Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Clin Oncol. Jul 10, 2012; 3(7): 104-110
Published online Jul 10, 2012. doi: 10.5306/wjco.v3.i7.104
Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells
Harun M Said, Buelent Polat, Susanne Stein, Mathias Guckenberger, Carsten Hagemann, Adrian Staab, Astrid Katzer, Jelena Anacker, Michael Flentje, Dirk Vordermark
Harun M Said, Buelent Polat, Mathias Guckenberger, Adrian Staab, Astrid Katzer, Michael Flentje, Dirk Vordermark, Department of Radiation Oncology, University of Wuerzburg, 97080 Würzburg, Germany
Susanne Stein, Department of Hematology and Oncology, Johannes Gutenberg University, III Medical School, 55101 Mainz, Germany
Carsten Hagemann, Jelena Anacker, Department of Neurosurgery, Tumorbiology Laboratory, University of Wuerzburg, 97080 Würzburg, Germany
Adrian Staab, Department of Radiation Oncology, Paul Scherer Institute, CH-5100 Villingen, Switzerland
Jelena Anacker, Department of Gynaecology and Obstetrics, University of Wuerzburg, 97080 Würzburg, Germany
Dirk Vordermark, Department of Radiation Oncology, University-Halle-Wittenberg, 06110 Halle, Germany
Author contributions: Said HM was the primary author of the manuscript, performed the in vitro hypoxia experiments, supplied the in vitro mRNA, protein lysates and nuclear extracts, performed the Western blotting, densitometric analysis of the results and participated in the study design; Polat B, Stein S, Guckenberger M and Hagemann C co-authored the manuscript and participated in the study design; Said HM, Stein S, Hagemann C and Vordermark D coordinated the group and contributed to the development of the experimental strategy; Anacker J designed the primers used for reverse transcription polymerase chain reaction and participated in the study design and evaluation; Said HM, Flentje M and Vordermark D also participated in the study design; all authors read and approved the manuscript.
Supported by Deutsche Forschungsgemeinschaft DFG, VO 871/2-3, to Vordermark D; and the IZKF Würzburg, B25, to Hagemann C
Correspondence to: Dr. Harun M Said, PhD, Department of Radiation Oncology, University of Würzburg, Josef-Schneider-Str. 11, 97080 Würzburg, Germany. said@scientist.com
Telephone: +49-163-7538317 Fax: +49-163-7531174
Received: August 30, 2011
Revised: December 10, 2011
Accepted: June 30, 2012
Published online: July 10, 2012
Abstract

AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation.

METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5’-GCATTATTGGCATGGGAAC-3’ and 5’-ATGCAGAGTAACGTGGAAG-3’. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant).

RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results.

CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.

Keywords: N-Myc down regulated gene 1; Short dsRNA oligonucleotides; Human cancer diseases; Brain cancer; Radiotherapy