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

Summary

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.

Abstract

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 Fe^2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe^2+/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.

Introduction

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 cancer^3,4, 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.

Protocol

1. Stock solution preparation When preparing Calcein stocks solutions, prevent photo degradation by keeping solutions in the dark. Prepare a 1 mM solution of Calcein in Dulbecco'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'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. …

Representative Results

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 µM. 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 µM of Calcein. In further experiment…

Discussion

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…

Materials

Ammonium iron(II) sulfate hexahydrate	Sigma-Aldrich	215406	other wise known as FAS
Calcein	Sigma-Aldrich	C0875
Deferiprone	Sigma-Aldrich	379409
Dulbecco′s Phosphate Buffered Saline	Sigma-Aldrich	D5652	magnesium and calcium free
Greiner CELLSTAR 96 well plates	Sigma-Aldrich	M0812	any optically transparent 96 well plate will work