Dataset Information

Proteomic study of a breast cancer cell line following hemin treatment

ABSTRACT: Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on protein expression modifications in a BC cell line to gain insight into the molecular mechanisms of hemin antitumor activity. For this purpose, we carried out proteome analysis by Mass Spectrometry (MS) which showed that 1309 proteins were significantly increased in hemin-treated cells, including HO-1 and the proteases that regulate HO-1 function, and 921 proteins were significantly decreased. Furthermore, the MS-data analysis showed that hemin regulates the expression of heme- and iron- related proteins, adhesion and cytoskeletal proteins, cancer signal transduction proteins and enzymes involved in lipid metabolism. By biochemical and cellular studies, we further corroborated the most relevant in-silico results. Altogether, these results show the multiple physiological effects that hemin treatment displays in BC and demonstrate its potential as anticancer agent.

INSTRUMENT(S): Thermo Scientific instrument model

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: GEORGINA COLO

LAB HEAD: Alejandro Curino

PROVIDER: PXD036701 | Pride | 2023-07-20

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20190513_coreQEP2_AnWe_LC12-17_SA_Orbi3208Sample01.raw	Raw
	20190513_coreQEP2_AnWe_LC12-17_SA_Orbi3208Sample02.raw	Raw
	20190513_coreQEP2_AnWe_LC12-17_SA_Orbi3208Sample03.raw	Raw
	20190513_coreQEP2_AnWe_LC12-17_SA_Orbi3208Sample04.raw	Raw
	20190513_coreQEP2_AnWe_LC12-17_SA_Orbi3208Sample05.raw	Raw

Items per page:

1 - 5 of 16

Publications

Proteomic analysis of the effect of hemin in breast cancer.

Coló G P GP Schweitzer K K Oresti G M GM Alonso E G EG Chávez L Fernández LF Mascaró M M Giorgi G G Curino A C AC Facchinetti M M MM

Scientific reports 20230621 1

Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on pr ...[more]

PMID: 37344532

Similar Datasets

Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.

Project description:Hemes are essential but potentially cytotoxic cofactors that participate in critical and diverse biological processes. Although the pathway and intermediates for heme biosynthesis have been well defined, the intracellular networks which mediate heme trafficking remain unknown. Caenorhabditis elegans and related helminths are natural heme auxotrophs requiring environmental heme for growth and development. We exploited this auxotrophy to identify HRG-1 and HRG-4 in C. elegans and show that they are essential for heme homeostasis and normal vertebrate development. We demonstrate that heme deficiency upregulates expression of hrg-4 and its evolutionarily conserved paralog hrg-1. Depletion of either HRG-1 or HRG-4 in worms results in disruption of organismal heme sensing and abnormal response to heme analogs. HRG-1 and HRG-4 are novel transmembrane proteins that bind heme and have evolutionarily conserved functions. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations, and, most strikingly, profound defects in erythropoiesis - phenotypes that are fully rescued by worm HRG-1. These findings reveal unanticipated and conserved pathways for cellular heme trafficking in animals that defines the paradigm for eukaryotic heme transport. Uncovering the mechanisms of heme transport in C. elegans will provide novel insights into human disorders of heme metabolism and generate unique anthelmintics to combat worm infestations. Experiment Overall Design: As a first-step toward understanding heme homeostasis at the molecular level, we performed genome-wide microarrays to identify genes that are transcriptionally regulated by heme. For microarray analysis, synchronized F2 larvae were re-inoculated in mCeHR-2 medium supplemented with 4, 20 or 500 uM hemin and harvested at the late L4 stage for mRNA prep and probe hybridization to Affymetrix C. elegans Whole Genome Expression Arrays. Total RNA from three biological replicates were used at each hemin concentration. Data from worms grown in mCeHR-2 medium with 4 and 500 uM hemin were compared to data from worms grown in 20 uM hemin. Microarray data were verified with Microarray Suite 5.0 (Affymetrix) and Robust Multichip Average Method (RMA, R package). Results from MAS 5.0 and RMA analyses provided with 375 genes that showed 1.6 fold change in 4 and 500 uM hemin when compared to data from 20 uM hemin samples. Statistical analyses identified changes in 375 genes from worms grown in either 4 or 500 ÂµM heme (see Methods). The microarray results were validated by qRT-PCR (Supplementary Fig. 1) and the 375 heme-responsive genes were classified into eight categories based on their relative changes in gene expression (Table I). The data from the microarray study show that â?°1.9 % of genes in the worm genome are transcriptionally responsive to heme. Notably, of the 375 genes, 164 had some sequence identity in human genome databases at the amino acid level, and >90 % of the genes had no ascribed function in the C.

Project description:We recently showed that the two-component system (TCS) HrrSA plays a central role in the control of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum. Here, we characterized the function of another TCS of this organism, ChrSA, which exhibits significant sequence similarity to HrrSA, and provide evidence for cross-regulation of the two systems. In this study ChrSA was shown to be crucial for heme resistance of C. glutamicum by activation of the putative heme-detoxifying ABC-transporter HrtBA in the presence of heme. Deletion of either hrtBA or chrSA resulted in a strongly increased sensitivity towards heme. DNA microarray analysis and gel retardation assays with the purified response regulator ChrA provided evidence for ChrA being a repressor of the heme biosynthesis operon hemAC and hemH and an activator of the ctaE-qcrCAB operon, encoding subunits of the cytochrome bc1-aa3 supercomplex of the respiratory chain. The heme oxygenase gene, hmuO, showed a strongly decreased mRNA level in the M-NM-^TchrSA mutant, but no significant binding of ChrA was observed in vitro. Promoter fusion studies of PchrSA with eyfp indicated positive autoregulation of the chrSA operon in the presence of heme. Interestingly, ChrA was also shown to bind to the hrrA promoter and to repress transcription of the paralog response regulator, suggesting a close link between HrrSA and ChrSA. Mutational analysis and in silico prediction resulted in the deduction of a 16-bp weakly conserved inverted repeat as consensus DNA-binding motif of ChrA. Altogether, the present study emphasizes ChrSA as a second TCS, besides HrrSA, involved in heme-dependent gene regulation in C. glutamicum. To identify the influence of ChrSA on global gene expression , DNA microarray analyses were performed with the M-NM-^TchrSA mutant compared to C. glutamicum wild type. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII minimal medium with 4% glucose and either 2.5 M-BM-5M FeSO4 or 2.5 M-BM-5M hemin as iron source. Three biological replicates were performed.