Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4-CXCL12 axisReportar como inadecuado




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Journal of Experimental and Clinical Cancer Research

, 36:104

First Online: 03 August 2017Received: 06 June 2017Accepted: 24 July 2017

Abstract

BackgroundFerritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit FHC is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition EMT is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms.

MethodsTargeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry.

ResultsExperimental data indicated that FHC-silenced MCF-7 and H460 cells MCF-7, H460 acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4-CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4-CXCL12 axis.

ConclusionsOur findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron-ROS metabolism and CXCR4-CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount-function might be an important additional tool to predict tumor aggressiveness.

KeywordsFerritin heavy chain EMT CXCR4 CXCL12 Cancer ROS AbbreviationsBSABovine serum albumin

CNSCentral nervous system

CXCL12C-X-C motif chemokine ligand 12

CXCR4C-X-C chemokine receptor type 4

DMEMDulbecco’s Modified Eagle Medium

ECLElectrochemiluminescence

EMTEpithelial to mesenchymal transition

FBSFetal bovine serum

FHCHeavy chain of ferritin

FTH or FHCFerritin heavy chain

FTLFerritin light chain

GAPDHGlyceraldehyde 3-phosphate dehydrogenase

GPCRG protein-coupled receptor

HEK-293 THuman embryonic kidney cells 293

HSHigh salt solution

LIPLabile Iron Pool

MORMu opioid receptor

MTT3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide

NACN-acetylcysteine

O.DOpticla Density

PBSPhosphate buffered saline

PFAParaformaldehyde

qRT-PCRQuantitative RT-PCR

ROSreactive species of oxygen

RPMIRoswell Park Memorial Institute medium

SDSSodium dodecyl sulfate

shRNAShort hairpin

TPBSTris buffered saline

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Autor: I. Aversa - F. Zolea - C. Ieranò - S. Bulotta - A. M. Trotta - M. C. Faniello - C. De Marco - D. Malanga - F. Biamonte

Fuente: https://link.springer.com/







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