Lytic Bacteriophages Against Bacterial Biofilms Formed by Multidrug-Resistant Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus Isolated from Burn Wounds.

Background: Use of bacteriophages as antibiofilm agents to tackle multidrug-resistant bacteria has gained importance in recent years. Materials and Methods: In this study, biofilm formation by Staphylococcus aureus, Pseudomona aeruginosa, Klebsiella pneumoniae, and Escherichia coli under different growth conditions was studied. Furthermore, the ability of bacteriophages to inhibit biofilm formation was analyzed. Results: Under dynamic growth condition, wherein the medium is renewed for every 12 h, the amount of biomass produced and log10 colony-forming unit counts of all bacterial species studied was highest when compared with other growth conditions tested. Biomass of biofilms produced was drastically reduced when incubated for 2 or 4 h with bacteriophages vB_SAnS_SADP1, vB_PAnP_PADP4, vB_KPnM_KPDP1, and vB_ECnM_ECDP3. Scanning electron microscopy and confocal laser scanning microscopy analyses indicated that the reduction in biomass was due to the lytic action of the bacteriophages. Conclusions: Results of our study reinforce the concept of developing bacteriophages as alternatives to antibiotics to treat bacterial infections.

TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

The cold and menthol receptor TRPM8 is highly expressed in prostate and prostate cancer (PC). Recently, we identified that TRPM8 is as an ionotropic testosterone receptor. The TRPM8 mRNA is expressed in early prostate tumors with high androgen levels, while anti-androgen therapy greatly reduces its expression. Here, from the chromatin-immunoprecipitation (ChIP) analysis, we found that an androgen response element (ARE) mediates androgen regulation of trpm8. Furthermore, using immunofluorescence, calcium-imaging and planar lipid bilayers, we identified that TRPM8 channel is functionally regulated by androgens in the prostate. Although TRPM8 mRNA is expressed at high levels, we found that the TRPM8 protein undergoes ubiquitination and degradation in PC cells. The mass-spectrometry analysis of TRPM8, immunoprecipitated from LNCaP cells identified ubiquitin-like modifier-activating enzyme 1 (UBA1). PYR-41, a potent inhibitor of initial enzyme in the ubiquitination cascade, UBA1, increased TRPM8 activity on the plasma membrane (PM) of LNCaP cells. Furthermore, PYR-41-mediated PMTRPM8 activity was accompanied by enhanced activation of p53 and Caspase-9. Interestingly, we found that the trpm8 promoter possesses putative binding sites for p53 and that the overexpression of p53 increased the TRPM8 mRNA levels. In addition to the genomic regulation of TRPM8 by AR and p53, our findings indicate that the testosterone-induced PMTRPM8 activity elicits Ca2+ uptake, subsequently causing apoptotic cell death. These findings support the strategy of rescuing PMTRPM8 expression as a new therapeutic application through the regulation of PC cell growth and proliferation.

The TRPM8 protein is a testosterone receptor: I. Biochemical evidence for direct TRPM8-testosterone interactions.

The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca(2+) responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.

The TRPM8 protein is a testosterone receptor: II. Functional evidence for an ionotropic effect of testosterone on TRPM8.

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

Nuclear translocation of Hand-1 acts as a molecular switch to regulate vascular radiosensitivity in medulloblastoma tumors: the protein uPAR is a cytoplasmic sequestration factor for Hand-1.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in the tumor-stromal invasive microenvironment in many human cancers, including medulloblastoma. The role of uPAR in tumor progression and angiogenesis has been well characterized. Previously, in medulloblastoma cells, we showed that ionizing radiation (IR)-induced uPAR is a potent activator of cancer stem cell (CSC)-like properties and is associated with various transcription factors that are involved during embryonic development and cancer. In the present study, we show that uPAR protein acts as a cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. The Hand-1 protein plays an essential role in the differentiation of trophoblast giant cells and cardiac morphogenesis, and yet its precise cellular function and its contribution to cancer remain mostly unknown. We also observed that the Hand-1 protein is upregulated in uPAR short hairpin RNA-treated medulloblastoma cells and accompanies sustained cell growth and angiogenesis. Furthermore, IR-induced uPAR overexpression negatively regulates Hand-1 activity and results in the stabilization of angiogenesis-promoting molecules, including hypoxia-inducible factor-1α. Finally, uPAR overexpression and its association with Hand-1 after IR treatment indicate that uPAR is capable of regulating Hand-1 and that uPAR has a role in the process of IR-induced tumor angiogenesis.

