Anticancer activity of the iron facilitator LS081
© Li et al; licensee BioMed Central Ltd. 2011
Received: 21 January 2011
Accepted: 31 March 2011
Published: 31 March 2011
Cancer cells have increased levels of transferrin receptor and lower levels of ferritin, an iron deficient phenotype that has led to the use of iron chelators to further deplete cells of iron and limit cancer cell growth. As cancer cells also have increased reactive oxygen species (ROS) we hypothesized that a contrarian approach of enhancing iron entry would allow for further increased generation of ROS causing oxidative damage and cell death.
A small molecule library consisting of ~11,000 compounds was screened to identify compounds that stimulated iron-induced quenching of intracellular calcein fluorescence. We verified the iron facilitating properties of the lead compound, LS081, through 55Fe uptake and the expression of the iron storage protein, ferritin. LS081-induced iron facilitation was correlated with rates of cancer cell growth inhibition, ROS production, clonogenicity, and hypoxia induced factor (HIF) levels.
Compound LS081 increased 55Fe uptake in various cancer cell lines and Caco2 cells, a model system for studying intestinal iron uptake. LS081 also increased the uptake of Fe from transferrin (Tf). LS081 decreased proliferation of the PC-3 prostate cancer cell line in the presence of iron with a lesser effect on normal prostate 267B1 cells. In addition, LS081 markedly decreased HIF-1α and -2α levels in DU-145 prostate cancer cell line and the MDA-MB-231 breast cancer cell lines, stimulated ROS production, and decreased clonogenicity.
We have developed a high through-put screening technique and identified small molecules that stimulate iron uptake both from ferriTf and non-Tf bound iron. These iron facilitator compounds displayed properties suggesting that they may serve as anti-cancer agents.
Iron is an essential element required for many biological processes from electron transport to ATP production to heme and DNA synthesis with the bulk of the iron being in the hemoglobin of circulating red blood cells [1, 2]. Too little iron leads to a variety of pleiotropic effects from iron deficiency anemia to abnormal neurologic development, while too much iron may result in organ damage including hepatic cirrhosis and myocardiopathies. The system for the maintenance of iron homeostasis is complex. Approximately 1 mg of the iron utilized daily for the synthesis of nascent red blood cells is newly absorbed in the intestine to replace the amount lost by shed epithelial cells and normal blood loss. The remainder of the iron incorporated into newly synthesized hemoglobin is derived from macrophages from catabolized senescent red blood cells. Hence, the uptake of iron for its final incorporation into hemoglobin or other ferriproteins requires 3 different transport pathways: intestinal iron absorption, iron release from macrophages, and iron uptake into erythroid precursors and other iron-requiring cells.
In vertebrates, iron entry into the body occurs primarily in the duodenum, where Fe3+ is reduced to the more soluble Fe2+ by a ferrireductase (DcytB), which transports electrons from cytosolic NADPH to extracellular acceptors such as Fe3+. The Fe2+ is transported across the brush border membrane (BBM) of duodenal enterocytes via the transmembrane protein, DMT1 (divalent metal transporter, also known as SLC11a2, DCT1, or Nramp2) [4, 5]. Subsequently, the internalized Fe2+ is transported across the basolateral membrane (BLM) by the transmembrane permease ferroportin (FPN1, also known as SLC40a1) [3, 6] in cooperation with the multicopper oxidase Hephaestin (Heph) [7, 8]. The exit of iron from macrophages onto plasma transferrin (Tf) is also mediated by the interaction of FPN1 and Heph . The efflux of iron into the systemic circulation from the enterocyte and the macrophage is negatively regulated by hepcidin, the iron-stores regulator. Hepcidin binds to FPN1 promoting phosphorylation, internalization, and subsequent catabolism of FPN1 via proteasomes .
In erythroid precursor cells, and indeed in all non-intestinal cells, iron uptake is mediated by receptor mediated endocytosis of ferri-transferrin (Fe-Tf) although routes for non-transferrin bound Fe (NTBI) also exist. Fe-Tf binds to the transferrin receptor (TfR) on the cell surface  and the Fe-Tf complex is internalized into endosomes with subsequent acidification of the endosome which releases Fe3+ from Tf. The Fe3+ is then reduced to Fe2+ by the ferrireductase STEAP 3  and the Fe2+ transported by DMT1 into the cytosol.
