http://hdl.handle.net/10027/7413 Tue, 21 May 2013 13:15:26 GMT 2013-05-21T13:15:26Z http://hdl.handle.net/10027/8607 Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma Dasari, Venkata Ramesh; Velpula, Kiran Kumar; Alapati, Kiranmai; Gujrati, Meena; Tsung, Andrew J. 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. © 2012 Dasari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi:10.1371/journal.pone.0031884 Wed, 01 Feb 2012 06:00:00 GMT http://hdl.handle.net/10027/8607 2012-02-01T06:00:00Z http://hdl.handle.net/10027/8601 TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer’s disease Song, Min; Jin, JingJi; Lim, Jeong-Eun; Kou, Jinghong; Pattanayak, Abhinandan; Rehman, Jamaal A; Kim, Hong-Duck; Tahara, Kazuki; Lalonde, Robert; Fukuchi, Ken-ichiro Background: Amyloid plaques, a pathological hallmark of Alzheimer’s disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs), a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start. Methods: Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. Results: While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg) was less than that in a TLR4 wild-type AD model (TLR4W Tg). At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes. Conclusion: This is the first demonstration of TLR4-dependent activation of microglia at the early stage of β-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits start, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity. © 2011 Song et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1186/1742-2094-8-92 Mon, 01 Aug 2011 05:00:00 GMT http://hdl.handle.net/10027/8601 2011-08-01T05:00:00Z http://hdl.handle.net/10027/8541 Combined treatment of Aβ immunization with statin in a mouse model of Alzheimer's disease Kou, Jinghong; Song, Min; Pattanayak, Abhinandan; Lim, Jeong-Eun; Yang, Junling; Cao, Dongfeng; Li, Ling; Fukuchi, Ken-ichiro We evaluated the therapeutic efficacy of combined treatment of Aβ-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aβ titers and simvastatin increased the seropositive rate. Aβ-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aβ deposits but increased soluble Aβ and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aβ titers can increase soluble Aβ and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models. NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Neuroimmunology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Neuroimmunology, [Vol 244, Issue 1-2, (March 2012)] DOI: doi.org/10.1016/j.jneuroim.2012.01.008 Thu, 01 Mar 2012 06:00:00 GMT http://hdl.handle.net/10027/8541 2012-03-01T06:00:00Z http://hdl.handle.net/10027/8508 Intermittent Hypoxia Regulates Stem-like Characteristics and Differentiation of Neuroblastoma Cells Bhaskara, Vasantha Kumar; Mohanam, Indra; Rao, Jasti S.; Mohanam, Sanjeeva Background: Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells. Methods and Findings: Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1a and HIF-2 a proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acidinduced differentiation of neuroblastoma cells. Conclusions: Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses ifferentiation propensities in neuroblastoma cells. © 2012 Bhaskara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi:10.1371/journal.pone.0030905 Wed, 01 Feb 2012 06:00:00 GMT http://hdl.handle.net/10027/8508 2012-02-01T06:00:00Z