<meta charset="utf8"></head><body>Dosage fluorescent pour la détection de nouveaux chélateurs du fer

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	<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. 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<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

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.

<meta charset="utf8"></head><body>Pleins feux sur l’auteur : Évaluation de l’impact de nouveaux chélateurs du fer sur le métabolisme des cellules cancéreuses

Méthode de fluorescence acellulaire quantifiable et peu coûteuse pour confirmer la capacité de nouveaux composés à chélater le fer 

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

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

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Research

JoVE Journal

Cancer Research

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

276 vues.  Northumbria University. Nous avons mis au point un test fluorescent qui peut confirmer rapidement et à peu de frais la capacité de nouveaux composés à chélater le fer. Les chélateurs du fer sont une classe prometteuse de composés qui peuvent être utilisés pour cibler les cancers. La perturbation des adaptations métaboliques du cancer par les chélateurs pourrait conduire à une nouvelle stratégie thérapeutique dans le traitement du cancer.La recherche se concentre...

Vidéo: Pleins feux sur l’auteur : Évaluation de l’impact de nouveaux chélateurs du fer sur le métabolisme des cellules cancéreuses

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.

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.