4)] for 30 min at 37°C, then analyzed by flow cytometry.
106 HCT116 and NCM460 cells were collected in 8 ml HNO3 65% + 2 ml H2O2 30% and digested in a closed vessel microwave (Milestone ETHOS PLUS with HPR-1000/10S high pressure rotor). Cell lysates were measured for ion concentration against standard solutions prepared for Copper, Zinc and Iron in deionized water in an atomic absorption spectrophotometer (Furnace).
UV/VIS and EPR spectroscopy
EPR spin-trapping experiments were carried out with a JEOL-RE1X spectrometer (Kyoto, Japan). Spectrometer settings were as follows: field center, 335.094 mT; microwave power, 10 mW; sweep time, 2.0 min; time constant, 0.3 s; and modulation width, 0.2 mT). UV/VIS spectra were recorded with Helios Alpha spectrophotometer (Thermo Fisher Scientific, Inc.; Pittsburgh, PA). All measurements were performed at room temperature.
NOD/SCID female mice, (6–8 weeks, ~20 g) (Charles River Laboratories, France) were divided into two groups of 8 mice each and maintained in Maxi-miser hepafiltered facility. 2-3×106 HCT-116 tumor cells in 100 μl of 0.9% NaCl were inoculated subcutaneously in the flank. An average age (6–8 weeks) and body weight of mice (18-22 g) were used for the experiments. Prior to manipulations, mice were anaesthetized with isoflurane (Forane®, Abbott) by inhalation. On day 7 post inoculation mice received i.p. injections of either saline (control) or 20 mg/kg TPEN every other day for 28 days. Tumor measurements were performed 3 times per week using a sterile Vernier caliper. Tumor volume was calculated by the formula: Volume = π/6 (length × width × height). A 5-10% loss of body weight was detected in mice receiving TPEN treatment in comparison to the control but they otherwise looked healthy and tolerated the drug treatment well. The use of laboratory animals was in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). The IACUC committee at the American University of Beirut where the animal studies were conducted reviewed and approved the studies described.
Immunohistochemical analysis of xenografts
Tissue sections (4 μm) were stained with and anti-Ki-67 antibody (Santa-Cruz, US), for 60 min followed by secondary and tertiary antibodies and incubated with the chromogen (Zymed, US) before counterstaining with Hematoxylin. To assess the extent of total cell death, tissue sections were stained by using the terminal deoxyribonucleotidyl transferase-mediated dUTP-nick-end labeling (TUNEL) assay, according to manufacturer’s instructions (in situ cell death detection kit, fluorescein; Roche) and counterstained with PI. Slides were analyzed under LSCM fluorescent confocal microscope (LSM 410, Zeiss, Germany).
Data are expressed as the mean ± standard deviation, and statistical significance between different groups was determined using a two-tailed Student’s t-test. Statistical significance was defined as a *p < 0.05 and **p < 0.01.
TPEN selectively kills HCT116 human colon cancer cells
TPEN induces apoptosis in HCT116 cells
The observed increases in the PreG1 population are associated with higher levels of apoptosis, as indicated by TUNEL and Annexin V assays in TPEN treated cells in a dose-dependent fashion (Figure 1D). An early hallmark of most apoptotic cell death is increased mitochondrial membrane permeability. TPEN induced permeabilization of the mitochondrial membrane, as indicated by changes in mitochondrial membrane potential (ΔΨm) (Figure 1E). Interestingly, this effect may be associated with TPEN’s ability to “extract” essential metals, particularly iron and copper from mitochondrial electron carriers, thus facilitating the loss of membrane potential and the deviation of electron flow to molecular oxygen O2 to generate superoxide and H2O2. Treatment with TPEN induced disruption of the mitochondrial membrane potential in a dose-dependent fashion, as detected by the reduced accumulation of the fluorescent dye rhodamine (Figure 1E). Consistently, disruption of the mitochondrial membrane is associated with the release of cytochrome c from the mitochondria and its accumulation in the cytosolic fraction (Figure 1F).