There are two situations in which one could envision a benefit from being able to accelerate or otherwise increase cellular uptake of iron. First, iron deficiency is endemic in much of the world resulting in decreased ability to work especially in women of child bearing age and in impaired neurologic development in children [13, 14]. Common factors leading to an imbalance in iron metabolism include insufficient iron intake and decreased absorption due to poor dietary sources of iron . In fact, Fe deficiency is the most common nutritional deficiency in children and the incidence of iron deficiency among adolescents is also rising . Iron deficiency ultimately leads to anemia, a major public health concern affecting up to a billion people worldwide, with iron deficiency anemia being associated with poorer survival in older adults . As much of iron deficiency is nutritional, drugs that promote iron uptake could be beneficial without the necessity of changing economic and cultural habits that dictate the use of iron poor diets.
A second, and separate, situation exists in malignancies. Cancer cells often have an iron deficient phenotype with increased expression of TfR, DMT1, and/or Dcytb and decreased expression of the iron export proteins FPN1 and Heph [18–20]. Since higher levels of ROS are observed in cancer cells compared to non-cancer cells drugs that stimulate iron uptake into cancer cells might further increase ROS levels via the Fenton reaction. The increased ROS might lead to oxidative damage of DNA, proteins, and lipids [21, 22] and cell death or potentiate cell killing by radiation or radiomimetic chemotherapeutic agents. Further, increased intracellular levels of Fe would increase the activity of prolyl hydroxylases potentiating hydroxylation of HIF-1α and HIF-2α, transcription factors that drive cancer growth, resulting in decreased HIF expression via ubiquination and proteasome digestion.
Wessling-Resnick and colleagues have used a cell-based fluorescence assay to identify chemicals in a small molecule chemical library that block iron uptake [23–25]. While some of the chemicals identified inhibited Tf-mediated iron uptake  more recent studies utilizing a HEK293T cell line that stably expresses DMT1 have identified chemicals that act specifically on the iron transporter [24, 25]. In the current study, we have used a similar assay to identify chemicals that increase iron uptake into cells and demonstrate that these chemicals are effective in increasing iron transport across Caco2 cells, a model system for studying intestinal iron absorption, and increasing iron uptake into various cancer cell lines, favourably altering several aspects of the malignant phenotype.
Cell lines and Chemicals
All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) except for rabbit anti-HIF-1α and -2α which were purchased from Novos Biologicals (Littleton, CO). All analytical chemicals were from Sigma-Aldrich (St. Louis, MO). The chemical libraries were obtained from ChemDiv (San Diego, CA) and TimTec (Newark, DE). CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester) or DCFDA and calcein-AM were from Invitrogen (Carlsbad, CA). The cell lines K562, PC-3, Caco2, MDA-MB231, and 267B1 were obtained from ATCC (Bethesda, MD). RPMI1640 and DMEM culture media and fetal calf serum (FCS) were obtained from Atlanta Biologicals (Lawrenceville, GA).
Screening for chemicals that increase iron uptake
where Δ Fn is the normalized quench observed after addition of iron, Fcompound is the Δ F observed with compound, Fmin is the average Δ F of the DMSO control; and Fmax is the average Δ F of the DTPA control. With this normalization 100% indicates that a test compound is as potent as DTPA in blocking iron-induced quenching and 0% indicates no inhibition of iron quenching by a test compound or the same quench as observed with the DMSO vehicle control. Compounds with Δ Fn between 0% and 100% are defined as inhibitors of iron uptake. Negative values for Δ Fn represent compounds that facilitate iron uptake into cells. Our criteria for active compounds to be further investigated was arbitrarily set as Δ Fn = 50-100% quenching for iron uptake inhibitors and < -50% quenching for iron uptake facilitators.
55Fe uptake into K562 cells
3 × 105 K562 cells in 300 μl NaCl-Hepes-0.1% BSA were incubated for 30 min with test compound at various concentrations as indicated in a humidified 37°C incubator with 5% CO2. A mixture of 55Fe- and AA was then added for a final concentration of 1 μM 55Fe -1 mM AA and the cells incubated for an additional 60 min. The reaction was stopped by the addition of ice-cold quench buffer (NaCl-Hepes with 2 mM EDTA) followed by extensive washing of the cells which were then dispersed in scintillation fluid and 55Fe radioactivity determined in a Tri-carb 2900 TR liquid scintillation analyzer (Packard BioScience Company, Meriden, CT).
