Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vectorexpressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (ip) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.

With technological advances in basic research, the intricate mechanism of secondary delayed spinal cord injury (SCI) continues to unravel at a rapid pace. However, despite our deeper understanding of the molecular changes occurring after initial insult to the spinal cord, the cure for paralysis remains elusive. Current treatment of SCI is limited to early administration of high dose steroids to mitigate the harmful effect of cord edema that occurs after SCI and to reduce the cascade of secondary delayed SCI. R ecent evident-based clinical studies have cast doubt on the clinical benefit of steroids in SCI and intense focus on stem cell-based therapy has yielded some encouraging results. An array of mesenchymal stem cells (MSCs) from various sources with novel and promising strategies are being developed to improve function after SCI. In this review, we briefly discuss the pathophysiology of spinal cord injuries and characteristics and the potential sources of MSCs that can be used in the treatment of SCI. We will discuss the progress of MSCs application in research, focusing on the neuroprotective properties of MSCs. Finally, we will discuss the results from preclinical and clinical trials involving stem cell-based therapy in SCI. Core tip: Despite our deeper understanding of the molecular changes that occurs after the spinal cord injury (SCI), the cure for paralysis remains elusive. In this review, the pathophysiology of SCI and characteristics and potential sources of mesenchymal stem cells (MSCs) that can be used in the treatment of SCI were discussed. We also discussed the progress of application of MSCs in research focusing on the neuroprotective properties of MSCs. Finally, we discussed the results from preclinical and clinical trials involving stem cell-based therapy in SCI.

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy. RNA interference (RNAi)1 is a sequence-specific, post-transcriptional gene silencing mechanism, which is triggered by double-stranded RNA and causes the degradation of mRNA homologous in sequence to the double-stranded RNA (1-3). This is an ancient and ubiquitous antiviral system used by organisms to maintain the integrity of the genome, to defend cells against viral infection, and to regulate expression of cellular genes (4). RNAi depends upon the formation of doublestrand RNA (double-stranded RNA) whose antisense strand is complementary to the transcript of a targeted gene. Recently, it has been shown that sequence-specific inhibition RNAi can also be induced in mammalian cells (4, 5). In one implementation of RNAi, selective degradation of target mRNAs in mammalian cells is achieved by transfection with double-stranded, short interfering RNAs (siRNAs), leading to rapid and efficient degradation of the target (4). These siRNAs were shown to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells.Glioblastoma multiforme is a highly malignant primary central nervous system neoplasm, which is extremely refractory to therapy. One property that makes glioblastoma resistant to treatment is the tendency of the tumor cells to invade normal brain tissue (6). Invasiveness is thus considered to be a major determinant of ...

BackgroundPTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumor suppressor gene implicated in a wide variety of human cancers, including glioblastoma. PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Most human gliomas show high levels of activated Akt, whereas less than half of these tumors carry PTEN mutations or homozygous deletions. The unique ability of mesenchymal stem cells to track down tumor cells makes them as potential therapeutic agents. Based on this capability, new therapeutic approaches have been developed using mesenchymal stem cells to cure glioblastoma. However, molecular mechanisms of interactions between glioma cells and stem cells are still unknown.Methodology/Principal FindingsIn order to study the mechanisms by which migration of glioma cells can be inhibited by the upregulation of the PTEN gene, we studied two glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310) alone and in co-culture with human umbilical cord blood-derived mesenchymal stem cells (hUCBSC). Co-cultures of glioma cells showed increased expression of PTEN as evaluated by immunofluorescence and immunoblotting assays. Upregulation of PTEN gene is correlated with the downregulation of many genes including Akt, JUN, MAPK14, PDK2, PI3K, PTK2, RAS and RAF1 as revealed by cDNA microarray analysis. These results have been confirmed by reverse-transcription based PCR analysis of PTEN and Akt genes. Upregulation of PTEN resulted in the inhibition of migration capability of glioma cells under in vitro conditions. Also, wound healing capability of glioma cells was significantly inhibited in co-culture with hUCBSC. Under in vivo conditions, intracranial tumor growth was inhibited by hUCBSC in nude mice. Further, hUCBSC upregulated PTEN and decreased the levels of XIAP and Akt, which are responsible for the inhibition of tumor growth in the mouse brain.Conclusions/SignificanceOur studies indicated that upregulation of PTEN by hUCBSC in glioma cells and in the nude mice tumors downregulated Akt and PI3K signaling pathway molecules. This resulted in the inhibition of migration as well as wound healing property of the glioma cells. Taken together, our results suggest hUCBSC as a therapeutic agent in treating malignant gliomas.