The ability of TPEN to induce death of HCT116 colon cancer cells was dependent on its metal chelation properties, as it was reversed when TPEN was added in the presence of ZnSO4 (Figure 2B, Zinc). TPEN is a metal chelator with high affinity for zinc, copper and iron. Addition of exogenous Zn2+ is expected to associate with and saturate TPEN, preventing it from chelating other metals in the cell. ZnSO4 addition effectively abrogated the toxic effects of TPEN as indicated by the cell viability assay (Figure 2B, Zinc), TUNEL and annexin staining (Figure 2C, Zinc) and PARP, caspase 3 and XIAP cleavage (Figure 2A, Zinc). In contrast, inhibition of caspases using the pan-caspase inhibitor z-VAD-fmk resulted in significant, yet only partial reversal of TPEN toxicity (Figure 2B and 2C). z-VAD treatment effectively blocked caspase 3 and PARP cleavage (Figure 2A), this however was not sufficient to fully reverse TPEN-dependent cell killing (Figure 2B and 2C, z-VAD). These results suggest that the toxic effects of TPEN are mediated only partially through the induction of apoptosis, and potentially invoke other death pathways such as necrosis and autophagy.
TPEN toxicity is associated with ROS generation
To assess whether ROS generation is involved in mediating TPEN-induced cell death, we pretreated HCT116 cells with antioxidants, including NAC and vitamin E, after which cell viability and apoptosis were assessed. As expected, the general ROS scavenger NAC and vitamin E significantly decreased H2O2- and TPEN-induced ROS generation (Additional file 2: Figure S2). Antioxidants effectively prevented TPEN toxicity as measured by cell viability (Figure 3C), TUNEL and annexin assays (Figure 3D), and reversed caspase 3 and 9 activation (Additional file 3: Figure S3). This argues that TPEN-mediated cell killing requires ROS production. Furthermore, the protective effects of antioxidants are also compatible with the idea of transition metal catalyzed oxidative stress. However, the observed incomplete protection by two different antioxidants – NAC and Vit E – may indicate contribution of non-redox driven pathways, such as Zn-dependent effects, given previous reports that argued for a role of Zn-homeostasis in TPEN toxicity. Nonetheless, the significant reversal of the TPEN toxicity using two different anti-oxidants strongly argues for an essential role for ROS production in TPEN toxicity.
TPEN-metal complex are redox active
(EPR) doublet of the semidehydroascorbyl radical (ascorbate + radicals → semidehydroascorbyl radical) was gradually substituted by the characteristic 6-line EPR spectrum of PBN/.CH3 (Figure 4C; CH3SOCH3 + HO. → .CH3 + CH3S (O) OH; .CH3 + PBN → PBN/.CH3 .). The formation of PBN/.CH3 was inhibited by depletion of ascorbic acid, as assessed by the disappearance of the semidehydroascorbyl radical. No PBN/.CH3 formation was observed if either H2O2 or ascorbic acid was omitted from the reaction system. Under these experimental conditions, the rate of PBN/.CH3 formation by [TPEN-Fe(III)] was 4 times slower than that observed with [TPEN-Cu (II)] (Figure 4D). The results obtained indicate that both [TPEN-Fe(II)] and [TPEN-Cu (I)] have the potential to generate HO. in biological systems ([TPEN-Me(n+)] + H2O2 → [TPEN-Me(n+1)] + HO. + HO-). These observations extend the reaction mechanism of the breakdown of H2O2 by ([TPEN-Fe(n+)], which includes the formation of high-valent oxo-species that can activate C-H bonds and epoxidate > C = C < bonds [28–30]. Hence the TPEN-Fe and TPEN-Cu complex engage in redox cycling resulting in the generation of ROS. As such chelation of cellular copper and/or iron by TPEN has the potential to produce ROS leading to cellular toxicity.