Preparation of medium containing 10% FCS with iron-saturated Tf
Iron on the Tf in FCS was removed from the Tf by lowering the pH to 4.5 followed by dialysis against 0.1 M citrate buffer, pH 4.5, in the presence of Chelex for 16 hours, and dialyzed again against HEPES buffered saline, pH 7.4, in the presence of Chelex. FeNTA (1:2 molar ratio for Fe: NTA) was then added to the now iron-free FCS at 1 mM final concentration followed by extensive dialysis against HEPES buffered saline, pH 7.4. The resulted FCS containing iron-saturated Tf was added into RPMI1640 to make the medium containing 10% iron-saturated FCS.
Western blot analysis of ferritin, TfR, and HIF-1α and -2α
PC-3 cells were plated into 6-well plates at cell density of 5 × 105 cells/well for overnight attachment before addition of test compound or vehicle control for 16 hours. The cells were then lysed with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, pH 7.4) and the lysates separated on SDS-PAGE with subsequent transfer to nitrocellulose for western blot analysis using the following antibodies: mouse anti-human ferritin-heavy chain, mouse anti-human TfR, anti-HIF-1α or -2α, and rabbit anti-human β-actin. Results were quantitated by densitometry and relative densitometric units expressed as the ratio of protein of interest to actin.
55Fe uptake and transport in Caco2 cells
Caco2 cells were seeded in 6.5 mm bicameral chambers in 24-well plates, grown in 10% FCS-minimum essential medium for ~2 week to reach a transepithelial electrical resistance (TEER) of 250 .cm2. The cells were incubated in serum-free DMEM with 0.1% BSA overnight and the inserts then transferred to fresh 24-well plates with the basal chambers containing 700 μL of 20 μM Apo-Tf in DMEM. Test compound at concentrations of 0-100 μM in a total volume of 150 μl were added to the top chamber, incubated for 60 min at 37°C, 5% CO2 incubator, followed by the addition of 55Fe to the top chamber at a final concentration of 0.125 μM 55Fe in 1 mM AA. At various times up to 2 hours, the top and bottom chamber buffer were removed, the cell layer washed extensively with Hepes-NaCl containing 0.1 mM EDTA, and 55Fe radioactivity determined in the upper and lower chamber buffers and the cell layer.
To determine if compound affected cellular production of ROS, 5 × 105 K562 cells were washed, treated for 30 min with compound in Hepes-NaCl buffer, and intracellular levels of ROS detected with CM-H2DCFDA by flow cytometry as described . ROS levels are presented as mean fluorescence intensity in the appropriate gated areas. K562 cells exposed to 10 μM H2O2 were used as positive control for ROS generation.
Cell proliferation and colony formation assays
To assess cell proliferation PC-3 cells were seeded into 96-well plates at 1 × 104/well for 24 hr to allow for cell attachment. Cells were treated with 0.1% DMSO, 10 μM ferric ammonium citrate, 10 μM LS081, or the combination of 10 μM Fe + 10 μM LS081 in RPMI1640-10% FCS for 24-72 hr with the treatment media being replenished every 24 hr. Cell proliferation was accessed 24, 48, or 72 hr after treatment. In separate experiments, PC-3 or 267B1 cells were plated in 96-well plates at 1 × 104/well in RPMI1640 containing 10% FCS overnight before 24 hr treatment with 0.1% DMSO, 2 μM ferric ammonium citrate, 3 or 10 μM LS081 ± Fe in serum-free-RPMI1640, with an additional 24 hr incubation in RPMI-1640-10% FCS without LS081. Cell proliferation was assayed with CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega) kit on a Synergy 2 Spectrophotometric Analyzer (BioTek Inc., Winooski, Vermont) with wavelength of 490 nM and the results standardized to the percentage of inhibition induced by DMSO alone. Cell viability was assessed by Trypan blue exclusion.
Colony formation was assayed in PC-3 cells by plating 500 cells/well in 6-well plates in 10% FCS-RPMI1640 for 48 hr, followed by incubation with 0.1% DMSO, 10 μM ferric ammonium citrate, 3 or 10 μM LS081 ± ferric ammonium citrate for an additional 48 hours, after which the media was replaced with 10% FCS-RPMI1640. The cells were cultured for an additional 10-14 days and then stained with Crystal violet before colonies consisting of more than 50 cells were enumerated.
A cell based fluorescence assay to screen small molecules that increase iron transport into cells
Caco2 cells grown in bicameral chambers for 2-3 weeks to reach the desired trans-epithelial electrical resistance were used as a model for intestinal iron absorption. Under these conditions the Caco2 cells differentiate to form a confluent, polarized monolayer with the brush border membrane of the apical surface in contact with the buffer of the top chamber which then mimics the intestinal lumen and the basal layer in contact with the bottom chamber which represents the systemic circulation. This model allows assaying in the presence of LS081 the transport of 55Fe from the apical chamber into the cells and then into the bottom chamber. In this model over 2 hours, LS081 increased 55Fe uptake into the Caco2 cells and into the basal chamber by 4.0 ± 0.66 and 3.71 ± 0.29 fold, respectively, compared to the DMSO-treated control (mean fold change ± SEM of 3 experiments) with P < 0.001 for both uptake and transport into the basal chamber.
Effect of the iron facilitator LS081 on intracellular levels of ferritin
Iron facilitation is cytotoxic to cancer cells
The effect of LS081 and iron on the proliferation of PC-3 cells
1.00 ± 0.00*
1.00 ± 0.00*
10 μM Fe
1.13 ± 0.04***
1.02 ± 0.06*
10 μM LS081
1.05 ± 0.05**
1.01 ± 0.03*
10 μM Fe and LS081
0.81 ± 0.01
0.80 ± 0.09
Effect of the iron facilitator LS081 on clonogenic potential on prostate cancer cells
Effect of the iron facilitator LS081 on the level of HIF-1α and -2α protein
As noted by Wessling-Resnik and colleagues in their search for iron uptake inhibitors chemical genetics, i.e. the use of small molecules to perturb a physiologic system, has the ability to shed light on mechanisms of the pathway that is being disturbed . Additionally, compounds that perturb iron uptake could have beneficial, medicinal effects. For example, small molecules which stimulate iron absorption might be used as adjuncts to diets that are iron-deficient. Conversely, molecules that blocked iron uptake might counter the increased iron absorption and resultant iron toxicity often seen in widely prevalent diseases such as sickle cell disease and the thalassemias. Wessling-Resnik has screened chemical libraries to identify chemicals that block iron uptake  but also found "activators" of iron uptake which were postulated to have potential as agents to relieve iron deficiency. In the current study we have adapted their calcein-based cell assay and identified compounds that increase iron uptake into Caco2 cells, as a model system for intestinal transport, and into various cancer cell lines, thereby altering several aspects of the malignant phenotype.
In our assay, intracellular calcein fluorescence in K562 cells was quenched upon extracellular iron being transported into the cells. Iron facilitation was defined as fluorescence quenching greater in the presence of a test compound compared to vehicle control. In addition, none of the facilitators appeared to be iron chelators as the chemicals did not compete with iron for calcein quenching in an in vitro assay and the iron facilitators affected the cell cycle differently from the iron chelator deferoxamine (data not shown). We did, however, find a number of chemicals that inhibited iron uptake and several of these chemicals appeared to be iron chelators by an in vitro assay. Notwithstanding that the faciltators inhibited cell proliferation there was no evidence that the chemicals caused cell lysis as cell number was not diminished during the screening assays or during subsequent measurements of 55Fe uptake.
In iron uptake whether from NTBI, in the case of enterocytes, or from ferri-Tf, in the case of all other cell types, the uptake occurs by iron being transported through DMT1. The facilitators could act by activating DMT1, repositioning DMT1 within the cell to more efficiently transport iron, or activating another transporter. DMT1 is a highly insoluble membrane protein making it difficult to determine the effect of the facilitators on DMT1 transport activity in an in vitro system; however, a clue to the mode of action of the facilitators comes from our observation that LS081 increased iron uptake when the sole source of iron was ferri-Tf. Iron uptake from Tf requires that the Tf undergo receptor mediated endocytosis and DMT1 is part of the internalized endosome. Hence, for more iron to be delivered to a cell by ferri-Tf the endosomes containing DMT1 must cycle into and out of the cell more rapidly. When iron is delivered by ferri-Tf the rate limiting step in iron uptake is the length of the transferrin cycle, that is the time for ferri-Tf to undergo endocytosis, release iron from Tf into the endosome, and for the now apo-Tf still bound to the TfR to undergo exocytosis and be released from the TfR at the cell surface. If the facilitator shortened the length of the Tf cycle then DMT1 would be internalized more rapidly and the iron from Tf could be delivered faster. Inhibitors of iron uptake from ferri-Tf have been shown to adversely affect the Tf cycle . In enterocytes we and others have shown that DMT1 is internalized upon exposure of the duodenum and Caco2 cells to Fe. Hence, increasing the rate of DMT1 internalization would also increase iron uptake in the enterocytes.
