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Sample records for human glioblastoma intracellular

  1. The mechanism of honokiol-induced intracellular Ca(2+) rises and apoptosis in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Chang, Hong-Tai; Cheng, Jin-Shiung; Kuo, Daih-Huang; Ko, Kuang-Chung; Chiang, Ni-Na; Wu, Ru-Fang; Shieh, Pochuen; Jan, Chung-Ren

    2014-09-25

    Honokiol, an active constituent of oriental medicinal herb Magnolia officinalis, caused Ca(2+) mobilization and apoptosis in different cancer cells. In vivo, honokiol crossed the blood-brain or -cerebrospinal fluid barrier, suggesting that it may be an effective drug for the treatment of brain tumors, including glioblastoma. This study examined the effect of honokiol on intracellular Ca(2+) concentration ([Ca(2+)]i) and apoptosis in DBTRG-05MG human glioblastoma cells. Honokiol concentration-dependently induced a [Ca(2+)]i rise. The signal was decreased partially by removal of extracellular Ca(2+). Honokiol-triggered [Ca(2+)]i rise was not suppressed by store-operated Ca(2+) channel blockers (nifedipine, econazole, SK&F96365) and the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was inhibited by the PKC inhibitor GF109203X. GF109203X-induced inhibition was not altered by removal of extracellular Ca(2+). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished honokiol-induced [Ca(2+)]i rise. Conversely, incubation with honokiol abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished honokiol-induced [Ca(2+)]i rise. Honokiol (20-80μM) reduced the cell viability, which was not reversed by prechelating cytosolic Ca(2+) with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Honokiol (20-60μM) enhanced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, released cytochrome c, and activated caspase-9/caspase-3. Together, honokiol induced a [Ca(2+)]i rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-dependent, non store-operated Ca(2+) channels. Moreover, honokiol activated the mitochondrial pathway of apoptosis in DBTRG-05MG human glioblastoma cells. Copyright © 2014

  2. The interplay between intracellular progesterone receptor and PKC plays a key role in migration and invasion of human glioblastoma cells.

    PubMed

    Marquina-Sánchez, Brenda; González-Jorge, Jesús; Hansberg-Pastor, Valeria; Wegman-Ostrosky, Talia; Baranda-Ávila, Noemi; Mejía-Pérez, Sonia; Camacho-Arroyo, Ignacio; González-Arenas, Aliesha

    2017-09-01

    Intracellular progesterone receptors (PRs) and protein kinases C (PKCs) are known regulators of cancer cell proliferation and metastasis. Both PRs and PKCs are found overexpressed in grade IV human astrocytomas, also known as glioblastomas, which are the most frequent and aggressive brain tumors. In the present study, we investigated whether PR activation by PKC induces the migration and invasion of glioblastoma derived cell lines and if PKCα and δ isoforms are involved in PR activation. We observed that PKC activation with tetradecanoylphorbol acetate (TPA) increases the migration and invasion capacity of two human glioblastoma derived human cell lines (U251 MG and U87) and that the treatment with the PR receptor antagonist RU486 blocks these processes. Interestingly, the pharmacological inhibition of the isoenzymes PKCα and PKCδ also resulted in a blocked PR transcriptional activity. Also, TPA-dependent PR activation increases the expression of progesterone-induced blocking factor (PIBF), a known PR target gene. These results hint to an existing cross-talk between PKCs and PRs in regulating the infiltration process of human glioblastomas. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Innovative fluorescent magnetic albumin microbead-assisted cell labeling and intracellular imaging of glioblastoma cells.

    PubMed

    Wang, Xueqin; Wei, Fang; Yan, Shuang; Zhang, Huiru; Tan, Xiaorong; Zhang, Lu; Zhou, Guangzhou; Cui, Liuqing; Li, Cuixiang; Wang, Liang; Li, Yatao

    2014-04-15

    Superparamagnetic nanoparticle-based polymer microbeads utilized as carriers are attractive materials widely applied in the biomedical field. However, the deficiency of toxicity, biocompatibility, and biodegradability for polymer materials often limits the application of these microbeads. In the present study, magnetic albumin microbeads (MAMbs), i.e., human serum albumin-coated γ-Fe2O3 nanoparticles, are synthesized to label human U251 glioblastoma multiforme cells. The effects of MAMbs on the biological behavior of U251 glioblastoma cells, including their proliferation, cell viability, cytoskeletal structure, cell cycle, and apoptosis rate, are investigated. Moreover, fluorescein isothiocyanate (FITC)-MAMbs are fabricated by reaction with fluorescent dye FITC used for intracellular imaging of U251 glioblastoma cells. MAMbs possess undetectable cytotoxicity and excellent biocompatibility with U251 glioblastoma cells, as demonstrated by the biological behavior and morphology of U251 cells exposed to MAMbs. Furthermore, the constructed fluorescent MAMbs allow effective intracellular imaging, as illustrated by fluorescence microscopic analysis. The fabricated fluorescent MAMbs have promising perspectives in biomedical research, especially in cell-targeted labeling and intracellular fluorescence magnetic dual-mode imaging in cancer-targeted diagnosis and therapy. © 2013 Published by Elsevier B.V.

  4. Intracellular Progesterone Receptor Mediates the Increase in Glioblastoma Growth Induced by Progesterone in the Rat Brain.

    PubMed

    Germán-Castelán, Liliana; Manjarrez-Marmolejo, Joaquín; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2016-08-01

    Progesterone (P) is a steroid hormone involved in the development of several types of cancer including astrocytomas, the most common and malignant brain tumors. We undertook this study to investigate the effects of P on the growth and infiltration of a tumor caused by the xenotransplant of U87 cells derived from a human astrocytoma grade IV (glioblastoma) in the cerebral cortex of male rats and the participation of intracellular progesterone receptor (PR) on these effects. Eight weeks after the implantation of U87 cells in the cerebral cortex, we administered phosphorothioated antisense oligodeoxynucleotides (ODNs) to silence the expression of PR. This treatment lasted 15 days and was administered at the site of glioblastoma cells implantation using Alzet osmotic pumps. Vehicle (propylene glycol) or P4 (400 μg/100 g) was subcutaneously injected for 14 days starting 1 day after the beginning of ODN administration. We observed that P significantly increased glioblastoma tumor area and infiltration length as compared with vehicle, whereas PR antisense ODNs blocked these effects. P, through the interaction with PR, increases the area and infiltration of a brain tumor formed from the xenotransplant of human glioblastoma-derived U87 cells in the cerebral cortex of the rat. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

  5. A tree shrew glioblastoma model recapitulates features of human glioblastoma.

    PubMed

    Tong, Yaohui; Hao, Junjun; Tu, Qiu; Yu, Hualin; Yan, Lanzhen; Li, Yuan; Lv, Longbao; Wang, Fei; Iavarone, Antonio; Zhao, Xudong

    2017-03-14

    Tupaia belangeri (tree shrew), an animal species whose genome has significantly higher similarity to primates than rodents, has been used in biomedical research. To generate animal models that reproduce the human tumors more faithfully than rodents, we present the first report of a cancer model mimicking human tumor genetics in tree shrew. By engineering a lentiviral system for the transduction of mutant H-Ras and a shRNA against tree shrew p53, we successfully generated malignant glioma in tree shrew. The tree shrew glioma exhibited aggressive behavior and a relatively short latency, and markedly reduced animal survival. Remarkably, the biological features of human high-grade glioma (necrosis, microvascular proliferation, pseudopalisading) were all present in tree shrew glioma. Furthermore, genetic analysis of tree shrew glioma revealed that the tumors were clustered within the mesenchymal subgroup of human glioblastoma multiforme. Compared with the corresponding mouse glioma, tree shrew gliomas were markedly more similar to human glioblastoma at gene expression profile. The tree shrew glioma model provides colleagues working in the field of gliomas and cancer in general with a more accurate animal model.

  6. Metformin selectively affects human glioblastoma tumor-initiating cell viability

    PubMed Central

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirana; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2013-01-01

    Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. PMID:23255107

  7. Glioblastoma

    MedlinePlus

    ... most common form of glioblastoma; it is very aggressive. Secondary: These tumors have a longer, somewhat slower growth history, but still are very aggressive. They may begin as lower-grade tumors which ...

  8. Glioblastoma.

    PubMed

    Wirsching, Hans-Georg; Galanis, Evanthia; Weller, Michael

    2016-01-01

    Glioblastoma is the most common and aggressive primary brain tumor in adults. Defining histopathologic features are necrosis and endothelial proliferation, resulting in the assignment of grade IV, the highest grade in the World Health Organization (WHO) classification of brain tumors. The classic clinical term "secondary glioblastoma" refers to a minority of glioblastomas that evolve from previously diagnosed WHO grade II or grade III gliomas. Specific point mutations of the genes encoding isocitrate dehydrogenase (IDH) 1 or 2 appear to define molecularly these tumors that are associated with younger age and more favorable outcome; the vast majority of glioblastomas are IDH wild-type. Typical molecular changes in glioblastoma include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide 3-kinase (PI3K), p53, and retinoblastoma protein (RB) signaling. Standard treatment of glioblastoma includes surgery, radiotherapy, and alkylating chemotherapy. Promoter methylation of the gene encoding the DNA repair protein, O(6)-methylguanyl DNA methyltransferase (MGMT), predicts benefit from alkylating chemotherapy with temozolomide and guides choice of first-line treatment in elderly patients. Current developments focus on targeting the molecular characteristics that drive the malignant phenotype, including altered signal transduction and angiogenesis, and more recently, various approaches of immunotherapy.

  9. Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

    PubMed Central

    Perez, Ana Paula; Cosaka, Maria Luz; Romero, Eder Lilia; Morilla, Maria Jose

    2011-01-01

    Background Gene silencing using small interfering RNA (siRNA) is a promising new therapeutic approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers. In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-β-cyclodextrin, and genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-β-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing

  10. Establishment of a human glioblastoma stemlike brainstem rodent tumor model.

    PubMed

    Siu, I-Mei; Tyler, Betty M; Chen, James X; Eberhart, Charles G; Thomale, Ulrich-Wilhelm; Olivi, Alessandro; Jallo, George I; Riggins, Gregory J; Gallia, Gary L

    2010-07-01

    Diffuse brainstem tumors are the most difficult type of pediatric CNS malignancy to treat. These inoperable lesions are treated with radiation alone or in combination with chemotherapy, and the survival rate is less than 10%. It is therefore essential to develop a reliable animal model to screen new therapeutic agents for the treatment of this type of tumor. A multipotent human glioblastoma stemlike neurosphere line, 060919, was established from a surgically resected glioblastoma specimen; when cells were implanted intracranially into athymic nude mice, they formed invasive, vascular tumors that exhibited the features of glioblastoma. Ten female Fischer 344 rats received an injection of 75,000 F98 rat glioma cells and 10 female athymic nude rats received an injection of 75,000 060919 human glioblastoma stemlike cells in the pontine tegmentum of the brainstem. A control group of 5 female Fischer rats received an injection of saline in the same location as the animals in the tumor groups. Kaplan-Meier curves were generated for survival, and brains were processed postmortem for histopathological investigation. Both F98 cells and 060919 cells grew in 100% of the animals injected. Median survival of animals injected with F98 was 15 days, consistent with the authors' previous reports on the establishment of the brainstem tumor model using the F98 rat glioma line. Median survival of animals injected with 060919 was 31 days. Histopathological analysis of the tumors confirmed the presence of brainstem lesions in animals that received brainstem injections of F98 and in animals that received brainstem injections of 060919. The 060919 brainstem tumors histologically resembled glioblastoma. Tumor take and median survival were consistent for animals injected in the brainstem with either the established F98 rat glioma cell line or the 060919 human glioblastoma stemlike neurosphere line. Histopathological features of the 060919 brainstem tumors resembled glioblastoma

  11. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  12. Comprehensive genomic characterization defines human glioblastoma genes and core pathways.

    PubMed

    2008-10-23

    Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas--the most common type of adult brain cancer--and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

  13. Delivery of doxorubicin-loaded PLGA nanoparticles into U87 human glioblastoma cells.

    PubMed

    Malinovskaya, Julia; Melnikov, Pavel; Baklaushev, Vladimir; Gabashvili, Anna; Osipova, Nadezhda; Mantrov, Sergey; Ermolenko, Yulia; Maksimenko, Olga; Gorshkova, Marina; Balabanyan, Vadim; Kreuter, Jörg; Gelperina, Svetlana

    2017-03-27

    The paramount problem in the therapy of brain tumors is the inability of most drugs to cross the blood-brain barrier. PLGA nanoparticles overcoated with poloxamer 188 could overcome this problem and enabled a high anti-tumoral effect against the very aggressive intracranial 101.8 glioblastoma in rats that closely resembles human grade IV glioblastomas. The basis for the transport of these particles across the blood-brain barrier appears to be adsorption of blood apolipoproteins (ApoE or ApoA-I) on the nanoparticle surface caused by the poloxamer 188-coating, followed by receptor-mediated transcytosis of the nanoparticles. The objective of the present study is the elucidation of the mechanism by which the poloxamer 188-coated nanoparticles then enter the brain tumor cells. Their intracellular fate, therefore, was investigated using the U87 human glioma cell line. The main mechanism of the PLGA nanoparticle internalization by U87 cells was clathrin-mediated endocytosis. Within 1h free doxorubicin was released from late endosomes and could reach its target site, i.e. the DNA in the nuclei without degradation, whereas the PLGA nanoparticles, which were labeled with Cy5.5, still were observed in the endo-lysosomal compartment. These results demonstrate that the underlying mechanism of action in the brain cells is by diffusive doxorubicin release from the nanoparticles rather than by their intracellular degradation.

  14. DIETARY ISOTHIOCYANATE IBERIN INHIBITS GROWTH AND INDUCES APOPTOSIS IN HUMAN GLIOBLASTOMA CELLS

    USDA-ARS?s Scientific Manuscript database

    In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in Brassicaceae species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition...

  15. Deregulation of a STAT3-IL8 Signaling Pathway Promotes Human Glioblastoma Cell Proliferation and Invasiveness

    PubMed Central

    de la Iglesia, Núria; Konopka, Genevieve; Lim, Kah Leong; Nutt, Catherine L.; Bromberg, Jacqueline F.; Frank, David A.; Mischel, Paul S.; Louis, David N.; Bonni, Azad

    2009-01-01

    Inactivation of the tumor suppressor PTEN is recognized as a major event in the pathogenesis of the brain tumor glioblastoma. However, the mechanisms by which PTEN loss specifically impacts the malignant behavior of glioblastoma cells including their proliferation and propensity for invasiveness remain poorly understood. Genetic studies suggest that the transcription factor STAT3 harbors a PTEN-regulated tumor suppressive function in mouse astrocytes. Here, we report that STAT3 plays a critical tumor suppressive role in PTEN-deficient human glioblastoma cells. Endogenous STAT3 signaling is specifically inhibited in PTEN-deficient glioblastoma cells. Strikingly, reactivation of STAT3 in PTEN-deficient glioblastoma cells inhibits their proliferation, invasiveness, and ability to spread on myelin. We also identify the chemokine IL8 as a novel target gene of STAT3 in human glioblastoma cells. Activated STAT3 occupies the endogenous IL8 promoter and directly represses IL8 transcription. Consistent with these results, IL8 is upregulated in PTEN-deficient human glioblastoma tumors. Importantly, IL8 repression mediates STAT3-inhibition of glioblastoma cell proliferation, invasiveness, and spreading on myelin. Collectively, our findings uncover a novel link between STAT3 and IL8 whose deregulation plays a key role in the malignant behavior of PTEN-deficient glioblastoma cells. These studies suggest that STAT3 activation or IL8 inhibition may have potential in patient-tailored treatment of PTEN-deficient brain tumors. PMID:18524891

  16. Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

    PubMed

    Ignarro, Raffaela Silvestre; Facchini, Gustavo; Vieira, André Schwambach; De Melo, Daniela Rodrigues; Lopes-Cendes, Iscia; Castilho, Roger Frigério; Rogerio, Fabio

    2016-07-01

    Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

  17. Comprehensive genomic characterization defines human glioblastoma genes and core pathways

    PubMed Central

    2008-01-01

    Human cancer cells typically harbor multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multidimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here, we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas (GBM), the most common type of adult brain cancer, and nucleotide sequence aberrations in 91 of the 206 GBMs. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the PI3 kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of GBM. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer. PMID:18772890

  18. Characterization of glioma stem-like cells from human glioblastomas

    PubMed Central

    YAMAMURO, SHUN; OKAMOTO, YUTAKA; SANO, EMIKO; OCHIAI, YUSHI; OGINO, AKIYOSHI; OHTA, TAKASHI; HARA, HIROYUKI; UEDA, TAKUYA; NAKAYAMA, TOMOHIRO; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2015-01-01

    Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs. PMID:25955568

  19. Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

    PubMed

    Lan, Xiaoyang; Jörg, David J; Cavalli, Florence M G; Richards, Laura M; Nguyen, Long V; Vanner, Robert J; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle M; Pellacani, Davide; Park, Nicole I; Coutinho, Fiona J; Whetstone, Heather; Selvadurai, Hayden J; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H; Mungall, Andrew J; Moore, Richard A; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J; Taylor, Michael D; Hirst, Martin; Eaves, Connie J; Simons, Benjamin D; Dirks, Peter B

    2017-09-14

    Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

  20. Effects of stratospheric radiations on human glioblastoma cells.

    PubMed

    Cerù, Maria Paola; Amicarelli, Fernanda; Cristiano, Loredana; Colafarina, Sabrina; Aimola, Pierpaolo; Falone, Stefano; Cinque, Benedetta; Ursini, Ornella; Moscardelli, Roberto; Ragni, Pietro

    2005-01-01

    The aim of this work was to evaluate the effect of stratospheric radiations on neural tumour cells. ADF human glioblastoma cells were hosted on a stratospheric balloon within the 2002 biological experiment campaign of the Italian Space Agency. The flight at an average height of 37 km lasted about 24 hrs. Cell morphology, number and viability, cell cycle and apoptosis, some antioxidant enzymes and proteins involved in cell cycle regulation, DNA repair and gene expression were studied. Stratospheric radiations caused a significant decrease in cell number, as well as a block of proliferation, but not apoptosis or necrosis. Radiations also induced activation and induction of some antioxidant enzymes, increase in DNA repair-related proteins (p53 and Proliferating Cell Nuclear Antigen) and variations of the transcription factors Peroxisome Proliferator-Activated Receptors. Morphologically, test cells exhibited more electron dense cytoplasm and less condensed chromatin than controls and modification of their surfaces. Our results indicate that glioblastoma cells, exposed to continuous stratospheric radiations for 24 hrs, show activation of cell cycle check point, decrease of cell number, variations of Peroxisome Proliferator-Activated Receptors and increase of Reactive Oxygen Species-scavenging enzymes.

  1. Metformin repositioning as antitumoral agent: selective antiproliferative effects in human glioblastoma stem cells, via inhibition of CLIC1-mediated ion current

    PubMed Central

    Barbieri, Federica; Peretti, Marta; Pizzi, Erika; Pattarozzi, Alessandra; Carra, Elisa; Sirito, Rodolfo; Daga, Antonio; Curmi, Paul M.G.; Mazzanti, Michele; Florio, Tullio

    2014-01-01

    Epidemiological and preclinical studies propose that metformin, a first-line drug for type-2 diabetes, exerts direct antitumor activity. Although several clinical trials are ongoing, the molecular mechanisms of this effect are unknown. Here we show that chloride intracellular channel-1 (CLIC1) is a direct target of metformin in human glioblastoma cells. Metformin exposure induces antiproliferative effects in cancer stem cell-enriched cultures, isolated from three individual WHO grade IV human glioblastomas. These effects phenocopy metformin-mediated inhibition of a chloride current specifically dependent on CLIC1 functional activity. CLIC1 ion channel is preferentially active during the G1-S transition via transient membrane insertion. Metformin inhibition of CLIC1 activity induces G1 arrest of glioblastoma stem cells. This effect was time-dependent, and prolonged treatments caused antiproliferative effects also for low, clinically significant, metformin concentrations. Furthermore, substitution of Arg29 in the putative CLIC1 pore region impairs metformin modulation of channel activity. The lack of drugs affecting cancer stem cell viability is the main cause of therapy failure and tumor relapse. We identified CLIC1 not only as a modulator of cell cycle progression in human glioblastoma stem cells but also as the main target of metformin's antiproliferative activity, paving the way for novel and needed pharmacological approaches to glioblastoma treatment. PMID:25361004

  2. VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

    SciTech Connect

    Oka, Naoki; Soeda, Akio . E-mail: ccd29400@nyc.odn.ne.jp; Inagaki, Akihito; Onodera, Masafumi; Maruyama, Hidekazu; Hara, Akira; Kunisada, Takahiro; Mori, Hideki; Iwama, Toru

    2007-08-31

    There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.

  3. Four cardiac hormones eliminate 4-fold more human glioblastoma cells than the green mamba snake peptide.

    PubMed

    Vesely, Brian A; Eichelbaum, Ehrentraud J; Alli, Abdel A; Sun, Ying; Gower, William R; Vesely, David L

    2007-08-28

    Within 24h four cardiac hormones, i.e., vessel dilator, kaliuretic peptide, atrial natriuretic peptide, and long acting natriuretic peptide decrease the number of human glioblastoma cells 75%, 68%, 67%, and 65% while Dendroaspis (green mamba) peptide caused a 17% decrease when each were utilized at 100 microM. The four cardiac hormones decreased DNA synthesis 65-87% and increased cyclic GMP 1.3- to 3.8-fold in the glioblastoma cells. Natriuretic peptide receptors (NPR)-A and -C were present. four cardiac hormones eliminate up to 75% of glioblastoma cells via cyclic GMP-mediated up to 87% decrease in DNA synthesis.

  4. The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Hsu, Shu-Shong; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Tseng, Hui-Wen; Kuo, Chun-Chi; Jan, Chung-Ren

    2015-01-05

    Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca(2+) levels ([Ca(2+)]i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca(2+)]i rises which were reduced by removing extracellular Ca(2+). Eugenol-induced [Ca(2+)]i rises were not altered by store-operated Ca(2+) channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca(2+)]i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca(2+)]i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca(2+)]i rises by inducing PLC-dependent release of Ca(2+) from the endoplasmic reticulum and caused Ca(2+) influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.

  5. IGF-IR: a new prognostic biomarker for human glioblastoma

    PubMed Central

    Maris, C; D'Haene, N; Trépant, A-L; Le Mercier, M; Sauvage, S; Allard, J; Rorive, S; Demetter, P; Decaestecker, C; Salmon, I

    2015-01-01

    Background: Glioblastomas (GBMs) are the most common malignant primary brain tumours in adults and are refractory to conventional therapy, including surgical resection, radiotherapy and chemotherapy. The insulin-like growth factor (IGF) system is a complex network that includes ligands (IGFI and IGFII), receptors (IGF-IR and IGF-IIR) and high-affinity binding proteins (IGFBP-1 to IGFBP-6). Many studies have reported a role for the IGF system in the regulation of tumour cell biology. However, the role of this system remains unclear in GBMs. Methods: We investigate the prognostic value of both the IGF ligands' and receptors' expression in a cohort of human GBMs. Tissue microarray and image analysis were conducted to quantitatively analyse the immunohistochemical expression of these proteins in 218 human GBMs. Results: Both IGF-IR and IGF-IIR were overexpressed in GBMs compared with normal brain (P<10−4 and P=0.002, respectively). Moreover, with regard to standard clinical factors, IGF-IR positivity was identified as an independent prognostic factor associated with shorter survival (P=0.016) and was associated with a less favourable response to temozolomide. Conclusions: This study suggests that IGF-IR could be an interesting target for GBM therapy. PMID:26291053

  6. p53 regulates the mevalonate pathway in human glioblastoma multiforme

    PubMed Central

    Laezza, C; D'Alessandro, A; Di Croce, L; Picardi, P; Ciaglia, E; Pisanti, S; Malfitano, A M; Comegna, M; Faraonio, R; Gazzerro, P; Bifulco, M

    2015-01-01

    The mevalonate (MVA) pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, we provided evidence that p53 induces the expression of a group of enzymes of the MVA pathway including 3′-hydroxy-3′-methylglutaryl-coenzyme A reductase, MVA kinase, farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, in the human glioblastoma multiforme cell line, U343 cells, and in normal human astrocytes, NHAs. Genetic and pharmacologic perturbation of p53 directly influences the expression of these genes. Furthermore, p53 is recruited to the gene promoters in designated p53-responsive elements, thereby increasing their transcription. Such effect was abolished by site-directed mutagenesis in the p53-responsive element of promoter of the genes. These findings highlight another aspect of p53 functions unrelated to tumor suppression and suggest p53 as a novel regulator of the MVA pathway providing insight into the role of this pathway in cancer progression. PMID:26469958

  7. A tumorigenic MLL-homeobox network in human glioblastoma stem cells.

    PubMed

    Gallo, Marco; Ho, Jenny; Coutinho, Fiona J; Vanner, Robert; Lee, Lilian; Head, Renee; Ling, Erick K M; Clarke, Ian D; Dirks, Peter B

    2013-01-01

    Glioblastoma growth is driven by cancer cells that have stem cell properties, but molecular determinants of their tumorigenic behavior are poorly defined. In cancer, altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here, we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn, HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma.

  8. Xanthohumol induces apoptosis in human malignant glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways.

    PubMed

    Festa, Michela; Capasso, Anna; D'Acunto, Cosimo W; Masullo, Milena; Rossi, Adriano G; Pizza, Cosimo; Piacente, Sonia

    2011-12-27

    The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme.

  9. Transforming fusions of FGFR and TACC genes in human glioblastoma.

    PubMed

    Singh, Devendra; Chan, Joseph Minhow; Zoppoli, Pietro; Niola, Francesco; Sullivan, Ryan; Castano, Angelica; Liu, Eric Minwei; Reichel, Jonathan; Porrati, Paola; Pellegatta, Serena; Qiu, Kunlong; Gao, Zhibo; Ceccarelli, Michele; Riccardi, Riccardo; Brat, Daniel J; Guha, Abhijit; Aldape, Ken; Golfinos, John G; Zagzag, David; Mikkelsen, Tom; Finocchiaro, Gaetano; Lasorella, Anna; Rabadan, Raul; Iavarone, Antonio

    2012-09-07

    The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.

  10. Core Canonical Pathways Involved in Developing Human Glioblastoma Multiforme (GBM).

    PubMed

    Ghosh, Somiranjan; Dutta, Sisir; Thorne, Gabriel; Boston, Ava; Barfield, Alexis; Banerjee, Narendra; Walker, Rayshawn; Banerjee, Hirendra Nath

    2017-02-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of the primary brain tumors with pathologic hallmarks of necrosis and vascular proliferation. The diagnosis of GBM is currently mostly based on histological examination of brain tumor tissues, after radiological characterization and surgical biopsy. The ability to characterize tumors comprehensively at the molecular level raises the possibility that diagnosis can be made based on molecular profiling with or without histological examination, rather than solely on histological phenotype. The development of novel genomic and proteomic techniques will foster in the identification of such diagnostic and prognostic molecular markers. We analyzed the global differential gene expression of a GBM cell line HTB15 in comparison to normal human Astrocytes, and established a few canonical pathways that are important in determining the molecular mechanisms of cancer using global gene expression microarray, coupled with the Ingenuity Pathway Analysis (IPA®). Overall, we revealed a discrete gene expression profile in the experimental model that resembled progression of GBM cancer. The canonical pathway analysis showed the involvement of genes that differentially expressed in such a disease condition that included Inositol pathway, Polo like kinases, nNOS signaling, and Tetrapyrrole biosynthesis. Our findings established that the gene expression pattern of this dreaded brain cancer will probably help the cancer research community by finding out newer therapeutic strategies to combat this dreaded cancer type that leads to the identification of high-risk population in this category, with almost hundred percent mortality rate.

  11. Delayed IFN response differentiates replication of West Nile virus and Japanese encephalitis virus in human neuroblastoma and glioblastoma cells.

    PubMed

    Takamatsu, Yuki; Uchida, Leo; Morita, Kouichi

    2015-08-01

    West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important causes of human encephalitis cases, which result in a high mortality ratio and neurological sequelae after recovery. Understanding the mechanism of neuropathogenicity in these viral infections is important for the development of specific antiviral therapy. Here, we focused on human-derived neuronal and glial cells to understand the cellular responses against WNV and JEV infection. It was demonstrated that early IFN-β induction regulated virus replication in glioblastoma tbl98G cells, whereas delayed IFN-β induction resulted in efficient virus replication in neuroblastoma SK-N-SH cells. Moreover, the concealing of viral dsRNA in the intracellular membrane resulted in the delayed IFN response in SK-N-SH cells. These results, which showed different IFN responses between human neuronal and glial cells after WNV or JEV infection, are expected to contribute to our understanding of the molecular mechanisms for neuropathology in these viral infections.

  12. Impact of Human Immunodeficiency Virus in the Pathogenesis and Outcome of Patients with Glioblastoma Multiforme

    PubMed Central

    Choy, Winward; Lagman, Carlito; Lee, Seung J.; Bui, Timothy T.; Safaee, Michael

    2016-01-01

    Background Improvement in antiviral therapies have been accompanied by an increased frequency of non-Acquired Immune Deficiency Syndrome (AIDS) defining malignancies, such as glioblastoma multiforme. Here, we investigated all reported cases of human immunodeficiency virus (HIV)-positive patients with glioblastoma and evaluated their clinical outcomes. A comprehensive review of the molecular pathogenetic mechanisms underlying glioblastoma development in the setting of HIV/AIDS is provided. Methods We performed a PubMed search using keywords “HIV glioma” AND “glioblastoma,” and “AIDS glioma” AND “glioblastoma.” Case reports and series describing HIV-positive patients with glioblastoma (histologically-proven World Health Organization grade IV astrocytoma) and reporting on HAART treatment status, clinical follow-up, and overall survival (OS), were included for the purposes of quantitative synthesis. Patients without clinical follow-up data or OS were excluded. Remaining articles were assessed for data extraction eligibility. Results A total of 17 patients met our inclusion criteria. Of these patients, 14 (82.4%) were male and 3 (17.6%) were female, with a mean age of 39.5±9.2 years (range 19–60 years). Average CD4 count at diagnosis of glioblastoma was 358.9±193.4 cells/mm3. Tumor progression rather than AIDS-associated complications dictated patient survival. There was a trend towards increased median survival with HAART treatment (12.0 vs 7.5 months, p=0.10) Conclusion Our data suggests that HAART is associated with improved survival in patients with HIV-associated glioblastoma, although the precise mechanisms underlying this improvement remain unclear. PMID:27867916

  13. Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

    PubMed Central

    García, Isabel; Pascual, Alvaro; Ballesta, Sofía; Joyanes, Providencia; Perea, Evelio J.

    2000-01-01

    The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus. PMID:11036051

  14. Response of intracerebral human glioblastoma xenografts to multifraction radiation exposures

    SciTech Connect

    Ozawa, Tomoko; Faddegon, Bruce A.; Hu, Lily J.; Bollen, Andrew W.; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: ddeen@itsa.ucsf.edu

    2006-09-01

    Purpose: We investigated the effects of fractionated radiation treatments on the life spans of athymic rats bearing intracerebral brain tumors. Methods and Materials: U-251 MG or U-87 MG human glioblastoma cells were implanted into the brains of athymic rats, and the resulting tumors were irradiated once daily with various doses of ionizing radiation for 5 consecutive days or for 10 days with a 2-day break after Day 5. Results: Five daily doses of 1 and 1.5 Gy, and 10 doses of 0.75 and 1 Gy, cured some U-251 MG tumors. However, five daily doses of 0.5 Gy increased the survival time of animals bearing U-251 MG tumors 5 days without curing any animals of their tumors. Ten doses of 0.3 Gy given over 2 weeks extended the lifespan of the host animals 9 days without curing any animals. For U-87 MG tumors, 5 daily doses of 3 Gy produced an increased lifespan of 8 days without curing any animals, and 10 doses of 1 Gy prolonged lifespan 5.5 days without curing any animals. The differences in extension of life span between the 5- and 10-fraction protocols were minor for either tumor type. Conclusion: The finding that the U-251 MG tumors are more sensitive than U-87 MG tumors, despite the fact that U-251 MG tumors contain many more hypoxic cells than U-87 MG tumors, suggests the intrinsic cellular radiosensitivities of these cell lines are more important than hypoxia in determining their in vivo radiosensitivities.

  15. Oxygen-dependent regulation of NDRG1 in human glioblastoma cells in vitro and in vivo.

    PubMed

    Said, Harun M; Stein, Susanne; Hagemann, Carsten; Polat, Buelent; Staab, Adrian; Anacker, Jelena; Schoemig, Beate; Theobald, Matthias; Flentje, Michael; Vordermark, Dirk

    2009-01-01

    NDRG1 is a member of the N-myc downregulated gene (NDRG) family. Its induction occurs via diverse physiological and pathological conditions (hypoxia, cellular differentiation, heavy metal, N-myc, neoplasia) which modulate NDRG1 transcription, mRNA stability and translation. Hypoxia, among other factors, induces NDRG1 expression and plays an important role in its regulation of expression. To date, the complete detailed function of this protein in humans remains unknown. Hypoxia represents a common feature of solid tumors. In our study, differences in NDRG1 expression between different WHO grades of astrocytic tumors were comparatively examined in vivo in human low-grade astrocytoma (WHO grade 2) and glioblastoma (WHO grade 4) at both the protein and mRNA level by Western blot analysis and semi-quantitative RT-PCR, respectively. Furthermore, the same proteins were determined in vitro in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1alpha protein and mRNA regulation under hypoxia was also determined in vitro in U251, U373 and GaMG cells. This regulation was shown at the same levels in vivo in human low-grade astrocytoma (WHO grade 2) and glioblastoma which showed a higher NDRG1 overexpression level in glioblastoma than in low-grade astrocytoma. siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements (HREs) bound by nuclear HIF-1alpha in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. Due to its clear regulatory behavior under hypoxic condition in human tumor cells, NDRG1 represents an additional

  16. MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

    PubMed Central

    2010-01-01

    Background Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). Methods We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. Results Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). Conclusions Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a

  17. Ultrastructural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of eicosanoid metabolism

    SciTech Connect

    Wilson, D.E.; Anderson, K.M. ); Seed, T.M. )

    1990-01-01

    Human glioblastoma cells incubated in the presence of inhibitors of eicosanoid biosynthesis show decreased cellular proliferation without cytotoxicity. The authors studied the ultrastructural morphology of a human glioblastoma cell line cultured with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, or 5,8,11,14-eicosatetraynoic acid, a cyclooxygenase and lipoxygenase inhibitor. When glioblastoma cells were treated for 3 days with antiproliferative concentrations of either agent, they shared many morphological characteristics, including evidence for increased astrocytic differentiation with only limited signs of toxicity. The inhibited glioma cells demonstrated an increase in the number and length of astrocytic processes containing greater numbers of glial filaments, and the NDGA-treated cells also demonstrated extensive lateral pseudopod formation along the processes. The glioblastoma cell shape also become more elongated, losing the usual nuclear lobularity and nuclear inclusions, especially in NDGA-treated cells. Many cytoplasmic organelles packed the cytosol of the inhibited glioma cells, including prominent Golgi apparatus, dilated smooth endoplasmic reticulum evolving into dilated vesicles, cytoplasmic vacuoles, and numerous concentric laminations. There was limited evidence for toxicity, however, as the mitochondria were more pleomorphic with some mitochondrial distension and disruption of the cristae along with an increase in cytoplasmic vacuolization. The authors conclude that the inhibitors of eicosanoid biosynthesis. NDGA and 5,8,11,14-eicosatetraynoic acid, not only suppress glioblastoma cell proliferation, but also include increased astrocytic differentiation.

