Abstract
BackgroundGlioblastoma 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.Methodology/Principal FindingsHere 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.Conclusion/SignificanceOur 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.
Highlights
Glioblastoma Multiforme (GBM) is a highly malignant tumor of the central nervous system with a median survival rate of less than 12 months and with a high rate of recurrence
We observed urokinase plasminogen activator receptor (uPAR) interaction with b1 integrin and coimmunoprecipitation of uPAR with matrix metalloprotease 9 (MMP9) and cathepsin B (Figure 1C). We anticipate that these interactions among MMP9, uPAR, cathepsin B and b1 integrin demonstrate b1 integrinmediated ECM signaling in reduced activity of non-homologous end joining (NHEJ) in pMUand pMC-treated 4910 and 5310 glioma xenograft cells compared to untreated cells
These result clearly demonstrates how protein levels in MMP9-uPAR (pMU) and pMC treatments orchestrate complex signaling in regulating key proteins leading to accumulation of double strand break (DSB). These results indicate that induction of apoptosis after pMU and pMC treatments is due to accumulated unrepaired DSBs in 4910 and 5310 glioma cells compared to untreated cells
Summary
Glioblastoma Multiforme (GBM) is a highly malignant tumor of the central nervous system with a median survival rate of less than 12 months and with a high rate of recurrence. NHEJ is the predominant DSB repair mechanism compared to HR [7] Both DNA protein kinase catalytic subunit (DNA PKcs) and ataxia talengiectasia mutated (ATM) belong to the phosphatidylinositol kinase family. ATM plays an important role in DNA damage response (DDR) by facilitating cell cycle arrest or triggering apoptosis based on the severity of the damage [10] Growth signaling proteins such as Epidermal growth factor receptor (EGFR) are over-expressed in various cancers including GBM [11,12]. 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. Nonhomologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells
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