Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

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.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.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

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of alphaVbeta3, alpha6beta1 and alpha9beta1 integrins in xenograft cells. Treatment with bicistronic constructs reduced alphaVbeta3, alpha6beta1 and alpha9beta1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma

MMP-9 and uPAR regulated glioma cell migration

Integrin dependent and independent MMP-9 and uPAR signaling plays a key role in glioma cell migration and invasion. In this article, we comment on all the possible pathways and molecules associated with MMP-9- and uPAR- mediated glioma cell migration with a special emphasis on integrins, a family of cell adhesion molecules. Our recent research investigations highlighted the substantial benefit of silencing both MMP-9 and uPAR together compared to their individual treatments in glioma. Simultaneous knockdown of both MMP-9 and uPAR regulated a majority of the molecules associated with glioma cell migration and significantly reduced the migration potential of glioma cells. Our results point out that the bicistronic construct, which can simultaneously silence both MMP-9 and uPAR offers a great therapeutic potential and is worth developing as a new drug for treating GBM patients

Regulation of Proteases after Spinal Cord Injury

Spinal cord injury is a major medical problem worldwide. Unfortunately, we still do not have suitable therapeutic agents for the treatment of spinal cord injury and prevention of its devastating consequences. Scientists and physicians are baffled by the challenges of controlling progressive neurodegeneration in spinal cord injury, which has not been healed with any currently-available treatments. Although extensive work has been carried out to better understand the pathophysiology of spinal cord injury, our current understanding of the repair mechanisms of secondary injury processes is still meager. Several investigators reported the crucial role played by various proteases after spinal cord injury. Understanding the beneficial and harmful roles these proteases play after spinal cord injury will allow scientists to plan and design appropriate treatment strategies to improve functional recovery after spinal cord injury. This review will focus on various proteases such as matrix metalloproteinases, cysteine proteases, and serine proteases and their inhibitors in the context of spinal cord injury

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Upregulate Myelin Basic Protein in Shiverer Mice

Human umbilical cord blood is a rich source of pluripotent mesenchymal stem cells and possesses significant advantages over other stem cell sources such as the embryo and bone marrow. In the present study, we aimed to investigate the potential of human umbilical cord blood-derived pluripotent mesenchymal stem cells (hUCB) to myelinate the axons of shiverer mice brains. We also investigated the effect of hUCB treatment on regulation of myelin basic protein in vitro in PC-12 cells, which are normally not myelinated. The results of our study clearly demonstrated that hUCB survive and migrate in vivo and has the potential to myelinate shiverer mice brains. The expression level of myelin basic protein, a major component of the myelin sheath, has been significantly increased in vivo and in vitro as revealed by Western blot, reverse transcription (RT)-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and fluorescent in situ hybridization results. Further, transmission electron microscopic images of hUCB-treated shiverer mice brains showed several layers of myelin around the axons compared with a thin and fragmented layer of myelin in untreated animals. Moreover, the frequency of shivering was diminished 1 month after hUCB treatment in shiverer mice. Our results strongly indicated that hUCB transplantation could be an effective means of treating demyelinating or hypomyelinating disorders

Mesenchymal Stem Cell Treatment Prevents Post-Stroke Dysregulation of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases

Background/Aims: Stem cell treatment is one of the potential treatment options for ischemic stroke. We recently demonstrated a protective effect of human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) in a rat model of ischemic stroke. The treatment attenuated apoptosis and prevented DNA damage. A collection of published studies, including several from our laboratory, indicated the induction and detrimental role for several matrix metalloproteinases (MMPs) in post-stroke brain injury. We hypothesized that the HUCB-MSCs treatment after focal cerebral ischemia prevents the dysregulation of MMPs and induces the expression of endogenous tissue inhibitors of metalloproteinases (TIMPs) to neutralize the elevated activity of MMPs. Methods: To test our hypothesis, we administered HUCB-MSCs (0.25 million cells/animal and 1 million cells/animal) intravenously via tail vein to male Sprague-Dawley rats that were subjected to a transient (two-hour) right middle cerebral artery occlusion (MCAO) and one-day reperfusion. Ischemic brain tissues obtained from various groups of rats seven days after reperfusion were subjected to real-time PCR, immunoblot, and immunofluorescence analysis. Results: HUCB-MSCs treatment prevented the induction of MMPs, which were upregulated in ischemia-induced rats that received no treatment. HUCB-MSCs treatment also prevented the induction of TIMPs expression. The extent of prevention of MMPs and TIMPs induction by HUCB-MSCs treatment is similar at both the doses tested. Conclusion: Prevention of stroke-induced MMPs upregulation after HUCB-MSCs treatment is not mediated through TIMPs upregulation