  18. CREB Regulates AChE-R-Induced Proliferation of Human Glioblastoma Cells1

    PubMed Central

    Perry, Chava; Sklan, Ella H; Soreq, Hermona

    2004-01-01

    Abstract The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) ε and the scaffold protein RACK1, enhanced PKCε phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors—but not CREB combined with PKC inhibition suppressed—this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors. PMID:15153340

  19. CREB regulates AChE-R-induced proliferation of human glioblastoma cells.

    PubMed

    Perry, Chava; Sklan, Ella H; Soreq, Hermona

    2004-01-01

    The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) epsilon and the scaffold protein RACK1, enhanced PKCepsilon phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors-but not CREB combined with PKC inhibition suppressed-this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors.

  20. Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane.

    PubMed

    Karmakar, S; Weinberg, M S; Banik, N L; Patel, S J; Ray, S K

    2006-09-01

    Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.

  1. Low concentration of quercetin antagonizes the invasion and angiogenesis of human glioblastoma U251 cells.

    PubMed

    Liu, Yue; Tang, Zhen-Gang; Yang, Jian-Quan; Zhou, Yi; Meng, Ling-Hu; Wang, Heng; Li, Cai-Li

    2017-01-01

    Glioblastoma is the most aggressive type of brain tumor with a very poor prognosis. Therefore, it is always of great importance to explore and develop new potential treatment for glioblastoma. Quercetin, a flavonoid present in a variety of human foods, has been shown to inhibit various tumor cell proliferation. In this study, we found that treating human glioblastoma U251 cells with 10 μg/mL quercetin for 24 hours, a concentration that was far below the IC50 (113.65 μg/mL) and at which quercetin failed to inhibit cell proliferation, inhibited cell migration (30%) and cell invasion as examined by wound scratch assay and transwell assay, respectively. We further showed that 10 μg/mL quercetin inhibited cell migration and tube formation of human umbilical vein endothelial cells induced by the conditioned medium derived from U251 cell culture. The inhibitory effect of quercetin on migration and angiogenesis is possibly mediated through the downregulation of protein levels of VEGFA, MMP9, and MMP2 as detected by Western blot. Our findings demonstrated that low concentration of quercetin antagonized glioblastoma cell invasion and angiogenesis in vitro.

  2. Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells

    PubMed Central

    Golebiewska, Anna; Bougnaud, Sébastien; Stieber, Daniel; Brons, Nicolaas H. C.; Vallar, Laurent; Hertel, Frank; Klink, Barbara; Schröck, Evelin; Bjerkvig, Rolf

    2013-01-01

    The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the ‘side population’ phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of

  3. FTY720 induces autophagy-related apoptosis and necroptosis in human glioblastoma cells.

    PubMed

    Zhang, Li; Wang, Handong; Ding, Ke; Xu, Jianguo

    2015-07-02

    FTY720 is a potent immunosuppressant which has preclinical antitumor efficacy in various cancer models. However, its role in glioblastoma remains unclear. In the present study, we found that FTY720 induced extrinsic apoptosis, necroptosis and autophagy in human glioblastoma cells. Inhibition of autophagy by either RNA interference or chemical inhibitors attenuated FTY720-induced apoptosis and necrosis. Furthermore, autophagy, apoptosis and necrosis induction were dependent on reactive oxygen species-c-Jun N-terminal kinase-protein 53 (ROS-JNK-p53) loop mediated phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) pathway. In addition, receptor-interacting protein 1 and 3 (RIP1 and RIP3) served as an upstream of ROS-JNK-p53 loop. However, the phosphorylation form of FTY720 induced autophagy but not apoptosis and necroptosis. Finally, the in vitro results were validated in vivo in xenograft mouse of glioblastoma cells. In conclusion, the current study provided novel insights into understanding the mechanisms and functions of FTY720-induced apoptosis, necroptosis and autophagy in human glioblastoma cells.

  4. CCAAT/enhancer binding protein β negatively regulates progesterone receptor expression in human glioblastoma cells.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2017-01-05

    Many progesterone (P4) actions are mediated by its intracellular receptor (PR), which has two isoforms (PR-A and PR-B) differentially transcribed from separate promoters of a single gene. In glioblastomas, the most frequent and aggressive brain tumors, PR-B is the predominant isoform. In an in silico analysis we showed putative CCAAT/Enhancer Binding Protein (C/EBP) binding sites at PR-B promoter. We evaluated the role of C/EBPβ in PR-B expression regulation in glioblastoma cell lines, which expressed different ratios of PR and C/EBPβ isoforms (LAP1, LAP2, and LIP). ChIP assays showed a significant basal binding of C/EBPβ, specific protein 1 (Sp1) and estrogen receptor alpha (ERα) to PR-B promoter. C/EBPβ knockdown increased PR-B expression and treatment with estradiol (E2) reduced C/EBPβ binding to the promoter and up-regulated PR-B expression. P4 induced genes were differently regulated when CEBP/β was silenced. These data show that C/EBPβ negatively regulates PR-B expression in glioblastoma cells.

  5. Functional expression of the serotonin 5-HT7 receptor in human glioblastoma cell lines

    PubMed Central

    Mahé, Cécile; Bernhard, Michel; Bobirnac, Ionel; Keser, Corinna; Loetscher, Erika; Feuerbach, Dominik; Dev, Kumlesh K; Schoeffter, Philippe

    2004-01-01

    Serotonin 5-HT7 receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT7 receptors and 5-HT7 receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT>5-HT=5-MeOT≫8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT7 receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89–1.13) and pA2 values of 8.69–9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT7 receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT7 receptor (5-HT7(a/b/d)) was visualized by RT–PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT7 receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT7 receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines. PMID:15339860

  6. Expression and stability of c-sis mRNA in human glioblastoma cells

    SciTech Connect

    Press, R.D.; Samols, D.; Goldthwait, D.A.

    1988-07-26

    The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, the authors have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.

  7. Cytotoxic and apoptotic effects of bortezomib and gefitinib compared to alkylating agents on human glioblastoma cells.

    PubMed

    Pédeboscq, Stéphane; L'Azou, Béatrice; Passagne, Isabelle; De Giorgi, Francesca; Ichas, François; Pometan, Jean-Paul; Cambar, Jean

    2008-01-01

    Glioblastoma is a malignant astrocytic tumor with a median survival of about 12 months for which new therapeutic strategies are required. We therefore examined the cytotoxicity of anticancer drugs with different mechanisms of action on two human glioblastoma cell lines expressing various levels of EGFR (epidermal growth factor receptor). Apoptosis induced by these anticancer agents was evaluated by flow cytometry. The cytotoxicity of alkylating drugs followed a dose-effect curve and cytotoxicity index values were lower with carboplatin than with BCNU and temozolomide. Anti-EGFR gefitinib (10 microM) cytotoxicity on DBTRG.05-MG expressing high levels of EGFR was significantly higher than on U87-MG expressing low levels of EGFR. Carboplatin and temozolomide cytotoxicity was potentiated with the addition of gefitinib on DBTRG.05-MG. Among the anticancer agents tested, the proteasome inhibitor bortezomib was the most cytotoxic with very low IC50 on the two cell lines. Moreover, all anticancer drugs tested induced apoptosis in a concentration-dependent manner. Bortezomib proved to be a more potent inductor of apoptosis than gefitinib and alkylating agents. These results show the efficacy of bortezomib and of the association between conventional chemotherapy and gefitinib on glioblastoma cells and therefore suggest the interest of these molecules in the treatment of glioblastoma.

  8. Histology-Based Expression Profiling Yields Novel Prognostic Markers in Human Glioblastoma

    PubMed Central

    Dong, Shumin; Nutt, Catherine L.; Betensky, Rebecca A.; Stemmer-Rachamimov, Anat O.; Denko, Nicholas C.; Ligon, Keith L.; Rowitch, David H.; Louis, David N.

    2006-01-01

    Although the prognosis for patients with glioblastoma is poor, survival is variable, with some patients surviving longer than others. For this reason, there has been longstanding interest in the identi-fication of prognostic markers for glioblastoma. We hypothesized that specific histologic features known to correlate with malignancy most likely express molecules that are directly related to the aggressive behavior of these tumors. We further hypothesized that such molecules could be used as biomarkers to predict behavior in a manner that might add prognostic power to sole histologic observation of the feature. We reasoned that perinecrotic tumor cell palisading, which denotes the most aggressive forms of malignant gliomas, would be a striking histologic feature on which to test this hypothesis. We therefore used laser capture microdissection and oligonucleotide arrays to detect molecules differentially expressed in perinecrotic palisades. A set of RNAs (including POFUT2, PTDSR, PLOD2, ATF5, and HK2) that were differentially expressed in 3 initially studied, micro-dissected glioblastomas also provided prognostic information in an independent set of 28 glioblastomas that did not all have perinecrotic palisades. On validation in a second, larger independent series, this approach could be applied to other human glioma types to derive tissue biomarkers that could offer ancillary prognostic and predictive information alongside standard histopathologic examination. PMID:16254489

  9. Direct effect of bevacizumab on glioblastoma cell lines in vitro.

    PubMed

    Simon, Thomas; Coquerel, Bérénice; Petit, Alexandre; Kassim, Yusra; Demange, Elise; Le Cerf, Didier; Perrot, Valérie; Vannier, Jean-Pierre

    2014-12-01

    Bevacizumab is a humanized monoclonal antibody directed against the pro-angiogenic factor vascular and endothelial growth factor-A (VEGF-A) used in the treatment of glioblastomas. Although most patients respond initially to this treatment, studies have shown that glioblastomas eventually recur. Several non-mutually exclusive theories based on the anti-angiogenic effect of bevacizumab have been proposed to explain these mechanisms of resistance. In this report, we studied whether bevacizumab can act directly on malignant glioblastoma cells. We observe changes in the expression profiles of components of the VEGF/VEGF-R pathway and in the response to a VEGF-A stimulus following bevacizumab treatment. In addition, we show that bevacizumab itself acts on glioblastoma cells by activating the Akt and Erks survival signaling pathways. Bevacizumab also enhances proliferation and invasiveness of glioblastoma cells in hyaluronic acid hydrogel. We propose that the paradoxical effect of bevacizumab on glioblastoma cells could be due to changes in the VEGF-A-dependent autocrine loop as well as in the intracellular survival pathways, leading to the enhancement of tumor aggressiveness. Investigation of how bevacizumab interacts with glioblastoma cells and the resulting downstream signaling pathways will help targeting populations of resistant glioblastoma cells.

  10. Ellagic acid inhibits human glioblastoma growth in vitro and in vivo.

    PubMed

    Wang, Dongliang; Chen, Qianxue; Tan, Yinqiu; Liu, Baohui; Liu, Chao

    2017-02-01

    Ellagic acid (EA) is present in various fruits and plants and has recently been found to possess anticarcinogenic effects. The objective of this study was to investigate the anti‑glioblastoma effect of EA and its mechanisms in vitro and in vivo. We first studied the anticancer activity of EA in U87 and U118 human glioblastoma cell lines. The cell viability and cell proliferation were examined by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine staining respectively. The cell cycle was detected with propidium iodide staining method by flow cytometry and the DNA damage of the cells caused by EA exposure was evaluated by detection of γ-H2AX foci. Then we examined the effect of EA on tumor growth in glioblastoma xenografted mice, and expression of Akt and Notch signaling and their target gene products were detected by immunohistochemistry and western blot analysis. As a result, we found that the cell viability and proliferation of glioblastoma cells treated with EA were significantly suppressed compared with the control; EA significantly increased the proportion of cells in the S phase accompanied by a decrease in the population in the G1 and G2/M phase in both cell lines. Meanwhile, the level of DNA damage in the EA-treated group was significantly higher than that of the control. Treatment of glioblastoma in xenografted mice by EA led to a significant suppression in tumor growth. EA upregulated the expression of E-cadherin and inhibited the expression of Snail, matrix metalloproteinase (MMP)-2 and MMP-9. EA also inhibited the expression of Bcl-2, cyclin D1, cyclin-dependent kinase (CDK)2 and CDK6 in U87 xenograft tissues. In addition, significant suppression of Akt and Notch was found in the xenografts of the tumor-bearing mice treated with EA. These data indicate that EA can suppress glioblastoma proliferation and invasion by inhibiting the Akt and Notch signaling pathways, which suggests that EA may be beneficial for the treatment of

  11. Contrast-enhancing tumor growth dynamics of preoperative, treatment-naive human glioblastoma.

    PubMed

    Ellingson, Benjamin M; Nguyen, Huytram N; Lai, Albert; Nechifor, Ruben E; Zaw, Okkar; Pope, Whitney B; Yong, William H; Nghiemphu, Phioanh L; Liau, Linda M; Cloughesy, Timothy F

    2016-06-01

    Little is known about the natural growth characteristics of untreated glioblastoma before surgical or therapeutic intervention, because patients are rapidly treated after preliminary radiographic diagnosis. Understanding the growth characteristics of uninhibited human glioblastoma may be useful for characterizing changes in response to therapy. Thus, the objective of the current study was to explore tumor growth dynamics in a cohort of patients with untreated glioblastoma before surgical or therapeutic intervention. Ninety-five patients with glioblastoma who had measurable enhancing disease on >2 magnetic resonance imaging scans before surgery were identified. Tumor growth rates were quantified in 4 different ways (the percentage change per day, the absolute rate of change per day, the estimated volumetric doubling time, and the radial expansion rate) using 3 different approaches (bidirectional product, enhancing disease, and total lesion volume). The median volumetric doubling time was 21.1 days, the percentage change in tumor volume was 2.1% per day, and the rate of change in total lesion volume was 0.18 cc per day. The length of follow-up between magnetic resonance imaging examinations should be >28 days to detect progressive disease with high specificity. Small initial tumor sizes (<3 cm in greatest dimension) are biased toward a large percentage change at follow-up. Presurgical, treatment-naive glioblastoma growth dynamics can be estimated in a variety of ways with similar results. The percentage changes in tumor size and volume depend on baseline tumor size and the time interval between scans. Cancer 2016;122:1718-27. © 2016 American Cancer Society. © 2016 American Cancer Society.

  12. Proliferative and Invasive Effects of Progesterone-Induced Blocking Factor in Human Glioblastoma Cells

    PubMed Central

    Hansberg-Pastor, Valeria

    2017-01-01

    Progesterone-induced blocking factor (PIBF) is a progesterone (P4) regulated protein expressed in different types of high proliferative cells including astrocytomas, the most frequent and aggressive brain tumors. It has been shown that PIBF increases the number of human astrocytoma cells. In this work, we evaluated PIBF regulation by P4 and the effects of PIBF on proliferation, migration, and invasion of U87 and U251 cells, both derived from human glioblastomas. PIBF mRNA expression was upregulated by P4 (10 nM) from 12 to 24 h. Glioblastoma cells expressed two PIBF isoforms, 90 and 57 kDa. The content of the shorter isoform was increased by P4 at 24 h, while progesterone receptor antagonist RU486 (10 μM) blocked this effect. PIBF (100 ng/mL) increased the number of U87 cells on days 4 and 5 of treatment and induced cell proliferation on day 4. Wound-healing assays showed that PIBF increased the migration of U87 (12–48 h) and U251 (24 and 48 h) cells. Transwell invasion assays showed that PIBF augmented the number of invasive cells in both cell lines at 24 h. These data suggest that PIBF promotes proliferation, migration, and invasion of human glioblastoma cells. PMID:28168193

  13. Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

    SciTech Connect

    Lopez, Carlos A. Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

    2007-09-01

    Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G{sub 1} arrest, increase in sub-G{sub 1} fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios ({+-} SE) of 1.5 ({+-} 0.2) and 1.3 ({+-} 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53.

  14. Three-dimensional Invasion of Human Glioblastoma Cells Remains Unchanged by X-ray and Carbon Ion Irradiation In Vitro

    SciTech Connect

    Eke, Iris; Storch, Katja; Kaestner, Ina; Vehlow, Anne; Faethe, Christina; Mueller-Klieser, Wolfgang; Taucher-Scholz, Gisela; Temme, Achim; Schackert, Gabriele

    2012-11-15

    Purpose: Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Methods and Materials: Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks ({gamma}H2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, {beta}1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Results: Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the {beta}1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. Conclusions: These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy.

  15. Three-dimensional invasion of human glioblastoma cells remains unchanged by X-ray and carbon ion irradiation in vitro.

    PubMed

    Eke, Iris; Storch, Katja; Kästner, Ina; Vehlow, Anne; Faethe, Christina; Mueller-Klieser, Wolfgang; Taucher-Scholz, Gisela; Temme, Achim; Schackert, Gabriele; Cordes, Nils

    2012-11-15

    Cell invasion represents one of the major determinants that treatment has failed for patients suffering from glioblastoma. Contrary findings have been reported for cell migration upon exposure to ionizing radiation. Here, the migration and invasion capability of glioblastoma cells on and in collagen type I were evaluated upon irradiation with X-rays or carbon ions. Migration on and invasion in collagen type I were evaluated in four established human glioblastoma cell lines exposed to either X-rays or carbon ions. Furthermore, clonogenic radiation survival, proliferation (5-bromo-2-deoxyuridine positivity), DNA double-strand breaks (γH2AX/53BP1-positive foci), and expression of invasion-relevant proteins (eg, β1 integrin, FAK, MMP2, and MMP9) were explored. Migration and invasion assays for primary glioblastoma cells also were carried out with X-ray irradiation. Neither X-ray nor carbon ion irradiation affected glioblastoma cell migration and invasion, a finding similarly observed in primary glioblastoma cells. Intriguingly, irradiated cells migrated unhampered, despite DNA double-strand breaks and reduced proliferation. Clonogenic radiation survival was increased when cells had contact with extracellular matrix. Specific inhibition of the β1 integrin or proliferation-associated signaling molecules revealed a critical function of JNK, PI3K, and p38 MAPK in glioblastoma cell invasion. These findings indicate that X-rays and carbon ion irradiation effectively reduce proliferation and clonogenic survival without modifying the migration and invasion ability of glioblastoma cells in a collagen type I environment. Addition of targeted agents against members of the MAPK and PI3K signaling axis to conventional chemoradiation therapy seems potentially useful to optimize glioblastoma therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. The effect of intracellular alkalinisation on intracellular Ca(2+) homeostasis in a human chondrocyte cell line.

    PubMed

    Browning, Joseph A; Wilkins, Robert J

    2002-09-01

    Intracellular pH (pH(i)) is a well-established determinant of cartilage matrix metabolism. Changes to chondrocyte pH(i), and therefore matrix turnover rates, arise following joint loading. It is not yet clear whether pH changes exert their effects on matrix metabolism directly, or by changing the concentration of another, as yet unidentified, intracellular factor. In this study the effect of intracellular alkalinisation on intracellular [Ca(2+)] has been examined using the human chondrocyte C-20/A4 cell line. pH(i) was manipulated by the addition of weak bases to suspensions of chondrocytes and fluorimetric techniques were employed to measure pH(i) and [Ca(2+)](i). The effect of pH(i) changes on intracellular inositol 1,4,5-trisphosphate (IP(3)) levels was also determined. The pH-sensitive properties of the Ca(2+)-sensitive fluoroprobe employed in this study, Fura-2, were investigated such that artefactual effects of pH changes upon the dye could be discounted. It was demonstrated that, for dye loaded into cells, alkalinisation resulted in a small increase in the affinity of the dye for Ca(2+) ions. Intracellular alkalinisation elicited by treatment with either of the weak bases trimethylamine or ammonium chloride initiated a rise in [Ca(2+)](i). This effect was too large to be explicable by the effects of pH changes on Fura-2 and was not dependent on the presence of extracellular Ca(2+) ions. Prior depletion of intracellular Ca(2+) stores by treatment with thapsigargin inhibited alkalinisation-induced increases in [Ca(2+)](i) and intracellular alkalinisation was also associated with increased levels of intracellular IP(3). These results confirm that alkaline pH(i) changes associated with dynamic loading of cartilage also result in knock-on alterations to [Ca(2+)](i). Given the sensitivity of cartilage matrix metabolism to [Ca(2+)](i) it is likely that this signalling cascade forms an important part of the mechanotransduction pathway that determines the response of

  17. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics

    PubMed Central

    Sottoriva, Andrea; Spiteri, Inmaculada; Piccirillo, Sara G. M.; Touloumis, Anestis; Collins, V. Peter; Marioni, John C.; Curtis, Christina; Watts, Colin; Tavaré, Simon

    2013-01-01

    Glioblastoma (GB) is the most common and aggressive primary brain malignancy, with poor prognosis and a lack of effective therapeutic options. Accumulating evidence suggests that intratumor heterogeneity likely is the key to understanding treatment failure. However, the extent of intratumor heterogeneity as a result of tumor evolution is still poorly understood. To address this, we developed a unique surgical multisampling scheme to collect spatially distinct tumor fragments from 11 GB patients. We present an integrated genomic analysis that uncovers extensive intratumor heterogeneity, with most patients displaying different GB subtypes within the same tumor. Moreover, we reconstructed the phylogeny of the fragments for each patient, identifying copy number alterations in EGFR and CDKN2A/B/p14ARF as early events, and aberrations in PDGFRA and PTEN as later events during cancer progression. We also characterized the clonal organization of each tumor fragment at the single-molecule level, detecting multiple coexisting cell lineages. Our results reveal the genome-wide architecture of intratumor variability in GB across multiple spatial scales and patient-specific patterns of cancer evolution, with consequences for treatment design. PMID:23412337

  18. MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin

    SciTech Connect

    Song, Yichen; Wang, Ping; Zhao, Wei; Yao, Yilong; Liu, Xiaobai; Ma, Jun; Xue, Yixue; Liu, Yunhui

    2014-05-15

    MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. - Highlights: • MiR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines. • Neogenin was identified as the target gene of miR-18a. • Neogenin expressions were decreased along with the rising pathological grades of glioblastoma. • Inhibition of miR-18a suppressed biological behavior of glioma cells by up-regulating neogenin.

  19. PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype

    PubMed Central

    Duan, Shunlei; Yuan, Guohong; Liu, Xiaomeng; Ren, Ruotong; Li, Jingyi; Zhang, Weizhou; Wu, Jun; Xu, Xiuling; Fu, Lina; Li, Ying; Yang, Jiping; Zhang, Weiqi; Bai, Ruijun; Yi, Fei; Suzuki, Keiichiro; Gao, Hua; Esteban, Concepcion Rodriguez; Zhang, Chuanbao; Belmonte, Juan Carlos Izpisua; Chen, Zhiguo; Wang, Xiaomin; Jiang, Tao; Qu, Jing; Tang, Fuchou; Liu, Guang-Hui

    2015-01-01

    PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs), but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs, binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7, which in turn promotes oncogenic transformation of NSCs and instates ‘aggressiveness' in human glioblastoma stem cells. In a large clinical database, we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore, we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together, we uncover a potential mechanism of how PTEN safeguards NSCs, and establish a cellular platform to identify factors involved in NSC transformation, potentially permitting personalized treatment of glioblastoma. PMID:26632666

  20. Dominant Negative Pleiotrophin Induces Tetraploidy and Aneuploidy in U87MG Human Glioblastoma Cells

    PubMed Central

    Chang, Yunchao; Berenson, James R.; Wang, Zhaoyi; Deuel, Thomas F.

    2007-01-01

    Summary Pleiotrophin (PTN, Ptn) is an 18 kD secretory cytokine that is expressed in many human cancers, including glioblastoma. In previous experiments, interruption of the constitutive PTN signaling in human U87MG glioblastoma cells that inappropriately express endogenous Ptn reversed their rapid growth in vitro and their malignant phenotype in vivo. To seek a mechanism for the effect of the dominant negative PTN, flow cytometry was used to compare the profiles of U87MG cells and four clones of U87MG cells that express the dominant negative PTN (U87MG/PTN 1–40 cells); here, we report that the dominant negative PTN in U87MG cells induces tetraploidy and aneuploidy and arrests the tetraploid and aneuploid cells in the G1 phase of the cell cycle. The data suggest that PTN signaling may have a critical role in chromosomal segregation and cell cycle progression; the data suggest induction of tetraploidy and aneuploidy in U87MG glioblastoma cells may be an important mechanism that contributes to the loss of the malignant phenotype of U87MG cells. PMID:17067552

  1. Opuntia humifusa partitioned extracts inhibit the growth of U87MG human glioblastoma cells.

    PubMed

    Hahm, Sahng-Wook; Park, Jieun; Son, Yong-Suk

    2010-09-01

    Opuntia humifusa, a member of the Cactaceae family widely distributed in the southern regions of the Korean peninsula, has potential bioactive functions and medicinal benefits. In the present study, we investigated the effect of hexane, ethyl acetate extracts and water partitioned fraction of O. humifusa on proliferation, G1 arrest and apoptosis in U87MG human glioblastoma cells. Glioblastoma cellular proliferation was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the effects of O. humifusa partitioned extracts on cell cycle and apoptosis were analyzed by flow cytometry. Our results revealed that when U87MG cells were treated with hexane extracts and water partitioned fraction of O. humifusa, the number of viable cells decreased in a concentration-dependent manner. In addition, water partitioned fractions of O. humifusa induced G1 arrest and non-apoptotic cell death as well as significant increases in ROS production in U87MG cells. In conclusion, water partitioned fractions of O. humifusa induce G1 arrest and inhibit U87MG human glioblastoma cell proliferation.

  2. Kynurenic acid inhibits proliferation and migration of human glioblastoma T98G cells.

    PubMed

    Walczak, Katarzyna; Deneka-Hannemann, Sylwia; Jarosz, Bożena; Zgrajka, Wojciech; Stoma, Filip; Trojanowski, Tomasz; Turski, Waldemar A; Rzeski, Wojciech

    2014-02-01

    Kynurenic acid (KYNA), tryptophan metabolite synthesized in the kynurenine pathway, is an endogenous antagonist of α-7 nicotinic receptor and all ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methyl-4-isoxasole propionate (AMPA) receptor and kainate receptor. The antiproliferative activity of KYNA toward colon and renal cancer cells has recently been discovered. The aim of the study was to verify whether human Glioblastoma tumors contain KYNA and if KYNA influences glioma cell proliferation and migration. KYNA content in Glioblastoma tumor samples was determined using HPLC. Proliferation of human glioblastoma T98G cells was measured by means of MTT and BrdU assays. Wound assay was used to evaluate the effect of KYNA on cancer cell migration. KYNA was detected in all tested Glioblastoma tumor samples (100.3 ± 17.6 pmol/g wet weight). In a series of experiments the antiproliferative activity of KYNA against T98G cells was revealed (IC(50) = 1.3 mM). Moreover, KYNA reversed the stimulatory effect of glutamate on glioma cell proliferation and enhanced antiproliferative effect of glutamate receptor antagonists MK801 and GYKI 52466. Next, KYNA at concentrations much lower than those needed to reduce cell proliferation elicited a prominent inhibitory effect on glioma cell motility. Moreover, co-incubation of temozolomide, a drug commonly used in antiglioblastoma therapy, with KYNA gave a superior effect than each of the substances applied alone. We demonstrate the antiproliferative and antimigrative potential of KYNA against glioma cells in vitro. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  4. Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

    PubMed Central

    Said, Harun M; Polat, Buelent; Stein, Susanne; Guckenberger, Mathias; Hagemann, Carsten; Staab, Adrian; Katzer, Astrid; Anacker, Jelena; Flentje, Michael; Vordermark, Dirk

    2012-01-01

    AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5’-GCATTATTGGCATGGGAAC-3’ and 5’-ATGCAGAGTAACGTGGAAG-3’. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human

  5. The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells.

    PubMed

    Atzori, Maria Grazia; Tentori, Lucio; Ruffini, Federica; Ceci, Claudia; Lisi, Lucia; Bonanno, Elena; Scimeca, Manuel; Eskilsson, Eskil; Daubon, Thomas; Miletic, Hrvoje; Ricci Vitiani, Lucia; Pallini, Roberto; Navarra, Pierluigi; Bjerkvig, Rolf; D'Atri, Stefania; Lacal, Pedro Miguel; Graziani, Grazia

    2017-08-10

    Glioblastoma (GBM) is a highly migratory, invasive, and angiogenic brain tumor. Like vascular endothelial growth factor-A (VEGF-A), placental growth factor (PlGF) promotes GBM angiogenesis. VEGF-A is a ligand for both VEGF receptor-1 (VEGFR-1) and VEGFR-2, while PlGF interacts exclusively with VEGFR-1. We recently generated the novel anti-VEGFR-1 monoclonal antibody (mAb) D16F7 that diminishes VEGFR-1 homodimerization/activation without affecting VEGF-A and PlGF binding. In the present study, we evaluated the expression of VEGFR-1 in human GBM tissue samples (n = 42) by immunohistochemistry, in cell lines (n = 6) and GBM stem cells (GSCs) (n = 18) by qRT-PCR and/or western blot analysis. In VEGFR-1 positive GBM or GSCs we also analyzed the ability of D16F7 to inhibit GBM invasiveness in response to VEGF-A and PlGF. Most of GBM specimens stained positively for VEGFR-1 and all but one GBM cell lines expressed VEGFR-1. On the other hand, in GSCs the expression of the receptor was heterogeneous. D16F7 reduced migration and invasion of VEGFR-1 positive GBM cell lines and patient-derived GSCs in response to VEGF-A and PlGF. Interestingly, this effect was also observed in VEGFR-1 positive GSCs transfected to over-express wild-type EGFR (EGFRwt(+)) or mutant EGFR (ligand binding domain-deficient EGFRvIII(+)). Furthermore, D16F7 suppressed intracellular signal transduction in VEGFR-1 over-expressing GBM cells by reducing receptor auto-phosphorylation at tyrosine 1213 and downstream Erk1/2 activation induced by receptor ligands. The results from this study suggest that VEGFR-1 is a relevant target for GBM therapy and that D16F7-derived humanized mAbs warrant further investigation.

  6. Cellular and molecular portrait of eleven human glioblastoma cell lines under photon and carbon ion irradiation.

    PubMed

    Ferrandon, S; Magné, N; Battiston-Montagne, P; Hau-Desbat, N-H; Diaz, O; Beuve, M; Constanzo, J; Chargari, C; Poncet, D; Chautard, E; Ardail, D; Alphonse, G; Rodriguez-Lafrasse, C

    2015-04-28

    This study aimed to examine the cellular and molecular long-term responses of glioblastomas to radiotherapy and hadrontherapy in order to better understand the biological effects of carbon beams in cancer treatment. Eleven human glioblastoma cell lines, displaying gradual radiosensitivity, were irradiated with photons or carbon ions. Independently of p53 or O(6)-methylguanine-DNA methyltransferase(1) status, all cell lines responded to irradiation by a G2/M phase arrest followed by the appearance of mitotic catastrophe, which was concluded by a ceramide-dependent-apoptotic cell death. Statistical analysis demonstrated that: (i) the SF2(2) and the D10(3) values for photon are correlated with that obtained in response to carbon ions; (ii) regardless of the p53, MGMT status, and radiosensitivity, the release of ceramide is associated with the induction of late apoptosis; and (iii) the appearance of polyploid cells after photon irradiation could predict the Relative Biological Efficiency(4) to carbon ions. This large collection of data should increase our knowledge in glioblastoma radiobiology in order to better understand, and to later individualize, appropriate radiotherapy treatment for patients who are good candidates.

  7. Effects of quercetin on proliferation and migration of human glioblastoma U251 cells.

    PubMed

    Liu, Yue; Tang, Zhen-Gang; Lin, Yi; Qu, Xin-Guo; Lv, Wei; Wang, Guo-Bin; Li, Cai-Li

    2017-08-01

    Quercetin is a flavonoid that has been shown to have anti-oxidation, anti-inflammation, anti-allergic, anti-viral, and anti-cancer activities. Here, we examined the effects of quercetin on cell viability, cell cycle progression, and migration in U251 cells, a human glioblastoma cell line. We found that quercetin inhibited cell proliferation after treating cells for 24 (IC50 of 113.65μg/ml) or 48h (IC50 of 48.61μg/ml). Quercetin treatment also induced apoptosis via deregulating the expression of apoptotic genes, including Bax and Bcl-2, and arrested cell cycle at G2/M phases. We further found that quercetin impaired cell migration and invasion via downregulating the expression of matrix metallopeptidases MMP9 and MMP2. Our results provide evidences that quercetin has inhibitory effects on glioblastoma cell proliferation and invasion, and suggest a potential clinical application for glioblastoma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Molecular mechanisms of the effect of TGF-β1 on U87 human glioblastoma cells

    PubMed Central

    Bryukhovetskiy, Igor; Shevchenko, Valeriy

    2016-01-01

    Glioblastoma multiforme (GBM) is the most widespread and aggressive type of primary brain tumor. The prognosis following diagnosis with GBM is poor, with a median survival time of 14 months. Tumor cell invasion, metastasis and proliferation are the major causes of mortality in patients with GBM. In order to develop effective GBM treatment methods it is necessary to identify novel targets involved in these processes. Recently, there has been increasing interest in investigating the signaling pathways involved in GBM development, and the transforming growth factor-β (TGF-β) signaling pathway is understood to be significant for regulating the behavior of GBM, as well as stimulating its invasion and metastatic development. Particular interest has been given to investigating the modulation of TGF-β-induced epithelial-to-mesenchymal transition (EMT); during this process, epithelial cells transdifferentiate into mobile cells with a mesenchymal phenotype. The induction of EMT increases the invasiveness of various types of carcinoma; however, the role of TGF-β in this process remains to be elucidated, particularly in the case of GBM. The current study presents a comparative proteome mapping of the U87 human glioblastoma cell line, with and without TGF-β1 treatment. Proteome analysis identified numerous proteins involved in the molecular mechanisms of GBM oncogenesis and TGF-β1 signaling in glioblastoma. The results of the present study facilitated the identification of novel potential markers of metastasis and candidates for targeted glioblastoma therapy, which may potentially be validated and used in clinical medicine to develop improved approaches for GBM diagnosis and treatment. PMID:27446475

  9. Yes-associated protein 1 is widely expressed in human brain tumors and promotes glioblastoma growth.

    PubMed

    Orr, Brent A; Bai, Haibo; Odia, Yazmin; Jain, Deepali; Anders, Robert A; Eberhart, Charles G

    2011-07-01

    The hippo pathway and its downstream mediator yes-associated protein 1 (YAP1) regulate mammalian organ size in part through modulating progenitor cell numbers. YAP1 has also been implicated as an oncogene in multiple human cancers. Currently, little is known about the expression of YAP1 either in normal human brain tissue or in central nervous system neoplasms. We used immunohistochemistry to evaluate nuclear YAP1 expression in the fetal and normal adult human brains and in 264 brain tumors. YAP1 was expressed in fetal and adult brain regions known to harbor neural progenitor cells, but there was little YAP1 immunoreactivity in the adult cerebral cortex. YAP1 protein was also readily detected in the nuclei of human brain tumors. In medulloblastoma, the expression varied between histologic subtypes and was most prominent in nodular/desmoplastic tumors. In gliomas, it was frequently expressed in infiltrating astrocytomas and oligodendrogliomas but rarely in pilocytic astrocytomas. Using a loss-of-function approach, we show that YAP1 promoted growth of glioblastoma cell lines in vitro. High levels of YAP1 messenger RNA expression were associated with aggressive molecular subsets of glioblastoma and with a nonsignificant trend toward reduced mean survival in human astrocytoma patients. These findings suggest that YAP1 may play an important role in normal human brain development and that it could represent a new target in human brain tumors.

  10. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  11. Phloretin induces cell cycle arrest and apoptosis of human glioblastoma cells through the generation of reactive oxygen species.

