Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.
BACKGROUND
Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.


RESULTS
Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.


CONCLUSIONS
Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
Shivani
S
Ponnala
Krishna Kumar
KK
Veeravalli
Chandramu
C
Chetty
Dzung H
DH
Dinh
Jasti S
JS
Rao
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D017209:Apoptosis / Q000528:radiation effects; D002401:Cathepsin B / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D059766:DNA End-Joining Repair / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D045643:DNA Repair Enzymes / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000528:radiation effects; D066246:ErbB Receptors / Q000378:metabolism; D005910:Glioma / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D000072200:Ku Autoantigen; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D011485:Protein Binding / Q000528:radiation effects; D011839:Radiation, Ionizing; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D055293:Receptors, Urokinase Plasminogen Activator; D066246:ErbB Receptors; D051747:DNA-Activated Protein Kinase; D002401:Cathepsin B; D020780:Matrix Metalloproteinase 9; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D045643:DNA Repair Enzymes