Venkata Ramesh Dasari

Upregulation of PTEN in Glioma Cells by Cord Blood Mesenchymal Stem Cells Inhibits Migration via Downregulation of the PI3K/Akt Pathway

Background PTEN (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 Findings In 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/Significance Our 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.

Mesenchymal stem cells in the treatment of spinal cord injuries: A review

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. Recent 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.

Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins α-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.

Neuroprotection by cord blood stem cells against glutamate-induced apoptosis is mediated by Akt pathway

The neurotransmitter glutamate mediates excitatory synaptic transmission in the brain and spinal cord. In pathological conditions massive glutamate release reaches near millimolar concentrations in the extracellular space and contributes to neuron degeneration and death. In the present study, we demonstrate a neuroprotective role for human umbilical cord blood stem cells (hUCB) against glutamate-induced apoptosis in cultured rat cortical neurons. Microarray analysis shows the upregulation of stress pathway genes after glutamate toxicity of neurons, while in cocultures with hUCB, survival pathway genes were upregulated. Real time-PCR analysis shows the expression of genes for NMDA receptors after glutamate toxicity in neurons. The neuroprotection of hUCB against glutamate toxicity is similar to the application of the glutamate receptor antagonist MK-801. Cocultures of hUCB protected neurons against glutamate-induced apoptosis as revealed by APO-BrdU TUNEL and FACS analyses. Immunoblot analysis shows that apoptosis is mediated by the cleavage of caspase-3 and caspase-7 in glutamate treated neurons. Cocultures with hUCB indicate the upregulation of Akt signaling pathway to protect neurons. Blocking of the Akt pathway by a dominant-negative Akt and using Akt-inhibitor IV, we confirm that the mechanism underlying hUCB neuroprotection involves activation of Akt signaling pathway. These results suggest the neuroprotective potential of hUCB against glutamate-induced apoptosis of cultured cortical neurons.

Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury.

Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in inflammation, wound healing and other pathological processes after neurological disorders. MMP-2 promotes functional recovery after spinal cord injury (SCI) by regulating the formation of a glial scar. In the present study, we aimed to investigate the expression and/or activity of several MMPs, after SCI and human umbilical cord blood mesenchymal stem cell (hUCB) treatment in rats with a special emphasis on MMP-2. Treatment with hUCB after SCI altered the expression of several MMPs in rats. MMP-2 is upregulated after hUCB treatment in spinal cord injured rats and in spinal neurons injured either with staurosporine or hydrogen peroxide. Further, hUCB induced upregulation of MMP-2 reduced formation of the glial scar at the site of injury along with reduced immunoreactivity to chondroitin sulfate proteoglycans. Blockade of MMP-2 activity in hUCB cocultured injured spinal neurons reduced the protection offered by hUCB which indicated the involvement of MMP-2 in the neuroprotection offered by hUCB. Based on these results, we conclude that hUCB treatment after SCI upregulates MMP-2 levels and reduces the formation of the glial scar thereby creating an environment suitable for endogenous repair mechanisms.

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser392. Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

Cord Blood Stem Cell-Mediated Induction of Apoptosis in Glioma Downregulates X-Linked Inhibitor of Apoptosis Protein (XIAP)

Background XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death. Methodology/Principal Findings We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO. Conclusions/Significance Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant gliomas.

Glioma stem cell invasion through regulation of the interconnected ERK, integrin α6 and N-cadherin signaling pathway

The recent characterization of glioma stem cells (GSCs) prompts a necessary examination of the signaling pathways that facilitate invasiveness. Molecular crosstalk between expression mechanisms has been identified in a range of cancers, including glioblastoma multiforme. However, hardly any literature exists that addresses whether cancer stem cells utilize these same interconnected pathways. Protein factors commonly implicated in malignant tumors include extracellular signal-regulated kinase (ERK), N-cadherin, and integrin α6. Although studies have reported the molecular crosstalk involved among these proteins, the present study illustrates the importance of the ERK transduction pathway in N-cadherin and integrin α6 regulated invasion in GSCs. Conversely, the data also suggests that GSCs rely on N-cadherin and integrin α6 interaction to regulate ERK signaling. Moreover, confocal visualization revealed the co-localization of N-cadherin and integrin α6 in GSCs and clinical surgical biopsies extracted from glioma patients. Interestingly, ERK knockdown reduced this co-localization. Upon co-culturing GSCs with human umbilical cord blood stem cells (hUCBSCs), we observed a subsequent decrease in pERK, N-cadherin and integrin α6 expression. In addition, co-culturing hUCBSCs with GSCs decreased co-localization of N-cadherin and integrin α6 in GSCs. Our results demonstrate the dynamic interplay among ERK, N-cadherin and integrin α6 in GSC invasion and also reveal the therapeutic potential of hUCBSCs in treating the molecular crosstalk observed in GSC-regulated invasion.

Epigenetic Upregulation of Urokinase Plasminogen Activator Promotes the Tropism of Mesenchymal Stem Cells for Tumor Cells

A major obstacle for the effective treatment of cancer is the invasive capacity of the tumor cells. Previous studies have shown the capability of mesenchymal stem cells (MSC) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. However, the molecular mechanisms that would enhance the migration of MSCs toward tumor areas are not well understood. In particular, very little is known about the role that epigenetic mechanisms play in cell migration and tropism of MSCs. In this study, we investigated whether histone deacetylation was involved in the repression of urokinase plasminogen activator (uPA) expression in MSCs derived from umbilical cord blood (CB) and bone marrow (BM). Induction of uPA expression by histone deacetylase inhibitors trichostatin A and sodium butyrate was observed in CB- and BM-derived MSCs examined. In vitro migration assays showed that induction of uPA expression by histone deacetylase inhibitors in CB- and BM-derived MSCs significantly enhanced tumor tropism of these cells. Furthermore, overexpression of uPA in CB-MSCs induced migration capacity toward human cancer cells in vitro. In addition, our results showed that uPA-uPAR knockdown in PC3 prostate cancer cells significantly inhibited tumor-specific migration of uPA-overexpressing MSCs. These results have significant implications for the development of MSC-mediated, tumor-selective gene therapies. Mol Cancer Res; 8(8); 1074–83. ©2010 AACR.

Regulation of glioblastoma progression by cord blood stem cells is mediated by downregulation of cyclin D1.

Background The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G1 phase. Methodology/Principal Findings In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G0-G1 phase arrest, but also reduced the number of cells at S and G2-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G0-G1 phase despite the reduction of G0-G1 regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G0-G1 regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC. Conclusions/Significance This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G0-G1 phase.