    PubMed

    Liu, Yuanyuan; Fan, Chenghe; Pu, Lv; Wei, Cui; Jin, Haiqiang; Teng, Yuming; Zhao, Mingming; Yu, Albert Cheung Hoi; Jiang, Feng; Shu, Junlong; Li, Fan; Peng, Qing; Kong, Jian; Pan, Bing; Zheng, Lemin; Huang, Yining

    2016-06-01

    Phloretin, a flavonoid present in various plants, has been reported to exert anticarcinogenic effects. However, the mechanism of its chemo-preventive effect on human glioblastoma cells is not fully understood. This study aimed to investigate the molecular mechanism of phloretin and its associated chemo-preventive effect in human glioblastoma cells. The results indicate that phloretin inhibited cell proliferation by inducing cell cycle arrest at the G0-G1 phase and induced apoptosis of human glioblastoma cells. Phloretin-induced cell cycle arrest was associated with increased expression of p27 and decreased expression of cdk2, cdk4, cdk6, cyclinD and cyclinE. Moreover, the PI3K/AKT/mTOR signaling cascades were suppressed by phloretin in a dose-dependent manner. In addition, phloretin triggered the mitochondrial apoptosis pathway and generated reactive oxygen species (ROS). This was accompanied by the up-regulation of Bax, Bak and c-PARP and the down-regulation of Bcl-2. The antioxidant agents N-acetyl-L-cysteine and glutathione weakened the effect of phloretin on glioblastoma cells. In conclusion, these results demonstrate that phloretin exerts potent chemo-preventive activity in human glioblastoma cells through the generation of ROS.

  12. Nanomelatonin triggers superior anticancer functionality in a human malignant glioblastoma cell line

    NASA Astrophysics Data System (ADS)

    Yadav, Sanjeev Kumar; Srivastava, Anup Kumar; Dev, Atul; Kaundal, Babita; Choudhury, Subhasree Roy; Karmakar, Surajit

    2017-09-01

    Melatonin (MEL) has promising medicinal value as an anticancer agent in a variety of malignancies, but there are difficulties in achieving a therapeutic dose due to its short half-life, low bioavailability, poor solubility and extensive first-pass metabolism. In this study chitosan/tripolyphosphate (TPP) nanoparticles were prepared by an ionic gelation method to overcome the therapeutic challenges of melatonin and to improve its anticancer efficacy. Characterization of the melatonin-loaded chitosan (MEL-CS) nanoformulation was performed using transmission and scanning electron microscopies, dynamic light scattering, Fourier transform infrared spectroscopy, Raman spectroscopy and x-ray diffraction. In vitro release, cellular uptake and efficacy studies were tested for their enhanced anticancer potential in human U87MG glioblastoma cells. Confocal studies revealed higher cellular uptake of MEL-CS nanoparticles and enhanced anticancer efficacy in human malignant glioblastoma cancer cells than in healthy non-malignant human HEK293T cells in mono- and co-culture models. Our study has shown for the first time that MEL-CS nanocomposites are therapeutically more effective as compared to free MEL at inducing functional anticancer efficacy in the human brain tumour U87MG cell line.

  13. Semiconductor Microcavity Flow Spectroscopy of Intracellular Protein in Human Cells

    NASA Astrophysics Data System (ADS)

    Gourley, Paul; Cox, Jim; Hendricks, Judy; McDonald, Anthony; Copeland, Guild; Sasaki, Darryl; Skirboll, Steve; Curry, Mark

    2001-03-01

    The speed of light through a biofluid or biological cell is inversely related to the biomolecular concentration of proteins and other complex molecules that modify the refractive index at wavelengths accessible to semiconductor lasers. By placing a fluid or cell into a semiconductor microcavity laser, these decreases in light speed can be sensitively recorded in picoseconds as frequency red-shifts in the laser output spectrum. This biocavity laser equipped with microfluidics for transporting cells at high speed through the laser microcavity has shown potential for rapid analysis of biomolecular mass of normal and malignant human cells in their physiologic condition without time-consuming fixing, staining, or tagging. We have used biocavity laser spectroscopy to measure the optical properties of solutions of standard biomolecules (sugars, proteins, DNA, and ions) and human cells. The technique determines the frequency shift, relative to that of water, of spontaneous or stimulated emission from cavity filled with a biomolecular solution. The shift was also measured in human glioblastoma cells that had been sorted by conventional fluorescence-activated cell sorting according to protein content. The results show a direct correlation between protein measured by fluorescence and the frequency shift observed in the microcavity laser.

  14. Molecular mechanism of inositol hexaphosphate-mediated apoptosis in human malignant glioblastoma T98G cells.

    PubMed

    Karmakar, Surajit; Banik, Naren L; Ray, Swapan K

    2007-12-01

    Glioblastoma is the deadliest brain tumor in humans. Current therapies are mostly ineffective and new agents need to be explored for controlling this devastating disease. Inositol hexaphosphate (IP6) is a phytochemical that is widely found in corns, cereals, nuts, and high fiber-content foods. Previous studies demonstrated anti-cancer properties of IP6 in several in vitro and in vivo tumor models. However, therapeutic efficacy of IP6 has not yet been evaluated in glioblastoma. Here, we explored the molecular mechanism of action of IP6 in human malignant glioblastoma T98G cells. The viability of T98G cells decreased following treatment with increasing doses of IP6. T98G cells exposed to 0.25, 0.5, and 1 mM IP6 for 24 h showed morphological and biochemical features of apoptosis. Western blotting indicated changes in expression of Bax and Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio and upregulation of cytosolic levels of cytochrome c and Smac/Diablo, suggesting involvement of mitochondria-dependent caspase cascade in apoptosis. IP6 downregulated cell survival factors such as baculovirus inhibitor-of-apoptosis repeat containing-2 (BIRC-2) protein and telomerase to promote apoptosis. Upregulation of calpain and caspase-9 occurred in course of apoptosis. Increased activities of calpain and caspase-3 cleaved 270 kD alpha-spectrin at specific sites generating 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Increased caspase-3 activity also cleaved inhibitor of caspase-3-activated DNase and poly(ADP-ribose) polymerase. Collectively, our results demonstrated that IP6 down regulated the survival factors BIRC-2 and telomerase and upregulated calpain and caspase-3 activities for apoptosis in T98G cells.

  15. Intracellular pH measured by (31) P-MR-spectroscopy might predict site of progression in recurrent glioblastoma under antiangiogenic therapy.

    PubMed

    Wenger, Katharina J; Hattingen, Elke; Franz, Kea; Steinbach, Joachim P; Bähr, Oliver; Pilatus, Ulrich

    2017-02-06

    In solid tumors, changes in the expression/activity of plasma membrane ion transporters facilitate proton efflux and enable tumor cells to maintain a higher intracellular pH (pHi ), while the microenvironment (pHe ) is commonly more acidic. This supports various tumor-promoting mechanisms. We propose that these changes in pH take place before a magnetic resonance imaging (MRI)-detectable brain tumor recurrence occurs. We enrolled 66 patients with recurrent glioblastoma, treated with bevacizumab. Patients received a baseline and 8-week follow-up MRI including (1) H/(31) P MRSI (spectroscopy) on a 3T clinical scanner, until progressive disease according to Response Assessment in Neuro-Oncology (RANO) criteria occurred. Fourteen patients showed a distant or diffuse tumor recurrence (subsequent tumor) during treatment and were therefore selected for further evaluation. At the site of the subsequent tumor, an area of interest for MRSI voxel selection was retrospectively defined on radiographically unaffected baseline MRI sequences. Before treatment, pHi in the area of interest (subsequent tumor) was significantly higher than pHi of the contralateral normal-appearing tissue (control; P < 0.001). It decreased at the time of best response (P = 0.06), followed by a significant increase at progression (P = 0.03; baseline mean: 7.06, median: 7.068, SD: 0.032; best response mean: 7.044, median: 7.036, SD: 0.025; progression mean: 7.08, median: 7.095, SD 0.035). Until progression, the subsequent tumor was not detectable on standard MRI sequences. The area of existing tumor responded similar, but changes were not significant (decrease P = 0.22; increase P = 0.28). Elevated pHi in radiographically unaffected tissue at baseline might precede MRI-detectable progression in patients with recurrent glioblastoma treated with bevacizumab. 2 J. Magn. Reson. Imaging 2017. © 2017 International Society for Magnetic Resonance in Medicine.

  16. RUNX1 Regulates Migration, Invasion, and Angiogenesis via p38 MAPK Pathway in Human Glioblastoma.

    PubMed

    Sangpairoj, Kant; Vivithanaporn, Pornpun; Apisawetakan, Somjai; Chongthammakun, Sukumal; Sobhon, Prasert; Chaithirayanon, Kulathida

    2016-12-24

    Runt-related transcription factor 1 (RUNX1) is essential for the establishment of fetal and adult hematopoiesis and neuronal development. Aberrant expression of RUNX1 led to proliferation and metastasis of several cancers. The aim of the present study was to investigate the role of RUNX1 in migration, invasion, and angiogenesis of human glioblastoma using IL-1β-treated U-87 MG human glioblastoma cells as a model. IL-1β at 10 ng/ml stimulated translocation of RUNX1 into the nucleus with increased expressions of RUNX1, MMP-1, MMP-2, MMP-9, MMP-19, and VEGFA in U-87 MG cells. In addition, silencing of RUNX1 gene significantly suppressed U-87 MG cell migration and invasion abilities. Moreover, knockdown of RUNX1 mRNA in U-87 MG cells reduced the tube formation of human umbilical vein endothelial cells. Further investigation revealed that IL-1β-induced RUNX1 expression might be mediated via the p38 mitogen-activated protein kinase (MAPK) signaling molecule for the expression of these invasion- and angiogenic-related molecules. Together with an inhibitor of p38 MAPK (SB203580) could decrease RUNX1 mRNA expression. Thus, RUNX1 may be one of the putative molecular targeted therapies against glioma metastasis and angiogenesis through the activation of p38 MAPK signaling pathway.

  17. Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

    PubMed Central

    Hau, Andrew M.; Greenwood, Jeffrey A.; Löhr, Christiane V.; Serrill, Jeffrey D.; Proteau, Philip J.; Ganley, Ian G.; McPhail, Kerry L.; Ishmael, Jane E.

    2013-01-01

    Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death

  18. Puerarin inhibits proliferation and induces apoptosis in human glioblastoma cell lines

    PubMed Central

    Yang, Ji-An; Li, Ji-Qiang; Shao, Ling-Min; Yang, Qian; Liu, Bao-Hui; Wu, Ting-Feng; Wu, Peng; Yi, Wei; Chen, Qian-Xue

    2015-01-01

    Puerarin has been widely used in clinical treatment and experiment research and is considered to exert an anticancer effect recently. The present study investigated the anticancer activity of puerarin in U251 and U87 human glioblastoma cells. The cells were treated with puerarin at various concentrations for different times. Cell viability and cell proliferation were detected by cell counting kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) staining respectively. Cell cycle and apoptosis were measured separately with PI staining and Annexin V-FITC/PI double staining method by flow cytometry. DNA damage of glioblastoma cells caused by puerarin exposure was evaluated by γ-H2AX foci detection, and the expressions of p-AKT, caspase-3 and apoptosis-related proteins were detected by Western blotting after puerarin treatment. Cell viability and proliferation of glioblastoma cells treated with puerarin were significantly lower than that of the control group; the apoptosis rate increased obviously compared to the control group. Puerarin significantly decreased the proportion at G1 phase of cell cycling accompanied by increased populations at the S and G2/M phases in both cell lines. At the same time, DNA damage level of puerarin treated cells was significantly higher than that in the control cells. Moreover, puerarin treatment suppressed the expression of p-Akt and Bcl-2 and promoted the expression of Bax and cleaved caspase-3 in U251 cells. These findings indicate that puerarin exerts antitumor effects both in U251 and U87 cells. PMID:26309712

  19. Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells

    PubMed Central

    Tang, Xiang-Jun; Huang, Kuan-Ming; Gui, Hui; Wang, Jun-Jie; Lu, Jun-Ti; Dai, Long-Jun; Zhang, Li; Wang, Gang

    2016-01-01

    As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR)/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs) and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence supportive of further development of MYR-MC formulation for preferentially targeting mitochondria of glioblastoma cells. PMID:27757032

  20. Expression profile of genes modulated by Aloe emodin in human U87 glioblastoma cells.

    PubMed

    Haris, Khalilah; Ismail, Samhani; Idris, Zamzuri; Abdullah, Jafri Malin; Yusoff, Abdul Aziz Mohamed

    2014-01-01

    Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence, approaches have been intensively investigated to targeti specific molecular pathways involved in glioblastoma development and progression. Aloe emodin is believed to modulate the expression of several genes in cancer cells. We aimed to understand the molecular mechanisms underlying the therapeutic effect of Aloe emodin on gene expression profiles in the human U87 glioblastoma cell line utilizing microarray technology. The gene expression analysis revealed that a total of 8,226 gene alterations out of 28,869 genes were detected after treatment with 58.6 μg/ml for 24 hours. Out of this total, 34 genes demonstrated statistically significant change (p<0.05) ranging from 1.07 to 1.87 fold. The results revealed that 22 genes were up-regulated and 12 genes were down-regulated in response to Aloe emodin treatment. These genes were then grouped into several clusters based on their biological functions, revealing induction of expression of genes involved in apoptosis (programmed cell death) and tissue remodelling in U87 cells (p<0.01). Several genes with significant changes of the expression level e.g. SHARPIN, BCAP31, FIS1, RAC1 and TGM2 from the apoptotic cluster were confirmed by quantitative real-time PCR (qRT-PCR). These results could serve as guidance for further studies in order to discover molecular targets for the cancer therapy based on Aloe emodin treatment.

  1. Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells.

    PubMed

    Tang, Xiang-Jun; Huang, Kuan-Ming; Gui, Hui; Wang, Jun-Jie; Lu, Jun-Ti; Dai, Long-Jun; Zhang, Li; Wang, Gang

    As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR)/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs) and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence supportive of further development of MYR-MC formulation for preferentially targeting mitochondria of glioblastoma cells.

  2. miR-22 inhibits the proliferation, motility, and invasion of human glioblastoma cells by directly targeting SIRT1.

    PubMed

    Chen, Hanchun; Lu, Qiong; Fei, Xifeng; Shen, Likui; Jiang, Dongyi; Dai, Dongwei

    2016-05-01

    Recently, microRNAs (miRNAs), a kind of small and non-coding RNA, can target the downstream molecules. Increasing evidence demonstrates that miRNAs meditate the onset and progression of a variety of tumors. In the present study, we carried out gene transfection, western blot, and reverse transcription PCR (RT-PCR) to explore the role of miR-22 in glioblastoma tissues and cell lines. Here, we verified that the expression of miR-22 was downregulated in glioblastoma tissues and cells rather than matched non-tumor tissues and normal human astrocyte (NHA) cells (p < 0.001). By contrast, SIRT1 messenger RNA (mRNA) and protein were upregulated in glioblastoma tissues and cells (p < 0.001). In vitro miR-22 mimics interfered with cell proliferation, migration, and invasion of U87 and U251 cells. Mechanically, the 3'-untranslated regions (3'-UTRs) of SIRT1 were a direct target of miR-22, leading to the decreased expression of SIRT1 protein in U87 and U251 cells. Meanwhile, miR-22 mimics also inhibited the expression of epidermal growth factor receptor (EGFR) and matrix metallopeptidase 9 (MMP9). In conclusion, miR-22 inhibited cell proliferation, migration, and invasion via targeting the 3'-UTR of SIRT1 in the progression of glioblastoma and miR-22-SIRT1 pathway can be recommended as a potential target for treatment of glioblastoma.

  3. Bortezomib sensitizes human glioblastoma cells to induction of apoptosis by type I interferons through NOXA expression and Mcl-1 cleavage.

    PubMed

    Wang, Ruishan; Davidoff, Andrew M; Pfeffer, Lawrence M

    2016-09-09

    Glioblastomas are highly invasive and aggressive primary brain tumors. Type I interferons have significant, pleiotropic anticancer activity. However, through various pathways many cancers become interferon-resistant, limiting interferon's clinical utility. In this study, we demonstrated that the proteasomal inhibitor bortezomib sensitized human glioblastoma cells to the antiproliferative action of interferons, which involved the induction of caspase-dependent apoptosis but not necroptosis. We found that death ligands such as TRAIL (TNF-related apoptosis-inducing ligand) were not involved in interferon/bortezomib-induced apoptosis, although interferon induced TRAIL expression. However, apoptosis was induced through an intrinsic pathway involving increased NOXA expression and Mcl-1 cleavage. Our findings may provide an important rationale for combining type I interferons with bortezomib for glioblastoma therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors.

    PubMed

    Moser, Joanna J; Fritzler, Marvin J; Rattner, Jerome B

    2014-01-01

    Primary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors. Seven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. We report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium. Primary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously

  5. Tapered microtract array platform for antimigratory drug screening of human glioblastoma multiforme.

    PubMed

    Cha, Junghwa; Koh, Ilkyoo; Choi, Yemuk; Lee, Jungwhoi; Choi, Chulhee; Kim, Pilnam

    2015-02-18

    Understanding the effects of topographic characteristics on tumor cell migration is important for the development of new anti-migratory therapies. However, simplified in vitro culture systems often lead to inaccurate results regarding the efficacy of drugs. Histopathologically, glioblastoma multiform (GBM) cells migrate along the orientation of thin, elongated anatomical structures, such as white-matter tracts. Here, a tapered microtract array platform which mimics the anatomical features of brain tissue is introduced. This platform enables optimization of design for platform fabrication depending on topographic effects. By monitoring the migration of GBM cells on a simple tapered microtract, a saltatory migration resembling the migratory phenotype of human GBM cells in vivo is observed. The platform effectively induces the native characteristics and behavior of cells by topographic cues, allowing to observe the critical point for crawling to saltatory transition. Furthermore, this platform can be applied to efficiently screen anti-cancer drug by inhibiting associated signaling pathways on GBM cells. In conclusion, the microtract array platform reported here may provide a better understanding of the effects of topographic characteristics on cell migration, and may also be useful to determine the efficacy of antimigratory drugs for glioblastoma cells with cellular and molecular research and high-throughput screening. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. PB-100: a potent and selective inhibitor of human BCNU resistant glioblastoma cell multiplication.

    PubMed

    Beljanski, M; Crochet, S; Beljanski, M S

    1993-01-01

    Major drawbacks to present-day cancer chemotherapy are its intrinsic lack of selectivity for tumour cells, resulting in severe damage to normal rapidly dividing cells, and the widespread emergence of drug resistance. Here experimental evidence is presented demonstrating that PB-100, a beta-carboline alkaloid, selectively inhibits in vitro multiplication of human BCNU-resistant glioblastoma cells (U251), but has no effect on normal astrocyte (CRL 1656) multiplication. PB-100 activity is dose-dependent. In the presence of ferritin or CaCl2, which are highly mitogenic for glioblastoma cells, higher doses of the alkaloid are required to inhibit multiplication completely. PB-100 is one of several compounds which were selected for their specific action on cancer DNA and cells, together with lack of activity on normal DNA and cells. Both the selectivity of PB-100 and its ability to overcome drug resistance stem from its effect on cancer DNA secondary structure. This activity is described and discussed, and therapeutic applications are mentioned.

  7. Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

    PubMed

    Wrzeszczynski, Kazimierz O; Frank, Mayu O; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A; Moore Vogel, Julia L; Bruce, Jeffrey N; Lassman, Andrew B; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V; Zody, Michael C; Jobanputra, Vaidehi; Royyuru, Ajay K; Darnell, Robert B

    2017-08-01

    To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. NCT02725684.

  8. 140 Genome-wide CRISPR/cas9 Knockout Screens in Human Glioblastoma Identify Genetic Vulnerabilities.

    PubMed

    Noorani, Imran

    2016-08-01

    Glioblastoma is the most common primary brain tumor with a poor prognosis. Identifying the genetic vulnerabilities of cancer is a novel method for discovering new cancer therapeutic targets. The genetic vulnerabilities of glioblastoma are poorly understood, however. The recent CRISPR/cas9 genetic knockout approach has been proposed as a useful method for high-throughput screening of such genetic liabilities. We established human glioblastoma stem cell lines from 5 patients with primary glioblastoma. The validity of these cell lines was confirmed by demonstrating expression of nestin and SOX2 with RT-PCR, and of CD133 with flow cytometry. Cell lines were subcutaneously injected into mice to track tumor growth in vivo. Lentiviral transfection of a cas9 vector followed by antibiotic selection led to generation of stable cas9-expressing cell lines. A genome-wide library containing 123 411 CRISPR guide RNAs (6 guide RNAs per gene, targeting 19 050 genes) was lentivirally transfected into these cell lines. DNA was extracted from cells and sequenced with Illumina Hi-Seq at day 1 and day 25 after transfection. These patient-derived stem cell lines displayed clonal sphere growth of between 42% and 61%, with a median time survival time of 40 days when transplanted into immunocompromised mice. High-throughput sequencing revealed significant depletion of CRISPR guide RNAs for 1190 genes, with a high degree of overlap between independent replicates for a given cell line. Analysis of gene function using ontology databases demonstrated that DNA repair pathways, RNA and protein synthesis, and regulators of cell proliferation were significantly depleted genes. Moreover, a number of genes specific for neuronal proliferation and differentiation were identified. Comparison with known genetic vulnerabilities of other cancer types revealed a specific profile of genetic liabilities unique to glioblastoma. We have identified a unique genetic vulnerability profile for glioblastoma

  9. Synergistic suppression of noscapine and conventional chemotherapeutics on human glioblastoma cell growth

    PubMed Central

    Qi, Qi; Liu, Xia; Li, Shiyong; Joshi, Harish C; Ye, Keqiang

    2013-01-01

    Aim: Noscapine (NOS) is a non-narcotic opium alkaloid with anti-tumor activity. The aim of this study was to investigate the effects of the combination of NOS with conventional chemotherapeutics temozolamide (TMZ), bis-chloroethylnitrosourea (BCNU), or cisplatin (CIS)on human glioblastoma cells. Methods: U87MG human glioblastoma cells were examined. Cell proliferation was quantified using MTT assay. Western blotting and flow cytometry were used to examine apoptosis and the expression of active caspase-3 and cleaved PARP. Mouse tumor xenograft model bearing U87MG cells was treated with TMZ (2 mg·kg−1·d−1, ip) or CIS (2 mg/kg, ip 3 times a week) alone or in combination with NOS (200 mg·kg−1·d−1, ig) for 3 weeks. Immunohistochemistry was used to investigate the expression of active caspase-3 and Ki67 following treatment in vivo. The safety of the combined treatments was evaluated based on the body weight and histological studies of the animal's organs. Results: NOS (10 or 20 mol/L) markedly increased the anti-proliferation effects of TMZ, BCNU, and CIS on U87MG cells in vitro. The calculated combination index (CI) values of NOS-CIS, NOS-TMZ, and NOS-BCNU (20 μmol/L) were 0.45, 0.51, and 0.57, respectively, demonstrating synergistic inhibition of the drug combinations. In tumor xenograft models, combined treatment with NOS robustly augmented the anti-cancer actions of TMZ and CIS, and showed no detectable toxicity. The combined treatments significantly enhanced the apoptosis, the activated caspase-3 and PARP levels in U87MG cells in vitro, and reduced Ki67 staining and increased the activated caspase-3 level in the shrinking xenografts in vivo. Conclusion: NOS synergistically potentiated the efficacy of FDA-approved anti-cancer drugs against human glioblastoma cells, thereby allowing them to be used at lower doses and hence minimizing their toxic side effects. PMID:23708557

  10. Synergistic suppression of noscapine and conventional chemotherapeutics on human glioblastoma cell growth.

    PubMed

    Qi, Qi; Liu, Xia; Li, Shiyong; Joshi, Harish C; Ye, Keqiang

    2013-07-01

    Noscapine (NOS) is a non-narcotic opium alkaloid with anti-tumor activity. The aim of this study was to investigate the effects of the combination of NOS with conventional chemotherapeutics temozolamide (TMZ), bis-chloroethylnitrosourea (BCNU), or cisplatin (CIS)on human glioblastoma cells. U87MG human glioblastoma cells were examined. Cell proliferation was quantified using MTT assay. Western blotting and flow cytometry were used to examine apoptosis and the expression of active caspase-3 and cleaved PARP. Mouse tumor xenograft model bearing U87MG cells was treated with TMZ (2 mg·kg(-1)·d(-1), ip) or CIS (2 mg/kg, ip 3 times a week) alone or in combination with NOS (200 mg·kg(-1)·d(-1), ig) for 3 weeks. Immunohistochemistry was used to investigate the expression of active caspase-3 and Ki67 following treatment in vivo. The safety of the combined treatments was evaluated based on the body weight and histological studies of the animal's organs. NOS (10 or 20 mol/L) markedly increased the anti-proliferation effects of TMZ, BCNU, and CIS on U87MG cells in vitro. The calculated combination index (CI) values of NOS-CIS, NOS-TMZ, and NOS-BCNU (20 μmol/L) were 0.45, 0.51, and 0.57, respectively, demonstrating synergistic inhibition of the drug combinations. In tumor xenograft models, combined treatment with NOS robustly augmented the anti-cancer actions of TMZ and CIS, and showed no detectable toxicity. The combined treatments significantly enhanced the apoptosis, the activated caspase-3 and PARP levels in U87MG cells in vitro, and reduced Ki67 staining and increased the activated caspase-3 level in the shrinking xenografts in vivo. NOS synergistically potentiated the efficacy of FDA-approved anti-cancer drugs against human glioblastoma cells, thereby allowing them to be used at lower doses and hence minimizing their toxic side effects.

  11. Novel animal glioma models that separately exhibit two different invasive and angiogenic phenotypes of human glioblastomas.

    PubMed

    Inoue, Satoshi; Ichikawa, Tomotsugu; Kurozumi, Kazuhiko; Maruo, Tomoko; Onishi, Manabu; Yoshida, Koichi; Fujii, Kentaro; Kambara, Hirokazu; Chiocca, E Antonio; Date, Isao

    2012-12-01

    Invasive behaviors of malignant gliomas are fundamental traits and major reasons for treatment failure. Delineation of invasive growth is important in establishing treatment for gliomas and experimental neuro-oncology could benefit from an invasive glioma model. In this study, we established two new cell line-based animal models of invasive glioma. Two cell lines, J3T-1 and J3T-2, were derived from the same parental canine glioma cell line, J3T. These cells were inoculated to establish brain tumors in athymic mice and rats. Pathologic samples of these animal gliomas were examined to analyze invasive patterns in relation to angiogenesis, and were compared with human glioblastoma samples. The molecular profiles of these cell lines were also shown. Histologically, J3T-1 and J3T-2 tumors exhibited different invasive patterns. J3T-1 cells clustered around newly developed vessels at tumor borders, whereas J3T-2 cells showed diffuse single cell infiltration into surrounding healthy parenchyma. In human malignant glioma samples, both types of invasion were observed concomitantly. Molecular profiles of these cell lines were analyzed by immunocytochemistry and with quantitative reverse transcription polymerase chain reaction. Vascular endothelial growth factor, matrix metalloproteinase-9, hypoxia-inducible factor-1, and platelet-derived growth factor were overexpressed in J3T-1 cells rather than in J3T-2 cells, whereas integrin αvβ3, matrix metalloproteinase-2, nestin, and secreted protein acidic and rich in cysteine were overexpressed in J3T-2 cells rather than in J3T-1 cells. These animal models histologically recapitulated two invasive and angiogenic phenotypes, namely angiogenesis-dependent and angiogenesis-independent invasion, also observed in human glioblastoma. These cell lines provided a reproducible in vitro and in vivo system to analyze the mechanisms of invasion and angiogenesis in glioma progression. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Human β-Cell Proliferation and Intracellular Signaling: Part 3

    PubMed Central

    Hussain, Mehboob A.; García-Ocaña, Adolfo; Vasavada, Rupangi C.; Bhushan, Anil; Bernal-Mizrachi, Ernesto

    2015-01-01

    This is the third in a series of Perspectives on intracellular signaling pathways coupled to proliferation in pancreatic β-cells. We contrast the large knowledge base in rodent β-cells with the more limited human database. With the increasing incidence of type 1 diabetes and the recognition that type 2 diabetes is also due in part to a deficiency of functioning β-cells, there is great urgency to identify therapeutic approaches to expand human β-cell numbers. Therapeutic approaches might include stem cell differentiation, transdifferentiation, or expansion of cadaver islets or residual endogenous β-cells. In these Perspectives, we focus on β-cell proliferation. Past Perspectives reviewed fundamental cell cycle regulation and its upstream regulation by insulin/IGF signaling via phosphatidylinositol-3 kinase/mammalian target of rapamycin signaling, glucose, glycogen synthase kinase-3 and liver kinase B1, protein kinase Cζ, calcium-calcineurin–nuclear factor of activated T cells, epidermal growth factor/platelet-derived growth factor family members, Wnt/β-catenin, leptin, and estrogen and progesterone. Here, we emphasize Janus kinase/signal transducers and activators of transcription, Ras/Raf/extracellular signal–related kinase, cadherins and integrins, G-protein–coupled receptors, and transforming growth factor β signaling. We hope these three Perspectives will serve to introduce these pathways to new researchers and will encourage additional investigators to focus on understanding how to harness key intracellular signaling pathways for therapeutic human β-cell regeneration for diabetes. PMID:25999530

  13. Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

    PubMed

    Binda, Elena; Visioli, Alberto; Giani, Fabrizio; Trivieri, Nadia; Palumbo, Orazio; Restelli, Silvia; Dezi, Fabio; Mazza, Tommaso; Fusilli, Caterina; Legnani, Federico; Carella, Massimo; Di Meco, Francesco; Duggal, Rohit; Vescovi, Angelo L

    2017-02-15

    Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5a(High)). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5a(Low) mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996-1007. ©2016 AACR. ©2016 American Association for Cancer Research.

  14. Evidence of association of human papillomavirus with prognosis worsening in glioblastoma multiforme

    PubMed Central

    Vidone, Michele; Alessandrini, Federica; Marucci, Gianluca; Farnedi, Anna; de Biase, Dario; Ricceri, Fulvio; Calabrese, Claudia; Kurelac, Ivana; Porcelli, Anna Maria; Cricca, Monica; Gasparre, Giuseppe

    2014-01-01

    Background Glioblastoma multiforme (GBM) is the most malignant brain tumor in adults, but its etiology still remains unknown. Recently, a role of viruses such as cytomegalovirus and JC virus in gliomagenesis has been suggested. Since human papillomavirus (HPV) is considered the most common oncogenic virus in humans, we evaluated its occurrence in GBM samples. Material and Methods Fifty-two formalin-fixed paraffin-embedded primary glioblastoma specimens were retrospectively analyzed. The presence of HPV genome on tumor DNA was assessed by MY/GP nested PCR. Confirmation of HPV detection was obtained by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) with an antibody directed against the L1 capsidic protein. Finally, univariate and multivariate proportional-hazards models were used to compare the risk of death among HPV-positive and HPV-negative patients. Results Strikingly, viral DNA was detected after PCR in 12 cases (23%). HPV16 genome was present in 25% infected samples, whereas the remaining samples tested positive for HPV6. CISH confirmed positivity in all infected samples for which enough material was available. Moreover, IHC positivity suggested that production of viral proteins from HPV genome is an ongoing process in GBM cancer cells. Finally an association between HPV infection and a worse prognosis was found in patients upon age stratification with a univariate analysis (HR, 2.10; 95% CI, 1.00–4.44; log-rank P = .045). Conclusions HPV infection status may be considered an independent prognostic factor in GBM patients and suggests that prevention may be considered, should HPV be recognized as a causative agent in gliomagenesis. PMID:24285549

  15. Glioblastoma-dependent differentiation and angiogenic potential of human mesenchymal stem cells in vitro.

    PubMed

    Birnbaum, Tobias; Hildebrandt, Jenna; Nuebling, Georg; Sostak, Petra; Straube, Andreas

    2011-10-01

    Tumor angiogenesis is of central importance in the malignancy of glioblastoma multiforme (GBM). As previously shown, human mesenchymal stem cells (hMSC) migrate towards GBM and are incorporated into tumor microvessels. However, phenotype and function of recruited hMSC remain unclear. We evaluated the differentiation and angiogenic potential of hMSC after stimulation with glioblastoma-conditioned medium in vitro. Immunostaining with endothelial, smooth muscle cell and pericyte markers was used to analyze hMSC differentiation in different concentrations of tumor-conditioned medium (CM), and the angiogenic potential was evaluated by matrigel-based tube-formation assay (TFA). Immunofluorescence staining revealed that tumor-conditioned hMSC (CM-hMSC) expressed CD 151, VE-cadherin, desmin, α-smooth muscle actin, nestin, and nerval/glial antigen 2 (NG2) in a CM concentration-dependent manner, whereas no expression of von-Willebrand factor (vWF) and smooth myosin could be detected. These findings are indicative of GBM-dependent differentiation of hMSC into pericyte-like cells, rather than endothelial or smooth muscle cells. Furthermore, TFA of hMSC and CM-hMSC revealed CM-dependent formation of capillary-like networks, which differed substantially from those formed by human endothelial cells (HUVEC), also implying pericyte-like tube formation. These results are indicative of GBM-derived differentiation of hMSC into pericyte-like mural cells, which might contribute to the neovascularization and stabilization of tumor vessels.

  16. Development of induced glioblastoma by implantation of a human xenograft in Yucatan minipig as a large animal model.

    PubMed

    Khoshnevis, Mehrdad; Carozzo, Claude; Bonnefont-Rebeix, Catherine; Belluco, Sara; Leveneur, Olivia; Chuzel, Thomas; Pillet-Michelland, Elodie; Dreyfus, Matthieu; Roger, Thierry; Berger, François; Ponce, Frédérique

    2017-04-15

    Glioblastoma is the most common and deadliest primary brain tumor for humans. Despite many efforts toward the improvement of therapeutic methods, prognosis is poor and the disease remains incurable with a median survival of 12-14.5 months after an optimal treatment. To develop novel treatment modalities for this fatal disease, new devices must be tested on an ideal animal model before performing clinical trials in humans. A new model of induced glioblastoma in Yucatan minipigs was developed. Nine immunosuppressed minipigs were implanted with the U87 human glioblastoma cell line in both the left and right hemispheres. Computed tomography (CT) acquisitions were performed once a week to monitor tumor growth. Among the 9 implanted animals, 8 minipigs showed significant macroscopic tumors on CT acquisitions. Histological examination of the brain after euthanasia confirmed the CT imaging findings with the presence of an undifferentiated glioma. Yucatan minipig, given its brain size and anatomy (gyrencephalic structure) which are comparable to humans, provides a reliable brain tumor model for preclinical studies of different therapeutic METHODS: in realistic conditions. Moreover, the short development time, the lower cyclosporine and caring cost and the compatibility with the size of commercialized stereotactic frames make it an affordable and practical animal model, especially in comparison with large breed pigs. This reproducible glioma model could simulate human anatomical conditions in preclinical studies and facilitate the improvement of novel therapeutic devices, designed at the human scale from the outset. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry

    SciTech Connect

    Shi, Zi-xuan; Rao, Wei; Wang, Huan; Wang, Nan-ding; Si, Jing-Wen; Zhao, Jiao; Li, Jun-chang; Wang, Zong-ren

    2015-02-13

    Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future. - Highlights: • Modeled microgravity (MMG) suppressed migration and invasion in U87 cells. • MMG downregulated the SOCE and the expression of Orai1. • SOCE inhibition mimicked the effects of MMG on migration and invasion potentials. • Restoration of SOCE diminished the effects of MMG on migration and invasion.

  18. Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry.

    PubMed

    Shi, Zi-xuan; Rao, Wei; Wang, Huan; Wang, Nan-ding; Si, Jing-wen; Zhao, Jiao; Li, Jun-chang; Wang, Zong-ren

    2015-02-13

    Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future.

  19. Anticancer potential and mechanism of action of mango ginger (Curcuma amada Roxb.) supercritical CO₂ extract in human glioblastoma cells.

