Radiation-inducible silencing of uPA and uPAR in vitro and in vivo in meningioma.
Stereospecific radiation treatment offers a distinct opportunity for temporal and spatial regulation of gene expression at tumor sites by means of inducible promoters. To this end, a plasmid, pCArG-U2, was constructed by incorporating nine CArG elements (in tandem) of EGR1 gene upstream to uPA and uPAR siRNA oligonucleotides in a pCi-neo vector. Radiation-induced siRNA expression was detected in a meningioma cell line (IOMM-Lee). Immunoblotting and RT-PCR analyses confirmed downregulation of uPA and uPAR. A similar effect was observed in transfected cells followed by H2O2 treatment. Moreover, pre-treatment of transfected cells with N-acetyl L-cysteine blocked the silencing of uPA and uPAR, which further confirmed the oxidative damage-mediated downregulation. Cell proliferation assays and Western blot analysis for apoptotic molecules confirmed cell death in a radiation-inducible fashion. Migration and matrigel invasion assays also revealed a marked decrease in migration and invasion. Immunocytochemistry showed a marked decrease in uPA and uPAR levels in transfected and irradiated cells. H&E staining revealed a decrease in the pre-established tumor volume among the animals treated with pCArG-U2 and radiation. Immunohistochemistry of the brain sections established with intracranial tumors also revealed a marked decrease in uPA and uPAR in a radiation-inducible fashion. Taken together, our data suggest pCArG-U2 as a suitable candidate for radiation-inducible gene therapy.
Rao Gogineni
Venkateswara
V
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.
Kumar Nalla
Arun
A
Gupta
Reshu
R
Gorantla
Bharathi
B
Gujrati
Meena
M
Dinh
Dzung H
DH
Rao
Jasti S
JS
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D051766:Early Growth Response Protein 1 / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008577:Meningeal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy / Q000628:therapy*; D008579:Meningioma / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy / Q000628:therapy*; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D034622:RNA Interference / Q000528:radiation effects*; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D018714:Radiotherapy, Adjuvant; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D051766:Early Growth Response Protein 1; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator