We would like to thank Northumbria University for their support.

1. Stock solution preparation
<ol>
	<li>When preparing Calcein stocks solutions, prevent photo degradation by keeping solutions in the dark. Prepare a 1 mM solution of Calcein in Dulbecco&#39;s PBS, without magnesium or calcium14. For 5 mL of 1 mM Calcein solution, add 3.1 mg of Calcein (622.5 g/M) to 5 mL of Dulbecco&#39;s PBS. Using the 1 mM sock of Calcein, prepare 1 mL aliquots of secondary stock solutions in microfuge tube as detailed in Table 1.</li>
	<li>...

Fluorescent Assay for Detection of Novel Iron Chelators

<ol>
	<li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Hsu, M. Y., Mina, E., Roetto, A., Porporato, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron:+An+essential+element+of+cancer+metabolism.">Iron: An essential element of cancer metabolism.</a> Cells. 9 (12), 2591 (2020).</li><li>Torti, S. V., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer:+More+ore+to+be+mined.">Iron and cancer: More ore to be mined.</a> Nat Rev Cancer. 13 (5), 342-355 (2013).</li><li>Zhang, C., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+homeostasis+and+tumorigenesis:+Molecular+mechanisms+and+therapeutic+opportunities.">Iron homeostasis and tumorigenesis: Molecular mechanisms and therapeutic opportunities.</a> Protein Cell. 6 (2), 88-100 (2015).</li><li>Torti, S. V., Manz, D. H., Paul, B. T., Blanchette-Farra, N., Torti, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer.">Iron and cancer.</a> Annu Rev Nutr. 38, 97-125 (2018).</li><li>Welch, D. R., Hurst, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+hallmarks+of+metastasis.">Defining the hallmarks of metastasis.</a> Cancer Res. 79 (12), 3011-3027 (2019).</li><li>Lehmann, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+defects+of+hepatocellular+carcinoma+are+already+found+in+non-neoplastic+liver+cells+from+patients+with+hereditary+haemochromatosis.">Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis.</a> Hum Mol Genet. 16 (11), 1335-1342 (2007).</li><li>Fonseca-Nunes, A., Jakszyn, P., Agudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+cancer+risk--a+systematic+review+and+meta-analysis+of+the+epidemiological+evidence.">Iron and cancer risk--a systematic review and meta-analysis of the epidemiological evidence.</a> Cancer Epidemiol Biomarkers Prev. 23 (1), 12-31 (2014).</li><li>Abdelaal, G., Veuger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+oncogenic+transformation+with+iron+chelation.">Reversing oncogenic transformation with iron chelation.</a> Oncotarget. 12 (2), 106-124 (2021).</li><li>Brown, R. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+iron+metabolism+and+impact+in+cancer+biology,+metastasis,+and+immunology.">Altered iron metabolism and impact in cancer biology, metastasis, and immunology.</a> Front Oncol. 10, 476 (2020).</li><li>Menyh&#225;rt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+selection+of+functional+assays+to+evaluate+the+hallmarks+of+cancer.">Guidelines for the selection of functional assays to evaluate the hallmarks of cancer.</a> Biochim Biophys Acta. 1866 (2), 300-319 (2016).</li><li>Basu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+novel+and+diverse+iron-chelators+in+silico.">Discovering novel and diverse iron-chelators in silico.</a> J Chem Inf Model. 56 (12), 2476-2485 (2016).</li><li>Prus, E., Fibach, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+measurement+of+the+labile+iron+pool+in+human+hematopoietic+cells.">Flow cytometry measurement of the labile iron pool in human hematopoietic cells.</a> Cytometry A. 73 (1), 22-27 (2008).</li><li>Cold Spring Harb. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phosphate-buffered saline (pbs). 2006 (1), (2006).</li><li>Kontoghiorghes, G. J., Pattichis, K., Neocleous, K., Kolnagou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+and+development+of+deferiprone+(l1)+and+other+iron+chelators+for+clinical+use:+Targeting+methods+and+application+prospects.">The design and development of deferiprone (l1) and other iron chelators for clinical use: Targeting methods and application prospects.</a> Curr Med Chem. 11 (16), 2161-2183 (2004).</li></ol>

The authors declare no conflict of interest.

The over reliance of cancers on iron to fuel their metabolism makes iron chelation a potential addition to therapeutic regimes4. However, there is a limited ability to quickly screen novel metal ion chelators for their ability to bind iron ions. The commonly used and widely available fluorescent probe Calcein is known to act as a weak iron chelator and binding by iron ions quenches Calcein fluorescence. Fluorescence can then be recovered by competing for binding to iron using a stronger iron chela...