    PubMed

    Ramachandran, Cheppail; Lollett, Ivonne V; Escalon, Enrique; Quirin, Karl-Werner; Melnick, Steven J

    2015-04-01

    Mango ginger (Curcuma amada Roxb.) is among the less-investigated species of Curcuma for anticancer properties. We have investigated the anticancer potential and the mechanism of action of a supercritical CO2 extract of mango ginger (CA) in the U-87MG human glioblastoma cell line. CA demonstrated higher cytotoxicity than temozolomide, etoposide, curcumin, and turmeric force with IC50, IC75, and IC90 values of 4.92 μg/mL, 12.87 μg/mL, and 21.30 μg/mL, respectively. Inhibitory concentration values of CA for normal embryonic mouse hypothalamus cell line (mHypoE-N1) is significantly higher than glioblastoma cell line, indicating the specificity of CA against brain tumor cells. CompuSyn analysis indicates that CA acts synergistically with temozolomide and etoposide for the cytotoxicity with combination index values of <1. CA treatment also induces apoptosis in glioblastoma cells in a dose-dependent manner and downregulates genes associated with apoptosis, cell proliferation, telomerase activity, oncogenesis, and drug resistance in glioblastoma cells.

  20. Effect of chlorpromazine on human and murine intracellular carboxylesterases.

    PubMed

    Radenovic, L; Kartelija, G

    2004-04-01

    Clinical use of chlorpromazine (CPZ), an antipsychotic drug, is limited due to its hepatotoxicity. CPZ is found to inhibit in vitro intracellular carboxylesterases (CE), such as alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase, and alpha-naphthyl butyrate esterase in polymorphonuclear neutrophils, hepatocytes, and neuronal brain cells from mice. CPZ inhibits CE of all these cell types, whereby the degree of the inhibition depends on the incubation time and CPZ concentration. The polymorphonuclear neutrophils are most sensitive to CPZ. Comparable results were obtained with polymorphonuclear neutrophils from mice and humans. Since leukocytes are much more available than hepatocytes or neuronal cells in humans, we assume that CE in peripheral blood leukocytes (neutrophils and monocytes) can be used as markers for indication of pending liver damage by CPZ.

  1. Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells

    PubMed Central

    Yang, C-H; Kao, Y-H; Huang, K-S; Wang, C-Y; Lin, L-W

    2012-01-01

    This study is the first report that investigated the apoptosis-inducing effects of Cordyceps militaris (CM) and its mycelial fermentation in human glioblastoma cells. Both fractions arrested the GBM8401 cells in the G0/G1 phase, whereas the U-87MG cells were arrested at the G2/M transitional stage. Western blot data suggested that upregulation of p53 and p21 might be involved in the disruption of cell cycle progression. Induction of chromosomal condensation and the appearance of a sub-G1 hypodipoid population further supported the proapoptogenicity, possibly through the activation of caspase-3 and caspase-8, and the downregulation of antiapoptotic Bcl-2 and the upregulation of proapoptotic Bax protein expression. Downregulation of mammalian target of rapamycin and upregulation of Atg5 and LC3 II levels in GBM8401 cells implicated the involvement of autophagy. The signaling profiles with mycelial fermentation treatment indicated that mycelial fermentation triggered rapid phosphorylation of Akt, p38 MAPK, and JNK, but suppressed constitutively high levels of ERK1/2 in GBM8401 cells. Mycelial fermentation treatment only significantly increased p38 MAPK phosphorylation, but decreased constitutively high levels of Akt, ERK1/2, and JNK phosphorylation in U-87MG cells. Pretreatment with PI3K inhibitor wortmannin and MEK1 inhibitor PD98059 prevented the mycelial fermentation-induced cytotoxicity in GBM8401 and U-87MG cells, suggesting the involvement of PI3K/Akt and MEK1 pathways in mycelial fermentation-driven glioblastoma cell apoptosis and autophagy. PMID:23190603

  2. Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma

    PubMed Central

    González-Gómez, P.; de la Fuente, M.; Hernández-Laín, Aurelio; Mira, H.; Sánchez-Gómez, P.; Garcia-Fuentes, M.

    2015-01-01

    Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133+, Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy. PMID:25860932

  3. Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma.

    PubMed

    González-Gómez, Pilar; Crecente-Campo, Jose; Zahonero, Cristina; de la Fuente, Maria; Hernández-Laín, Aurelio; Mira, Helena; Sánchez-Gómez, Pilar; Garcia-Fuentes, Marcos

    2015-05-10

    Glioblastoma tumor initiating cells are believed to be the main drivers behind tumor recurrence, and therefore therapies that specifically manage this population are of great medical interest. In a previous work, we synthesized controlled release microspheres optimized for intracranial delivery of BMP7, and showed that these devices are able to stop the in vitro growth of a glioma cell line. Towards the translational development of this technology, we now explore these microspheres in further detail and characterize the mechanism of action and the in vivo therapeutic potential using tumor models relevant for the clinical setting: human primary glioblastoma cell lines. Our results show that BMP7 can stop the proliferation and block the self-renewal capacity of those primary cell lines that express the receptor BMPR1B. BMP7 was encapsulated in poly (lactic-co-glycolic acid) microspheres in the form of a complex with heparin and Tetronic, and the formulation provided effective release for several weeks, a process controlled by carrier degradation. Data from xenografts confirmed reduced and delayed tumor formation for animals treated with BMP7-loaded microspheres. This effect was coincident with the activation of the canonical BMP signaling pathway. Importantly, tumors treated with BMP7-loaded microspheres also showed downregulation of several markers that may be related to a malignant stem cell-like phenotype: CD133(+), Olig2, and GFAPδ. We also observed that tumors treated with BMP7-loaded microspheres showed enhanced expression of cell cycle inhibitors and reduced expression of the proliferation marker PCNA. In summary, BMP7-loaded controlled release microspheres are able to inhibit GBM growth and reduce malignancy markers. We envisage that this kind of selective therapy for tumor initiating cells could have a synergistic effect in combination with conventional cytoreductive therapy (chemo-, radiotherapy) or with immunotherapy.

  4. Influenza vaccine induces intracellular immune memory of human NK cells.

    PubMed

    Dou, Yaling; Fu, Binqing; Sun, Rui; Li, Wenting; Hu, Wanfu; Tian, Zhigang; Wei, Haiming

    2015-01-01

    Influenza vaccines elicit antigen-specific antibodies and immune memory to protect humans from infection with drift variants. However, what supports or limits vaccine efficacy and duration is unclear. Here, we vaccinated healthy volunteers with annual vaccine formulations and investigated the dynamics of T cell, natural killer (NK) cell and antibody responses upon restimulation with heterologous or homologous influenza virus strains. Influenza vaccines induced potential memory NK cells with increased antigen-specific recall IFN-γ responses during the first 6 months. In the absence of significant changes in other NK cell markers (CD45RO, NKp44, CXCR6, CD57, NKG2C, CCR7, CD62L and CD27), influenza vaccines induced memory NK cells with the distinct feature of intracellular NKp46 expression. Indeed, surface NKp46 was internalized, and the dynamic increase in NKp46(intracellular)+CD56dim NK cells positively correlated with increased IFN-γ production to influenza virus restimulation after vaccination. In addition, anti-NKp46 antibodies blocked IFN-γ responses. These findings provide insights into a novel mechanism underlying vaccine-induced immunity and NK-related diseases, which may help to design persisting and universal vaccines in the future.

  5. New role of osteopontin in DNA repair and impact on human glioblastoma radiosensitivity

    PubMed Central

    Henry, Aurélie; Nokin, Marie-Julie; Leroi, Natacha; Lallemand, François; Lambert, Jérémy; Goffart, Nicolas; Roncarati, Patrick; Bianchi, Elettra; Peixoto, Paul; Blomme, Arnaud; Turtoi, Andrei; Peulen, Olivier; Habraken, Yvette; Scholtes, Félix; Martinive, Philippe; Delvenne, Philippe; Rogister, Bernard; Castronovo, Vincent; Bellahcène, Akeila

    2016-01-01

    Glioblastoma (GBM) represents the most aggressive and common solid human brain tumor. We have recently demonstrated the importance of osteopontin (OPN) in the acquisition/maintenance of stemness characters and tumorigenicity of glioma initiating cells. Consultation of publicly available TCGA database indicated that high OPN expression correlated with poor survival in GBM patients. In this study, we explored the role of OPN in GBM radioresistance using an OPN-depletion strategy in U87-MG, U87-MG vIII and U251-MG human GBM cell lines. Clonogenic experiments showed that OPN-depleted GBM cells were sensitized to irradiation. In comet assays, these cells displayed higher amounts of unrepaired DNA fragments post-irradiation when compared to control. We next evaluated the phosphorylation of key markers of DNA double-strand break repair pathway. Activating phosphorylation of H2AX, ATM and 53BP1 was significantly decreased in OPN-deficient cells. The addition of recombinant OPN prior to irradiation rescued phospho-H2AX foci formation thus establishing a new link between DNA repair and OPN expression in GBM cells. Finally, OPN knockdown improved mice survival and induced a significant reduction of heterotopic human GBM xenograft when combined with radiotherapy. This study reveals a new function of OPN in DNA damage repair process post-irradiation thus further confirming its major role in GBM aggressive disease. PMID:27563812

  6. Detection of Intracellular Bacterial Communities in Human Urinary Tract Infection

    PubMed Central

    Rosen, David A; Hooton, Thomas M; Stamm, Walter E; Humphrey, Peter A; Hultgren, Scott J

    2007-01-01

    Background Urinary tract infections (UTIs) are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC). While UTIs are typically considered extracellular infections, it has been recently demonstrated that UPEC bind to, invade, and replicate within the murine bladder urothelium to form intracellular bacterial communities (IBCs). These IBCs dissociate and bacteria flux out of bladder facet cells, some with filamentous morphology, and ultimately establish quiescent intracellular reservoirs that can seed recurrent infection. This IBC pathogenic cycle has not yet been investigated in humans. In this study we sought to determine whether evidence of an IBC pathway could be found in urine specimens from women with acute UTI. Methods and Findings We collected midstream, clean-catch urine specimens from 80 young healthy women with acute uncomplicated cystitis and 20 asymptomatic women with a history of UTI. Investigators were blinded to culture results and clinical history. Samples were analyzed by light microscopy, immunofluorescence, and electron microscopy for evidence of exfoliated IBCs and filamentous bacteria. Evidence of IBCs was found in 14 of 80 (18%) urines from women with UTI. Filamentous bacteria were found in 33 of 80 (41%) urines from women with UTI. None of the 20 urines from the asymptomatic comparative group showed evidence of IBCs or filaments. Filamentous bacteria were present in all 14 of the urines with IBCs compared to 19 (29%) of 66 samples with no evidence of IBCs (p < 0.001). Of 65 urines from patients with E. coli infections, 14 (22%) had evidence of IBCs and 29 (45%) had filamentous bacteria, while none of the gram-positive infections had IBCs or filamentous bacteria. Conclusions The presence of exfoliated IBCs and filamentous bacteria in the urines of women with acute cystitis suggests that the IBC pathogenic pathway characterized in the murine model may occur in humans. The findings

  7. Evaluation of the potential of mobile phone specific electromagnetic fields (UMTS) to produce micronuclei in human glioblastoma cell lines.

    PubMed

    Al-Serori, Halh; Kundi, Michael; Ferk, Franziska; Mišík, Miroslav; Nersesyan, Armen; Murbach, Manuel; Lah, Tamara T; Knasmüller, Siegfried

    2017-04-01

    Some epidemiological studies indicate that mobile phones cause glioblastomas in humans. Since it is known that genomic instability plays a key role in the etiology of cancer, we investigated the effects of the universal mobile telecommunications system radiofrequency (UMTS-RF) signal, which is used in "smart" phones, on micronucleus (MN) formation and other anomalies such as nuclear buds (NBUDs) and nucleoplasmatic bridges (NPBs). MN are formed by structural and numerical aberrations, NBs reflect gene amplification and NPBs are formed from dicentric chromosomes. The experiments were conducted with human glioblastoma cell lines, which differ in regard to their p53 status, namely U87 (wild-type) and U251 (mutated). The cells were cultivated for 16h in presence and absence of fetal calf serum and exposed to different SAR doses (0.25, 0.50 and 1.00W/kg), which reflect the exposure of humans, in presence and absence of mitomycin C as former studies indicate that RF may cause synergistic effects in combination with this drug. We found no evidence for induction of MN and other anomalies. However, with the highest dose, induction of apoptosis was observed in U251 cells on the basis of the morphological features of the cells. Our findings indicate that the UMTS-RF signal does not cause chromosomal damage in glioblastoma cells; the mechanisms which lead to induction of programmed cell death will be investigated in further studies.

  8. Dehydroleucodine Induces a TP73-dependent Transcriptional Regulation of Multiple Cell Death Target Genes in Human Glioblastoma Cells.

    PubMed

    Ratovitski, Edward A

    2017-01-01

    Dehydroleucodine, a natural sesquiterpene lactone from Artemisia douglassiana Besser (Argentine) and Gynoxys verrucosa (Ecuador). To define the molecular mechanisms underlying the effect of dehydroleucodine on the human glioblastoma cells. Various techniques (cDNA expression array, real-time quantitative PCR, chromatin immunprecipitation, luciferase reporter assay, use of phosphospecific antibodies, immunoprecipitation, immunoblotting, apoptosis and autophagy assays) were employed to define and validate multiple molecular gene targets affected in human glioblastoma cells upon dehydroleucodine exposure. Dehydroleucodine exposure upregulated the total and phosphorylated (p-Y99) levels of TP73 in U87- MG glioblastoma cells. We found that TP73 silencing led to a partial rescue of U87-MG cells from the cell death induced by dehydroleucodine. Upon the dehydroleucodine exposure numerous gene targets were upregulated and downregulated through a TP73-dependent transcriptional mechanism. Some of these gene targets are known to be involved in cell cycle arrest, apoptosis, autophagy and necroptosis. Dehydroleucodine induced the TP73 binding to the specific genes promoters (CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, and APG5L). Moreover, the exposure of U87-MG cells to dehydroleucodine upregulated the protein levels of CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, APG5L, and downregulated the CASP8 level. The formation of RIPK1 protein complexes and phosphorylation of MLKL were induced by dehydroleucodine supporting the notion of multiple cell death mechanisms implicated in the tumor cell response to dehydroleucodine. This multifaceted study led to a conclusion that dehydroleucodine induces the phosphorylation of tumor protein TP73 and in turn activates numerous TP73-target genes regulating apoptosis, autophagy and necroptosis in human glioblastoma cells.. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Modulation of intracellular pH in human ovarian cancer.

    PubMed

    Sanhueza, C; Araos, J; Naranjo, L; Villalobos, R; Westermeier, F; Salomon, C; Beltrán, A R; Ramírez, M A; Gutiérrez, J; Pardo, F; Leiva, A; Sobrevia, L

    2016-01-01

    To sustain tumor growth, the cancer cells need to adapt to low levels of oxygen (i.e., hypoxia) in the tumor tissue and to the tumor-associated acidic microenvironment. In this phenomenon, the activation of the sodium/proton exchanger 1 (NHE1) at the plasma membrane and the hypoxia-inducible factor (HIF) are critical for the control of the intracellular pH (pHi) and for hypoxia adaptation, respectively. Interestingly, both of these mechanisms end in sustaining cancer cell proliferation. However, regulatory mechanisms of pHi in human ovary tissue and in malignant ascites are unknown. Additionally, a potential role of NHE1 in the modulation of H(+) efflux in human ovarian cancer cells is unknown. In this review, we discussed the characteristics of tumor microenvironment of primary human ovarian tumors and tumor ascites, in terms of pHi regulatory mechanisms and oxygen level. The findings described in the literature suggest that NHE1 may likely play a role in pHi regulation and cell proliferation in human ovarian cancer, potentially involving HIF2α activation. Since ovarian cancer is the fifth cause of prevalence of women cancer in Chile and is usually of late diagnosis, i.e., when the disease jeopardizes peritoneal cavity and other organs, resulting in reduced patient survival, new efforts are required to improve patient-life span and for a better understanding of the pathophysiology of the disease. The potential advantage of the use of amiloride and amiloride-derivatives for cancer treatment in terms of NHE1 expression and activity is also discussed as a therapeutic approach in human ovarian cancer.

  10. Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization.

    PubMed

    Hayashi, Keiichiro; Michiue, Hiroyuki; Yamada, Hiroshi; Takata, Katsuyoshi; Nakayama, Hiroki; Wei, Fan-Yan; Fujimura, Atsushi; Tazawa, Hiroshi; Asai, Akira; Ogo, Naohisa; Miyachi, Hiroyuki; Nishiki, Tei-ichi; Tomizawa, Kazuhito; Takei, Kohji; Matsui, Hideki

    2016-03-18

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood-brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

  11. Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma

    PubMed Central

    Wrzeszczynski, Kazimierz O.; Frank, Mayu O.; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A.; Moore Vogel, Julia L.; Bruce, Jeffrey N.; Lassman, Andrew B.; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V.; Zody, Michael C.; Jobanputra, Vaidehi; Royyuru, Ajay K.

    2017-01-01

    Objective: To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Methods: Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. Results: More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. Conclusions: The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. ClinicalTrials.gov identifier: NCT02725684. PMID:28740869

  12. Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

    PubMed

    Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2016-06-21

    Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

  13. Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

    PubMed Central

    Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2016-01-01

    Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

  14. Copper induces cellular senescence in human glioblastoma multiforme cells through downregulation of Bmi-1.

    PubMed

    Li, Yuan; Hu, Jifan; Guan, Fangxia; Song, Laijun; Fan, Ruitai; Zhu, Huaijie; Hu, Xiang; Shen, Eileen; Yang, Bo

    2013-05-01

    Most human tumor cells, including glioblastoma multiforme (GBM) cells, have aberrant control of cell aging and apoptosis. Subcytotoxic concentrations of oxidative or stress‑causing agents, such as hydrogen peroxide, may induce human cell senescence. Thus, induction of tumor cells into premature senescence may provide a useful in vitro model for developing novel therapeutic strategy to combat tumors. In the present study, we assessed the molecular mechanism(s) underlying senescence in GBM cells induced by copper sulfate. Following pretreatment with subcytotoxic concentrations of copper sulfate, U87-MG tumor cells showed typical aging characteristics, including reduced cell proliferation, cell enlargement, increased level of senescence-associated β-galactosidase (SA β-gal) activity, and overexpression of several senescence-associated genes, p16, p21, transforming growth factor β-1 (TGF-β1), insulin growth factor binding protein 3 (IGFBP3) and apolipoprotein J (ApoJ). We further demonstrated that the Bmi-1 pathway was downregulated in GBM cells in parallel with the induced senescence. The present study for the first time demonstrates the ability of copper to induce GBM cell senescence by downregulating Bmi-1.

  15. Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells

    PubMed Central

    Li, Hui-Fang; Kim, Jung-Sik; Waldman, Todd

    2009-01-01

    Background Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance. Methods Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays. Results Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells. Conclusion These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients. PMID:19828040

  16. Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

    PubMed Central

    Wiedemeyer, Ruprecht; Brennan, Cameron; Heffernan, Timothy P.; Xiao, Yonghong; Mahoney, John; Protopopov, Alexei; Zheng, Hongwu; Bignell, Graham; Furnari, Frank; Cavenee, Webster K.; Hahn, William C.; Ichimura, Koichi; Collins, V. Peter; Chu, Gerald C.; Stratton, Michael R.; Ligon, Keith L.; Futreal, P. Andrew; Chin, Lynda

    2008-01-01

    Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation. PMID:18394558

  17. Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells.

    PubMed

    Liang, Wei Zhe; Lu, Cheng Hsien

    2012-05-15

    This study examined whether the essential oil component carvacrol altered cytosolic free Ca(2+) level ([Ca(2+)](i)) and viability in human glioblastoma cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Carvacrol at concentrations of 400-1000 μM induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Carvacrol-induced Ca(2+) signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca(2+)](i) rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca(2+)](i) rise. At concentrations of 200-800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N--tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS. In human glioblastoma cells, carvacrol induced a [Ca(2+)](i) rise by inducing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive, non store-operated Ca(2+) channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  18. In vitro expansion of human glioblastoma cells at non-physiological oxygen tension irreversibly alters subsequent in vivo aggressiveness and AC133 expression

    PubMed Central

    BOURSEAU-GUILMAIN, ERIKA; LEMAIRE, LAURENT; GRIVEAU, AUDREY; HERVOUET, ERIC; VALLETTE, FRANÇOIS; BERGER, FRANÇOIS; MENEI, PHILIPPE; BENOIT, JEAN-PIERRE; WION, DIDIER; GARCION, EMMANUEL

    2012-01-01

    Among markers of glioblastoma initiating cells, AC133 has been shown to be associated with glioblastoma resistance and malignancy. Recently, it was demonstrated that increasing oxygen tension (pO2) down-regulated AC133 expression in glioblastoma cells in vitro. In order to better understand extrinsic factor regulation of AC133, this work aimed to investigate the relationship between cell culture pO2, AC133 expression, and tumor development and phenotype. Using treatments with CoCl2 and HIF-1α shRNA knockdowns on non-sorted human primary glioblastoma cells cultured at low (3%) versus high (21%) oxygen tension, we established a responsibility for low pO2 in the maintenance of high levels of AC133 expression, with a major but non-exclusive role for HIF-1α. We also demonstrated that human glioblastoma cells previously cultured under high oxygen tension can lose part of their aggressiveness when orthotopically engrafted in SCID mice or lead to tumors with distinct phenotypes and no re-expression of AC133. These observations showed that the specific pO2 microenvironment irreversibly impacts glioblastoma cell phenotypes, highlighting the pertinence of culture conditions when extrapolating data from xenogenic models to human cells in their source environment. They also raised AC133 as a marker of non-exposure to oxygenated areas rather than a marker of aggressiveness or low pO2 niches. PMID:22134773

  19. Oligomer procyanidins from grape seeds induce a paraptosis-like programmed cell death in human glioblastoma U-87 cells.

    PubMed

    Zhang, Feng-Jiao; Yang, Jing-Yu; Mou, Yan-Hua; Sun, Bao-Shan; Wang, Ji-Ming; Wu, Chun-Fu

    2010-08-01

    We recently reported that F2, an oligomer procyanidin fraction isolated from grape seeds, triggered an original form of cell death in U-87 human glioblastoma cells with a phenotype resembling morphological characteristics of paraptosis. However, the specific death mode induced by F2 and the mechanism of its action have not been assessed so far. In the present work, we therefore further investigated the death mode of human glioblastoma cells induced by F2 and gained insight into the nature of the signaling pathways activated by F2 in glioblastoma cells. Cell viability assay using MTT, (AO/EB) double staining, Western blot analysis, and Ca2+ assay using fura-2. Morphology studies revealed extensive cytoplasmic vacuolization in dying cells and no apoptotic body formation, membrane bleb formation, or nuclear fragmentation, though some was accompanied by MAPK activation and new protein synthesis, and was independent of caspase activation. Moreover, we demonstrated the involvement of calcium mobilization in F2-induced U-87 cell signaling. Altogether we showed that F2 induced a kind of cell death resembling paraptosis in U-87 cells. The current report complements previous studies on the characterization of F2-induced U-87 cell death, enhances our understanding of the action mechanism of F2 on glioma, and helps in the development of novel antitumor therapeutics.

  20. (68)Ga-labeled 3PRGD2 for dual PET and Cerenkov luminescence imaging of orthotopic human glioblastoma.

    PubMed

    Fan, Di; Zhang, Xin; Zhong, Lijun; Liu, Xujie; Sun, Yi; Zhao, Huiyun; Jia, Bing; Liu, Zhaofei; Zhu, Zhaohui; Shi, Jiyun; Wang, Fan

    2015-06-17

    β-Emitters can produce Cerenkov radiation that is detectable by Cerenkov luminescence imaging (CLI), allowing the combination of PET and CLI with one radiotracer for both tumor diagnosis and visual guidance during surgery. Recently, the clinical feasibility of CLI with the established therapeutic reagent Na(131)I and the PET tracer (18)F-FDG was demonstrated. (68)Ga possesses a higher Cerenkov light output than (18)F and (131)I, which would result in higher sensitivity for CLI and improve the outcome of CLI in clinical applications. However, the research on (68)Ga-based tumor-specific tracers for CLI is limited. In this study, we examined the use of (68)Ga-radiolabeled DOTA-3PRGD2 ((68)Ga-3PRGD2) for dual PET and CLI of orthotopic U87MG human glioblastoma. For this purpose, the Cerenkov efficiencies of (68)Ga and (18)F were measured with the IVIS Spectrum system (PerkinElmer, USA). The CLI signal intensity of (68)Ga was 15 times stronger than that of (18)F. PET and CLI of (68)Ga-3PRGD2 were performed in U87MG human glioblastoma xenografts. Both PET and CLI revealed a remarkable accumulation of (68)Ga-3PRGD2 in the U87MG human glioblastoma xenografts at 1 h p.i. with an extremely low background in the brain when compared with (18)F-FDG. Furthermore, (68)Ga-3PRGD2 was used for dual PET and CLI of orthotopic human glioblastoma. The orthotopic human glioblastoma was clearly visualized by both imaging modalities. In addition, the biodistribution of (68)Ga-3PRGD2 was assessed in normal mice to estimate the radiation dosimetry. The whole-body effective dose is 20.1 ± 3.3 μSv/MBq, which is equal to 3.7 mSv per whole-body PET scan with a 5 mCi injection dose. Thus, (68)Ga-3PRGD2 involves less radiation exposure in patients when compared with (18)F-FDG (7.0 mSv). The use of (68)Ga-3PRGD2 in dual PET and CLI shows great promise for tumor diagnosis and image-guided surgery.

  1. Inhibition of AKT signaling by supercritical CO2 extract of mango ginger (Curcuma amada Roxb.) in human glioblastoma cells.

    PubMed

    Ramachandran, Cheppail; Portalatin, Gilda; Quirin, Karl-W; Escalon, Enrique; Khatib, Ziad; Melnick, Steven J

    2015-12-01

    Mango ginger (Curcuma amada Roxb.) is a less-investigated herb for anticancer properties than other related Curcuma species. AKT (a serine/threonine protein kinase B, originally identified as an oncogene in the transforming retrovirus AKT8) plays a central role in the development and promotion of cancer. In this investigation, we have analyzed the effect of supercritical CO2 extract of mango ginger (CA) on the genetic pathways associated with AKT signaling in human glioblastoma cells. The inhibitory effect of supercritical CO2 extract of mango ginger (Curcuma amada) on AKT signaling was investigated in U-87MG glioblastoma cells. CA was highly cytotoxic to glioblastoma cell line (IC50=4.92±0.81 µg/mL) compared to mHypoE-N1 normal mouse hypothalamus cell line (IC50=40.57±0.06 µg/mL). CA inhibits AKT (protein Kinase B) and adenosine monophophate -activated protein kinase α (AMPKα) phosphorylation significantly in a dose-dependent manner. The cell migration which is necessary for invasion and metastasis was also inhibited by CA treatment, with about 43% reduction at 20 µg/mL concentration. Analysis of mRNA and protein expression of genes associated with apoptosis, cell proliferation and angiogenesis showed that CA modulates expression of genes associated with apoptosis (Bax, Bcl-2, Bcl-X, BNIP3, caspase-3, mutant p53 and p21), cell proliferation (Ki67) and angiogenesis vascular endothelial growth factor (VEGF). Additionally, heat shock protein 90 (HSP90) and AMPKα genes interacting with the AKT signaling pathway were also downregulated by CA treatment. These results indicate the molecular targets and mechanisms underlying the anticancer effect of CA in human glioblastoma cells.

  2. Intracellular and circulating neuronal antinuclear antibodies in human epilepsy

    PubMed Central

    Iffland, Philip H.; Carvalho-Tavares, Juliana; Trigunaite, Abhishek; Man, Shumei; Rasmussen, Peter; Alexopoulos, Andreas; Ghosh, Chaitali; Jørgensen, Trine N.; Janigro, Damir

    2013-01-01

    There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n= 4/4). In brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated albumin. All

  3. Intracellular and circulating neuronal antinuclear antibodies in human epilepsy.

    PubMed

    Iffland, Philip H; Carvalho-Tavares, Juliana; Trigunaite, Abhishek; Man, Shumei; Rasmussen, Peter; Alexopoulos, Andreas; Ghosh, Chaitali; Jørgensen, Trine N; Janigro, Damir

    2013-11-01

    There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, the human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from the brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in the serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n=4/4). In the brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated

  4. REST Controls Self-Renewal and Tumorigenic Competence of Human Glioblastoma Cells

    PubMed Central

    Conti, Luciano; Crisafulli, Laura; Brilli, Elisa; Conforti, Paola; Zunino, Franco; Magrassi, Lorenzo; Schiffer, Davide; Cattaneo, Elena

    2012-01-01

    The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets. PMID:22701651

  5. Bax Activation Blocks Self-Renewal and Induces Apoptosis of Human Glioblastoma Stem Cells.

    PubMed

    Daniele, Simona; Pietrobono, Deborah; Costa, Barbara; Giustiniano, Mariateresa; La Pietra, Valeria; Giacomelli, Chiara; La Regina, Giuseppe; Silvestri, Romano; Taliani, Sabrina; Trincavelli, Maria Letizia; Da Settimo, Federico; Novellino, Ettore; Martini, Claudia; Marinelli, Luciana

    2017-04-11

    Glioblastoma (GBM) is characterized by a poor response to conventional chemotherapeutic agents, attributed to the insurgence of drug resistance mechanisms and to the presence of a subpopulation of glioma stem cells (GSCs). GBM cells and GSCs present, among others, an overexpression of antiapoptotic proteins and an inhibition of pro-apoptotic ones, which help to escape apoptosis. Among pro-apoptotic inducers, the Bcl-2 family protein Bax has recently emerged as a promising new target in cancer therapy along with first BAX activators (BAM7, Compound 106, and SMBA1). Herein, a derivative of BAM-7, named BTC-8, was employed to explore the effects of Bax activation in different human GBM cells and in their stem cell subpopulation. BTC-8 inhibited GBM cell proliferation, arrested the cell cycle, and induced apoptosis through the induction of mitochondrial membrane permeabilization. Most importantly, BTC-8 blocked proliferation and self-renewal of GSCs and induced their apoptosis. Notably, BTC-8 was demonstrated to sensitize both GBM cells and GSCs to the alkylating agent Temozolomide. Overall, our findings shed light on the effects and the relative molecular mechanisms related to Bax activation in GBM, and they suggest Bax-targeting compounds as promising therapeutic tools against the GSC reservoir.

  6. Oxymatrine Inhibits Proliferation and Migration While Inducing Apoptosis in Human Glioblastoma Cells

    PubMed Central

    Wang, Baocheng; Wang, Jiajia; Li, Qifeng; Meng, Wei

    2016-01-01

    Oxymatrine (OMT), an alkaloid derived from the traditional Chinese medicine herb Sophora flavescens Aiton, has been shown to exhibit anticancer properties on various types of cancer cells. In this study, we investigate the anticancer properties of OMT on human glioblastoma (GBM) cells and evaluate their underlying mechanisms. MTT assays were performed and demonstrated that OMT significantly inhibits the proliferation of GBM cells. Flow cytometry suggested that OMT at a concentration of 10−5 M may induce apoptosis in U251 and A172 cells. Western blot analyses demonstrated a significant increase in the expression of Bax and caspase-3 and a significant decrease in expression of Bcl-2 in both U251 and A172 cells. Additionally, OMT was found by transwell and high-content screening assays to decrease the migratory ability of the evaluated GBM cells. These findings suggest that the antitumor effects of OMT may be the result of inhibition of cell proliferation and migration and the induction of apoptosis by regulating the expression of apoptosis-associated proteins. OMT may represent a novel anticancer therapy for the treatment of GBM. PMID:27957488

  7. Detection of complex genetic alterations in human glioblastoma multiforme using comparative genomic hybridization

    SciTech Connect

    Schlegel, J.; Stumm, G.; Scherthan, H.; Arens, N.

    1996-01-01

    The aim of the present study was to detect complex genetic alterations in human glioblastoma multiforme (GBM) by comparative genomic in situ hybridization (CGH). Of the 24 GBM that were examined, increased fluorescence intensities indicating chromosomal polysomy of chromosome 7 and gene amplification at chromosome 7p were found in 42% of the tumors. In addition, signal enhancement of chromosome 19 was present in 29% and at 12q13-15 in 21% of the tumors. We also detected reduction of fluorescence intensities indicating gross deletions on chromosomes 10 (58%), 9p (46%), and 13 (29%). There was a close correlation of CGH results when compared with Southern analysis of the EGFR gene localized on chromosome 7 and loss of heterozygosity detection of chromosome 9 and 10 by microsatellite PCR. A close correlation was also observed between copy number changes of chromosome 7 and deletions of chromosome 10. Amplification of chromosome 12q and deletions of chromosomes 9p and 13 seemed to be complementary in the tumors investigated in the present study. 44 refs., 3 figs., 1 tab.

  8. REST controls self-renewal and tumorigenic competence of human glioblastoma cells.

    PubMed

    Conti, Luciano; Crisafulli, Laura; Caldera, Valentina; Tortoreto, Monica; Brilli, Elisa; Conforti, Paola; Zunino, Franco; Magrassi, Lorenzo; Schiffer, Davide; Cattaneo, Elena

    2012-01-01

    The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

  9. Vitamin D receptor expression is associated with improved overall survival in human glioblastoma multiforme.

    PubMed

    Salomón, Débora G; Fermento, María E; Gandini, Norberto A; Ferronato, María J; Arévalo, Julián; Blasco, Jorge; Andrés, Nancy C; Zenklusen, Jean C; Curino, Alejandro C; Facchinetti, María M

    2014-05-01

    Vitamin D and its analogs have been shown to display anti-proliferative effects in a wide variety of cancer types including glioblastoma multiforme (GBM). These anticancer effects are mediated by its active metabolite, 1α, 25-dihydroxyvitamin D3 (calcitriol) acting mainly through vitamin D receptor (VDR) signaling. In addition to its involvement in calcitriol action, VDR has also been demonstrated to be useful as a prognostic factor for some types of cancer. However, to our knowledge, there are no studies evaluating the expression of VDR protein and its association with outcome in gliomas. Therefore, we investigated VDR expression by using immunohistochemical analysis in human glioma tissue microarrays, and analyzed the association between VDR expression and clinico-pathological parameters. We further investigated the effects of genetic and pharmacologic modulation of VDR on survival and migration of glioma cell lines. Our data demonstrate that VDR is increased in tumor tissues when compared with VDR in non-malignant brains, and that VDR expression is associated with an improved outcome in patients with GBM. We also show that both genetic and pharmacologic modulation of VDR modulates GBM cellular migration and survival and that VDR is necessary for calcitriol-mediated effects on migration. Altogether these results provide some limited evidence supporting a role for VDR in glioma progression.

  10. High-Throughput Chemical Screens Identify Disulfiram as an Inhibitor of Human Glioblastoma Stem Cells

    PubMed Central

    Hothi, Parvinder; Martins, Timothy J.; Chen, LiPing; Deleyrolle, Loic; Yoon, Jae-Geun; Reynolds, Brent; Foltz, Greg

    2012-01-01

    Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. PMID:23165409

  11. Towards precision medicine-based therapies for glioblastoma: interrogating human disease genomics and mouse phenotypes.

    PubMed

    Chen, Yang; Gao, Zhen; Wang, Bingcheng; Xu, Rong

    2016-08-22

    Glioblastoma (GBM) is the most common and aggressive brain tumors. It has poor prognosis even with optimal radio- and chemo-therapies. Since GBM is highly heterogeneous, drugs that target on specific molecular profiles of individual tumors may achieve maximized efficacy. Currently, the Cancer Genome Atlas (TCGA) projects have identified hundreds of GBM-associated genes. We develop a drug repositioning approach combining disease genomics and mouse phenotype data towards predicting targeted therapies for GBM. We first identified disease specific mouse phenotypes using the most recently discovered GBM genes. Then we systematically searched all FDA-approved drugs for candidates that share similar mouse phenotype profiles with GBM. We evaluated the ranks for approved and novel GBM drugs, and compared with an existing approach, which also use the mouse phenotype data but not the disease genomics data. We achieved significantly higher ranks for the approved and novel GBM drugs than the earlier approach. For all positive examples of GBM drugs, we achieved a median rank of 9.2 45.6 of the top predictions have been demonstrated effective in inhibiting the growth of human GBM cells. We developed a computational drug repositioning approach based on both genomic and phenotypic data. Our approach prioritized existing GBM drugs and outperformed a recent approach. Overall, our approach shows potential in discovering new targeted therapies for GBM.