While we presume that LS081 acts via DMT1 by altering the kinetics of DMT1 internalization there are other routes for iron uptake that could be affected. For example, lipocalin (also known as NGAL or 24p3), the L-type Ca2+ channel, and Zip14, a member of zinc transporter family, all have been demonstrated to be iron transporters or channels [28–30]. Whether these potential routes of iron entry are affected by the iron facilitators is not known but these alternative minor routes for iron transport function with NTBI and not with ferri-Tf and could not explain, therefore, how the facilitators affect uptake from ferri-Tf.
Whatever the mechanism(s) by which iron uptake facilitation occurs the Fe that gains entry to the cell enters a pool of metabolically active iron as evidenced by several observations. First, cellular ferritin levels increased in the presence of LS081 whether iron was offered as non-Tf or Tf-bound iron. Second, HIF1α and 2α protein expression was decreased. Third, the colony forming ability of prostate cancer cell lines was decreased. Fourth, LS081 increased the level of ROS.
It is interesting to consider the effects of iron facilitation on the levels of ROS as a possible explanation for the decreased cell proliferation and clonogenicity we observed in cancer cells. ROS levels are increased in cancer cells and it is possible that the additional ROS generation by LS081 exceeds cellular defences. Elevated ROS might then make LS081 treated cells more sensitive to radiation therapy and radiomimetic drugs, a hypothesis that is being actively pursued. The idea of disturbing the redox balance in cancer cells as a therapeutic approach for cancer has been postulated by other investigators [31–33]. Some conventional chemotherapy agents such as melphalan, cisplatin, anthracyclines, or bleomycin, are known to increase ROS by compromising the ROS scavenging capability of cancer cells [34–36]. Dicholoracetate, an inhibitor of pyruvate dehydrogenase kinase, stimulates ROS production and elicits apoptosis in cancer but not in normal cells . Moreover, reducing ROS scavengers by inhibition of glutamate-cysteine ligase, the rate limiting enzyme in glutathione synthesis, increases radiosensitivity of cancer cells . In addition, metal-binding compounds have been considered to be potential anti-cancer agents and have demonstrated anticancer activity . Although some compounds appear to act via metal chelation, others appear to increase intracellular metal concentrations, suggesting different mechanisms of action. For example, clioquinol induces apoptosis of prostate cancer cells by increasing intracellular zinc levels , and the anti-malarial drug artemisinin has anti-cancer activity that may be mediated by Fe2+ and/or heme [41, 42]. The potential toxicity of excess of iron in cancer cells suggests the benefit of identifying molecules that promote iron uptake into cancer cells triggering more efficient cell death.
Hypoxia is a common feature of most solid tumors with concomitant increased expression of the HIF-1α or HIF-2α components of the HIF transcription factor [43, 44]. Elevated levels of HIF-1α or HIF-2α are poor prognostic indicators in a variety of tumors . Under normoxic conditions, both HIF-1α and -2α are hydroxylated by an iron-dependent prolyl hydroxylase (PHD), which requires a ferrous ion at the active site, with subsequent hydroxylation ubiquitination by the von Hipple-Lindau tumor suppressor (VHL) and then proteasome degradation. Higher levels of intracellular iron could facilitate hydroxylation leading to increased ubiquitization and subsequent proteosome degradation of HIF-1α and -2α. HIF expression is important in cancer growth via several mechanisms including neo-vascularization. While HIF-1α and -2α have been targets for drug development [46, 47] there is as yet no clinically active drug that specifically targets HIF expression. Presumably LS081 induced reduction in HIF-1α and -2α is directly related to iron facilitation with increased activity of PHD from increased cellular iron, an hypothesis supported by loss of PHD activity and HIF1α stabilization when cellular Fe uptake is limited by TfR knockdown .
In summary, we identified a series of compounds capable of increasing iron uptake into cells. The lead compound, LS081, facilitated iron uptake which resulted in reduced cancer cell growth, colony formation, and decreased HIF-1α and -2α protein levels, suggests that this class of compounds could be a useful anti-cancer agent. In addition, the ability of these compounds to affect iron uptake in a model system of intestinal iron absorption suggests, also, that these compounds have a more general clinical utility for the management of iron deficiency.
Acknowledgements and Funding
This study was supported by Feist-Weiller Cancer Center at Louisiana State University Health Sciences Center-Shreveport and Message Pharmaceutical Inc.
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