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Proteomic Study to Survey the CIGB-552 Antitumor Effect.

ABSTRACT: CIGB-552 is a cell-penetrating peptide that exerts in vitro and in vivo antitumor effect on cancer cells. In the present work, the mechanism involved in such anticancer activity was studied using chemical proteomics and expression-based proteomics in culture cancer cell lines. CIGB-552 interacts with at least 55 proteins, as determined by chemical proteomics. A temporal differential proteomics based on iTRAQ quantification method was performed to identify CIGB-552 modulated proteins. The proteomic profile includes 72 differentially expressed proteins in response to CIGB-552 treatment. Proteins related to cell proliferation and apoptosis were identified by both approaches. In line with previous findings, proteomic data revealed that CIGB-552 triggers the inhibition of NF-?B signaling pathway. Furthermore, proteins related to cell invasion were differentially modulated by CIGB-552 treatment suggesting new potentialities of CIGB-552 as anticancer agent. Overall, the current study contributes to a better understanding of the antitumor action mechanism of CIGB-552.

SUBMITTER: Rodriguez-Ulloa A

PROVIDER: S-EPMC4630370 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomic Study to Survey the CIGB-552 Antitumor Effect.

Rodríguez-Ulloa Arielis A Gil Jeovanis J Ramos Yassel Y Hernández-Álvarez Lilian L Flores Lisandra L Oliva Brizaida B García Dayana D Sánchez-Puente Aniel A Musacchio-Lasa Alexis A Fernández-de-Cossio Jorge J Padrón Gabriel G González López Luis J LJ Besada Vladimir V Guerra-Vallespí Maribel M

BioMed research international 20151020

CIGB-552 is a cell-penetrating peptide that exerts in vitro and in vivo antitumor effect on cancer cells. In the present work, the mechanism involved in such anticancer activity was studied using chemical proteomics and expression-based proteomics in culture cancer cell lines. CIGB-552 interacts with at least 55 proteins, as determined by chemical proteomics. A temporal differential proteomics based on iTRAQ quantification method was performed to identify CIGB-552 modulated proteins. The proteom ...[more]

PMID: 26576414

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Project description:Nucleoid-associated proteins (NAPs) are small, highly abundant transcriptional regulators with low sequence specificity which are involved in multiple DNA-related processes including gene expression, DNA protection, recombination/repair and nucleoid structuring. Through these functions they are able to regulate important phenotypic properties including virulence, secondary metabolism and stress resistance. However the set of NAPs known within the Actinobacteria is small and incomplete. The missing proteins are likely to be key regulators of virulence in pathogens such as Mycobacterium tuberculosis and also of development and secondary metabolism in industrially-important species such as Streptomyces. Here, we use label-free LC-MS/MS to systematically search for novel NAPs in isolated nucleoids of the model actinomycete Streptomyces coelicolor. Based on the criteria of high abundance (emPAI score) and predicted DNA-binding ability (DNAbinder score) we identified a set of 24 proteins with a high predicted likelihood of being NAPs. The approach was deemed successful as the set included known major NAPs HupA, HupS, sIHF and Lsr2 as well as the global transcriptional regulators BldD and CRP and the pleiotropic response regulator AfsQ1. It also included a number of proteins whose functions are not yet known from recognisable classes of transcription factor (SCO2140, SCO4493, SCO1839, SCO1210, SCO5405, SCO4229, SCO3198) or from uncharacterised protein families (SCO5783, SCO5592, SCO3793, SCO6482) which comprise a valuable set of candidates for further study.In this paper we establish a robust protocol for preparing S. coelicolor nucleoids for mass spectrometric analysis and develop a workflow for identifying novel nucleoid-associated proteins (NAPs) by combining LC-MS/MS with a bioinformatical analysis. The nucleoid-associated proteins of many species are known to be key regulators of virulence, stress tolerance and global patterns of gene expression. Identifying the "missing" nucleoid proteins of S. coelicolor is likely to have important implications for manipulating the production of secondary metabolites such as antibiotics. Candidate NAPs were identified. Several of these are highly conserved in clinically important species such as Mycobacterium and in many commercially important species such as Salinispora and Micromonospora which represent a vital source of novel drugs such as antibiotics, antifungals and anticancer agents.

Project description:Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5'-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chromatography-electrospray tandem mass spectrometry of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.

Dataset Information

Proteomic Study to Survey the CIGB-552 Antitumor Effect.

Publications

Proteomic Study to Survey the CIGB-552 Antitumor Effect.

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