  12. Proton and photon beams interaction with radiosensitizing agents in human glioblastoma cells

    NASA Astrophysics Data System (ADS)

    Lafiandra, M.

    2016-03-01

    In oncological field, chemoradiotherapy treatments that combine radiations to radiosensitizing chemical agents are spreading out. The aim of this kind of treatment is to obtain a better tumor local control and at the same time to reduce the distant failure. The combination of radiation with microtubule-stabilizing agents is very promising in cancer therapy. In the present study, the combination of clinical proton beams and the microtubule-stabilizing agent Epothilone B has been investigated in human glioblastoma cells cultured in vitro. Photon beams have been used for comparison. Cell survival has been evaluated by colony forming assay and the interaction mechanism between radiation and Epothilone B has been investigated: survival curves relative to the combined treatment (protons or photons with Epothilone B) showed a linear trend, different from the linear quadratic behavior found with radiation alone. The analysis performed showed a synergism in the radiation-drug interaction. Thus, Epothilone B in conjunction with radiation acts as a radiosensitizer. Finally proton Relative Biological Effectiveness has been determined and results are reported in this paper.

  13. Cytopathic Effects of X-ray Irradiation and MnO Nanoparticles on Human Glioblastoma (U87)

    NASA Astrophysics Data System (ADS)

    Kuper, K. E.; Zavjalov, E. L.; Razumov, I. A.; Romaschenko, A. V.; Stupak, A. S.; Troicky, S. Yu; Goldenberg, B. G.; Legkodymov, A. G.; Lemzyakov, A. A.; Moshkin, M. P.

    Glioblastoma is a leader among the most malignant brain tumors with the average lifespan of patients around 9-12 months. For prevention and treatment of neuropathology, a variety of therapeutic and surgical approaches are being developed and improved, including radiation and chemical therapy methods. In our work, we investigated cytopathic effect of X-ray irradiation with application of metal oxides nanoparticles such as manganese oxide (MnO) on U87 human glioblastoma cells. We used the X-ray irradiation dose of 0.5, 4, 40 and 100 Gy in combination with nanoparticles at the concentration of 0.5 ng/ml. The irradiation of glioma cell was carried out at the synchrotron radiation source VEPP-4. After cells treatments with nanoparticles for about 24 h and radiation the results were assessed by MTT assay test with 106/ml cells densities. We demonstrate that preincubation of the glioblastoma cell lines U87 with MnO nanoparticles allows reducing dose of irradiation. This combination of nanoparticles and X-ray irradiation provides new possibilities for the treatment of brain tumors.

  14. Adult, embryonic and fetal hemoglobin are expressed in human glioblastoma cells.

    PubMed

    Emara, Marwan; Turner, A Robert; Allalunis-Turner, Joan

    2014-02-01

    Hemoglobin is a hemoprotein, produced mainly in erythrocytes circulating in the blood. However, non-erythroid hemoglobins have been previously reported in other cell types including human and rodent neurons of embryonic and adult brain, but not astrocytes and oligodendrocytes. Human glioblastoma multiforme (GBM) is the most aggressive tumor among gliomas. However, despite extensive basic and clinical research studies on GBM cells, little is known about glial defence mechanisms that allow these cells to survive and resist various types of treatment. We have shown previously that the newest members of vertebrate globin family, neuroglobin (Ngb) and cytoglobin (Cygb), are expressed in human GBM cells. In this study, we sought to determine whether hemoglobin is also expressed in GBM cells. Conventional RT-PCR, DNA sequencing, western blot analysis, mass spectrometry and fluorescence microscopy were used to investigate globin expression in GBM cell lines (M006x, M059J, M059K, M010b, U87R and U87T) that have unique characteristics in terms of tumor invasion and response to radiotherapy and hypoxia. The data showed that α, β, γ, δ, ζ and ε globins are expressed in all tested GBM cell lines. To our knowledge, we are the first to report expression of fetal, embryonic and adult hemoglobin in GBM cells under normal physiological conditions that may suggest an undefined function of those expressed hemoglobins. Together with our previous reports on globins (Ngb and Cygb) expression in GBM cells, the expression of different hemoglobins may constitute a part of series of active defence mechanisms supporting these cells to resist various types of treatments including chemotherapy and radiotherapy.

  15. Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells.

    PubMed

    Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A

    2015-01-01

    Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively "glues" cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our results

  16. FRMD6 inhibits human glioblastoma growth and progression by negatively regulating activity of receptor tyrosine kinases

    PubMed Central

    Xu, Yin; Wang, Kaiqiang; Yu, Qin

    2016-01-01

    FRMD6 is an Ezrin/Radixin/Moesin (ERM) family protein and a human homologue of Drosophila expanded (ex). Ex functions in parallel of Drosophila merlin at upstream of the Hippo signaling pathway that controls proliferation, apoptosis, tissue regeneration, and tumorigenesis. Even though the core kinase cascade (MST1/2-Lats1/2-YAP/TAZ) of the Hippo pathway has been well established, its upstream regulators are not well understood. Merlin promotes activation of the Hippo pathway. However, the effect of FRMD6 on the Hippo pathway is controversial. Little is known about how FRMD6 functions and the potential role of FRMD in gliomagenesis and glioblastoma (GBM) progression. We demonstrate for the first time that FRMD6 is down-regulated in human GBM cells and tissues and that increased FRMD6 expression inhibits whereas FRMD6 knockdown promotes GBM cell proliferation/invasion in vitro and GBM growth/progression in vivo. Furthermore, we demonstrate that unlike increased expression of merlin, which enhances the stress induced activation of the Hippo pathway, increased FRMD6 expression displays little effect on the pathway. In contrast, we show that FRMD6 inhibits activation of a couple of receptor tyrosine kinases (RTKs) including c-Met and PDGFR and their downstream Erk and AKT kinases. Moreover, we show that expression of constitutively active c-Met, the TPR-Met fusion protein, largely reverses the anti-GBM effect of FRMD6 in vivo, suggesting that FRMD6 functions at least partially through inhibiting activity of RTKs especially c-Met. These results establish a novel function of FRMD6 in inhibiting human GBM growth and progression and uncover a novel mechanism by which FRMD6 exerts its anti-GBM activity. PMID:27661120

  17. Gefitinib selectively inhibits tumor cell migration in EGFR-amplified human glioblastoma.

    PubMed

    Parker, Jonathon J; Dionne, Kalen R; Massarwa, Rada; Klaassen, Marci; Foreman, Nicholas K; Niswander, Lee; Canoll, Peter; Kleinschmidt-Demasters, B K; Waziri, Allen

    2013-08-01

    Tissue invasion is a hallmark of most human cancers and remains a major source of treatment failure in patients with glioblastoma (GBM). Although EGFR amplification has been previously associated with more invasive tumor behavior, existing experimental models have not supported quantitative evaluation of interpatient differences in tumor cell migration or testing of patient-specific responses to therapies targeting invasion. To explore these questions, we optimized an ex vivo organotypic slice culture system allowing for labeling and tracking of tumor cells in human GBM slice cultures. With use of time-lapse confocal microscopy of retrovirally labeled tumor cells in slices, baseline differences in migration speed and efficiency were determined and correlated with EGFR amplification in a cohort of patients with GBM. Slices were treated with gefitinib to evaluate anti-invasive effects associated with targeting EGFR. Migration analysis identified significant patient-to-patient variation at baseline. EGFR amplification was correlated with increased migration speed and efficiency compared with nonamplified tumors. Critically, gefitinib resulted in a selective and significant reduction of tumor cell migration in EGFR-amplified tumors. These data provide the first identification of patient-to-patient variation in tumor cell migration in living human tumor tissue. We found that EGFR-amplified GBM are inherently more efficient in their migration and can be effectively targeted by gefitinib treatment. These data suggest that stratified clinical trails are needed to evaluate gefitinib as an anti-invasive adjuvant for patients with EGFR-amplified GBM. In addition, these results provide proof of principle that primary slice cultures may be useful for patient-specific screening of agents designed to inhibit tumor invasion.

  18. Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

    PubMed Central

    Costa, Barbara; Bendinelli, Sara; Gabelloni, Pamela; Da Pozzo, Eleonora; Daniele, Simona; Scatena, Fabrizio; Vanacore, Renato; Campiglia, Pietro; Bertamino, Alessia; Gomez-Monterrey, Isabel; Sorriento, Daniela; Del Giudice, Carmine; Iaccarino, Guido; Novellino, Ettore; Martini, Claudia

    2013-01-01

    Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients. PMID:23977270

  19. Early Intracellular Trafficking of Granulibacter bethesdensis in Human Macrophages.

    PubMed

    Chu, Jessica; Smelkinson, Margery G; Dorward, David W; Zarember, Kol A; Gallin, John I

    2017-06-01

    Granulibacter bethesdensis is a Gram-negative bacterium that infects patients with chronic granulomatous disease (CGD), a primary immunodeficiency marked by a defect in NOX2, the phagocyte NADPH oxidase. Previous studies have shown that NOX2 is essential for killing of G. bethesdensis by neutrophils and monocytes and that the bacteriostatic activity of monocyte-derived macrophages (MDM) requires NOX2 and gamma interferon (IFN-γ) pretreatment. To determine whether G. bethesdensis evades phagolysosomal killing, a host defense pathway intact in both normal and CGD MDM, or whether it occupies a distinct intracellular niche in CGD MDM, we assessed the trafficking patterns of this organism. We observed colocalization of G. bethesdensis with an early endosome antigen 1 (EEA1)-positive compartment, followed by colocalization with lysosome-associated membrane protein 1 (LAMP1)-positive and LysoTracker-positive late phagosomes; these characteristics were similar in both normal and CGD MDM. Despite localization to acidified late phagosomes, viable G. bethesdensis cells were recovered from viable MDM in numbers greater than in the initial input up to 6 days after infection. G. bethesdensis remains, and in some cases appears to divide, within a membrane-bound compartment for the entire 6-day time course. These findings indicate that this organism resists both oxygen-dependent and oxygen-independent phagolysosomal antimicrobial systems of human macrophages. Copyright © 2017 American Society for Microbiology.

  20. Intracellular pH regulation during spreading of human neutrophils

    PubMed Central

    1996-01-01

    The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results

  1. Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.

    PubMed

    Iacopino, Fortunata; Angelucci, Cristiana; Piacentini, Roberto; Biamonte, Filippo; Mangiola, Annunziato; Maira, Giulio; Grassi, Claudio; Sica, Gigliola

    2014-01-01

    Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP); Ca(2+) signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca(2+)-channels but they exhibited increased intracellular Ca(2+) levels in response to ATP. These Ca(2+) signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca(2+)-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

  2. Down-regulation of ribosomal protein S15A inhibits proliferation of human glioblastoma cells in vivo and in vitro via AKT pathway.

    PubMed

    Yao, Yiqun; Liu, Yongjian; Lv, Xiupeng; Dong, Bin; Wang, Feng; Li, Jun; Zhang, Qiuping; Xu, Ruixue; Xu, Yinghui

    2016-04-01

    Ribosomal protein s15a (RPS15A), a highly conserved cytoplasmic protein, promotes mRNA/ribosome interaction in translation. Recent evidence showed that RPS15A is essential for tumor growth. RPS15A expression level was measured in glioblastoma tissue samples and normal brain (NB) tissue samples. RPS15A RNAi stable cell line U87 and U251 was generated by the pLVTHM-GFP lentiviral RNAi expression system. The knockdown efficiency was confirmed by quantitative real-time PCR and western blot. Molecular mechanisms and the effect of RPS15A on cell growth and migration were investigated by using western blot, MTT assay, wound healing assay, transwell migration assay, and tumorigenesis in nude mice. Here, we report that RPS15A is overexpressed in human glioblastoma tumor tissues. RPS15A knockdown inhibits proliferation and migration of glioblastoma cells in vitro. Knocking down RPS15A leads to the level of p-Akt decrease and cell cycle arrested in G0/G1 phase in U87 and U251 cells. Furthermore, the growth of glioblastoma cell-transplanted tumors in nude mice is inhibited by transduction with Lv-shRPS15A. Our findings indicate that RPS15A promotes cell proliferation and migration in glioblastoma for the first time. RPS15A might play a distinct role in glioblastoma and serve as a potential target for therapy.

  3. Allopregnanolone promotes proliferation and differential gene expression in human glioblastoma cells.

    PubMed

    Zamora-Sánchez, Carmen J; Hansberg-Pastor, Valeria; Salido-Guadarrama, Ivan; Rodríguez-Dorantes, Mauricio; Camacho-Arroyo, Ignacio

    2017-03-01

    Allopregnanolone (3α-THP) is one of the main reduced progesterone (P4) metabolites that is recognized as a neuroprotective and myelinating agent. 3α-THP also induces proliferation of different neural cells. It has been shown that P4 favors the progression of glioblastomas (GBM), the most common and aggressive primary brain tumors. However, the role of 3α-THP in the growth of GBMs is unknown. Here, we studied the effects of 3α-THP on the number of cells, proliferation and gene expression in U87 cell line derived from a human GBM. 3α-THP (10, 100nM and 1μM) increased the number of U87 cells, and at 10nM exerted a similar increase in both the number of total and proliferative U87 cells as compared with P4 (10nM). Interestingly, finasteride (F; 100nM), an inhibitor of 5α-reductase (5αR), an enzyme necessary to metabolize P4 and produce 3α-THP, blocked the increase in the number of U87 cells induced by P4. By using RT-qPCR, we determined that U87 cells express 5α-R isoenzymes 1 and 2 (5αR1 and 5αR2), being 5αR1 the predominant one in these cells. 3α-THP (10nM) increased the expression of TGFβ1, EGFR, VEGF and cyclin D1 genes. P4 increased TGFβ1 and EGFR expression, and this effect was blocked by F. These data provide evidence that P4, through its metabolite 3α-THP, can promote in part cell proliferation of human GBM cells by changing the expression of genes involved in tumor progression.

  4. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

    PubMed

    Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

    2015-02-18

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

  5. Biodegradable Polymeric Nanoparticles Show High Efficacy and Specificity at DNA Delivery to Human Glioblastoma in Vitro and in Vivo

    PubMed Central

    2015-01-01

    Current glioblastoma therapies are insufficient to prevent tumor recurrence and eventual death. Here, we describe a method to treat malignant glioma by nonviral DNA delivery using biodegradable poly(β-amino ester)s (PBAEs), with a focus on the brain tumor initiating cells (BTICs), the tumor cell population believed to be responsible for the formation of new tumors and resistance to many conventional therapies. We show transfection efficacy of >60% and low biomaterial-mediated cytotoxicity in primary human BTICs in vitro even when the BTICs are grown as 3-D oncospheres. Intriguingly, we find that these polymeric nanoparticles show intrinsic specificity for nonviral transfection of primary human BTICs over primary healthy human neural progenitor cells and that this specificity is not due to differences in cellular growth rate or total cellular uptake of nanoparticles. Moreover, we demonstrate that biodegradable PBAE/DNA nanoparticles can be fabricated, lyophilized, and then stored for at least 2 years without losing efficacy, increasing the translational relevance of this technology. Using lyophilized nanoparticles, we show transgene expression by tumor cells after intratumoral injection into an orthotopic murine model of human glioblastoma. PBAE/DNA nanoparticles were more effective than naked DNA at exogenous gene expression in vivo, and tumor cells were transfected more effectively than noninvaded brain parenchyma in vivo. This work shows the potential of nonviral gene delivery tools to target human brain tumors. PMID:24766032

  6. Intracellular accumulation of potent amiloride analogues by human neutrophils

    SciTech Connect

    Simchowitz, L.; Woltersdorf, O.W. Jr.; Cragoe, E.J. Jr.

    1987-11-25

    The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using (/sup 14/C)amiloride and the /sup 14/C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids.

  7. Comparison of microarray and sage techniques in gene expression analysis of human glioblastoma.

    PubMed

    Kavsan, V M; Dmitrenko, V V; Shostak, K O; Bukreieva, T V; Vitak, N Y; Simirenko, O E; Malisheva, T A; Shamayev, M I; Rozumenko, V D; Zozulya, Y A

    2007-01-01

    To enhance glioblastoma (GB) marker discovery we compared gene expression in GB with human normal brain (NB) by accessing SAGE Genie web site and compared obtained results with published data. Nine GB and five NB SAGE-libraries were analyzed using the Digital Gene Expression Displayer (DGED), the results of DGED were tested by Northern blot analysis and RT-PCR of arbitrary selected genes. Review of available data from the articles on gene expression profiling by microarray-based hybridization showed as few as 35 overlapped genes with increased expression in GB. Some of them were identified in four articles, but most genes in three or even in two investigations. There was found also some differences between SAGE results of GB analysis. Digital Gene Expression Displayer approach revealed 676 genes differentially expressed in GB vs. NB with cut-off ratio: twofold change and P < or = 0.05. Differential expression of selectedgenes obtained by DGED was confirmed by Northern analysis and RT-PCR. Altogether, only 105 of 955 genes presented in published investigations were among the genes obtained by DGED. Comparison of the results obtained by microarrays and SAGE is very complicated because authors present only the most prominent differentially expressed genes. However, even available data give quite poor overlapping of genes revealed by microarrays. Some differences between results obtained by SAGE in different investigations can be explained by high dependence on the statistical methods used. As for now, the best solution to search for molecular tumor markers is to compare all available results and to select only those genes, which significant expression in tumor combined with very low expression in normal tissues was reproduced in several articles. 105 differentially expressed genes, common to both methods, can be included in the list of candidates for the molecular typing of GBs. Some genes, encoded cell surface or extra-cellular proteins may be useful for targeting

  8. IL22/IL-22R pathway induces cell survival in human glioblastoma cells.

    PubMed

    Akil, Hussein; Abbaci, Amazigh; Lalloué, Fabrice; Bessette, Barbara; Costes, Léa M M; Domballe, Linda; Charreau, Sandrine; Guilloteau, Karline; Karayan-Tapon, Lucie; Bernard, François-Xavier; Morel, Franck; Jauberteau, Marie-Odile; Lecron, Jean-Claude

    2015-01-01

    Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.

  9. SV40 DNA in a carrier system of human glioblastoma cells.

    PubMed

    Steinberg, V I; Norkin, L C

    1988-04-01

    The state of the SV40 DNA in a stable carrier system of A172 human glioblastoma cells was examined by Southern blot hybridization analysis. At a sensitivity of 0.1 viral genome equivalents per cell, we detected only free, apparently nondefective, viral genomes. However, when we overexposed our autoradiograms or examined cloned cell populations, integrated viral sequences were observed. Furthermore, aberrant forms of free viral DNA were seen as well. Four clones, isolated at 15 weeks, produced T antigen and displayed enhanced saturation density and plating efficiency characteristic of SV40 transformation. None of these clones produced capsid proteins or infectious virus, even upon fusion with CV-1 cells, Viral DNA in the clones ranged from 0.5 to 50 equivalents per cell, on the average. Two of the Week-15 clones contained a similar (but not identical) predominant truncated SV40 sequence which was present both in a free state and integrated at a single major site in a reiterated head-to-tail array. These clones also contained other minor integrated sequences. Another Week-15 clone contained viral sequences integrated at two major sites as well as heterogeneous free DNA. Only free aberrant DNA was detected in the fourth Week-15 clone. Seven of eight clones isolated at 23 weeks produced no infectious virus or T antigen. No viral DNA was detected in those clones. The eighth clone did produce infectious virus and contained a predominance of free viral DNA. All of the clones were susceptible to superinfection with wild-type SV40, although less so than uninfected A172 cultures.

  10. Marker-independent Method for Isolating Slow-Dividing Cancer Stem Cells in Human Glioblastoma12

    PubMed Central

    Richichi, Cristina; Brescia, Paola; Alberizzi, Valeria; Fornasari, Lorenzo; Pelicci, Giuliana

    2013-01-01

    Glioblastoma (GBM) is a devastating brain tumor with a poor survival outcome. It is generated and propagated by a small subpopulation of rare and hierarchically organized cells that share stem-like features with normal stem cells but, however, appear dysregulated in terms of self-renewal and proliferation and aberrantly differentiate into cells forming the bulk of the disorganized cancer tissues. The complexity and heterogeneity of human GBMs underlie the lack of standardized and effective treatments. This study is based on the assumption that available markers defining cancer stem cells (CSCs) in all GBMs are not conclusive and further work is required to identify the CSC. We implemented a method to isolate CSCs independently from cell surface markers: four patient-derived GBM neurospheres containing stem, progenitors, and differentiated cells were labeled with PKH-26 fluorescent dye that reliably selects for cells that divide at low rate. Through in vitro and in vivo assays, we investigated the growth and self-renewal properties of the two different compartments of high- and slow-dividing cells. Our data demonstrate that only slow-dividing cells retain the ability of a long-lasting self-renewal capacity after serial in vitro passaging, while high-dividing cells eventually exhaust. Moreover, orthotopic transplantation assay revealed that the incidence of tumors generated by the slow-dividing compartment is significantly higher in the four patient-derived GBM neurospheres analyzed. Importantly, slow-dividing cells feature a population made up of homogeneous stem cells that sustain tumor growth and therefore represent a viable target for GBM therapy development. PMID:23814495

  11. Therapeutic Efficacy of Aldoxorubicin in an Intracranial Xenograft Mouse Model of Human Glioblastoma12

    PubMed Central

    Marrero, Luis; Wyczechowska, Dorota; Musto, Alberto E.; Wilk, Anna; Vashistha, Himanshu; Zapata, Adriana; Walker, Chelsey; Velasco-Gonzalez, Cruz; Parsons, Christopher; Wieland, Scott; Levitt, Daniel; Reiss, Krzysztof; Prakash, Om

    2014-01-01

    Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a median survival of 12 to 15 months after diagnosis. Acquired chemoresistance, high systemic toxicity, and low penetration of the blood brain barrier by many anticancer drugs contribute to the failure of anti-GBM therapies. To circumvent some of these obstacles, we tested a novel prodrug approach to evaluate anti-GBM efficacy by utilizing serum albumin-binding doxorubicin (Doxo), aldoxorubicin (Aldoxo), which is less toxic, is released from albumin in an acidic environment and accumulates in tumor tissues. A human GBM cell line that expresses a luciferase reporter (U87-luc) was stereotactically injected into the left striatum of the brain of immunodeficient mice. Following initial tumor growth for 12 days, mice were injected once a week in the tail-vein with Aldoxo [24 mg/kg or 18 mg/kg of doxorubicin equivalents—3/4 maximum tolerated dose (MTD)], Doxo [6 mg/kg (3/4 MTD)], or vehicle. Aldoxo-treated mice demonstrated significantly slower growth of the tumor when compared to vehicle-treated or Doxo-treated mice. Five out of eight Aldoxo-treated mice remained alive more than 60 days with a median survival of 62 days, while the median survival of vehicle- and Doxo-treated mice was only 26 days. Importantly, Aldoxo-treated mice exhibited high levels of Doxo within the tumor tissue, accompanied by low tumor cell proliferation (Ki67) and abundant intratumoral programmed cell death (cleaved caspase-3). Effective accumulation of Aldoxo in brain tumor tissues but not normal brain, its anti-tumor efficacy, and low toxicity, provide a strong rationale for evaluating this novel drug conjugate as a treatment for patients afflicted with GBM. PMID:25379024

  12. Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM).

    PubMed

    Demeure, Kevin; Fack, Fred; Duriez, Elodie; Tiemann, Katja; Bernard, Amandine; Golebiewska, Anna; Bougnaud, Sébastien; Bjerkvig, Rolf; Domon, Bruno; Niclou, Simone P

    2016-02-01

    Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future

  13. Human Cytomegalovirus-Infected Glioblastoma Cells Display Stem Cell-Like Phenotypes

    PubMed Central

    Liu, Che; Clark, Paul A.; Kuo, John S.

    2017-01-01

    ABSTRACT Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Human cytomegalovirus (HCMV) genomes are present in GBM tumors, yielding hope that antiviral treatments could prove therapeutic and improve the poor prognosis of GBM patients. We discovered that GBM cells infected in vitro with HCMV display properties of cancer stem cells. HCMV-infected GBM cells grow more slowly than mock-infected controls, demonstrate a higher capacity for self-renewal determined by a sphere formation assay, and display resistance to the chemotherapeutic drug temozolomide. Our data suggest that HCMV, while present in only a minority of the cells within a tumor, could contribute to the pathogenesis of GBMs by promoting or prolonging stem cell-like phenotypes, thereby perpetuating tumors in the face of chemotherapy. Importantly, we show that temozolomide sensitivity is restored by the antiviral drug ganciclovir, indicating a potential mechanism underlying the positive effects observed in GBM patients treated with antiviral therapy. IMPORTANCE A role for HCMV in GBMs remains controversial for several reasons. Some studies find HCMV in GBM tumors, while others do not. Few cells within a GBM may harbor HCMV, making it unclear how the virus could be contributing to the tumor phenotype without infecting every cell. Finally, HCMV does not overtly transform cells in vitro. However, tumors induced by other viruses can be treated with antiviral remedies, and initial results indicate that this may be true for anti-HCMV therapies and GBMs. With such a poor prognosis for GBM patients, any potential new intervention deserves exploration. Our work here describes an evidence-based model for how HCMV could contribute to GBM biology while infecting very few cells and without transforming them. It also illuminates why anti-HCMV treatments may be beneficial to GBM patients. Our observations provide blueprints for future in vitro studies examining how HCMV manipulates stem cell

  14. STAT3 Serine 727 Phosphorylation: A Relevant Target to Radiosensitize Human Glioblastoma.

    PubMed

    Ouédraogo, Zangbéwendé Guy; Müller-Barthélémy, Mélanie; Kemeny, Jean-Louis; Dedieu, Véronique; Biau, Julian; Khalil, Toufic; Raoelfils, Lala Ines; Granzotto, Adeline; Pereira, Bruno; Beaudoin, Claude; Guissou, Innocent Pierre; Berger, Marc; Morel, Laurent; Chautard, Emmanuel; Verrelle, Pierre

    2016-01-01

    Radiotherapy is an essential component of glioma standard treatment. Glioblastomas (GBM), however, display an important radioresistance leading to tumor recurrence. To improve patient prognosis, there is a need to radiosensitize GBM cells and to circumvent the mechanisms of resistance caused by interactions between tumor cells and their microenvironment. STAT3 has been identified as a therapeutic target in glioma because of its involvement in mechanisms sustaining tumor escape to both standard treatment and immune control. Here, we studied the role of STAT3 activation on tyrosine 705 (Y705) and serine 727 (S727) in glioma radioresistance. This study explored STAT3 phosphorylation on Y705 (pSTAT3-Y705) and S727 (pSTAT3-S727) in glioma cell lines and in clinical samples. Radiosensitizing effect of STAT3 activation down-modulation by Gö6976 was explored. In a panel of 15 human glioma cell lines, we found that the level of pSTAT3-S727 was correlated to intrinsic radioresistance. Moreover, treating GBM cells with Gö6976 resulted in a highly significant radiosensitization associated to a concomitant pSTAT3-S727 down-modulation only in GBM cell lines that exhibited no or weak pSTAT3-Y705. We report the constitutive activation of STAT3-S727 in all GBM clinical samples. Targeting pSTAT3-S727 mainly in pSTAT3-Y705-negative GBM could be a relevant approach to improve radiation therapy. © 2015 International Society of Neuropathology.

  15. Long non-coding RNA taurine upregulated 1 enhances tumor-induced angiogenesis through inhibiting microRNA-299 in human glioblastoma.

    PubMed

    Cai, H; Liu, X; Zheng, J; Xue, Y; Ma, J; Li, Z; Xi, Z; Li, Z; Bao, M; Liu, Y

    2017-01-19

    Angiogenesis is one of the critical biological elements affecting the development and progression of cancer. Long non-coding RNAs (lncRNAs) are important regulators and aberrantly expressed in various types of human cancer. Our previous studies indicated that lncRNA taurine upregulated 1 (TUG1) implicated in the regulation of blood-tumor barrier permeability; however, its role in glioblastoma angiogenesis still unclear. Here we demonstrated that TUG1 was up-expressed in human glioblastoma tissues and glioblastoma cell lines. Knockdown of TUG1 remarkably suppressed tumor-induced endothelial cell proliferation, migration and tube formation as well as reducing spheroid-based angiogenesis ability in vitro, which are the critical steps for tumor angiogenesis. Besides, knockdown of TUG1 significantly increased the expression of mircroRNA-299 (miR-299), which was down-expressed in glioblastoma tissues and glioblastoma cell lines. Bioinformatics analysis and luciferase reporter assay revealed that TUG1 influenced tumor angiogenesis via directly binding to the miR-299 and there was a reciprocal repression between TUG1 and miR-299 in the same RNA-induced silencing complex. Moreover, knockdown of TUG1 reduced the expression of vascular endothelial growth factor A (VEGFA), which was defined as a functional downstream target of miR-299. In addition, knockdown of TUG1, shown in the in vivo studies, has effects on suppressing tumor growth, reducing tumor microvessel density and decreasing the VEGFA expression by upregulating miR-299 in xenograft glioblastoma model. Overall, the results demonstrated that TUG1 enhances tumor-induced angiogenesis and VEGF expression through inhibiting miR-299. Also, the inhibition of TUG1 could provide a novel therapeutic target for glioblastoma treatment.

  16. Cannabidiol enhances the inhibitory effects of Δ9-tetrahydrocannabinol on human glioblastoma cell proliferation and survival

    PubMed Central

    Marcu, Jahan P.; Christian, Rigel T.; Lau, Darryl; Zielinski, Anne J.; Horowitz, Maxx P.; Lee, Jasmine; Pakdel, Arash; Allison, Juanita; Limbad, Chandani; Moore, Dan H.; Yount, Garret L.; Desprez, Pierre-Yves; McAllister, Sean D.

    2009-01-01

    The cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor agonist, Δ9-tetrahydrocannabinol (THC), has been shown to be a broad range inhibitor of cancer in culture and in vivo, and is currently being used in a clinical trial for the treatment of glioblastoma. It has been suggested that other plant-derived cannabinoids, which do not interact efficiently with CB1 and CB2 receptors, can modulate the actions of Δ9-THC. However, there are conflicting reports as to what extent other cannabinoids can modulate Δ9-THC activity, and most importantly, it is not clear whether other cannabinoid compounds can either potentiate or inhibit the actions of Δ9-THC. We therefore tested cannabidiol (CBD), the second most abundant plant derived cannabiniod, in combination with Δ9-THC. In U251 and SF126 glioblastoma cell lines, Δ9-THC and CBD acted synergistically to inhibit cell proliferation. The treatment of glioblastoma cells with both compounds led to significant modulations of the cell cycle and induction of reactive oxygen species (ROS) and apoptosis as well as specific modulations of extracellular signal-regulated kinase (ERK) and caspase activities. These specific changes were not observed with either compound individually, indicating that the signal transduction pathways affected by the combination treatment were unique. Our results suggest that the addition of CBD to Δ9-THC may improve the overall effectiveness of Δ9-THC in the treatment of glioblastoma in cancer patients. PMID:20053780

  17. BAG3 Protein Is Overexpressed in Human Glioblastoma and Is a Potential Target for Therapy

    PubMed Central

    Festa, Michelina; Del Valle, Luis; Khalili, Kamel; Franco, Renato; Scognamiglio, Giosuè; Graziano, Vincenzo; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra

    2011-01-01

    Glioblastoma multiforme, which represents 80% of malignant gliomas, is characterized by aggressiveness and high recurrence rates. Despite therapeutic advances, patients with glioblastoma multiforme show a poor survival, and identification of novel markers and molecular targets for therapy is needed. A role for BAG3, a member of the BAG family of HSC/HSP70 co-chaperones, in promoting tumor cell growth in vivo has recently been described. We analyzed BAG3 levels by IHC in specimens from patients affected by brain tumors and we found that BAG3, although negative in normal brain tissues, was highly expressed in astrocytic tumors and increasingly expressed in more aggressive types of cancer; it was particularly high in glioblastomas. Down-regulating BAG3 both in vitro and in vivo in a rat glioblastoma model resulted in increased sensitivity to apoptosis, suggesting that BAG3 is a potential target for novel therapies. Finally, we determined that the underlying molecular mechanism requires the formation of a complex of BAG3, HSP70, and BAX that prevents BAX translocation to mitochondria, thus protecting tumor cells from apoptosis. Our data identify BAG3 as a potential marker of glial brain tumor sensitivity to therapy and thus also an attractive candidate for new molecular therapies. PMID:21561597

  18. The malignancy of miR-18a in human glioblastoma via directly targeting CBX7

    PubMed Central

    Wu, Weining; Zhou, Xu; Yu, Tianfu; Bao, Zhongyuan; Zhi, Tongle; Jiang, Kuan; Nie, Er; Wang, Yingyi; Zhang, Junxia; You, Yongping

    2017-01-01

    Aberrant levels of microRNAs (miRNAs) are linked to tumorigenesis and tumor progression. Here we analyzed the expression of microRNA 18a in glioblastoma multiforme (GBM) within groups of coexpressed groups of genes through analysis of expression profiling databases and clinical tissues. Cell proliferation and flow cytometry experiments were performed to determine the roles of miR-18a in the proliferation of glioblastoma cells in vitro. We employed bioinformatics analyses, luciferase reporter assays, and immunoblotting to identify chromobox protein homolog 7 (CBX7) as the target gene of miR-18a. A significant inverse correlation was observed between miR-18a and CBX7 expression in GBM tissues (r = -0.6264, P = 0.0094). Bioinformatics analyses revealed that most CBX7-associated genes were enriched in terms associated with cell cycle pathways. Upregulated expression of CBX7 inhibited the growth of GBM cells and reduced the expression of CDK2 and cyclin A2 (CCNA2). Rescue experiments indicated that overexpression of CBX7 significantly recovered the increase in cell proliferation and cell cycle distribution induced by miR-18a overexpression. In vivo studies revealed that decreased levels of miR-18a delayed the growth of intracranial tumors, which was accompanied by increased CBX7 expression. We suggest that miR-18a promotes glioblastoma progression via altering CBX7 expression and therefore may serve as a potential target for treating glioblastoma. PMID:28123848

  19. Dexamethasone-Mediated Activation of Fibronectin Matrix Assembly Reduces Dispersal of Primary Human Glioblastoma Cells

    PubMed Central

    Shannon, Stephen; Vaca, Connan; Jia, Dongxuan; Entersz, Ildiko; Schaer, Andrew; Carcione, Jonathan; Weaver, Michael; Avidar, Yoav; Pettit, Ryan; Nair, Mohan; Khan, Atif; Foty, Ramsey A.

    2015-01-01

    Despite resection and adjuvant therapy, the 5-year survival for patients with Glioblastoma multiforme (GBM) is less than 10%. This poor outcome is largely attributed to rapid tumor growth and early dispersal of cells, factors that contribute to a high recurrence rate and poor prognosis. An understanding of the cellular and molecular machinery that drive growth and dispersal is essential if we are to impact long-term survival. Our previous studies utilizing a series of immortalized GBM cell lines established a functional causation between activation of fibronectin matrix assembly (FNMA), increased tumor cohesion, and decreased dispersal. Activation of FNMA was accomplished by treatment with Dexamethasone (Dex), a drug routinely used to treat brain tumor related edema. Here, we utilize a broad range of qualitative and quantitative assays and the use of a human GBM tissue microarray and freshly-isolated primary human GBM cells grown both as conventional 2D cultures and as 3D spheroids to explore the role of Dex and FNMA in modulating various parameters that can significantly influence tumor cell dispersal. We show that the expression and processing of fibronectin in a human GBM tissue-microarray is variable, with 90% of tumors displaying some abnormality or lack in capacity to secrete fibronectin or assemble it into a matrix. We also show that low-passage primary GBM cells vary in their capacity for FNMA and that Dex treatment reactivates this process. Activation of FNMA effectively “glues” cells together and prevents cells from detaching from the primary mass. Dex treatment also significantly increases the strength of cell-ECM adhesion and decreases motility. The combination of increased cohesion and decreased motility discourages in vitro and ex vivo dispersal. By increasing cell-cell cohesion, Dex also decreases growth rate of 3D spheroids. These effects could all be reversed by an inhibitor of FNMA and by the glucocorticoid receptor antagonist, RU-486. Our

  20. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  1. Diversified expression of NG2/CSPG4 isoforms in glioblastoma and human foetal brain identifies pericyte subsets.

    PubMed

    Girolamo, Francesco; Dallatomasina, Alice; Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  2. PROX1 promotes human glioblastoma cell proliferation and invasion via activation of the nuclear factor-κB signaling pathway.

    PubMed

    Xu, Xuchang; Wan, Xuefeng; Wei, Xinting

    2017-02-01

    Prospero homeobox protein 1 (PROX1) is highly expressed in high-grade malignant astrocytic gliomas. However, the role of PROX1 in the pathogenesis of glioblastoma multiforme (GBM) remains unclear. The present study overexpressed PROX1 in human GBM cell lines and examined its effects on cell growth, tumorigenesis, and invasiveness. In addition, the involvement of the nuclear factor‑κB (NF‑κB) signaling pathway in the action of PROX1 was examined. It was identified that overexpression of PROX1 significantly increased the proliferation and colony formation of glioblastoma cells, compared with empty vector‑transfected controls. Furthermore, ectopic expression of PROX1 promoted the growth of GBM xenograft tumors. Western blot analysis revealed that PROX1 overexpression induced nuclear accumulation of NF‑κB p65 and upregulated the expression levels of the NF‑κB responsive genes cyclin D1 and matrix metallopeptidase 9. An NF‑κB reporter assay demonstrated that PROX1‑overexpressing glioblastoma cells had significantly greater NF‑κB‑dependent reporter activities compared with empty vector‑transfected controls. Transfection of a dominant inhibitor of κBα mutant into PROX1‑overexpressing cells significantly impaired their proliferation and invasion capacities, which was accompanied by reduced levels of nuclear NF‑κB p65. Collectively, these data indicated that PROX1 serves an oncogenic role in GBM and promotes cell proliferation and invasiveness potentially via activation of the NF‑κB signaling pathway. Therefore, PROX1 may represent a potential target for the treatment of GBM.

  3. miR-221/222 overexpession in human glioblastoma increases invasiveness by targeting the protein phosphate PTPμ

    PubMed Central

    Quintavalle, C; Garofalo, M; Zanca, C; Romano, G; Iaboni, M; De Caro, M del Basso; Martinez-Montero, JC; Incoronato, M; Nuovo, G; Croce, CM; Condorelli, G

    2015-01-01

    Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. In order to identify microRNAs (miRs) involved in glioma tumorigenesis, we evaluated, by a miRarray, differential expression of miRs in the tumorigenic glioma LN-18, LN-229 and U87MG cells compared with the non-tumorigenic T98G cells. Among different miRs we focused our attention on miR-221 and -222. We demonstrated the presence of a binding site for these two miRs in the 3′ untranslated region of the protein tyrosine phosphatase μ (PTPμ). Previous studies indicated that PTPμ suppresses cell migration and is downregulated in glioblastoma. Significantly, we found that miR-221 and -222 over-expression induced a downregulation of PTPμ as analyzed by both western blot and real-time PCR. Furthermore, miR-222 and -221 induced an increase in cell migration and growth in soft agar in glioma cells. Interestingly, the re-expression of PTPμ gene was able to revert the miR-222 and -221 effects on cell migration. Furthermore, we found an inverse correlation between miR-221 and -222 and PTPμ in human glioma cancer samples. In conclusion, our results suggest that miR-221 and -222 regulate glioma tumorigenesis at least in part through the control of PTPμ protein expression. PMID:21743492

  4. Irradiation combined with SU5416: Microvascular changes and growth delay in a human xenograft glioblastoma tumor line

    SciTech Connect

    Schuuring, Janneke; Bussink, Johan . E-mail: J.Bussink@rther.umcn.nl; Bernsen, Hans; Peeters, Wenny; Kogel, Albert J. van der

    2005-02-01

    Purpose: The combination of irradiation and the antiangiogenic compound SU5416 was tested and compared with irradiation alone in a human glioblastoma tumor line xenografted in nude mice. The aim of this study was to monitor microenvironmental changes and growth delay. Methods and materials: A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without SU5416. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after imunohistochemical staining. Tumor growth delay was monitored for up to 200 days after treatment. Results: SU5416, when combined with irradiation, has an additive effect over treatment with irradiation alone. Analysis of the tumor microenvironment showed a decreased vascular density during treatment with SU5416. In tumors regrowing after reaching only a partial remission, vascular characteristics normalized shortly after cessation of SU5416. However, in tumors regrowing after reaching a complete remission, permanent microenvironmental changes and an increase of tumor necrosis with a subsequent slower tumor regrowth was found. Conclusions: Permanent vascular changes were seen after combined treatment resulting in complete remission. Antiangiogenic treatment with SU5416 when combined with irradiation has an additive effect over treatment with irradiation or antiangiogenic treatment alone.

  5. BC3EE2,9B, a synthetic carbazole derivative, upregulates autophagy and synergistically sensitizes human GBM8901 glioblastoma cells to temozolomide

    PubMed Central

    CHEN, CHIEN-MIN; SYU, JHIH-PU; WAY, TZONG-DER; HUANG, LI-JIAU; KUO, SHENG-CHU; LIN, CHUNG-TIEN; LIN, CHIH-LI

    2015-01-01

    Glioblastoma multiforme (GBM) is the most fatal form of human brain cancer. Although temozolomide (TMZ), an oral alkylating chemotherapeutic agent, improves the survival rate, the prognosis of patients with GBM remains poor. Naturally occurring carbazole alkaloids isolated from curry leaves (Murraya koenigii Spreng.) have been shown to possess a wide range of anticancer properties. However, the effects of carbazole derivatives on glioblastoma cells remain poorly understood. In the present study, anti-glioblastoma profiles of a series of synthetic carbazole derivatives were evaluated in vitro. The most promising derivative in this series was BC3EE2,9B, which showed significant anti-proliferative effects in GBM8401 and GBM8901 cells. BC3EE2,9B also triggered cell-cycle arrest, most prominently at the G1 stage, and suppressed glioblastoma cell invasion and migration. Furthermore, BC3EE2,9B induced autophagy-mediated cell death and synergistically sensitized GBM cells to TMZ cytotoxicity. The possible mechanism underlying BC3EE2,9B-induced autophagy may involve activation of adenosine monophosphate-activated protein kinase and the attenuation of the Akt and mammalian target of the rapamycin downstream signaling pathway. Taken together, the present results provide molecular evidence for the mode of action governing the ability of BC3EE2,9B to sensitize drug-resistant glioblastoma cells to the chemotherapeutic agent TMZ. PMID:26329365

  6. Human chorionic gonadotropin β induces cell motility via ERK1/2 and MMP-2 activation in human glioblastoma U87MG cells.

    PubMed

    Li, Zongwen; Du, Lianlian; Li, Chunliu; Wu, Wei

    2013-02-01

    Human chorionic gonadotropin β (hCGβ) promotes tumorigenesis in a variety of tumors including glioblastoma, breast and prostate cancer cells, etc. However, the involved mechanisms remain elusive. Distinct from the other tumors, glioblastoma is a highly invasive brain tumor; invasion causes high recurrence and mortality. Characterization of hCGβ signaling is to determine therapeutic targets to inhibit invasion and lower recurrence. Through both a stable cell line over-expressing hCGβ and hCGβ standards, we tested hCGβ signaling, migration and invasion in human glioblastoma U87MG cells. ELISA showed that hCGβ secreted into culture medium at an amount of 237.8 ± 7.8 ng/10(7) cells in hCGβ transfected stable cells after the cells were grown for 24 h. Through Western blot and Gelatin zymography, we found that hCGβ standards phosphorylated ERK1/2 and upregulated MMP-2 expression in dose- and time-dependent manners. Meanwhile, overexpressed hCGβ phosphorylated ERK1/2, and upregulated MMP-2 expression and activity, whereas ERK1/2 blocker PD98059 (25 μM) significantly decreased both ERK1/2 and MMP-2 expression and activity. In addition, in the same conditions as the signaling test, hCGβ promoted cell migration and invasion, whereas the PD98059 diminished these effects. These findings demonstrated that hCGβ phosphorylated ERK1/2 upregulating MMP-2 expression and activity leading to cell migration and invasion, suggesting that hCGβ, ERK1/2 and MMP-2 are the potential targets to inhibit glioblastoma invasion.

  7. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

    PubMed Central

    Meyer, Mona; Reimand, Jüri; Lan, Xiaoyang; Head, Renee; Zhu, Xueming; Kushida, Michelle; Bayani, Jane; Pressey, Jessica C.; Lionel, Anath C.; Clarke, Ian D.; Cusimano, Michael; Squire, Jeremy A.; Scherer, Stephen W.; Bernstein, Mark; Woodin, Melanie A.; Bader, Gary D.; Dirks, Peter B.

    2015-01-01

    Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies. PMID:25561528

  8. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.

    PubMed

    Meyer, Mona; Reimand, Jüri; Lan, Xiaoyang; Head, Renee; Zhu, Xueming; Kushida, Michelle; Bayani, Jane; Pressey, Jessica C; Lionel, Anath C; Clarke, Ian D; Cusimano, Michael; Squire, Jeremy A; Scherer, Stephen W; Bernstein, Mark; Woodin, Melanie A; Bader, Gary D; Dirks, Peter B

    2015-01-20

    Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

  9. Down regulation of Akirin-2 increases chemosensitivity in human glioblastomas more efficiently than Twist-1

    PubMed Central

    Krossa, Sebastian; Schmitt, Anne Dorothée; Hattermann, Kirsten; Fritsch, Jürgen; Scheidig, Axel J.; Mehdorn, Hubertus Maximilian; Held-Feindt, Janka

    2015-01-01

    The Twist-1 transcription factor and its interacting protein Akirin-2 regulate apoptosis. We found that in glioblastomas, highly malignant brain tumors, Akirin-2 and Twist-1 were expressed in glial fibrillary acidic protein positive tumor regions as well as in tumor endothelial cells and infiltrating macrophages / microglia. Temozolomide (TMZ) induced the expression of both molecules, partly shifting their nuclear to cytosolic localization. The knock-down (kd) of Akirin-2 increased the activity of cleaved (c)Caspase-3/-7, the amounts of cCaspases-3, -7 and cPARP-1 and resulted in an increased number of apoptotic cells after TMZ exposure. Glioblastoma cells containing decreased amounts of Akirin-2 after kd contained increased amounts of cCaspase-3 as determined by the ImageStreamx Mark II technology. For Twist-1, similar results were obtained with the exception that the combination of TMZ treatment and Twist-1 kd failed to significantly reduce chemoresistance compared with controls. This could be attributed to a cell population containing only slightly increased cCaspase-3 together with decreased Twist-1 levels, which was clearly larger than the respective population observed under Akirin-2 kd. Our results showed that, compared with Twist-1, Akirin-2 is the more promising target for RNAi strategies antagonizing Twist-1/Akirin-2 facilitated glioblastoma cell survival. PMID:26036627

  10. Columbia University: Computational Human High-grade Glioblastoma Multiforme Interactome - miRNA (Post-transcriptional) Layer | Office of Cancer Genomics

    Cancer.gov

    The Human High-Grade Glioma Interactome (HGi) contains a genome-wide complement of molecular interactions that are Glioblastoma Multiforme (GBM)-specific. HGi v3 contains the post-transcriptional layer of the HGi, which includes the miRNA-target (RNA-RNA) layer of the interactome. Read the Abstract

  11. Intracranial elimination of human glioblastoma brain tumors in nude rats using the bispecific ligand-directed toxin, DTEGF13 and convection enhanced delivery.

    PubMed

    Oh, Seunguk; Ohlfest, John R; Todhunter, Deborah A; Vallera, Vincent D; Hall, Walter A; Chen, Hua; Vallera, Daniel A

    2009-12-01

    A bispecific ligand-directed toxin (BLT) consisting of human interleukin-13, epithelial growth factor, and the first 389 amino acids of diphtheria toxin was assembled in order to target human glioblastoma. In vitro, DTEGF13 selectively killed the human glioblastoma cell line U87-luc as well as other human glioblastomas. DTEGF13 fulfilled the requirement of a successful BLT by having greater activity than either of its monospecific counterparts or their mixture proving it necessary to have both ligands on the same single chain molecule. Aggressive brain tumors established intracranially (IC) in nude rats with U87 glioma genetically marked with a firefly luciferase reporter gene were treated with two injections of DTEGF13 using convection enhanced delivery resulting in tumor eradication in 50% of the rats which survived with tumor free status at least 110 days post tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The bispecific DTEGF13 MTD dose was measured at 2 microg/injection or 0.5 microg/kg and toxicity studies indicated safety in this dose. Combination of monospecific DTEGF and DTIL13 did not inhibit tumor growth. ELISA assay indicated that anti-DT antibodies were not generated in normal immunocompetent rats given identical intracranial DTEGF13 therapy. Thus, DTEGF13 is safe and efficacious as an alternative drug for glioblastoma therapy and warrants further study.

  12. D11-Mediated Inhibition of Protein Kinase CK2 Impairs HIF-1α-Mediated Signaling in Human Glioblastoma Cells

    PubMed Central

    Schaefer, Susanne; Svenstrup, Tina H.; Fischer, Mette; Guerra, Barbara

    2017-01-01

    Compelling evidence indicates that protein kinase CK2 plays an important role in many steps of cancer initiation and progression, therefore, the development of effective and cell-permeable inhibitors targeting this kinase has become an important objective for the treatment of a variety of cancer types including glioblastoma. We have recently identified 1,3-dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl]dibenzo(b,d)furan-2,7-diol (D11) as a potent and selective inhibitor of protein kinase CK2. In this study, we have further characterized this compound and demonstrated that it suppresses CK2 kinase activity by mixed type inhibition (KI 7.7 nM, KI′ 42 nM). Incubation of glioblastoma cells with D11 induces cell death and upon hypoxia the compound leads to HIF-1α destabilization. The analysis of differential mRNA expression related to human hypoxia signaling pathway revealed that D11-mediated inhibition of CK2 caused strong down-regulation of genes associated with the hypoxia response including ANGPTL4, CA9, IGFBP3, MMP9, SLC2A1 and VEGFA. Taken together, the results reported here support the notion that including D11 in future treatment regimens might turn out to be a promising strategy to target tumor hypoxia to overcome resistance to radio- and chemotherapy. PMID:28045438

  13. Highly efficient radiosensitization of human glioblastoma and lung cancer cells by a G-quadruplex DNA binding compound

    PubMed Central

    Merle, Patrick; Gueugneau, Marine; Teulade-Fichou, Marie-Paule; Müller-Barthélémy, Mélanie; Amiard, Simon; Chautard, Emmanuel; Guetta, Corinne; Dedieu, Véronique; Communal, Yves; Mergny, Jean-Louis; Gallego, Maria; White, Charles; Verrelle, Pierre; Tchirkov, Andreï

    2015-01-01

    Telomeres are nucleoprotein structures at the end of chromosomes which stabilize and protect them from nucleotidic degradation and end-to-end fusions. The G-rich telomeric single-stranded DNA overhang can adopt a four-stranded G-quadruplex DNA structure (G4). Stabilization of the G4 structure by binding of small molecule ligands enhances radiosensitivity of tumor cells, and this combined treatment represents a novel anticancer approach. We studied the effect of the platinum-derived G4-ligand, Pt-ctpy, in association with radiation on human glioblastoma (SF763 and SF767) and non-small cell lung cancer (A549 and H1299) cells in vitro and in vivo. Treatments with submicromolar concentrations of Pt-ctpy inhibited tumor proliferation in vitro with cell cycle alterations and induction of apoptosis. Non-toxic concentrations of the ligand were then combined with ionizing radiation. Pt-ctpy radiosensitized all cell lines with dose-enhancement factors between 1.32 and 1.77. The combined treatment led to increased DNA breaks. Furthermore, a significant radiosensitizing effect of Pt-ctpy in mice xenografted with glioblastoma SF763 cells was shown by delayed tumor growth and improved survival. Pt-ctpy can act in synergy with radiation for efficient killing of cancer cells at concentrations at which it has no obvious toxicity per se, opening perspectives for future therapeutic applications. PMID:26542881

  14. D11-Mediated Inhibition of Protein Kinase CK2 Impairs HIF-1α-Mediated Signaling in Human Glioblastoma Cells.

    PubMed

    Schaefer, Susanne; Svenstrup, Tina H; Fischer, Mette; Guerra, Barbara

    2017-01-01

    Compelling evidence indicates that protein kinase CK2 plays an important role in many steps of cancer initiation and progression, therefore, the development of effective and cell-permeable inhibitors targeting this kinase has become an important objective for the treatment of a variety of cancer types including glioblastoma. We have recently identified 1,3-dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl]dibenzo(b,d)furan-2,7-diol (D11) as a potent and selective inhibitor of protein kinase CK2. In this study, we have further characterized this compound and demonstrated that it suppresses CK2 kinase activity by mixed type inhibition (KI 7.7 nM, KI' 42 nM). Incubation of glioblastoma cells with D11 induces cell death and upon hypoxia the compound leads to HIF-1α destabilization. The analysis of differential mRNA expression related to human hypoxia signaling pathway revealed that D11-mediated inhibition of CK2 caused strong down-regulation of genes associated with the hypoxia response including ANGPTL4, CA9, IGFBP3, MMP9, SLC2A1 and VEGFA. Taken together, the results reported here support the notion that including D11 in future treatment regimens might turn out to be a promising strategy to target tumor hypoxia to overcome resistance to radio- and chemotherapy.

  15. Icaritin induces apoptotic and autophagic cell death in human glioblastoma cells

    PubMed Central

    Li, Zhaopei; Meng, Xiangwen; Jin, Lin

    2016-01-01

    Background: GBM represents the most aggressive type of glioma which is featured by extremely aggressive invasion and destructive malignancy with a high proliferation rate. The aim of this study was to investigate the in vitro anti-tumor effect of icaritin in human GBM cell line U87. Methods: The effect of icaritin on In vitro cell viability was determined by MTT assay and colony formation assay. The inducing effect of icaritin on cell cycle arrest, mitochondrial membrane potential loss, apoptosis, autophagy and intracellular ROS generation was assessed by flow cytometry. The apoptotic cell death was also confirmed by TUNEL assay. The expression levels of target or marker molecules were examined by western blot. The activity of caspase-3, -8 and -9 was detected with ELISA kit. Results: Our results showed that icaritin significantly induced both caspase-dependent apoptosis and autophagy in human GBM cell line U87. Additionally, our findings revealed that icaritin exerted anti-tumor effect by modulating Stat3 through generating ROS and subsequent activation of AMPK and inhibition of mTOR. Further investigation also showed that icaritin-induced autophagy served as a pro-death function and possibly contributed to icaritin-induced apoptosis. Conclusion: Icaritin potently inhibit the cell growth of human GBM cell line U87 through inducing both caspase-dependent apoptosis and autophagy. Base on our findings, icaritin can be considered as a promising candidate therapeutic agent for treatment of GBM, though further studies are needed. PMID:27904667

  16. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

    PubMed Central

    Friesen, Claudia; Hormann, Inis; Roscher, Mareike; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf; Debatin, Klaus-Michael; Miltner, Erich

    2014-01-01

    Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells. PMID:24626197

  17. Pre-clinical analysis of changes in intra-cellular biochemistry of glioblastoma multiforme (GBM) cells due to c-Myc silencing.

    PubMed

    Rajagopalan, Vishal; Vaidyanathan, Muthukumar; Janardhanam, Vanisree Arambakkam; Bradner, James E

    2014-10-01

    Glioblastoma Multiforme (GBM) is an aggressive form of brain Tumor that has few cures. In this study, we analyze the anti-proliferative effects of a new molecule JQ1 against GBMs induced in Wistar Rats. JQ1 is essentially a Myc inhibitor. c-Myc is also known for altering the biochemistry of a tumor cell. Therefore, the study is intended to analyze certain other oncogenes associated with c-Myc and also the change in cellular biochemistry upon c-Myc inhibition. The quantitative analysis of gene expression gave a co-expressive pattern for all the three genes involved namely; c-Myc, Bcl-2, and Akt. The cellular biochemistry analysis by transmission electron microscopy revealed high glycogen and lipid aggregation in Myc inhibited cells and excessive autophagy. The study demonstrates the role of c-Myc as a central metabolic regulator and Bcl-2 and Akt assisting in extending c-Myc half-life as well as in regulation of autophagy, so as to regulate cell survival on the whole. The study also demonstrates that transient treatment by JQ1 leads to aggressive development of tumor and therefore, accelerating death, emphasizing the importance of dosage fixation, and duration for clinical use in future.

  18. Galectin-3 Guides Intracellular Trafficking of Some Human Serotransferrin Glycoforms*

    PubMed Central

    Carlsson, Michael C.; Bengtson, Per; Cucak, Helena; Leffler, Hakon

    2013-01-01

    Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity. PMID:23926108

  19. Molecular profiling indicates orthotopic xenograft of glioma cell lines simulate a subclass of human glioblastoma.

    PubMed

    Shankavaram, Uma T; Bredel, Markus; Burgan, William E; Carter, Donna; Tofilon, Philip; Camphausen, Kevin

    2012-03-01

    Cell line models have been widely used to investigate glioblastoma multiforme (GBM) pathobiology and in the development of targeted therapies. However, GBM tumours are molecularly heterogeneous and how cell lines can best model that diversity is unknown. In this report, we investigated gene expression profiles of three preclinical growth models of glioma cell lines, in vitro and in vivo as subcutaneous and intracerebral xenografts to examine which cell line model most resembles the clinical samples. Whole genome DNA microarrays were used to profile gene expression in a collection of 25 high-grade glioblastomas, and comparisons were made to profiles of cell lines under three different growth models. Hierarchical clustering revealed three molecular subtypes of the glioblastoma patient samples. Supervised learning algorithm, trained on glioma subtypes predicted the intracerebral cell line model with one glioma subtype (r = 0.68; 95% bootstrap CI -0.41, 0.46). Survival analysis of enriched gene sets (P < 0.05) revealed 19 biological categories (146 genes) belonging to neuronal, signal transduction, apoptosis- and glutamate-mediated neurotransmitter activation signals that are associated with poor prognosis in this glioma subclass. We validated the expression profiles of these gene categories in an independent cohort of patients from 'The Cancer Genome Atlas' project (r = 0.62, 95% bootstrap CI: -0.42, 0.43). We then used these data to select and inhibit a novel target (glutamate receptor) and showed that LY341595, a glutamate receptor specific antagonist, could prolong survival in intracerebral tumour-implanted mice in combination with irradiation, providing an in vivo cell line system of preclinical studies. © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  20. Non–invasive Quantification of Intracellular Sodium in Human Brain using Ultra–High–Field MRI

    PubMed Central

    Fleysher, Lazar; Oesingmann, Niels; Brown, Ryan; Sodickson, Daniel K.; Wiggins, Graham C.; Inglese, Matilde

    2012-01-01

    In vivo sodium magnetic resonance imaging (MRI) provides a measure of the tissue sodium content in living human brain, but current methods do not allow non–invasive quantitative assessment of intracellular sodium concentration (ISC) – the most useful marker of tissue viability. In this study we report the first non–invasive quantitative in–vivo measurement of the ISC and of the intracellular sodium volume fraction (ISVF) in the healthy human brain made possible by measuring the tissue sodium concentration (TSC) and the intracellular sodium molar fraction (ISMF) at ultra–high–field MRI. The method features the use of single–quantum (SQ) and triple–quantum filtered (TQF) imaging at 7 Tesla to separate intracellular and extracellular sodium signals and to provide quantification of ISMF, ISC and ISVF. This novel method allows non–invasive quantitative measurement of ISC and ISVF, opening many possibilities for structural and functional metabolic studies in the healthy and diseased brain. PMID:22714793

  1. β-Arrestin 1 has an essential role in neurokinin-1 receptor-mediated glioblastoma cell proliferation and G2/M phase transition.

    PubMed

    Zhang, Yi-Xin; Li, Xiao-Fang; Yuan, Guo-Qiang; Hu, Hui; Song, Xiao-Yun; Li, Jing-Yi; Miao, Xiao-Kang; Zhou, Tian-Xiong; Yang, Wen-Le; Zhang, Xiao-Wei; Mou, Ling-Yun; Wang, Rui

    2017-05-26

    Glioblastoma is the most common malignant brain tumor and has a poor prognosis. Tachykinin receptor neurokinin-1 (NK1R) is a promising target in glioblastoma therapy because of its overexpression in human glioblastoma. NK1R agonists promote glioblastoma cell growth, whereas NK1R antagonists efficiently inhibit cell growth both in vitro and in vivo However, the molecular mechanisms involved in these effects are incompletely understood. β-Arrestins (ARRBs) serve as scaffold proteins and adapters to mediate intracellular signal transduction. Here we show that the ARRB1-mediated signaling pathway is essential for NK1-mediated glioblastoma cell proliferation. ARRB1 knockdown significantly inhibited NK1-mediated glioblastoma cell proliferation and induced G2/M phase cell cycle arrest. ARRB1 knockdown cells showed remarkable down-regulation of CDC25C/CDK1/cyclin B1 activity. We also demonstrated that ARRB1 mediated prolonged phosphorylation of ERK1/2 and Akt in glioblastoma cells induced by NK1R activation. ERK1/2 and Akt phosphorylation are involved in regulating CDC25C/CDK1/cyclin B1 activity. The lack of long-term ERK1/2 and Akt activation in ARRB1 knockdown cells was at least partly responsible for the delayed cell cycle progression and proliferation. Moreover, we found that ARRB1-mediated ERK1/2 and Akt phosphorylation regulated the transcriptional activity of both NF-κB and AP-1, which were involved in cyclin B1 expression. ARRB1 deficiency increased the sensitivity of glioblastoma cells to the treatment of NK1R antagonists. Taken together, our results suggest that ARRB1 plays an essential role in NK1R-mediated cell proliferation and G2/M transition in glioblastoma cells. Interference with ARRB1-mediated signaling via NK1R may have potential significance for therapeutic strategies targeting glioblastoma. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    PubMed

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  3. Inhibition of T-type Ca2+ channels by endostatin attenuates human glioblastoma cell proliferation and migration

    PubMed Central

    Zhang, Yuan; Zhang, Junhong; Jiang, Dongsheng; Zhang, Dong; Qian, Zhiyuan; Liu, Chunfeng; Tao, Jin

    2012-01-01

    BACKGROUND AND PURPOSE Endostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions. EXPERIMENTAL APPROACH Using cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca2+ channels. KEY RESULTS Extracellular application of ES reversibly inhibited T-type Ca2+ channel currents (T-currents) in U87 cells, whereas L-type Ca2+ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three α1 subunits of T-type Ca2+ channels (CaV3), α1G (CaV3.1), α1H (CaV3.2) and α1I (CaV3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only CaV3.1 and CaV3.2, but not CaV3.3 or CaV1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca2+ channel blocker mibefradil occluded these inhibitory effects of ES. CONCLUSION AND IMPLICATIONS This study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca2+ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells. LINKED ARTICLE This article is commented on by Santoni et al., pp. 1244–1246 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01908.x PMID:22233416

  4. Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse, human glioblastomas in the mouse brain in vivo

    PubMed Central

    Marin-Valencia, Isaac; Yang, Chendong; Mashimo, Tomoyuki; Cho, Steve; Baek, Hyeonman; Yang, Xiao-Li; Rajagopalan, Kartik N.; Maddie, Melissa; Vemireddy, Vamsidhara; Zhao, Zhenze; Cai, Ling; Good, Levi; Tu, Benjamin P.; Hatanpaa, Kimmo J.; Mickey, Bruce E.; Matés, José M.; Pascual, Juan M.; Maher, Elizabeth A.; Malloy, Craig R.; DeBerardinis, Ralph J.; Bachoo, Robert M.

    2012-01-01

    SUMMARY Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused 13C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo. PMID:22682223

  5. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

    PubMed Central

    Carr, Lynn; Bardet, Sylvia M.; Burke, Ryan C.; Arnaud-Cormos, Delia; Leveque, Philippe; O’Connor, Rodney P.

    2017-01-01

    High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules. PMID:28117459

  6. Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells.

    PubMed

    Carr, Lynn; Bardet, Sylvia M; Burke, Ryan C; Arnaud-Cormos, Delia; Leveque, Philippe; O'Connor, Rodney P

    2017-01-24

    High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10 ns, 44 kV/cm pulses at a frequency of 10 Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules.

  7. The effect of gallic acid on cytotoxicity, Ca(2+) homeostasis and ROS production in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes.

    PubMed

    Hsu, Shu-Shong; Chou, Chiang-Ting; Liao, Wei-Chuan; Shieh, Pochuen; Kuo, Daih-Huang; Kuo, Chun-Chi; Jan, Chung-Ren; Liang, Wei-Zhe

    2016-05-25

    Gallic acid, a polyhydroxylphenolic compound, is widely distributed in various plants, fruits and foods. It has been shown that gallic acid passes into blood brain barrier and reaches the brain tissue of middle cerebral artery occlusion rats. However, the effect of gallic acid on Ca(2+) signaling in glia cells is unknown. This study explored whether gallic acid affected Ca(2+) homeostasis and induced Ca(2+)-associated cytotoxicity in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. Gallic acid (20-40 μM) concentration-dependently induced cytotoxicity and intracellular Ca(2+) level ([Ca(2+)]i) increases in DBTRG-05MG cells but not in CTX TNA2 cells. In DBTRG-05MG cells, the Ca(2+) response was decreased by half by removal of extracellular Ca(2+). In Ca(2+)-containing medium, gallic acid-induced Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (2-APB, econazole and SKF96365). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished gallic acid-induced [Ca(2+)]i increases. Conversely, incubation with gallic acid also abolished thapsigargin-induced [Ca(2+)]i increases. Inhibition of phospholipase C with U73122 abolished gallic acid-induced [Ca(2+)]i increases. Gallic acid significantly caused cytotoxicity in DBTRG-05MG cells, which was partially prevented by prechelating cytosolic Ca(2+) with BAPTA-AM. Moreover, gallic acid activated mitochondrial apoptotic pathways that involved ROS production. Together, in DBTRG-05MG cells but not in CTX TNA2 cells, gallic acid induced [Ca(2+)]i increases by causing Ca(2+) entry via 2-APB, econazole and SKF96365-sensitive store-operated Ca(2+) entry, and phospholipase C-dependent release from the endoplasmic reticulum. This Ca(2+) signal subsequently evoked mitochondrial pathways of apoptosis that involved ROS production.

  8. Advanced Magnetic Resonance Imaging of the Physical Processes in Human Glioblastoma

    PubMed Central

    Emblem, Kyrre E.; Andronesi, Ovidiu; Rosen, Bruce

    2014-01-01

    The most common malignant primary brain tumor, glioblastoma (GBM) is a devastating disease with a grim prognosis. Patient survival is typically less than 2 years and fewer than 10% of patients survive more than 5 years. Magnetic Resonance Imaging (MRI) can have great utility in the diagnosis, grading and management of patients with GBM as many of the physical manifestations of the pathological processes in GBM can be visualized and quantified using MRI. Newer MRI techniques such as dynamic contrast enhanced (DCE) and dynamic susceptibility contrast (DSC) MRI provide functional information regarding the tumor hemodynamic status. Diffusion MRI can shed light on tumor cellularity and the disruption of white matter tracts in the proximity of tumors. MR spectroscopy can be used to study new tumor tissue markers such as IDH mutations. MRI is helping to noninvasively explore the link between the molecular basis of gliomas and the imaging characteristics of their physical processes. We will review several approaches to MR-based imaging and discuss the potential for these techniques to quantify the physical processes in glioblastoma including tumor cellularity and vascularity, metabolite expression, and patterns of tumor growth and recurrence. We will conclude with challenges and opportunities for further research in applying physical principles to better understand the biological process in this deadly disease. PMID:25183787

  9. Interindividual differences in anticancer drug cytotoxicity in primary human glioblastoma cells.

    PubMed

    Pédeboscq, Stéphane; L'Azou, Béatrice; Liguoro, Dominique; Pometan, Jean-Paul; Cambar, Jean

    2007-01-01

    Glioblastoma multiforme is a malignant astrocytic tumor characterized by rapid growth, extensive invasiveness and high vascularity. Despite advances in surgical techniques and in the development of new protocols in radio- and chemotherapy, the prognosis for patients suffering from this malignancy remains poor. Since the clinical response to chemotherapy varies greatly owing to different interindividual gene expression profiles, it would be of considerable interest to develop an in vitro model able to evaluate anticancer drug toxicity and the effectiveness of therapeutic strategies on cells obtained from individual patients. In the protocol for obtaining primary cultures of glioblastoma cells described in this report, a confluent monolayer of cells can be obtained within 1 or 2 weeks. A complementary immunocytochemical assay using glial fibrillary acidic protein (GFAP) to reliably mark glial cells confirms the glial origin of the cultured cells. A cytotoxicity test based on mitochondrial activity is then used to evaluate in vitro drug efficacy. Cell dedifferentiation as evidenced by loss of GFAP expression after a few passages requires determination of drug toxicity before the fourth passage. Data show a wide range of response to temozolomide (1000 microM) after 72 h with 24-81% cell death depending on patients. Results presented confirm the heterogeneity of response to anticancer drugs between the patients and methods described allow to carry out cytotoxicity studies in order to determine the individualized most effective treatment.

  10. Downregulation of solute carriers of glutamate in gliosomes and synaptosomes may explain local brain metastasis in anaplastic glioblastoma.

    PubMed

    Tong, Huaiyu; Yu, Xinguang; Lu, Xuechun; Wang, Peng

    2015-04-01

    Advanced grades of glioblastoma are highly aggressive, especially in terms of multisite spread within the brain or even to distant sites at the spinal cord. In advanced grades of glioblastoma, glutamate and glutamine are reported to be increased in concentration in the extracellular fluid. It has been reported that glutamate acts as an extracellular signaling molecule for facilitating local spread of advanced grades of glioblastoma. In the present study, we aimed to examine whether glutamate uptake mechanisms is impaired in advanced glioblastoma. The possible downregulated mechanisms of glutamate uptake would facilitate persistence of glutamate in the extracellular environment, rather than intracellular uptake. We obtained biobanked human specimens of glioblastoma and tested expression of proteins belonging to the solute carrier families of proteins that are known to function as membrane-located excitatory amino acid like glutamate transporters. The present study provides preliminary evidence of the downregulation of membrane expression of excitatory amino acid transporters solute carrier family 1 member 3 (SLC1A3) and its palmitoylated form in gliosomes, as well as SLC1A2 in the glio-synaptosomes. Compounds like riluzole used in the treatment of amyotrophic lateral sclerosis and the antibiotic ceftriaxone have the potential to facilitate glutamate uptake. These medications may be examined as adjunct chemotherapy in the massively aggressive tumor glioblastoma multiforme. © 2015 International Union of Biochemistry and Molecular Biology.

  11. SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

    p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6 h infusion of BPA, instead of a 2 h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6 h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500 nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na-K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)- molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN- from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6 h exposures

  12. Honokiol inhibits U87MG human glioblastoma cell invasion through endothelial cells by regulating membrane permeability and the epithelial-mesenchymal transition.

    PubMed

    Joo, Young Nak; Eun, So Young; Park, Sang Won; Lee, Jae Heun; Chang, Ki Churl; Kim, Hye Jung

    2014-01-01

    Glioblastoma is one of the most lethal and prevalent malignant human brain tumors, with aggressive proliferation and highly invasive properties. There is still no effective cure for patients with glioblastoma. Honokiol, derived from Magnolia officinalis, can cross the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), making it a strong candidate for an effective drug for the treatment of brain tumors, including glioblastoma. In our previous study, we demonstrated that honokiol effectively induced apoptotic cell death in glioblastoma. Metastasis poses the largest problem to cancer treatment and is the primary cause of death in cancer patients. Thus, in this study, we investigated the effect of honokiol on the cell invasion process of U87MG human glioblastoma cells through brain microvascular endothelial cells (BMECs) and its possible mechanisms. Honokiol dose-dependently inhibited TNF-α-induced VCAM-1 expression in BMECs and adhesion of U87MG to BMECs. Moreover, honokiol effectively blocked U87MG invasion through BMEC-Matrigel-coated transwell membranes. Increased phosphorylation of VE-cadherin and membrane permeability by TNF-α were suppressed by honokiol in BMECs. Furthermore, we investigated the effect of honokiol on the epithelial-mesenchymal transition (EMT) in U87MG cells. Honokiol reduced the expression levels of Snail, N-cadherin and β-catenin, which are mesenchymal markers, but increased E-cadherin, an epithelial marker. In conclusion, these results suggest that honokiol inhibits metastasis by targeting the interaction between U87MG and BMECs, regulating the adhesion of U87MG to BMECs by inhibiting VCAM-1, and regulating the invasion of U87MG through BMECs by reducing membrane permeability and EMT processes of U87MG cells.

  13. Chemical Library Screening and Structure-Function Relationship Studies Identify Bisacodyl as a Potent and Selective Cytotoxic Agent Towards Quiescent Human Glioblastoma Tumor Stem-Like Cells

    PubMed Central

    Mameri, Samir; Dong, Jihu; Salomé, Christophe; Chen, Wanyin; El-Habr, Elias A.; Bousson, Fanny; Sy, Mohamadou; Obszynski, Julie; Boh, Alexandre; Villa, Pascal; Assad Kahn, Suzana; Didier, Bruno; Bagnard, Dominique; Junier, Marie-Pierre; Chneiweiss, Hervé; Haiech, Jacques; Hibert, Marcel; Kilhoffer, Marie-Claude

    2015-01-01

    Cancer stem-like cells reside in hypoxic and slightly acidic tumor niches. Such microenvironments favor more aggressive undifferentiated phenotypes and a slow growing "quiescent state" which preserves them from chemotherapeutic agents that essentially target proliferating cells. Our objective was to identify compounds active on glioblastoma stem-like cells, including under conditions that mimick those found in vivo within this most severe and incurable form of brain malignancy. We screened the Prestwick Library to identify cytotoxic compounds towards glioblastoma stem-like cells, either in a proliferating state or in more slow-growing "quiescent" phenotype resulting from non-renewal of the culture medium in vitro. Compound effects were assessed by ATP-level determination using a cell-based assay. Twenty active molecules belonging to different pharmacological classes have thus been identified. Among those, the stimulant laxative drug bisacodyl was the sole to inhibit in a potent and specific manner the survival of quiescent glioblastoma stem-like cells. Subsequent structure-function relationship studies led to identification of 4,4'-dihydroxydiphenyl-2-pyridyl-methane (DDPM), the deacetylated form of bisacodyl, as the pharmacophore. To our knowledge, bisacodyl is currently the only known compound targeting glioblastoma cancer stem-like cells in their quiescent, more resistant state. Due to its known non-toxicity in humans, bisacodyl appears as a new potential anti-tumor agent that may, in association with classical chemotherapeutic compounds, participate in tumor eradication. PMID:26270679

  14. Chemical Library Screening and Structure-Function Relationship Studies Identify Bisacodyl as a Potent and Selective Cytotoxic Agent Towards Quiescent Human Glioblastoma Tumor Stem-Like Cells.

    PubMed

    Zeniou, Maria; Fève, Marie; Mameri, Samir; Dong, Jihu; Salomé, Christophe; Chen, Wanyin; El-Habr, Elias A; Bousson, Fanny; Sy, Mohamadou; Obszynski, Julie; Boh, Alexandre; Villa, Pascal; Assad Kahn, Suzana; Didier, Bruno; Bagnard, Dominique; Junier, Marie-Pierre; Chneiweiss, Hervé; Haiech, Jacques; Hibert, Marcel; Kilhoffer, Marie-Claude

    2015-01-01

    Cancer stem-like cells reside in hypoxic and slightly acidic tumor niches. Such microenvironments favor more aggressive undifferentiated phenotypes and a slow growing "quiescent state" which preserves them from chemotherapeutic agents that essentially target proliferating cells. Our objective was to identify compounds active on glioblastoma stem-like cells, including under conditions that mimick those found in vivo within this most severe and incurable form of brain malignancy. We screened the Prestwick Library to identify cytotoxic compounds towards glioblastoma stem-like cells, either in a proliferating state or in more slow-growing "quiescent" phenotype resulting from non-renewal of the culture medium in vitro. Compound effects were assessed by ATP-level determination using a cell-based assay. Twenty active molecules belonging to different pharmacological classes have thus been identified. Among those, the stimulant laxative drug bisacodyl was the sole to inhibit in a potent and specific manner the survival of quiescent glioblastoma stem-like cells. Subsequent structure-function relationship studies led to identification of 4,4'-dihydroxydiphenyl-2-pyridyl-methane (DDPM), the deacetylated form of bisacodyl, as the pharmacophore. To our knowledge, bisacodyl is currently the only known compound targeting glioblastoma cancer stem-like cells in their quiescent, more resistant state. Due to its known non-toxicity in humans, bisacodyl appears as a new potential anti-tumor agent that may, in association with classical chemotherapeutic compounds, participate in tumor eradication.

  15. Morphine modulates doxorubicin uptake and improves efficacy of chemotherapy in an intracranial xenograft model of human glioblastoma

    PubMed Central

    da Ros, Martina; Iorio, Anna Lisa; Consolante, Dario; Cardile, Francesco; Muratori, Monica; Fantappiè, Ornella; Lucchesi, Maurizio; Guidi, Milena; Pisano, Claudio; Sardi, Iacopo

    2016-01-01

    Morphine may alter the permeability of Blood-Brain Barrier (BBB), enhancing the access of molecules normally unable to cross it, as Doxorubicin (Dox). In addition, morphine seems to mediate the uptake of Dox into the brain by its reduced efflux mediated by P-glycoprotein (P-gp). We evaluated the antitumor efficacy of Dox plus morphine treatment by an orthotopic glioblastoma xenograft model. Foxn1 mice were injected with U87MG-luc cells in the left lobe of the brain and treated with Dox (5 mg/kg and 2.5 mg/kg, weekly) with or without morphine pretreatment (10 mg/kg, weekly). Bioluminescence imaging (BLI) was used to monitoring tumor growth and response to therapy. Additionally, we investigated the role of morphine on the uptake of Dox by MDCKII cells transfected with human MDR1 gene encoding for P-gp. The data demonstrate that only Dox 5 mg/kg determined a significant tumor regression while the lower dose (2.5 mg/kg) was not effective. However, if combined with morphine, the group treated with Dox 2.5 mg/kg showed a decreasing tumor growth. The average BLI for Dox 2.5 mg/kg plus morphine was 5 fold lower than Dox 2.5 mg/kg alone (P=0.0053) and 8 fold lower than vehicle (P=0.0004). Additionally, Dox increased in MDCKII-P-gp transfected cells only in the presence of morphine with a significantly higher level comparing control group (3.84) vs Dox plus morphine group (12.29, P<0.05). Our results indicate that Dox alone and in combination with morphine appear to be effective in controlling the growth of glioblastoma in a xenograft mouse model. PMID:27152241

  16. Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma.

    PubMed

    Attia, Amalina Binte Ebrahim; Ho, Chris Jun Hui; Chandrasekharan, Prashant; Balasundaram, Ghayathri; Tay, Hui Chien; Burton, Neal C; Chuang, Kai-Hsiang; Ntziachristos, Vasilis; Olivo, Malini

    2016-07-01

    Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.

  17. Most human non-GCIMP glioblastoma subtypes evolve from a common proneural-like precursor glioma

    PubMed Central

    Ozawa, Tatsuya; Riester, Markus; Cheng, Yu-Kang; Huse, Jason T; Squatrito, Massimo; Helmy, Karim; Charles, Nikki; Michor, Franziska; Holland, Eric C.

    2014-01-01

    SUMMARY To understand the relationships between the non-GCIMP glioblastoma (GBM) subgroups, we performed mathematical modeling to predict the temporal sequence of driver events during tumorigenesis. The most common order of evolutionary events is 1) chromosome (chr) 7 gain and chr10 loss, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes. We then developed a computational methodology to identify drivers of broad copy number changes, identifying PDGFA (chr7) and PTEN (chr10) as driving initial non-disjunction events. These predictions were validated using mouse modeling, showing that PDGFA is sufficient to induce proneural-like gliomas, and additional NF1 loss converts proneural to the mesenchymal subtype. Our findings suggest most non-GCIMP-mesenchymal GBMs arise as, and evolve from, a proneural-like precursor. PMID:25117714

  18. PKCδ activated by c-MET enhances infiltration of human glioblastoma cells through NOTCH2 signaling

    PubMed Central

    Kang, Seok-Gu; Kim, Rae-Kwon; Cui, Yan-Hong; Lee, Hae-June; Kim, Min-Jung; Lee, Jae-Seong; Kim, In-Gyu; Suh, Yongjoon; Lee, Su-Jae

    2016-01-01

    Poor prognosis of glioblastoma (GBM) is attributable to the propensity of tumor cells to infiltrate into the brain parenchyma. Protein kinase C (PKC) isozymes are highly expressed or aberrantly activated in GBM. However, how this signaling node translates to GBM cell invasiveness remains unknown. Here, we report that among PKC isoforms, PKCδ is strongly associated with infiltration of GBM cells. Notably, PKCδ enhanced Tyr418 phosphorylation of the non-receptor tyrosine kinase SRC, which in turn activated STAT3 and subsequent NOTCH2 signaling, ultimately leading to GBM cell invasiveness. Furthermore, we showed that PKCδ was aberrantly activated in GBM cells by c-MET, a receptor tyrosine kinase hyperactivated in GBM. In agreement, inhibition either component in the c-MET/PKCδ/SRC/STAT3 signaling axis effectively blocked the NOTCH2 signaling and invasiveness of GBM cells. Taken together, our findings shed a light on the signaling mechanisms behind the constitutive activation of PKCδ signaling in GBM. PMID:26700818

  19. Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line

    PubMed Central

    2013-01-01

    Background Propolis is a honey bee product which contains many active compounds, such as CAPE or chrysin, and has many beneficial activities. Recently, its anti-tumor properties have been discussed. We have tested whether the ethanolic extract of propolis (EEP) interferes with temozolomide (TMZ) to inhibit U87MG cell line growth. Methods The U87MG glioblastoma cell line was exposed to TMZ (10-100 μM), EEP (10-100 μg/ml) or a mixture of TMZ and EEP during 24, 48 or 72 hours. The cell division was examined by the H3-thymidine incorporation, while the western blot method was used for detection of p65 subunit of NF-κB and ELISA test to measure the concentration of its p50 subunit in the nucleus. Results We have found that both, TMZ and EEP administrated alone, had a dose- and time-dependent inhibitory effect on the U87MG cell line growth, which was manifested by gradual reduction of cell viability and alterations in proliferation rate. The anti-tumor effect of TMZ (20 μM) was enhanced by EEP, which was especially well observed after a short time of exposition, where simultaneous usage of TMZ and EEP resulted in a higher degree of growth inhibition than each biological factor used separately. In addition, cells treated with TMZ presented no changes in NF-κB activity in prolonged time of treatment and EEP only slightly reduced the nuclear translocation of this transcription factor. In turn, the combined incubation with TMZ and EEP led to an approximately double reduction of NF-κB nuclear localization. Conclusions We conclude that EEP presents cytotoxic properties and may cooperate with TMZ synergistically enhancing its growth inhibiting activity against glioblastoma U87MG cell line. This phenomenon may be at least partially mediated by a reduced activity of NF-κB. PMID:23445763

  20. Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line.

    PubMed

    Markiewicz-Żukowska, Renata; Borawska, Maria H; Fiedorowicz, Anna; Naliwajko, Sylwia K; Sawicka, Diana; Car, Halina

    2013-02-27

    Propolis is a honey bee product which contains many active compounds, such as CAPE or chrysin, and has many beneficial activities. Recently, its anti-tumor properties have been discussed. We have tested whether the ethanolic extract of propolis (EEP) interferes with temozolomide (TMZ) to inhibit U87MG cell line growth. The U87MG glioblastoma cell line was exposed to TMZ (10-100 μM), EEP (10-100 μg/ml) or a mixture of TMZ and EEP during 24, 48 or 72 hours. The cell division was examined by the H3-thymidine incorporation, while the western blot method was used for detection of p65 subunit of NF-κB and ELISA test to measure the concentration of its p50 subunit in the nucleus. We have found that both, TMZ and EEP administrated alone, had a dose- and time-dependent inhibitory effect on the U87MG cell line growth, which was manifested by gradual reduction of cell viability and alterations in proliferation rate. The anti-tumor effect of TMZ (20 μM) was enhanced by EEP, which was especially well observed after a short time of exposition, where simultaneous usage of TMZ and EEP resulted in a higher degree of growth inhibition than each biological factor used separately. In addition, cells treated with TMZ presented no changes in NF-κB activity in prolonged time of treatment and EEP only slightly reduced the nuclear translocation of this transcription factor. In turn, the combined incubation with TMZ and EEP led to an approximately double reduction of NF-κB nuclear localization. We conclude that EEP presents cytotoxic properties and may cooperate with TMZ synergistically enhancing its growth inhibiting activity against glioblastoma U87MG cell line. This phenomenon may be at least partially mediated by a reduced activity of NF-κB.

  1. Pharmacologic blockade of FAK autophosphorylation decreases human glioblastoma tumor growth and synergizes with temozolomide.

    PubMed

    Golubovskaya, Vita M; Huang, Grace; Ho, Baotran; Yemma, Michael; Morrison, Carl D; Lee, Jisook; Eliceiri, Brian P; Cance, William G

    2013-02-01

    Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide.

  2. Analysis of physical characteristics of Tumor Treating Fields for human glioblastoma.

    PubMed

    Lok, Edwin; San, Pyay; Hua, Van; Phung, Melissa; Wong, Eric T

    2017-06-01

    Tumor Treating Fields (TTFields) therapy is an approved treatment that has known clinical efficacy against recurrent and newly diagnosed glioblastoma. However, the distribution of the electric fields and the corresponding pattern of energy deposition in the brain are poorly understood. To evaluate the physical parameters that may influence TTFields, postacquisition MP-RAGE, T1 and T2 MRI sequences from a responder with a right parietal glioblastoma were anatomically segmented and then solved using finite-element method to determine the distribution of the electric fields and rate of energy deposition at the gross tumor volume (GTV) and other intracranial structures. Electric field-volume histograms (EVH) and specific absorption rate-volume histograms (SARVH) were constructed to numerically evaluate the relative and/or absolute magnitude volumetric differences between models. The electric field parameters EAUC , VE150 , E95% , E50% , and E20% , as well as the SAR parameters SARAUC , VSAR7.5 , SAR95% , SAR50% , and SAR20% , facilitated comparisons between models derived from various conditions. Specifically, TTFields at the GTV were influenced by the dielectric characteristics of the adjacent tissues as well as the GTV itself, particularly the presence or absence of a necrotic core. The thickness of the cerebrospinal fluid on the convexity of the brain and the geometry of the tumor were also relevant factors. Finally, the position of the arrays also influenced the electric field distribution and rate of energy deposition in the GTV. Using EVH and SARVH, a personalized approach for TTFields treatment can be developed when various patient-related and tumor-related factors are incorporated into the planning procedure. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  3. Magnolol and honokiol exert a synergistic anti-tumor effect through autophagy and apoptosis in human glioblastomas.

    PubMed

    Cheng, Yu-Chen; Hueng, Dueng-Yuan; Huang, Hua-Yin; Chen, Jang-Yi; Chen, Ying

    2016-05-17

    Glioblastoma (GBM) is a malignant brain tumor associated with a high mortality rate. The aim of this study is to investigate the synergistic effects of honokiol (Hono) and magnolol (Mag), extracted from Magnolia officinalis, on cytotoxicity and inhibition of human GBM tumor progression in cellular and animal models. In comparison with Hono or Mag alone, co-treatment with Hono and Mag (Hono-Mag) decreased cyclin A, D1 and cyclin-dependent kinase 2, 4, 6 significantly, leading to cell cycle arrest in U87MG and LN229 human glioma cells. In addition, phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, and Ki67 were decreased after Hono-Mag treatment, showing proliferation inhibition. Hono-Mag treatment also reduced p-p38 and p-JNK but elevated p-ERK expression. Besides, Hono-Mag treatment induced autophagy and intrinsic and extrinsic apoptosis. Both ERK and autophagy inhibitors enhanced Hono-Mag-induced apoptosis in LN229 cells, indicating a rescuer role of ERK. In human GBM orthotopic xenograft model, the Hono-Mag treatment inhibited the tumor progression and induced apoptosis more efficiently than Temozolomide, Hono, or Mag group. In conclusion, the Hono-Mag exerts a synergistic anti-tumor effect by inhibiting cell proliferation and inducing autophagy and apoptosis in human GBM cells. The Hono-Mag may be applied as an adjuvant therapy to improve the therapeutic efficacy of GBM treatment.

  4. Nanotechnology Applications for Glioblastoma

    PubMed Central

    Nduom, Edjah; Bouras, Alexandros; Kaluzova, Milota; Hadjipanayis, Costas G.

    2012-01-01

    Synopsis Glioblastoma remains one of the most difficult cancers to treat and represents the most common primary malignancy of the brain. While conventional treatments have found modest success in reducing the initial tumor burden, infiltrating cancer cells beyond the main mass are responsible for tumor recurrence and ultimate patient demise. Targeting the residual infiltrating cancer cells requires the development of new treatment strategies. The emerging field of cancer nanotechnology holds much promise in the use of multifunctional nanoparticles for the imaging and targeted therapy of GBM.. Nanoparticles have emerged as potential “theranostic” agents that can permit the diagnosis and therapeutic treatment of GBM tumors. A recent human clinical trial with magnetic nanoparticles has provided feasibility and efficacy data for potential treatment of GBM patients with thermotherapy. Here we examine the current state of nanotechnology in the treatment of glioblastoma and interesting directions of further study. PMID:22748656

  5. Human islet amyloid polypeptide expression in COS-1 cells. A model of intracellular amyloidogenesis.

    PubMed Central

    O'Brien, T. D.; Butler, P. C.; Kreutter, D. K.; Kane, L. A.; Eberhardt, N. L.

    1995-01-01

    Non-insulin-dependent diabetes mellitus is characterized by concurrent loss of beta-cells and deposition of islet amyloid derived from islet amyloid polypeptide (IAPP). We have previously demonstrated that IAPP-derived amyloid forms intracellularly in humans with chronic excess insulin expression (eg, insulinoma and insulin receptor antibody-induced insulin resistance). To determine whether overexpression of IAPP results in intracellular amyloid in mammalian cells, we transfected COS cells with vectors expressing amyloidogenic human IAPP or non-amyloidogenic rat IAPP. Transfected COS-1 cells secreted comparable amounts of human IAPP and rat IAPP (2.1 to 2.8 nmol/L/48 hours). After 96 hours, 90% of cells expressing human IAPP contained amyloid fibrils and were degenerating or dead, whereas cells transfected with rat IAPP lacked amyloid and were viable. Thus, overexpression of human IAPP can result in intracellular amyloid formation that is associated with cell death, suggesting that intracellular amyloid may play a role in beta-cell loss in non-insulin-dependent diabetes mellitus. Images Figure 1 Figure 2 PMID:7677175

  6. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells

    PubMed Central

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting

    2015-01-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  7. 18F-FET and 18F-FCH uptake in human glioblastoma T98G cell lines

    PubMed Central

    Persico, Marco Giovanni; Buroni, Federica Eleonora; Pasi, Francesca; Aprile, Carlo; Nano, Rosanna; Hodolic, Marina

    2016-01-01

    Abstract Background Despite complex treatment of surgery, radiotherapy and chemotherapy, high grade gliomas often recur. Differentiation between post-treatment changes and recurrence is difficult. 18F-methyl-choline (18F-FCH) is frequently used in staging and detection of recurrent prostate cancer disease as well as some brain tumours; however accumulation in inflammatory tissue limits its specificity. The 18F-ethyl-tyrosine (18F-FET) shows a specific uptake in malignant cells, resulting from increased expression of amino acid transporters or diffusing through the disrupted blood-brain barrier. 18F-FET exhibits lower uptake in machrophages and other inflammatory cells. Aim of this study was to evaluate 18F-FCH and 18F-FET uptake by human glioblastoma T98G cells. Material and methods Human glioblastoma T98G or human dermal fibroblasts cells, seeded at a density to obtain 2 × 105 cells per flask when radioactive tracers were administered, grew adherent to the plastic surface at 37°C in 5% CO2 in complete medium. Equimolar amounts of radiopharmaceuticals were added to cells for different incubation times (20 to 120 minutes) for 18F-FCH and 18F-FET respectively. The cellular radiotracer uptake was determined with a gamma counter. All experiments were carried out in duplicate and repeated three times. The uptake measurements are expressed as the percentage of the administered dose of tracer per 2 × 105 cells. Data (expressed as mean values of % uptake of radiopharmaceuticals) were compared using parametric or non-parametric tests as appropriate. Differences were regarded as statistically significant when p<0.05. Results A significant uptake of 18F-FCH was seen in T98G cells at 60, 90 and 120 minutes. The percentage uptake of 18F-FET in comparison to 18F-FCH was lower by a factor of more than 3, with different kinetic curves.18F-FET showed a more rapid initial uptake up to 40 minutes and 18F-FCH showed a progressive rise reaching a maximum after 90 minutes

  8. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells.

    PubMed

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting; Chen, Yu-Jen

    2015-03-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  9. Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-β) Signaling Transducers in Human Glioblastoma.

    PubMed

    Akbari, Abolfazl; Farahnejad, Zohreh; Akhtari, Javad; Abastabar, Mahdi; Mobini, Gholam Reza; Mehbod, Amir Seied Ali

    2016-05-01

    It has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies. We examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β). A human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test. We reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 μg/mL and 2 μg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner. We suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation.

  10. Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-β) Signaling Transducers in Human Glioblastoma

    PubMed Central

    Akbari, Abolfazl; Farahnejad, Zohreh; Akhtari, Javad; Abastabar, Mahdi; Mobini, Gholam Reza; Mehbod, Amir Seied Ali

    2016-01-01

    Background It has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies. Objectives We examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β). Materials and Methods A human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test. Results We reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 μg/mL and 2 μg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner. Conclusions We suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation. PMID:27540448

  11. The effect of methylprednisolone on intracellular calcium of normal and dystrophic human skeletal muscle cells.

    PubMed

    Vandebrouck, C; Imbert, N; Duport, G; Cognard, C; Raymond, G

    1999-07-09

    Clinical trials have shown that a glucocorticoid, the methyiprednisolone (PDN), has a beneficial effect on muscle strength and function in Duchenne muscular dystrophy (DMD) patients. The aim of this study was to test if the effect of PDN could be mediated via a possible action on intracellular calcium. The intracellular calcium activity, at rest and during calcium mobilizing drug superfusion protocols was recorded in normal and dystrophic human cocultured muscle cells. PDN (10 microM) pretreatment induced an elevation of the resting calcium concentration of 51, 34 and 38% in proliferating normal myoblasts, DMD myoblasts and DMD myotubes, respectively, while normal myotubes resting [Ca2+]i was not altered.

  12. Neural Stem Cells and Glioblastoma

    PubMed Central

    Rispoli, Rossella; Conti, Carlo; Celli, Paolo; Caroli, Emanuela; Carletti, Sandro

    2014-01-01

    Summary Glioblastoma multiforme represents one of the most common brain cancers with a rather heterogeneous cellular composition, as indicated by the term “multiforme". Recent reports have described the isolation and identification of cancer neural stem cells from human adult glioblastoma multiforme, which possess the capacity to establish, sustain, and expand these tumours, even under the challenging settings posed by serial transplantation experiments. Our study focused on the distribution of neural cancer stem cells inside the tumour. The study is divided into three phases: removal of tumoral specimens in different areas of the tumour (centre, periphery, marginal zone) in an operative room equipped with a 1.5 T scanner; isolation and characterization of neural cancer stem cells from human adult glioblastoma multiforme; identification of neural cancer stem cell distribution inside the tumour. PMID:24750704

  13. Kukoamine A inhibits human glioblastoma cell growth and migration through apoptosis induction and epithelial-mesenchymal transition attenuation

    PubMed Central

    Wang, Qiaoping; Li, Haiyan; Sun, Zhen; Dong, Lihua; Gao, Ling; Liu, Chunlan; Wang, Xiujie

    2016-01-01

    Cortex lycii radicis is the dried root bark of Lycium chinense, a traditional Chinese herb used in multiple ailments. The crude extract of Cortex lycii radicis has growth inhibition effect on GBM cells. Kukoamine A (KuA) is a spermine alkaloid derived from it. KuA possesses antioxidant, anti-inflammatory activities, but its anticancer activity is unknown. In this study, the growth and migration inhibition effect of KuA on human GBM cells and the possible mechanism of its activity were investigated. After KuA treatment, proliferation and colony formation of GBM cells were decreased significantly; apoptotic cells were increased; the cell cycle was arrested G0/G1 phase; the migration and invasion were decreased, the growth of tumors initiated from GBM cells was inhibited significantly; the expressions of 5-Lipoxygenase (5-LOX) were decreased, apoptotic proteins, Bax and caspase-3 were increased, and antiapoptotic protein Bcl-2 was decreased significantly; The expressions of CCAAT/enhancer binding protein β (C/EBPβ), N-cadherin, vimentin, twist and snail+slug were decreased significantly, while the expression of E-cadherin was increased significantly in KuA treated GBM cells and tumor tissues. KuA inhibited human glioblastoma cell growth and migration in vitro and in vivo through apoptosis induction and epithelial-mesenchymal transition attenuation by downregulating expressions of 5-LOX and C/EBPβ. PMID:27824118

  14. Methylglyoxal (MGO) inhibits proliferation and induces cell death of human glioblastoma multiforme T98G and U87MG cells.

    PubMed

    Paul-Samojedny, Monika; Łasut, Barbara; Pudełko, Adam; Fila-Daniłow, Anna; Kowalczyk, Małgorzata; Suchanek-Raif, Renata; Zieliński, Michał; Borkowska, Paulina; Kowalski, Jan

    2016-05-01

    Glioblastoma multiforme (GBM) is the most malignant and invasive human brain tumor and it is characterized by a poor prognosis and short survival time. Current treatment strategies for GBM using surgery, chemotherapy and/or radiotherapy are ineffective. Thus new therapeutic strategies to target GBM are urgently needed. The effect of methylglyoxal (MGO) on the cell cycle, cell death and proliferation of human GBM cells was investigated. The T98G and U87MG cell lines were cultured in modified EMEM supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were exposed to methylglyoxal (0.025mM) per 72h. The influence of MGO on T98G and U87MG cell cycle, proliferation and apoptosis was evaluated as well. Cell cycle phase distribution, proliferation, apoptosis were analyzed by flow cytometry. MGO causes changes in cell cycle and induces accumulation of G1/G0-phase cells and reduced fraction of cells in S and G2/M phases. We have also observed inhibition of cell proliferation and induction of apoptosis in cancer cells. We have also revealed that MGO induces senescence of U87MG but not T98G cells, but further studies are necessary in order to clarify and check mechanism of action of methylglyoxal and it Is a positive phenomenon for the treatment of GBM.

  15. Acetate is a Bioenergetic Substrate for Human Glioblastoma and Brain Metastases

    PubMed Central

    Mashimo, Tomoyuki; Pichumani, Kumar; Vemireddy, Vamsidhara; Hatanpaa, Kimmo J.; Singh, Dinesh Kumar; Sirasanagandla, Shyam; Nannepaga, Suraj; Piccirillo, Sara G.; Kovacs, Zoltan; Foong, Chan; Huang, Zhiguang; Barnett, Samuel; Mickey, Bruce E.; DeBerardinis, Ralph J.; Tu, Benjamin P.; Maher, Elizabeth A.; Bachoo, Robert M.

    2015-01-01

    Glioblastomas and brain metastases are highly proliferative brain tumors with short survival times. Previously, using 13C-NMR analysis of brain tumors resected from patients during infusion of 13C-glucose, we demonstrated that there is robust oxidation of glucose in the citric acid cycle, yet glucose contributes less than 50% of the carbons to the acetyl-CoA pool. Here we show that primary and metastatic mouse orthotopic brain tumors have the capacity to oxidize [1,2-13C]acetate and can do so simultaneously with [1,6-13C]glucose oxidation. The tumors do not oxidize [U-13C]glutamine. In vivo oxidation of [1,2-13C]acetate was validated in brain tumor patients and was correlated with expression of acetyl-CoA synthetase enzyme 2, ACSS2. Together the data demonstrate a strikingly common metabolic phenotype in diverse brain tumors that includes the ability to oxidize acetate in the citric acid cycle. This adaptation may be important for meeting the high biosynthetic and bioenergetic demands of malignant growth. PMID:25525878

  16. Static magnetic fields modulate X-ray-induced DNA damage in human glioblastoma primary cells.

    PubMed

    Teodori, Laura; Giovanetti, Anna; Albertini, Maria Cristina; Rocchi, Marco; Perniconi, Barbara; Valente, Maria Giovanna; Coletti, Dario

    2014-03-01

    Although static magnetic fields (SMFs) are used extensively in the occupational and medical fields, few comprehensive studies have investigated their possible genotoxic effect and the findings are controversial. With the advent of magnetic resonance imaging-guided radiation therapy, the potential effects of SMFs on ionizing radiation (IR) have become increasingly important. In this study we focused on the genotoxic effect of 80 mT SMFs, both alone and in combination with (i.e. preceding or following) X-ray (XR) irradiation, on primary glioblastoma cells in culture. The cells were exposed to: (i) SMFs alone; (ii) XRs alone; (iii) XR, with SMFs applied during recovery; (iv) SMFs both before and after XR irradiation. XR-induced DNA damage was analyzed by Single Cell Gel Electrophoresis assay (comet assay) using statistical tools designed to assess the tail DNA (TD) and tail length (TL) as indicators of DNA fragmentation. Mitochondrial membrane potential, known to be affected by IR, was assessed using the JC-1 mitochondrial probe. Our results showed that exposure of cells to 5 Gy of XR irradiation alone led to extensive DNA damage, which was significantly reduced by post-irradiation exposure to SMFs. The XR-induced loss of mitochondrial membrane potential was to a large extent averted by exposure to SMFs. These data suggest that SMFs modulate DNA damage and/or damage repair, possibly through a mechanism that affects mitochondria.

  17. Static magnetic fields modulate X-ray-induced DNA damage in human glioblastoma primary cells

    PubMed Central

    Teodori, Laura; Giovanetti, Anna; Albertini, Maria Cristina; Rocchi, Marco; Perniconi, Barbara; Valente, Maria Giovanna; Coletti, Dario

    2014-01-01

    Although static magnetic fields (SMFs) are used extensively in the occupational and medical fields, few comprehensive studies have investigated their possible genotoxic effect and the findings are controversial. With the advent of magnetic resonance imaging-guided radiation therapy, the potential effects of SMFs on ionizing radiation (IR) have become increasingly important. In this study we focused on the genotoxic effect of 80 mT SMFs, both alone and in combination with (i.e. preceding or following) X-ray (XR) irradiation, on primary glioblastoma cells in culture. The cells were exposed to: (i) SMFs alone; (ii) XRs alone; (iii) XR, with SMFs applied during recovery; (iv) SMFs both before and after XR irradiation. XR-induced DNA damage was analyzed by Single Cell Gel Electrophoresis assay (comet assay) using statistical tools designed to assess the tail DNA (TD) and tail length (TL) as indicators of DNA fragmentation. Mitochondrial membrane potential, known to be affected by IR, was assessed using the JC-1 mitochondrial probe. Our results showed that exposure of cells to 5 Gy of XR irradiation alone led to extensive DNA damage, which was significantly reduced by post-irradiation exposure to SMFs. The XR-induced loss of mitochondrial membrane potential was to a large extent averted by exposure to SMFs. These data suggest that SMFs modulate DNA damage and/or damage repair, possibly through a mechanism that affects mitochondria. PMID:24345558

  18. 5-Iodo-2-Pyrimidinone-2'-Deoxyribose-Mediated Cytotoxicity and Radiosensitization in U87 Human Glioblastoma Xenografts

    SciTech Connect

    Kinsella, Timothy J. Kinsella, Michael T.; Seo, Yuji; Berk, Gregory

    2007-11-15

    Purpose: 5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is a novel orally administered (p.o.) prodrug of 5-iododeoxyuridine. Because p.o. IPdR is being considered for clinical testing as a radiosensitizer in patients with high-grade gliomas, we performed this in vivo study of IPdR-mediated cytotoxicity and radiosensitization in a human glioblastoma xenograft model, U87. Methods and Materials: Groups of 8 or 9 athymic male nude mice (6-8 weeks old) were implanted with s.c. U87 xenograft tumors (4 x 10{sup 6} cells) and then randomized to 10 treatment groups receiving increasing doses of p.o. IPdR (0, 100, 250, 500, and 1000 mg/kg/d) administered once daily (q.d.) x 14 days with or without radiotherapy (RT) (0 or 2 Gy/d x 4 days) on days 11-14 of IPdR treatment. Systemic toxicity was determined by body weight measurements during and after IPdR treatment. Tumor response was assessed by changes in tumor volumes. Results: IPdR alone at doses of {>=}500 mg/kg/d resulted in moderate inhibition of tumor growth. The combination of IPdR plus RT resulted in a significant IPdR dose-dependent tumor growth delay, with the maximum radiosensitization using {>=}500 mg/kg/d. IPdR doses of 500 and 1000 mg/kg/d resulted in transient 5-15% body weight loss during treatment. Conclusions: In U87 human glioblastoma s.c. xenografts, p.o. IPdR given q.d. x 14 days and RT given 2 Gy/d x 4 days (days 11-14 of IPdR treatment) results in a significant tumor growth delay in an IPdR dose-dependent pattern. The use of p.o. IPdR plus RT holds promise for Phase I/II testing in patients with high-grade gliomas.

  19. Human SERPINB12 Is an Abundant Intracellular Serpin Expressed in Most Surface and Glandular Epithelia.

    PubMed

    Niehaus, Jason Z; Good, Misty; Jackson, Laura E; Ozolek, John A; Silverman, Gary A; Luke, Cliff J

    2015-11-01

    The intracellular serine protease inhibitors (serpins) are an important family of proteins that protect cells form proteinase-mediated injury. Understanding the tissue and cellular expression pattern of this protein family can provide important insights into their physiologic roles. For example, high expression in epithelial tissues, such as lung, may suggest a biologic function in cellular defense, secretion, or selective absorption. Although the expression pattern of many of the intracellular serpins has been well described, one member of this class, SERPINB12, has not been carefully examined. We generated a mouse monoclonal antibody directed against human SERPINB12 and delineated its specificity and tissue and cell type distribution pattern through immunoblotting and immunohistochemistry, respectively. This monoclonal antibody was human specific and did not cross-react with other human intracellular serpins or mouse Serpinb12. SERPINB12 was found in nearly all the tissues investigated. In addition, this serpin was found in multiple cell types within individual tissues but primarily the epithelium. These data suggest that SERPINB12, like some other intracellular serpins, may play a vital role in barrier function by providing protection of epithelial cells.

  20. Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

    PubMed

    Opländer, Christian; Wetzel, Wiebke; Cortese, Miriam M; Pallua, Norbert; Suschek, Christoph V

    2008-05-01

    Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

  1. A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

    PubMed

    Kinoshita, Yusuke; Kamitani, Hideki; Mamun, Mahabub Hasan; Wasita, Brian; Kazuki, Yasuhiro; Hiratsuka, Masaharu; Oshimura, Mitsuo; Watanabe, Takashi

    2010-05-01

    Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

  2. Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression

    PubMed Central

    Song, Xiang; Liu, Bing-Chen; Lu, Xiao-Yu; Yang, Li-Li; Zhai, Yu-Jia; Eaton, Amity F.; Thai, Tiffany L.; Eaton, Douglas C.; Ma, He-Ping; Shen, Bao-Zhong

    2014-01-01

    Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or a NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca2+-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca2+ also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca2+. PMID:24518247

  3. Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression.