Functional and structural analysis of mice TRPC6 with human analogue through homology modelling.

Homology models are increasingly used to determine structural and functional relationships of genes and proteins in biomedical research. In the current study, for the first time, we compared the TRPC6 gene in mouse and human. The protein encoded by this gene forms a receptor activated calcium channel in cell membrane. Defects in this gene have been implicated in a wide range of diseases including glioblastomas. To determine the structural similarities in mouse and human TRPC6, we used standard bioinformatics tools such as fold prediction to identify the protein 3D structure, sequence-structure comparison, and prediction of template and protein structure. We also used glioblastoma cell line U373MG and human glioblastoma tumour tissues to study the expression of TRPC6 in disease conditions to implicate this gene in pathological ailment. Based on the results we conclude that human TRPC6 contains 90% identity and 93% similarity with mouse TRPC6, suggesting that this protein is well conserved in these two species. These isoforms likely demonstrate similar mechanisms in regulating gene expression; thus TRPC6 studies in mice may be extrapolated to humans.

Targeting DUSPs in glioblastomas - wielding a double-edged sword?

Several dual-specificity phosphatases (DUSPs) that play key roles in the direct or indirect inactivation of different MAP kinases (MAPKs) have been implicated in human cancers over the past decade. This has led to a growing interest in identifying DUSPs and their specific inhibitors for further testing and validation as therapeutic targets in human cancers. However, the lack of understanding of the complex regulatory mechanisms and cross-talks between MAPK signaling pathways, combined with the fact that DUSPs can act as a double-edged sword in cancer progression, calls for a more careful and thorough investigation. Among the various types of brain cancer, glioblastoma multiforme (GBM) is notorious for its aggressiveness and resistance to current treatment modalities. This has led to the search for new molecular targets, particularly those involving various signaling pathways. DUSPs appear to be a promising target, but much more information on DUSP targets and their effects on GBM is needed before potential therapies can be developed, tested, and validated. This review identifies and summarize the specific roles of DUSP1, DUSP4, DUSP6 and DUSP26 that have been implicated in GBM.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration.

BACKGROUND: Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9ß1 signaling. Our recent studies have clearly demonstrated the role of α9ß1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9ß1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. METHODS: MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. CONCLUSIONS: Taken together, our results from the present and earlier studies clearly demonstrate that α9ß1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9ß1 integrin and iNOS levels.

Combined targeting of PDK1 and EGFR triggers regression of glioblastoma by reversing the Warburg effect.

Glioblastoma multiforme is the most aggressive primary brain tumor in adults. Overexpression of the EGF receptor (EGFR) is recognized as a widespread oncogenic signature in glioblastoma multiforme, but the complexity of its contributions is not fully understood, nor the most effective ways to leverage anti-EGFR therapy in this setting. Hypoxia is known to drive the aggressive character of glioblastoma multiforme by promoting aerobic glycolysis rather than pyruvate oxidation carried out in mitochondria (OXPHOS), a phenomenon termed the Warburg effect, which is a general feature of oncogenesis. In this study, we report that hypoxia drives expression of the pyruvate dehydrogenase kinase (PDK1) and EGFR along with the hypoxia-inducing factor (HIF)-1α in human glioblastoma multiforme cells. PDK1 is a HIF-1-regulated gene and our findings indicated that hypoxia-induced PDK1 expression may promote EGFR activation, initiating a feed-forward loop that can sustain malignant progression. RNAi-mediated attenuation of PDK1 and EGFR lowered PDK1-EGFR activation and decreased HIF-1α expression, shifting the Warburg phenotype to OXPHOS and inhibiting glioblastoma multiforme growth and proliferation. In clinical specimens of glioblastoma multiforme, we found that immunohistochemical expression of PDK1, EGFR, and HIF-1α were elevated in glioblastoma multiforme specimens when compared with normal brain tissues. Collectively, our studies establish PDK1 as a key driver and candidate therapeutic target in glioblastoma multiforme.