Colon cancer cells accumulate higher levels of copper and zinc
Given the potential of TPEN-Cu (II) and TPEN-Fe(II) to redox-cycle in the presence of H2O2 and a reductant (eg, ascorbate) to yield hydroxyl radicals, the ability of these TPEN-metal complexes to produce cellular damage is likely to be proportional to the amount of chelatable metals in cells. Tumor tissues from multiple cancers have been reported to have elevated copper content . This is also the case for HCT116 cells as shown by atomic absorption spectroscopy studies, where both copper (enriched ~7-folds) and zinc (enriched ~5.5-folds) accumulate to significantly higher levels as compared to NCM460 (Figure 4E). In contrast, iron content was similar between the two cell types (Figure 4E). Similar accumulation of copper and zinc but not iron were observed in two additional colon cancer cell lines, LoVo and SW480, where the measured levels for Cu were 120.25 and 99.56 μg/L; Zn 20.34 and 15.26 μg/L; Fe 44.24 and 46.25 μg/L in LoVo and SW480 cells respectively. Given that zinc is not redox active and that the levels of iron are similar in both cell types, the mechanisms of action of TPEN is likely to involve formation of a TPEN-Cu complex that engages in redox cycling to generate ROS and cause cellular damage. Interestingly the fold enrichment of copper in HCT116 cells (Figure 4E) mirrors the fold increase in redox-cycling activity revealed by DCFH oxidation measured in these cells as compared to NCM460 following TPEN treatment (Figure 3A).
Cellular copper is required for TPEN toxicity
The above data support a model where TPEN induces selective death of cancer cells through its ability to chelate copper away from cellular proteins, resulting in the formation of a TPEN-copper complex able to engage in redox cycling thus generating ROS and inducing cell death. Interestingly, the TPEN-Cu complex is predicted to be impermeant to the cell membrane, given the positive charges contributed by the copper ion, thus trapping the complex intracellularly and increasing the levels of ROS produced. This model makes two testable predictions. First, it predicts that TPEN is capable of stripping a significant proportion of copper away from cellular proteins. To determine whether this is the case, we measured the fraction of total cellular copper that is chelatable by TPEN under our experimental conditions. As shown in Figure 4F, ~50% of the total cellular copper can be chelated by TPEN following incubation with 5μM TPEN for 24 hrs.
To test whether neocuproine reverses TPEN toxicity by chelating intracellular copper, we incubated cells with a membrane impermeant copper specific chelator, bathocuproine . In contrast to neocuproine, bathocuproine did not reverse the cellular toxicity due to TPEN treatment (Figure 5C). Collectively these data show that TPEN toxicity requires cellular copper. When intracellular copper is chelated with neocuproine, a copper chelator that is effective at chelating copper but does not allow the TPEN-Cu complex to engage in redox reactions, TPEN becomes ineffective at producing ROS and killing colon cancer cells. Furthermore, as shown in Figure 2A with zinc, copper was also effective at reversing the toxic effects of TPEN and its ability to generate ROS in a dose dependent fashion (Figure 5A and B), showing that TPEN toxicity is dependent on its metal chelation properties.
Tumoristatic activity of TPEN in vivo
Histological H & E staining of tumor sections from untreated mice showed malignant cell infiltration and retention in the tissue (Figure 6B). However, TPEN-treated group showed a significant decrease of malignant cell nuclei staining relative to the control mice, consistent with the tumor growth profile (Figure 6A and B). In addition, the expression of Ki-67, a nuclear protein proliferative marker, was reduced in TPEN-tissue section as depicted by immunostaining (Figure 6B). Furthermore, in vivo TPEN treatment induced apoptosis in tumor tissue as reported by TUNEL staining (Figure 6C). Collectively these data show that TPEN is effective at inhibiting the growth of colon cancer cells in vivo, arguing that metal chelation is a promising intervention for colon cancer treatment.
Metals such as iron, copper and zinc are essential elements in mammalian cells and are used as cofactors or as structural components of many enzymes. However, an excess of these metals causes toxicity. Therefore a balance between metal accumulation, their sequestration within cellular compartments, and their association with cellular proteins is essential for the maintenance of cell viability [35, 36]. Transient intracellular elevations of free iron or copper is toxic because of their redox reactivity and participation in ROS metabolism. In particular, low molecular mass complexes of Fe (II) and Cu (II) can react with hydrogen peroxide to generate hydroxyl radical, which is believed to be the most toxic form of ROS encountered in cells.
