title: Knockdown of the AKT3 (PKBγ), PI3KCA, and VEGFR2 genes by RNA interference suppresses glioblastoma multiforme T98G cells invasiveness in vitro.
abstract: Glioblastoma multiforme (GBM) is the most common primary brain malignancy, having a very poor prognosis and is characterized by extensive brain invasion as well as resistance to the therapy. The phosphoinositide 3-kinase (PI3K)/Akt/PTEN signaling pathway is deregulated in GBM. Besides, florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF) occur very often. The present study was designed to examine the inhibitory effect of AKT3, PI3KCA, and VEGFR2 small interfering RNAs (siRNAs) on GBM cell invasiveness. T98G cells were transfected with AKT3, PI3KCA, and/or VEGFR2 siRNAs. VEGFR2 protein-positive cells were identified by flow cytometry using specific monoclonal anti-VEGFR2 antibodies. Alterations in messenger RNA (mRNA) expression of VEGF, VEGFR2, matrix metalloproteinases (MMPs) (MMP-2, MMP-9, MMP-13, MMP-14), tissue inhibitors of metalloproteinases (TIMPs) (TIMP-1, TIMP-3), c-Fos, c-Jun, hypoxia-inducible factor-1α (HIF-1α), ObRa, and cathepsin D genes were analyzed by qRT-PCR. Cells treated with specific siRNA were also analyzed for invasion using the Matrigel invasion assay. We have found significantly lower mRNA levels of MMPs, cathepsin D, VEGF, VEGFR2, HIF-1α, and c-Fos/c-Jun ratio, as well as significantly higher mRNA level of TIMPs in AKT3 and PI3KCA siRNA transfected cells compared to untransfected cells, while significantly lower mRNA levels of MMPs (MMP-2, MMP-9, MMP-14) and TIMP-1, as well as significantly higher mRNA level of TIMP-3, were shown only in cells transfected with VEGFR2 siRNA. The positive correlation between MMP-13 and ObRa mRNA copy number has been found. Summarizing, transfection of T98G cells with AKT3, PI3KCA, or VEGFR2 siRNAs leads to a significant reduction in cell invasiveness. The siRNA-induced AKT3, PI3KCA, and VEGFR2 mRNA knockdown may offer a novel therapeutic strategy to reduce the invasiveness of GBM cells.
authors.affiliation: Department of Medical Genetics, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia, Jednosci 8 Street, 41-200, Katowice, Sosnowiec, Poland, mpaul@sum.edu.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Paul-Samojedny
authors.forename: Adam
authors.initials: A
authors.lastname: Pudełko
authors.forename: Renata
authors.initials: R
authors.lastname: Suchanek-Raif
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kowalczyk
authors.forename: Anna
authors.initials: A
authors.lastname: Fila-Daniłow
authors.forename: Paulina
authors.initials: P
authors.lastname: Borkowska
authors.forename: Jan
authors.initials: J
authors.lastname: Kowalski
mesh_terms: D001932:Brain Neoplasms / Q000201:enzymology* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D055785:Gene Knockdown Techniques; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; C503859:PI3KCA protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D014157:Transcription Factors; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2; C491754:AKT3 protein, human; D051057:Proto-Oncogene Proteins c-akt; D053505:Matrix Metalloproteinases, Secreted