Radiation-induced hypomethylation triggers urokinase plasminogen activator transcription in meningioma cells.

Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5)-methyltransferase 1 (DNMT1) and methyl-CpG binding domain protein (MBD) expression. However, oxidative damage by H(2)O(2) or pretreatment of irradiated cells with N-acetyl cysteine (NAC) did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA)-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in radiation-treated cells, while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation triggers uPA expression in meningioma cells.

The secreted protein acidic and rich in cysteine (SPARC) induces endoplasmic reticulum stress leading to autophagy-mediated apoptosis in neuroblastoma.

Our previous studies showed that overexpression of secreted protein acidic and rich in cysteine (SPARC) induced autophagy-mediated apoptosis in PNET cells. In the present study, we attempted to elucidate the molecular mechanisms and signaling cascades associated with SPARC overexpression in combination with radiation therapy that eventually leads to autophagy-mediated apoptosis in neuroblastoma. SPARC expression in SK-N-AS and NB-1691 cells demonstrated the activation of caspase 3, cleavage of PARP and induction of apoptosis. The experiments to unravel the mechanisms associated with autophagy-apoptosis illustrated that SPARC overexpression triggered endoplasmic reticulum (ER) stress and thereby unfolded protein response (UPR). This was apparent with the activation of stress receptors, inositol-requiring enzyme (IRE 1α), RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and BiP. This study further demonstrated the induction of transcription factor CHOP as a result of IRE-JNK activation in response to increased SPARC levels. Inhibition of ER stress and JNK activation led to inhibition of autophagy-mediated apoptosis. Further, the apparent expression of ER stress molecules among the orthotopic tumors treated by SPARC overexpression plasmids substantiated our in vitro observations. Taken together, these results illustrate the critical role of ER stress in regulating autophagy-mediated apoptosis in SPARC-overexpressed neuroblastoma cells and radiation treatment.

Epigenetic regulation of miRNA-211 by MMP-9 governs glioma cell apoptosis, chemosensitivity and radiosensitivity.

Glioblastoma multiforme (GBM) is the most aggressive brain cancer, and to date, no curative treatment has been developed. In this study, we report that miR-211, a microRNA predicted to target MMP-9, is suppressed in grade IV GBM specimens. Furthermore, we found that miR-211 suppression in GBM involves aberrant methylation-mediated epigenetic silencing of the miR-211 promoter. Indeed, we observed a highly significant inverse correlation between miR-211 expression and MMP-9 protein levels, which is indicative of post-transcriptional control of gene expression. Additionally, shRNA specific for MMP-9 (pM) promoted miR-211 expression via demethylation of miR-211 promoter-associated CpG islands (-140 to +56). In independent experiments, we confirmed that miR-211 overexpression and pM treatments led to the activation of the intrinsic mitochondrial/Caspase-9/3-mediated apoptotic pathway in both glioma cells and cancer stem cells (CSC). We also investigated whether miR-211 is involved in the regulation of MMP-9 and thus plays a functional role in GBM. We found an acute inhibitory effect of miR-211 on glioma cell invasion and migration via suppression of MMP-9. Given the insensitivity of some GBMs to radiation and chemotherapy (temozolomide) along with the hypothesis that glioma CSC cause resistance to therapy, our study indicates that miR-211 or pM in combination with ionizing radiation (IR) and temozolomide significantly induces apoptosis and DNA fragmentation. Of note, miR-211- and pM-treated CSC demonstrated increased drug retention capacity, as observed by MDR1/P-gp mediated-Rhodamine 123 drug efflux activity assay. These results suggest that either rescuing miR-211 expression or downregulation of MMP-9 may have a new therapeutic application for GBM patients in the future.

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells.

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.

Computational analysis of human and mouse CREB3L4 Protein.

CREB3L4 is a member of the CREB/ATF transcription factor family, characterized by their regulation of gene expression through the cAMP-responsive element. Previous studies identified this protein in mice and humans. Whereas CREB3L4 in mice (referred to as Tisp40) is found in the testes and functions in spermatogenesis, human CREB3L4 is primarily detected in the prostate and has been implicated in cancer. We conducted computational analyses to compare the structural homology between murine Tisp40α human CREB3L4. Our results reveal that the primary and secondary structures of the two proteins contain high similarity. Additionally, predicted helical transmembrane structure reveals that the proteins likely have similar structure and function. This study offers preliminary findings that support the translation of mouse Tisp40α findings into human models, based on structural homology.