Here we show that this detrimental effect of transition metals can be used effectively to selectively eliminate cancer cells. TPEN is efficient at selectively eliminating colon cancer cells both in the dish and in vivo in mice. The mechanism of action of TPEN depends first on its high affinity for metals and as such its ability to strip intracellular metals from cellular proteins. Second colon cancer cells accumulate copper to significantly higher levels than normal colon cells. Finally, the TPEN-copper complex engages in redox cycling and the generation of ROS (hydroxyl radicals). Hence, TPEN selectively eliminates colon cancer cells using an elegant mechanism that relies on the biology of the cancer through its tendency to accumulate copper, a feature that is common to many other cancers including breast, ovarian, stomach, colorectal and leukemia . As such the TPEN-dependent selective killing observed in this study is likely to be applicable to other cancers that accumulate redox active metals.
However, TPEN anti-tumor activity is likely to involve additional mechanisms since antioxidants treatment was not fully effective at reversing TPEN toxicity (Figure 3). TPEN toxicity is likely to be partly due to the removal of zinc from essential Zn-dependent proteins as previously reported by others [7–15].
Another metal chelator that shows selective antitumor activity is di-2-pyridylk-etone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT). Dp44mT is thought to act in a similar fashion to TPEN through iron chelation and redox cycling to generate ROS [37, 38]. Dp44mT toxicity is also believed to involve activation of the lysosomal apoptotic pathway through its copper binding capacity .
Interestingly, cancer cells are known to have an altered redox status with an up-regulation of oxidative stress and an augmentation of antioxidant capacity (reviewed in ). It is also thought that a moderate increase in ROS enhances cell survival and proliferation . Therefore the observed increase in ROS in cancer cells may promote tumorigenesis . However, the increased basal ROS in cancer cells brings them closer to the toxicity threshold where the intrinsic antioxidant capacity, although enhanced, is not sufficient to contain toxic ROS levels . Therefore increased oxidative stress in cancer cells represents an effective mechanism to eliminate cancer cells .
In summary, selective killing of colon cancer cells can be achieved using TPEN, where the transition metal-chelator complex engages in redox cycling and the generation of hydroxyl radicals. This is an attractive potential anti-tumor therapeutic approach because its mechanism of action depends on the biology of cancer cells, including the significant accumulation of copper and their endogenous enhanced oxidative stress. Therefore, selectivity toward elimination of cancer cells without affecting normal cells is inherent in this approach because of different homeostatic mechanisms in cancer versus normal cells. In that context it would be attractive to systematically explore the redox cycling potential of metal chelators already in the clinic for the treatment of other diseases.
TPEN, a membrane permeant metal chelator, selectively kills colon cancer cells without affecting the viability of normal cells. TPEN is effective at preventing tumor growth in a xenograft model. The mechanism of action of TPEN involves chelation of intracellular copper, making it available for redox cycling thus leading to the generation of ROS and cell death. Because colon cancer cells accumulate copper to 7 folds higher levels than control cells, this endows TPEN with its selective killing ability. Therefore, the mechanism of action of TPEN offers an attractive anti-tumor therapeutic approach potentially for a multitude of cancers that accumulate copper.
2 ′,7′-Dichlorofluorescin diacetate
Poly aDP ribose polymerase
Reactive oxygen species
N, N, N’, N’-tetrakis-[2-pyridylmethyl]-ethylenediamine
Terminal deoxynucleotidyl transferase dUTP nick end labeling
X-Linked inhibitor of apoptosis.
This work was funded by NPRP grant # 09-047-3-012 from the Qatar National Research Fund (QNRF). The statements made herein are solely the responsibility of the authors. Additional support for KM’s Lab comes from the Biomedical Research Program at Weill Cornell Medical College in Qatar, a program funded by Qatar Foundation. We are grateful to Dr. Hussein Charif for his help with tissue immunostaining and to the members of the Kamal Shair Central Science Research Laboratory for their technical help.
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