title: uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells.
abstract: Glioblastomas present as diffuse tumors with invasion into normal brain tissue and frequently recur or progress after radiation as focal masses because of glioma-initiating cells. The role of the urokinase-type plasminogen activator receptor (uPAR) and cathepsin B in stem-like phenotype has been extensively studied in several solid tumors. In the present study, we demonstrated that selection of glioma-initiating cells using CD133 expression leads to a specific enrichment of CD133(+) cells in both U87 and 4910 cells. In addition, CD133(+) cells exhibited a considerable amount of other stem cell markers, such as Nestin and Sox-2. Radiation treatment significantly enhanced uPAR and cathepsin B levels in glioma-initiating cells. To downregulate radiation-induced uPAR and cathepsin B expression, we used a bicistronic shRNA construct that simultaneously targets both uPAR and cathepsin B (pCU). Downregulation of uPAR and cathepsin B using pCU decreased radiation-enhanced uPAR and cathepsin B levels and caused DNA damage-induced apoptosis in glioma cell lines and glioma-initiating cells. The most striking finding of this study is that knockdown of uPAR and cathepsin B inhibited ongoing transcription by suppressing BrUTP incorporation at γH2AX foci. In addition, uPAR and cathepsin B gene silencing inversely regulated survivin and H2AX expression in both glioma cells and glioma-initiating cells. Pretreatment with pCU reduced radiation-enhanced expression of uPAR, cathepsin B, and survivin and enhanced DNA damage in pre-established glioma in nude mice. Taken together, our in vitro and in vivo findings suggest that uPAR and cathepsin B inhibition might serve as an adjunct to radiation therapy to target glioma-initiating cells and, therefore, for the treatment of glioma.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605, USA.
authors.forename: Rama Rao
authors.initials: RR
authors.lastname: Malla
authors.forename: Sreelatha
authors.initials: S
authors.lastname: Gopinath
authors.forename: Kiranmai
authors.initials: K
authors.lastname: Alapati
authors.forename: Bharathi
authors.initials: B
authors.lastname: Gorantla
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Gondi
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000528:radiation effects*; D015153:Blotting, Western; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D005910:Glioma / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000528:radiation effects*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D055293:Receptors, Urokinase Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D014158:Transcription, Genetic
chemical_list: C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D000077022:Survivin; D002401:Cathepsin B