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.

The homing of human cord blood stem cells to sites of inflammation: unfolding mysteries of a novel therapeutic paradigm for glioblastoma multiforme.

Efficient homing of human umbilical cord blood mesenchymal stem cells (hUCBSC) to inflammation sites is crucial for therapeutic use. In glioblastoma multiforme, soluble factors released by the tumor facilitate the migratory capacity of mesenchymal stem cells toward glioma cells. These factors include chemokines and growth inducers. Nonetheless, the mechanistic details of these factors involved in hUCBSC homing have not been clearly delineated. The present study is aimed to deduce specific factors involved in hUCBSC homing by utilizing a glioma stem cell-induced inflammatory lesion model in the mouse brain. Our results show that hUCBSC do not form tumors in athymic nude mice brains and do not elicit immune responses in immunocompetent SKH1 mice. Further, hUCBSC spheroids migrate and invade glioma spheroids, while no effect was observed on rat fetal brain aggregates. Several cytokines, including GRO, MCP-1, IL-8, IL-3, IL-10, Osteopontin and TGF-ß2, were constitutively secreted in the naive hUCBSC-conditioned medium, while significant increases of IL-8, GRO, GRO-α, MCP-1 and MCP-2 were observed in glioma stem cell-challenged hUCBSC culture filtrates. Furthermore, hUCBSC showed a stronger migration capacity toward glioma stem cells in vitro and exhibited enhanced migration to glioma stem cells in an intracranial human malignant glioma xenograft model. Our results indicate that multiple cytokines are involved in recruitment of hUCBSC toward glioma stem cells, and that hUCBSC are a potential candidate for glioma therapy.

Urokinase-type plasminogen activator receptor (uPAR)-mediated regulation of WNT/ß-catenin signaling is enhanced in irradiated medulloblastoma cells.

Urokinase plasminogen activator receptor (uPAR) is known to promote invasion, migration, and metastasis in cancer cells. In this report, we showed that ionizing radiation (IR)-induced uPAR has a role in WNT-ß-catenin signaling and mediates induction of cancer stem cell (CSC)-like properties in medulloblastoma cell lines UW228 and D283. We observed that IR induced the expression of uPAR and CSC markers, such as Musashi-1 and CD44, and activated WNT-7a-ß-catenin signaling molecules. Overexpression of uPAR alone or with IR treatment led to increased WNT-7a-ß-catenin-TCF/LEF-mediated transactivation, thereby promoting cancer stemness. In contrast, treatment with shRNA specific for uPAR (pU) suppressed WNT-7a-ß-catenin-TCF/LEF-mediated transactivation both in vitro and in vivo. Quercetin, a potent WNT/ß-catenin inhibitor, suppressed uPAR and uPAR-mediated WNT/ß-catenin activation, and furthermore, addition of recombinant human WNT-7a protein induced uPAR, indicating the existence of a mutual regulatory relationship between uPAR and WNT/ß-catenin signaling. We showed that uPAR was physically associated with the WNT effector molecule ß-catenin on the membrane, cytoplasm, and nucleus of IR-treated cells and CSC. Most interestingly, we demonstrated for the first time that localization of uPAR in the nucleus was associated with transcription factors (TF) and their specific response elements. We observed from uPAR-ChIP, TF protein, and protein/DNA array analyses that uPAR associates with activating enhancer-binding protein 2α (AP2a) and mediates ß-catenin gene transcription. Moreover, association of uPAR with the ß-catenin·TCF/LEF complex and various other TF involved during embryonic development and cancer indicates that uPAR is a potent activator of stemness, and targeting of uPAR in combination with radiation has significant therapeutic implications.

Cord blood stem cells inhibit epidermal growth factor receptor translocation to mitochondria in glioblastoma.

BACKGROUND: Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date. METHODOLOGY/PRINCIPLE FINDINGS: In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling proteins in glioma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies.

Transcriptional repression of Mad-Max complex by human umbilical cord blood stem cells downregulates extracellular signal-regulated kinase in glioblastoma.

Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr(202)/Tyr(204) retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression.