Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells

AbstractOur 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 H2O2 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

Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma

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.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.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

Desalination of Water

Water is very essential for all living beings. It covers nearly 70% of earth’s surface. Even though the major portion of earth is covered by water, there is severe shortage of drinking water in most of the countries across the world. Safe drinking water is vital for all forms of life though it does not provide any calories. Desalination of sea water appears as a solution for this problem. Advanced desalination technologies that are applied to seawater and brackish water prove to be effective alternatives in a variety of situations. This study mainly focuses on upcoming trends in modern desalination technologies and emphasizing the options offered by them. Desalination is a technique where the excess salts are removed from sea water or brackish water converting it into safe potable or usable water. Desalination methods are categorized into thermal processes and membrane processes. In this chapter we discuss about different thermal processes like multistage flash distillation, multiple effect distillation, vapour compression evaporation, cogeneration and solar water desalination. We also discuss about various categories of membrane processes like reverse osmosis, electro dialysis and membrane distillation methods. This chapter also concentrates on advantages and disadvantages and economical parameters involved in each of these methods

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

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.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.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

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

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.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.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

Inhibition of cell proliferation by hUCBSC treatments.

<p>(A) Control and hUCBSC-treated glioma cells at different time points were analyzed for cell proliferation by incorporation of BrdU. *Significant at <i>p</i><0.05 compared to respective control cells. **Significant at <i>p</i><0.01 compared to respective control cells (by <i>t</i>-test). (B) Control, EGF-treated and EGF+hUCBSC treated glioma cells were analyzed for cell proliferation. *Significant at <i>p</i><0.05 both for control <i>vs.</i> EGF+hUCBSC treatment and EGF <i>vs.</i> EGF+hUCBSC treatments (one-way ANOVA followed by Bonferroni's post hoc test). (C) Control, TMZ treated, TMZ+hUCBSC treated glioma cells were subjected to cell proliferation analysis by BrdU incorporation. *Significant at <i>p</i><0.05; **Significant at <i>p</i><0.01 both for control <i>vs.</i> TMZ treatment and control <i>vs.</i> TMZ+hUCBSC treatments (one-way ANOVA followed by Bonferroni's post hoc test). For all these tests, raw data was taken into consideration to perform statistical analyses and then results were presented as % control values. Each bar represents n≥6.</p

Downregulation of pEGFR and phospho-c-Src in mitochondria in hUCBSC treatments.

<p>(A) Control and hUCBSC-treated U251, U87 and 5310 cells were labeled with pEGFR and Mito Tracker green and processed for immunofluorescence. (B) Control and hUCBSC-treated U251 and 5310 tissue sections were labeled with pEGFR and Mito Tracker green and processed for immunofluorescence. pEGFR was conjugated with Alexa Fluor 594 (red) secondary antibody (n≥3; bar = 100 µm). (C) Control and hUCBSC-treated U251, U87 and 5310 cells were labeled with phospho-c-Src and Mito Tracker green and processed for immunofluorescence. (D) Control and hUCBSC-treated U251 and 5310 tissue sections were labeled with phospho-c-Src and Mito Tracker green and processed for immunofluorescence. Phospho-c-Src was conjugated with Alexa Fluor 594 (red) secondary antibody (n≥3; bar = 100 µm).</p