Original Article
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World J Stem Cells. Jan 26, 2013; 5(1): 9-25
Published online Jan 26, 2013. doi: 10.4252/wjsc.v5.i1.9
Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome
Gry H Dihazi, Asima Bibi, Olaf Jahn, Jessica Nolte, Gerhard A Mueller, Wolfgang Engel, Hassan Dihazi
Gry H Dihazi, Asima Bibi, Gerhard A Mueller, Hassan Dihazi, Department of Nephrology and Rheumatology, Georg-August University Goettingen, D-37075 Goettingen, Germany
Olaf Jahn, Proteomics Group, Max-Planck-Institute of Experimental Medicine, D-37075 Goettingen, Germany
Olaf Jahn, Deutsche Forschungsgemeinschaft Research Center for Molecular Physiology of the Brain, D-37073 Goettingen, Germany
Jessica Nolte, Wolfgang Engel, Institute of Human Genetics, Georg-August University Goettingen, D-37073 Goettingen, Germany
Author contributions: Dihazi GH and Bibi A performed the majority of experiments, interpreted the data and wrote the article; Nolte J and Engel W provided the stem cell lines and were also involved in revising the manuscript; Jahn O was responsible for the acquisition of the mass spectrometry data and revised the article critically; Mueller GA helped by the study design and manuscript writing; Dihazi H designed and concepted the study and revised the article critically; Dihazi GH and Bibi A contributed equally to this work.
Correspondence to: Hassan Dihazi, PhD, Department of Nephrology and Rheumatology, Georg-August University Goettingen, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany. dihazi@med.uni-goettingen.de
Telephone: +49-551-3991221 Fax: +49-551-3991039
Received: February 7, 2012
Revised: September 19, 2012
Accepted: December 20, 2012
Published online: January 26, 2013

AIM: To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells.

METHODS: Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics.

RESULTS: Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that CPX treatment of stem cells, with subsequent proliferation inhibition, resulted in an alteration of the expression of 56 proteins compared to non-treated cells, and 54 proteins compared to RA treated cells. Bioinformatics analysis of the regulated proteins demonstrated their involvement in various biological processes. To our interest, a number of proteins have potential roles in the regulation of cell proliferation either directly or indirectly. Furthermore the classification of the altered polypeptides according to their main known/postulated functions revealed that the majority of these proteins are involved in molecular functions like nucleotide binding and metal ion binding, and biological processes like nucleotide biosynthetic processes, gene expression, embryonic development, regulation of transcription, cell cycle processes, RNA and mRNA processing. Proteins, which are involved in nucleotide biosynthetic process and proteolysis, were downregulated in CPX treated cells compared to control, as well as in RA treated cells, which may explain the cell cycle arrest. Moreover, proteins which were involved in cell death, positive regulation of biosynthetic process, response to organic substance, glycolysis, anti-apoptosis, and phosphorylation were downregulated in RA treated cells compared to control and CPX treated cells.

CONCLUSION: The CPX treatment of SCs results in downregulation of nucleotide binding proteins and leads to cell cycle stop without impairment of pluripotency.

Keywords: Stem cells, Differentiation, Hypusination, Ciclopirox olamine, Proteomics, Retinoic acid