Phenotypic changes to cells that relate to the development of cancer through a common set of altered biological capabilities are now commonly referred to as the hallmarks of cancer. Amongst them are changes resulting from the reprogramming of energy metabolism, which are widespread in cancer cell biology1. Such metabolic reprogramming includes an increased requirement for iron to support rapid proliferation and tumor growth2. This thirst for iron leads to dysregulated iron metabolism, which in and of itself is considered a hallmark of cancer3,4, with dysregulation occurring at all stages5. The hallmarks of metastasis, more recently proposed by Welch and Hurst, include a role for iron6 since iron can induce oxidative stress, and this can, in turn, mediate changes to the genome, epigenome, and proteome, enhancing the possibility of metastasis7. A link between iron levels and an increased occurrence of cancer has been demonstrated through epidemiological studies8.
Since cancer cells require large amounts of iron, they are susceptible to iron deficiency and, therefore, iron chelation. We have recently published a review article highlighting the potential for iron chelation in reversing several hallmarks of cancer through NDRG1 disrupting oncogenic signaling pathways9. However, the use of iron chelation as stand-alone cancer therapy has not yielded positive results in clinical trials due to their toxicity, short half-life, rapid metabolism, and emerging resistance mechanisms. Nevertheless, iron chelators have shown promise in in vitro and in vivo investigations, indicating that more work is needed to develop effective iron chelators for cancer therapy. Specific iron chelation is a validated strategy in anticancer drug discovery, but only a few classes have been reported to date10.
The identification and characterization of novel iron chelators requires the ability to measure their effect on several endpoints. Many of these (such as proliferation, apoptosis, reactive oxygen species formation) are routinely measured and have been outlined and reviewed in the literature as methods to evaluate the hallmarks of cancer11. When evaluating a novel iron chelator, many groups routinely examine the effect on anti-proliferative and redox activities as well as effects on iron influx or efflux. In silico prediction techniques12 further increase the growing pool of iron chelators that can be screened.
Screening of iron chelators requires the ability to measure their effect on iron levels as a means to effectively demonstrate proof of principle. Currently, the most common method to do so is flow cytometry13 which is costly, time consuming and poorly quantifiable. The choice of assay is often based on the availability of experimental equipment, speed, and cost of an assay. Therefore, the ability to chelate iron can be an overlooked end point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell free fluorescent method to confirm the ability of novel compounds to chelate iron.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium iron(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>215406</td>
			<td>other wise known as FAS</td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Deferiprone</td>
			<td>Sigma-Aldrich</td>
			<td>379409</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D5652</td>
			<td>magnesium and calcium free</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 96 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>M0812</td>
			<td>any optically transparent 96 well plate will work</td>
		</tr>
	</tbody>
</table>

quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron 

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron 

We developed a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. Iron chelators are a promising class of compound that can be used to target cancers. Chelators'disruption of cancer's metabolic adaptions may lead to a new therapeutic strategy in cancer treatment.There is a research focus on the development of iron chelators to disrupt the metabolic adaptions of cancers. However, there are no inexpensive and simple screening techniques for the observation of a novel molecule's ability to chelate iron. There is an unmet need for a simple, inexpensive assay for screening novel iron chelators.This assay will allow cancer biologists to observe the chelation ability of novel compounds before investing time and resource into cellular screens of biological activity. Using this assay, we were able to observe the iron chelation ability of newly developed iron chelators. We have then focused on the question of how these chelators disrupt metabolism in cancer cells and how these cells respond to this disruption.We are fascinated with how cancer cells respond to the metabolic disruptions created by the treatment with iron chelators. Understanding iron metabolism in cancers is fundamental to the development of novel therapeutics and new treatment regimes.

For the method shown in step 2, this first experiment (Figure 1) established the linear range of the microtiter fluorescence plate reader when detecting Calcein fluorescent emissions. Our representative results show a wide linear range of Calcein fluorescence from 0-100 &#181;M. ANOVA with LSD post hoc analysis demonstrates that there is a statistically significant differences in mean RFU for all Calcein concentrations when compared to a control of 0 &#181;M of Calcein. In further experiment...

Read this Scientific Article Protocol about Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron at JoVE.com

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

quantifiable-inexpensive-cell-free-fluorescent-method-to-confirm

Research

JoVE Journal

Cancer Research

Fluorescence Assay for the Detection of Iron Chelators Using Calcein and Ferrous Ions

To begin, use a multi-channel pipette to distribute 50 microliters of PBS into columns 2 to 10 of rows A to E on a 96-well plate. Pipette 100 microliters of two millimolar ferric ammonium sulfate stock solution into column 11. With a multi-channel pipette, transfer 50 microliters of the ferric ammonium sulfate stock from column 11 into column 10.Pipette the solutions multiple times to mix well. Perform step-wise double dilution across the columns from 9 to 2 and row A to 2. Now pipette out 50 microliters of the solution from column 2 to discard.After adding PBS into all the wells, pipette 10 microliters of 10 micromolar Calcein stock into each well. For the standalone PBS control, pipette 100 microliters of PBS into row F of columns 1 to 5. Then add 100 microliters of two millimolar ferric ammonium sulfate to row F of columns 6 to 10 for the FAS control.Incubate the plate at room temperature in the dark for 10 minutes. Analyze the fluorescence with a microplate reader. Pipette 50 microliters of PBS to columns 2 to 10 of rows A to E of a 96-well plate.To column 11, add 100 microliters of two micromolar stock solution of the iron chelator solution. With a multichannel manual pipette, remove 50 microliters of the iron chelator solution from column 11. Then transfer it into the PBS in column 10 from rows A to E.Pipette up and down to mix the solution well.Continue to dilute the chelator solution across the remaining columns. Discard 50 microliters of the solution from the wells of column 2, then pipette 30 microliters of PBS into all wells. Using a multichannel pipette, add 10 microliters of 100 micromolar ferric ammonium sulfate stock into columns 2 to 12 of rows A to E.Add 10 microliters of 10 micromolar Calcein stock into the same wells.Next for the positive control, pipette PBS, Calcein stock, and ferric ammonium sulfate stock into column 1 of rows A to E.Mix the samples well by pipetting the solutions up and down. Then add 100 microliters of PBS into row F of columns 1 to 5 to create the PBS negative control. Pipette the same volume of the iron chelator solution to row F from column 6 to 10 to create the standalone chelator control.Place the well plate at room temperature for 10 minutes in the dark. Then analyze on a multimodal microplate reader as before. The addition of ferrous ions to Calcein resulted in a linear decrease in Calcein fluorescence.The iron chelator outcompeted Calcein for the ferrous ions. The chelator increased the Calcein fluorescence up to a peak.

237 Views.  Northumbria University.  We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs. 

Fluorescent Assay for Detection of Novel Iron Chelators

Summary