title: siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma.
abstract: Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, One Illini Drive, Peoria, IL 61605, USA.
authors.forename: Ganji Purna Chandra
authors.initials: GP
authors.lastname: Nagaraju
authors.forename: Arun Kumar
authors.initials: AK
authors.lastname: Nalla
authors.forename: Reshu
authors.initials: R
authors.lastname: Gupta
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Mohanam
authors.forename: Meena
authors.initials: M
authors.lastname: Gujrati
authors.forename: Dzung H
authors.initials: DH
authors.lastname: Dinh
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000528:radiation effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D015536:Down-Regulation; D005434:Flow Cytometry; D016195:G2 Phase / Q000528:radiation effects; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D008527:Medulloblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering; D055293:Receptors, Urokinase Plasminogen Activator; D016159:Tumor Suppressor Protein p53; D019869:Phosphatidylinositol 3-Kinases; D016203:CDC2 Protein Kinase; D051357:Cyclin-Dependent Kinase 2; D020928:Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9; D018631:ras Proteins