CD44 is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. CD44 cleavage correlates with the tumor burden and metastatic potential in various cancers. In this study, we demonstrate that matrix metalloproteinase-9 (MMP-9) acts as a processing enzyme for CD44 cleavage. Further, this processing event stimulates cell motility and inhibition of either CD44 or MMP-9 inhibited cell migration. MMP-9 and CD44 co-localization on the cell surface was observed in the histological sections of human glioblastoma (GBM) tissues. Confocal microscopy and co-immunoprecipitation studies in GBM xenograft cells further confirm this interaction. The interaction of MMP-9 with CD44 induced CD44 cleavage which was inhibited by both transcriptional knockdown of MMP-9 and with MMP-9 specific inhibitor. Further, supplementation of purified and activated human MMP-9 (hMMP-9) in MMP-9-knockdown cells resumed CD44 cleavage and migration. Additionally, activated hMMP-9 protein induced cleavage of recombinant human CD44 (rhCD44) in an in vitro assay. Selective overexpression of either extracellular domain (CD44ECD) or intracellular domain (CD44ICD) confirmed that CD44ECD played a role in cell migration and invasion. Taken together, our results suggest that MMP-9 is involved in the shedding of CD44 from cancer cells, which would promote the malignant potential of tumor cells.

MMP-2 plays pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 siRNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared to mock- and pSV-(scrambled vector)treatments, as determined by BrDU incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and Western blotting using tumor conditioned media displayed modulated secretory levels of various cytokines including GM-CSF, IL-6, IL-8, IL-10, TNF-α, angiogenin, VEGF and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of JAK/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin mediated Stat3 phosphorylation and nuclear translocation. EMSA and ChIP assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in rhMMP-2 treatments resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM-treatment compared to mock- or pSV-treatments. Immunoflorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM-treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicates the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

Human umbilical cord blood stem cells (hUCB) hold great promise for therapeutic repair after spinal cord injury (SCI). Here, we present our preliminary investigations on axonal remyelination of injured spinal cord by transplanted hUCB. Adult male rats were subjected to moderate SCI using NYU Impactor, and hUCB were grafted into the site of injury one week after SCI. Immunohistochemical data provides evidence of differentiation of hUCB into several neural phenotypes including neurons, oligodendrocytes and astrocytes. Ultrastructural analysis of axons reveals that hUCB form morphologically normal appearing myelin sheaths around axons in the injured areas of spinal cord. Colocalization studies prove that oligodendrocytes derived from hUCB secrete neurotrophic hormones neurotrophin-3 (NT3) and brain-derived neurotrophic factor (BDNF). Cord blood stem cells aid in the synthesis of myelin basic protein (MBP) and proteolipid protein (PLP) of myelin in the injured areas, thereby facilitating the process of remyelination. Elevated levels of mRNA expression were observed for NT3, BDNF, MBP and PLP in hUCB-treated rats as revealed by fluorescent in situ hybridization (FISH) analysis. Recovery of hind limb locomotor function was also significantly enhanced in the hUCB-treated rats based on Basso-Beattie-Bresnahan (BBB) scores assessed 14 days after transplantation. These findings demonstrate that hUCB, when transplanted into the spinal cord 7 days after weight-drop injury, survive for at least 2 weeks, differentiate into oligodendrocytes and neurons, and enable improved locomotor function. Therefore, hUCB facilitate functional recovery after moderate SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.

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