    PubMed

    Song, Xiang; Liu, Bing-Chen; Lu, Xiao-Yu; Yang, Li-Li; Zhai, Yu-Jia; Eaton, Amity F; Thai, Tiffany L; Eaton, Douglas C; Ma, He-Ping; Shen, Bao-Zhong

    2014-05-01

    Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca(2+)-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca(2+) also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca(2+). Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Derivatives of the pyrazolo[1,5-a]pyrimidine acetamide DPA-713 as translocator protein (TSPO) ligands and pro-apoptotic agents in human glioblastoma.

    PubMed

    Werry, Eryn L; King, Victoria A; Barron, Melissa L; Banister, Samuel D; Sokias, Renee; Kassiou, Michael

    2017-01-01

    The 18kDa translocator protein (TSPO) is a target for novel glioblastoma therapies due to its upregulation in this cancer and relatively low levels of expression in the healthy cortex. The pyrazolo[1,5-a]pyrimidine acetamides, exemplified by DPA-713 and DPA-714, are a class of high affinity TSPO ligands with selectivity over the central benzodiazepine receptor. In this study we have explored the potential anti-glioblastoma activity of a library of DPA-713 and DPA-714 analogues, and investigated the effect of amending the alkyl ether chain on TSPO affinity and functional potential. All ligands demonstrated nanomolar affinity for TSPO, but showed diverse functional activity, for example DPA-713 and DPA-714 did not affect the proliferation or viability of human T98G glioblastoma cells, while the hexyl ether and benzyl ether derivatives decreased proliferation of T98G cells without affecting proliferation in human fetal glial SVGp12 cells. These ligands also induced apoptosis and dissipated T98G mitochondrial membrane potential. This suggests that the nature of the alkyl ether chain of pyrazolo[1,5-a]pyrimidine acetamides has little influence on TSPO affinity but is important for functional activity of this class of TSPO ligands. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells.

    PubMed

    Chou, Yu-Cheng; Chang, Meng-Ya; Wang, Mei-Jen; Liu, Hsin-Chung; Chang, Shu-Jen; Harnod, Tomor; Hung, Chih-Huang; Lee, Hsu-Tung; Shen, Chiung-Chyi; Chung, Jing-Gung

    2017-01-01

    Glioblastoma is the most common and aggressive primary brain malignancy. Phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, can induce apoptosis in many human cancer cells. Our previous study disclosed that PEITC induces apoptosis through the extrinsic pathway, dysfunction of mitochondria, reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress, and intrinsic (mitochondrial) pathway in human brain glioblastoma multiforme (GBM) 8401 cells. To the best of our knowledge, we first investigated the effects of PEITC on the genetic levels of GBM 8401 cells in vitro. PEITC may induce G0/G1 cell-cycle arrest through affecting the proteins such as cdk2, cyclin E, and p21 in GBM 8401 cells. Many genes associated with cell-cycle regulation of GBM 8401 cells were changed after PEITC treatment: 48 genes were upregulated and 118 were downregulated. The cell-division cycle protein 20 (CDC20), Budding uninhibited by benzimidazole 1 homolog beta (BUB1B), and cyclin B1 were downregulated, and clusterin was upregulated in GBM 8401 cells treated with PEITC. These changes of gene expression can provide the effects of PEITC on the genetic levels and potential biomarkers for glioblastoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 176-187, 2017. © 2015 Wiley Periodicals, Inc.

  6. Label-Free Delineation of Brain Tumors by Coherent Anti-Stokes Raman Scattering Microscopy in an Orthotopic Mouse Model and Human Glioblastoma

    PubMed Central

    Tamosaityte, Sandra; Leipnitz, Elke; Geiger, Kathrin D.; Schackert, Gabriele; Koch, Edmund; Steiner, Gerald; Kirsch, Matthias

    2014-01-01

    Background Coherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors. Methods Different human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor's localization, cell proliferation and vascularization. Results The morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma. Conclusions CARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation. PMID:25198698

  7. Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model

    PubMed Central

    d'Angelo, Michele; Cristiano, Loredana; Galzio, Renato; Destouches, Damien; Florio, Tiziana Marilena; Dhez, Anne Chloé; Astarita, Carlo; Cinque, Benedetta; Fidoamore, Alessia; Rosati, Floriana; Cifone, Maria Grazia; Ippoliti, Rodolfo; Giordano, Antonio; Courty, José; Cimini, Annamaria

    2015-01-01

    Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma. PMID:26540346

  8. Carbon ion beam is more effective to induce cell death in sphere-type A172 human glioblastoma cells compared with X-rays.

    PubMed

    Takahashi, Momoko; Hirakawa, Hirokazu; Yajima, Hirohiko; Izumi-Nakajima, Nakako; Okayasu, Ryuichi; Fujimori, Akira

    2014-12-01

    To obtain human glioblastoma cells A172 expressing stem cell-related protein and comparison of radiosensitivity in these cells with X-rays and carbon beam. Human monolayer-type A172 glioblastoma cells were maintained in normal medium with 10% bovine serum. In order to obtain sphere-type A172 cells the medium was replaced with serum-free medium supplemented with growth factors. Both types of A172 cells were irradiated with either X-rays or carbon ion beams and their radiosensitivity was evaluated. Serum-free medium induced expression of stem cell-related proteins in A172 cells along with the neurosphere-like appearance. These sphere-type cells were found resistant to both X-rays and carbon ion beams. Phosphorylation of histone H2A family member X persisted for a longer period in the cells exposed to carbon ion beams than in those exposed to X-rays and it disappeared quicker in the sphere type than in the monolayer type. Relative radioresistance of the sphere type cells was smaller for carbon ion beams than for X-rays. We demonstrated that glioblastoma A172 cells with induced stem cell-related proteins turned resistant to irradiation. Accelerated heavy ion particles may have advantage over X-rays in overcoming the tumor resistance due to cell stemness.

  9. Molecular determinants of the human α2C-adrenergic receptor temperature-sensitive intracellular traffic.

    PubMed

    Filipeanu, Catalin M; Pullikuth, Ashok K; Guidry, Jessie J

    2015-05-01

    The human α2C-adrenergic receptor (α2C-AR) is localized intracellularly at physiologic temperature. Decreasing the environmental temperature strongly stimulates the receptor transport to the cell surface. In contrast, rat and mouse α2C-AR plasma membrane levels are less sensitive to decrease in temperature, whereas the opossum α2C-AR cell surface levels are not changed in these conditions. Structural analysis demonstrated that human α2C-AR has a high number of arginine residues in the third intracellular loop and in the C-terminus, organized as putative RXR motifs. Although these motifs do not affect the receptor subcellular localization at 37°C, deletion of the arginine clusters significantly enhanced receptor plasma membrane levels at reduced temperature. We found that this exaggerated transport of the human receptor is mediated by two functional arginine clusters, one in the third intracellular loop and one in the C-terminus. This effect is mediated by interactions with COPI vesicles, but not by 14-3-3 proteins. In rat α2C-AR, the arginine cluster from the third intracellular loop is shifted to the left due to three missing residues. Reinsertion of these residues in the rat α2C-AR restored the same temperature sensitivity as in the human receptor. Proteomic and coimmunoprecipitation experiments identified pontin as a molecule having stronger interactions with human α2C-AR compared with rat α2C-AR. Inhibition of pontin activity enhanced human receptor plasma membrane levels and signaling at 37°C. Our results demonstrate that human α2C-AR has a unique temperature-sensitive traffic pattern within the G protein-coupled receptor class due to interactions with different molecular chaperones, mediated in part by strict spatial localization of specific arginine residues. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Molecular Determinants of the Human α2C-Adrenergic Receptor Temperature-Sensitive Intracellular Traffic

    PubMed Central

    Pullikuth, Ashok K.; Guidry, Jessie J.

    2015-01-01

    The human α2C-adrenergic receptor (α2C-AR) is localized intracellularly at physiologic temperature. Decreasing the environmental temperature strongly stimulates the receptor transport to the cell surface. In contrast, rat and mouse α2C-AR plasma membrane levels are less sensitive to decrease in temperature, whereas the opossum α2C-AR cell surface levels are not changed in these conditions. Structural analysis demonstrated that human α2C-AR has a high number of arginine residues in the third intracellular loop and in the C-terminus, organized as putative RXR motifs. Although these motifs do not affect the receptor subcellular localization at 37°C, deletion of the arginine clusters significantly enhanced receptor plasma membrane levels at reduced temperature. We found that this exaggerated transport of the human receptor is mediated by two functional arginine clusters, one in the third intracellular loop and one in the C-terminus. This effect is mediated by interactions with COPI vesicles, but not by 14-3-3 proteins. In rat α2C-AR, the arginine cluster from the third intracellular loop is shifted to the left due to three missing residues. Reinsertion of these residues in the rat α2C-AR restored the same temperature sensitivity as in the human receptor. Proteomic and coimmunoprecipitation experiments identified pontin as a molecule having stronger interactions with human α2C-AR compared with rat α2C-AR. Inhibition of pontin activity enhanced human receptor plasma membrane levels and signaling at 37°C. Our results demonstrate that human α2C-AR has a unique temperature-sensitive traffic pattern within the G protein–coupled receptor class due to interactions with different molecular chaperones, mediated in part by strict spatial localization of specific arginine residues. PMID:25680754

  11. Endocytosis and intracellular traffic of cholesterol-PDMAEMA liposome complexes in human epithelial-like cells.

    PubMed

    Szymanowski, F; Hugo, A A; Alves, P; Simões, P N; Gómez-Zavaglia, A; Pérez, Pablo F

    2017-08-01

    Liposomes are generally used as delivery systems, as they are capable of encapsulating a wide variety of molecules (i.e. plasmids, recombinant proteins, therapeutic drugs). However, liposomal drug delivery have to fulfill different requirements, such as the effective internalization by the target cells and avoidance of the degradative activity of the intracellular compartments. The use of polymer lipid complexes (PLCs), by including different polymers in the liposome formulation, could improve internalization and intracellular release of drugs. The aim of the present work is to study the mechanisms of cellular uptaking and the intracellular trafficking of PLCs formed with cholesterol-poly(2-(dimethylamino)ethyl methacrylate) CHO-PDMAEMA and lecithin (LC CHO-PD). Calcein-loaded liposomes were used to determine cellular uptake and intracellular localization by flow cytometry and confocal microscopy. Incorporation of CHO-PDMAEMA to lecithin liposomes enhanced the internalization capacity of PLCs. Internalization of PLCs by human epithelial-like cells (HEK-293) diminished at 4°C, suggesting uptake by endocytosis. PLCs showed no co-localization with acidic compartments after internalization. Experiments with endocytosis inhibitors and co-localization of liposomes and albumin, suggested the caveolae endocytic pathway as the most probable route for intracellular trafficking of PLCs. In this work, we demonstrated an efficient uptake of LC CHO-PDs by human epithelial-like cells (HEK-293) through the non-degradative caveolae endocytic pathway. The mode of internalization and the intracellular fate of liposomes under study, suggest a promising use of LC CHO-PDs as drug delivery systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

    PubMed

    Castaneda, Julie T; Harui, Airi; Kiertscher, Sylvia M; Roth, Jeffrey D; Roth, Michael D

    2013-03-01

    mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

  13. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses

    PubMed Central

    Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

    2017-01-01

    Abstract Background. Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. Methods. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. Results. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Conclusions. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM. PMID:27576873

  14. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

    PubMed

    Gomez-Roman, Natividad; Stevenson, Katrina; Gilmour, Lesley; Hamilton, Graham; Chalmers, Anthony J

    2017-02-01

    Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug-radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures. We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available. In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells. Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug-radiation combinations for GBM.

  15. Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

    PubMed Central

    Sesen, Julie; Dahan, Perrine; Scotland, Sarah J.; Saland, Estelle; Dang, Van-Thi; Lemarié, Anthony; Tyler, Betty M.; Brem, Henry; Toulas, Christine; Cohen-Jonathan Moyal, Elizabeth; Sarry, Jean-Emmanuel; Skuli, Nicolas

    2015-01-01

    High-grade gliomas, glioblastomas (GB), are refractory to conventional treatment combining surgery, chemotherapy, mainly temozolomide, and radiotherapy. This highlights an urgent need to develop novel therapies and increase the efficacy of radio/chemotherapy for these very aggressive and malignant brain tumors. Recently, tumor metabolism became an interesting potential therapeutic target in various cancers. Accordingly, combining drugs targeting cell metabolism with appropriate chemotherapeutic agents or radiotherapy has become attractive. In light of these perspectives, we were particularly interested in the anti-cancer properties of a biguanide molecule used for type 2 diabetes treatment, metformin. In our present work, we demonstrate that metformin decreases mitochondrial-dependent ATP production and oxygen consumption and increases lactate and glycolytic ATP production. We show that metformin induces decreased proliferation, cell cycle arrest, autophagy, apoptosis and cell death in vitro with a concomitant activation of AMPK, Redd1 and inhibition of the mTOR pathway. Cell sensitivity to metformin also depends on the genetic and mutational backgrounds of the different GB cells used in this study, particularly their PTEN status. Interestingly, knockdown of AMPK and Redd1 with siRNA partially, but incompletely, abrogates the induction of apoptosis by metformin suggesting both AMPK/Redd1-dependent and –independent effects. However, the primary determinant of the effect of metformin on cell growth is the genetic and mutational backgrounds of the glioma cells. We further demonstrate that metformin treatment in combination with temozolomide and/or irradiation induces a synergistic anti-tumoral response in glioma cell lines. Xenografts performed in nude mice demonstrate in vivo that metformin delays tumor growth. As current treatments for GB commonly fail to cure, the need for more effective therapeutic options is overwhelming. Based on these results, metformin could

  16. Double suicide gene therapy using human neural stem cells against glioblastoma: double safety measures.

    PubMed

    Lee, Ji Yeoun; Lee, Do-Hun; Kim, Hyung A; Choi, Seung-Ah; Lee, Hong Jun; Park, Chul-Kee; Phi, Ji Hoon; Wang, Kyu-Chang; Kim, Seung U; Kim, Seung-Ki

    2014-01-01

    With recent advancements in stem cell-based gene therapy, concerns about safety have grown. Stem cell-based gene therapies may pose the risk of immunological problems and oncogenesis. We investigated the feasibility of treating glioblastomas with neural stem cells [(NSCs), HB1.F3 cells] expressing double prodrug enzymes [cytosine deaminase (CD) and tyrosine kinase (TK)] to eliminate the NSCs following treatment for safety purposes. First, the in vitro and in vivo therapeutic efficacies of NSCs engineered with double prodrug enzymes (HB1.F3-CD.TK cells) were compared to cells expressing a single prodrug enzyme (HB1.F3-CD). Second, the degree of safety achieved by NSC elimination was compared with an in vitro viability assay of the NSCs after treatment with the double prodrugs. We further compared the differences in in vivo proliferation of control, single prodrug enzyme and double prodrug enzyme expressing NSCs. HB1.F3-CD.TK cells showed a better or comparable treatment outcome than HB1.F3-CD cells in vitro and in vivo. For safety, HB1.F3-CD.TK cells showed the least viability in vitro after treatment with prodrugs compared to HB1.F3 and HB1.F3-CD cells. Additionally, the in vivo proliferation among the injected NSCs found in the tumor was the smallest for HB1.F3-CD.TK cells. Double-prodrug enzyme-directed gene therapy shows good therapeutic efficacy as well as efficient eradication of the NSCs to ensure safety for clinical applications of stem cell-based gene therapies.

  17. Direct transfection of miR-137 mimics is more effective than DNA demethylation of miR-137 promoter to augment anti-tumor mechanisms of delphinidin in human glioblastoma U87MG and LN18 cells.

    PubMed

    Chakrabarti, Mrinmay; Ray, Swapan K

    2015-11-15

    Glioblastoma is the deadliest brain tumor in humans. Recent studies suggested that 5-aza-2-deoxycytidine (AzaC) could inhibit cell cycle progression in human glioblastoma stem cells by an indirect increase in expression of the tumor suppressor microRNA-137 (miR-137). Delphinidin (DPN), a new anthocyanidin, inhibits cell growth in different cancers. We investigated inhibition of glioblastoma growth after indirect or direct overexpression of miR-137 and then DPN treatment. The highest inhibition of cell growth occurred due to treatment with combination of 10 μM AzaC and 50 μM DPN in human glioblastoma U87MG and LN18 cells. The methylation sensitive-polymerase chain reaction (MS-PCR) results showed that AzaC inhibited methylation of miR-137 promoter region, which was hypermethylated in both glioblastoma cell lines, to cause indirect increase in miR-137 expression. Our results also indicated the highest miR-137 expression after direct transfection of miR-137 mimics and DPN treatment. Combination of miR-137 mimics transfection and DPN treatment caused the highest inhibition of cell invasion and prevented angiogenic network formation due to the least expression of angiogenic factor (VEGF) in human glioblastoma cells in co-culture with human microvascular endothelial cells. This combination strategy most effectively inhibited survival factors (p-Akt and NF-κB), angiogenic factors (VEGF and b-FGF), growth factor receptor (EGFR), and invasive factors (MMP-9 and MMP-2). Direct overexpression of miR-137 most effectively augmented efficacy of DPN to induce apoptosis with activation of extrinsic and intrinsic pathways. So, sequential miR-137 overexpression and DPN treatment could be a promising combination treatment to inhibit growth of human glioblastoma cells.

  18. Adherence and intracellular survival within human macrophages of Enterococcus faecalis isolates from coastal marine sediment.

    PubMed

    Sabatino, Raffaella; Di Cesare, Andrea; Pasquaroli, Sonia; Vignaroli, Carla; Citterio, Barbara; Amiri, Mehdi; Rossi, Luigia; Magnani, Mauro; Mauro, Alessandro; Biavasco, Francesca

    2015-09-01

    Enterococcus faecalis is part of the human intestinal microbiota and an important nosocomial pathogen. It can be found in the marine environment, where it is also employed as a fecal indicator. To assess the pathogenic potential of marine E. faecalis, four strains isolated from marine sediment were analyzed for their ability to survive in human macrophages. Escherichia coli DH5α was used as a negative control. The number of adherent and intracellular bacteria was determined 2.5 h after the infection (T0) and after further 24h (T24) by CFU and qPCR counts. At T24 adherent and intracellular enterococcal CFU counts were increased for all strains, the increment in intracellular bacteria being particularly marked. No CFU of E. coli DH5α were detected. In contrast, qPCR counts of intracellular enterococcal and E. coli bacteria were similar at both time points. These findings suggest that whereas E. coli was killed within macrophages (no CFU, positive qPCR), the E. faecalis isolates not only escaped killing, but actually multiplied, as demonstrated by the increase in the viable cell population. These findings support earlier data by our group, further documenting that marine sediment can be a reservoir of pathogenic enterococci.

  19. Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models

    PubMed Central

    Jain, Meenu; Gamage, Nipuni-Dhanesha H.; Alsulami, Meshal; Shankar, Adarsh; Achyut, Bhagelu R.; Angara, Kartik; Rashid, Mohammad H.; Iskander, Asm; Borin, Thaiz F.; Wenbo, Zhi; Ara, Roxan; Ali, Meser M.; Lebedyeva, Iryna; Chwang, Wilson B.; Guo, Austin; Bagher-Ebadian, Hassan; Arbab, Ali S.

    2017-01-01

    Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model. PMID:28139732

  20. Human cytomegalovirus encoded chemokine receptor US28 activates the HIF-1α/PKM2 axis in glioblastoma cells

    PubMed Central

    van Senten, Jeffrey R.; Fraile-Ramos, Alberto; Siderius, Marco; Smit, Martine J.

    2016-01-01

    The human cytomegalovirus (HCMV) encoded chemokine receptor US28 promotes tumorigenesis through activation of various proliferative and angiogenic signaling pathways. Upon infection, US28 displays constitutive activity and signals in a G protein-dependent manner, hijacking the host's cellular machinery. In tumor cells, the hypoxia inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) axis plays an important role by supporting proliferation, angiogenesis and reprogramming of energy metabolism. In this study we show that US28 signaling results in activation of the HIF-1α/PKM2 feedforward loop in fibroblasts and glioblastoma cells. The constitutive activity of US28 increases HIF-1 protein stability through a Gαq-, CaMKII- and Akt/mTOR-dependent mechanism. Furthermore, we found that VEGF and lactate secretion are increased and HIF-1 target genes, glucose transporter type 1 (GLUT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), involved in glucose metabolism, are upregulated in US28 expressing cells. In addition, PKM2 is phosphorylated and found to be in a tumor-associated dimeric state upon US28 expression. Also in HCMV-infected cells HIF-1 activity is enhanced, which in part is US28-dependent. Finally, increased proliferation of cells expressing US28 is abolished upon inhibition of the HIF-1α/PKM2 cascade. These data highlight the importance of HIF-1α and PKM2 in US28-induced proliferation, angiogenesis and metabolic reprogramming. PMID:27602585

  1. Evaluation of the cytotoxic activity of Hypericum spp. on human glioblastoma A1235 and breast cancer MDA MB-231 cells.

    PubMed

    Madunić, Josip; Matulić, Maja; Friščić, Maja; Pilepić, Kroata Hazler

    2016-11-09

    Cytotoxic activity of 16 Hypericum ethanolic extracts was evaluated by MTT assay on two human cancer cell lines: glioblastoma A1235 and breast cancer MDA MB-231. Morphology and the type of induced cell death were determined using light and fluorescence microscopy. The majority of Hypericum extracts had no significant cytotoxic effect on MDA MB-231 cells. Eight extracts exhibited mild cytotoxic effect on A1235 cells after 24 h incubation, ranging from 8.0% (H. patulum) to 21.7% (H. oblongifolium). After 72 h of treatment, the strongest inhibition of A1235 viability was observed for extracts of H. androsaemum (26.4-43.9%), H. balearicum (25.8-36.3%), H. delphicum (14.8-27.4%) and H. densiflorum (11.2-24.1%). Micro-scopic examination of cells showed apoptosis as the dominant type of cell death. Due to observed high viability of treated cells, we propose that cytotoxic effects of Hypericum extracts could be related to alternations/interruptions in the cell cycle.

  2. Heterogeneous intratumoral distribution of gadolinium nanoparticles within U87 human glioblastoma xenografts unveiled by micro-PIXE imaging.

    PubMed

    Carmona, Asuncion; Roudeau, Stéphane; L'Homel, Baptiste; Pouzoulet, Frédéric; Bonnet-Boissinot, Sarah; Prezado, Yolanda; Ortega, Richard

    2017-04-15

    Metallic nanoparticles have great potential in cancer radiotherapy as theranostic drugs since, they serve simultaneously as contrast agents for medical imaging and as radio-therapy sensitizers. As with other anticancer drugs, intratumoral diffusion is one of the main limiting factors for therapeutic efficiency. To date, a few reports have investigated the intratumoral distribution of metallic nanoparticles. The aim of this study was to determine the quantitative distribution of gadolinium (Gd) nanoparticles after direct intratumoral injection within U87 human glioblastoma tumors grafted in mice, using micro-PIXE (Particle Induced X-ray Emission) imaging. AGuIX (Activation and Guiding of Irradiation by X-ray) 3 nm particles composed of a polysiloxane network surrounded by gadolinium chelates were used. PIXE results indicate that the direct injection of Gd nanoparticles in tumors results in their heterogeneous diffusion, probably related to variations in tumor density. All tumor regions contain Gd, but with markedly different concentrations, with a more than 250-fold difference. Also Gd can diffuse to the healthy adjacent tissue. This study highlights the usefulness of mapping the distribution of metallic nanoparticles at the intratumoral level, and proposes PIXE as an imaging modality to probe the quantitative distribution of metallic nanoparticles in tumors from experimental animal models with micrometer resolution. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Implicated Survival Benefit in Rat Xenograft Models.

    PubMed

    Jain, Meenu; Gamage, Nipuni-Dhanesha H; Alsulami, Meshal; Shankar, Adarsh; Achyut, Bhagelu R; Angara, Kartik; Rashid, Mohammad H; Iskander, Asm; Borin, Thaiz F; Wenbo, Zhi; Ara, Roxan; Ali, Meser M; Lebedyeva, Iryna; Chwang, Wilson B; Guo, Austin; Bagher-Ebadian, Hassan; Arbab, Ali S

    2017-01-31

    Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPßCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPßCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p < 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p < 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p < 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and αSMA), in addition to inflammation and angiogenesis markers in the treatment group (p < 0.05). Our results indicate that HPßCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPßCD-HET0016 significantly prolonged survival in PDX GBM811 model.

  4. Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells

    PubMed Central

    Momeny, Majid; Moghaddaskho, Farima; Gortany, Narges K.; Yousefi, Hassan; Sabourinejad, Zahra; Zarrinrad, Ghazaleh; Mirshahvaladi, Shahab; Eyvani, Haniyeh; Barghi, Farinaz; Ahmadinia, Leila; Ghazi-Khansari, Mahmoud; Dehpour, Ahmad R.; Amanpour, Saeid; Tavangar, Seyyed M.; Dardaei, Leila; Emami, Amir H.; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H.

    2017-01-01

    Glioblastoma (GBM) remains one of the most fatal human malignancies due to its high angiogenic and infiltrative capacities. Even with optimal therapy including surgery, radiotherapy and temozolomide, it is essentially incurable. GBM is among the most neovascularised neoplasms and its malignant progression associates with striking neovascularisation, evidenced by vasoproliferation and endothelial cell hyperplasia. Targeting the pro-angiogenic pathways is therefore a promising anti-glioma strategy. Here we show that tivozanib, a pan-inhibitor of vascular endothelial growth factor (VEGF) receptors, inhibited proliferation of GBM cells through a G2/M cell cycle arrest via inhibition of polo-like kinase 1 (PLK1) signalling pathway and down-modulation of Aurora kinases A and B, cyclin B1 and CDC25C. Moreover, tivozanib decreased adhesive potential of these cells through reduction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Tivozanib diminished GBM cell invasion through impairing the proteolytic cascade of cathepsin B/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase-2 (MMP-2). Combination of tivozanib with EGFR small molecule inhibitor gefitinib synergistically increased sensitivity to gefitinib. Altogether, these findings suggest that VEGFR blockade by tivozanib has potential anti-glioma effects in vitro. Further in vivo studies are warranted to explore the anti-tumour activity of tivozanib in combinatorial approaches in GBM. PMID:28287096

  5. Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation.

    PubMed

    Zhang, Xianqin; Bogunovic, Dusan; Payelle-Brogard, Béatrice; Francois-Newton, Véronique; Speer, Scott D; Yuan, Chao; Volpi, Stefano; Li, Zhi; Sanal, Ozden; Mansouri, Davood; Tezcan, Ilhan; Rice, Gillian I; Chen, Chunyuan; Mansouri, Nahal; Mahdaviani, Seyed Alireza; Itan, Yuval; Boisson, Bertrand; Okada, Satoshi; Zeng, Lu; Wang, Xing; Jiang, Hui; Liu, Wenqiang; Han, Tiantian; Liu, Delin; Ma, Tao; Wang, Bo; Liu, Mugen; Liu, Jing-Yu; Wang, Qing K; Yalnizoglu, Dilek; Radoshevich, Lilliana; Uzé, Gilles; Gros, Philippe; Rozenberg, Flore; Zhang, Shen-Ying; Jouanguy, Emmanuelle; Bustamante, Jacinta; García-Sastre, Adolfo; Abel, Laurent; Lebon, Pierre; Notarangelo, Luigi D; Crow, Yanick J; Boisson-Dupuis, Stéphanie; Casanova, Jean-Laurent; Pellegrini, Sandra

    2015-01-01

    Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi-Goutières syndrome and spondyloenchondrodysplasia. We further show that an absence of intracellular ISG15 in the patients' cells prevents the accumulation of USP18, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation.

  6. Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation

    PubMed Central

    Zhang, Xianqin; Bogunovic, Dusan; Payelle-Brogard, Béatrice; Francois-Newton, Véronique; Speer, Scott D.; Yuan, Chao; Volpi, Stefano; Li, Zhi; Sanal, Ozden; Mansouri, Davood; Tezcan, Ilhan; Rice, Gillian I.; Chen, Chunyuan; Mansouri, Nahal; Alireza Mahdaviani, Seyed; Itan, Yuval; Boisson, Bertrand; Okada, Satoshi; Zeng, Lu; Wang, Xing; Jiang, Hui; Liu, Wenqiang; Han, Tiantian; Liu, Delin; Ma, Tao; Wang, Bo; Liu, Mugen; Liu, Jing-Yu; Wang, Qing K.; Yalnizoglu, Dilek; Radoshevich, Lilliana; Uzé, Gilles; Gros, Philippe; Rozenberg, Flore; Zhang, Shen-Ying; Jouanguy, Emmanuelle; Bustamante, Jacinta; Garcìa-Sastre, Adolfo; Abel, Laurent; Lebon, Pierre; Notarangelo, Luigi D.; Crow, Yanick J.; Boisson-Dupuis, Stèphanie; Casanova, Jean-Laurent; Pellegrini, Sandra

    2015-01-01

    Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice1–4. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases5. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi–Goutières syndrome and spondyloenchondrodysplasia6–9.We further show that an absence of intracellular ISG15 in the patients’ cells prevents the accumulation of USP1810,11, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation. PMID:25307056

  7. 1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro

    PubMed Central

    Liu, Shan-shan; Wang, Yan-feng; Ma, Li-sha; Zheng, Bei-bei; Li, Lin; Xie, Wei-dong; Li, Xia

    2013-01-01

    Aim: To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL), a novel eudesmane-type sesquiterpene isolated from Aster himalaicus, on the cell cycle and apoptosis in human glioblastoma cells in vitro. Methods: Human malignant glioblastoma cell lines U87 and A172 were used. The cytotoxicity of OEL was examined using the MTT assay. Cell apoptosis was assessed with DAPI staining and flow cytometry. DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting. Cell cycle profiles were measured with flow cytometry. The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR. The protein expression of γ-H2AX, caspase-9, caspase-3, p53, p21Waf1/Cip1, cyclin B1, and cdc2 was analyzed with Western blotting. Results: Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose- and time-dependent manners (the values of IC50 at 48 and 72 h were 29.5 and 16.99 μmol/L, respectively, in U87 cells; 7.2 and 9.5 μmol/L, respectively, in A172 cells). OEL (10–30μmol/L) induced apoptosis and G2/M phase arrest in both U87 and A172 cells. OEL induced the phosphorylation of cdc2, a G2/M phase cyclin-dependent kinase, and decreased the expression of cyclin B1 required for progression through the G2/M phase in U87 cells. The compound remarkably increased the phosphorylation of H2AX in U87 cells. Moreover, OEL increased the mRNA and protein levels of p53 and its target gene p21Waf1/Cip1 in U87 cells. The compound also induced p53 phosphorylation. Pretreatment with PFT-α, a specific inhibitor of p53 transcriptional activity, could partially reverse the inhibition of OEL on the viability of U87 and A172 cells. Conclusion: OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage, p53-mediated cell cycle arrest and apoptosis, thus warrants further studies as a lead compound of anti-glioblastoma drug. PMID:23160341

  8. 1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro.

    PubMed

    Liu, Shan-shan; Wang, Yan-feng; Ma, Li-sha; Zheng, Bei-bei; Li, Lin; Xie, Wei-dong; Li, Xia

    2013-02-01

    To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL), a novel eudesmane-type sesquiterpene isolated from Aster himalaicus, on the cell cycle and apoptosis in human glioblastoma cells in vitro. Human malignant glioblastoma cell lines U87 and A172 were used. The cytotoxicity of OEL was examined using the MTT assay. Cell apoptosis was assessed with DAPI staining and flow cytometry. DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting. Cell cycle profiles were measured with flow cytometry. The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR. The protein expression of γ-H2AX, caspase-9, caspase-3, p53, p21Waf1/Cip1, cyclin B1, and cdc2 was analyzed with Western blotting. Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose- and time-dependent manners (the values of IC(50) at 48 and 72 h were 29.5 and 16.99 μmol/L, respectively, in U87 cells; 7.2 and 9.5 μmol/L, respectively, in A172 cells). OEL (10-30 μmol/L) induced apoptosis and G(2)/M phase arrest in both U87 and A172 cells. OEL induced the phosphorylation of cdc2, a G(2)/M phase cyclin-dependent kinase, and decreased the expression of cyclin B1 required for progression through the G(2)/M phase in U87 cells. The compound remarkably increased the phosphorylation of H2AX in U87 cells. Moreover, OEL increased the mRNA and protein levels of p53 and its target gene p21(Waf1/Cip1) in U87 cells. The compound also induced p53 phosphorylation. Pretreatment with PFT-α, a specific inhibitor of p53 transcriptional activity, could partially reverse the inhibition of OEL on the viability of U87 and A172 cells. OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage, p53-mediated cell cycle arrest and apoptosis, thus warrants further studies as a lead compound of anti-glioblastoma drug.

  9. Human glioblastoma ADF cells express tyrosinase, L-tyrosine hydroxylase and melanosomes and are sensitive to L-tyrosine and phenylthiourea.

    PubMed

    Bonfigli, Antonella; Zarivi, Osvaldo; Colafarina, Sabrina; Cimini, Anna Maria; Ragnelli, Anna Maria; Aimola, Pierpaolo; Natali, Pier Giorgio; Cerù, Maria Paola; Amicarelli, Fernanda; Miranda, Michele

    2006-06-01

    Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.

  10. Role of intracellular free calcium in killing Penicillium marneffei within human macrophages.

    PubMed

    Chen, Renqiong; Ji, Guangquan; Ma, Tuan; Huang, Xiaowen; Ren, Hong; Xi, Liyan

    2015-01-01

    Increases in cytosolic Ca(2+) concentration ([Ca(2+)]c) promote phagocyte antimicrobial responses. Here, we investigated macrophages stimulated by Penicillium marneffei (P. marneffei). [Ca(2+)]c was determined in macrophages loaded with the fluorescent calcium probe Fura 2/AM as they were stimulated by P. marneffei. We found that P. marneffei induced an increase in [Ca(2+)]c in human macrophages. Further, increased [Ca(2+)]c with the ionophore A23187 promoted phagosomal acidification and maturation and reduced intracellular replication of P. marneffei in P. marneffei-infected human macrophages, whereas decreased [Ca(2+)]c with the chelation MAPTAM decreased TNF-α production, inhibited phagosomal acidification and maturation and increased intracellular replication of P. marneffei. These data indicate that Ca(2+) signaling may play an important role in controlling the replication of P. marneffei within macrophages.

  11. Differential intracellular efficacies of ciprofloxacin and cefixime against Neisseria gonorrhoeae in human fallopian tube organ culture.

    PubMed

    Phanucharas, J P; Gorby, G L

    1997-07-01

    This study compared the abilities of ciprofloxacin and cefixime to kill intracellular Neisseria gonorrhoeae in a human fallopian tube organ culture assay. When invasion was inhibited by cytochalasin D, 0.996% of the tissue-associated gonococci survived ciprofloxacin exposure compared to 1.70% of gonococci exposed to cefixime (95% confidence interval for the ratio of the means, 0.267 to 1.30), indicating that the two antibiotics did not significantly differ in the ability to kill extracellular attached organisms. In the absence of cytochalasin D, 1.63% survived ciprofloxacin exposure while 9.76% survived cefixime treatment (95% confidence interval for the ratio of the means, 0.067 to 0.418). These results suggest that ciprofloxacin penetrated epithelial cells and killed intracellular gonococci better than did cefixime. Thus, at concentrations achievable in serum, ciprofloxacin was more effective in total gonococcal killing than cefixime in this human fallopian tube organ culture model.

  12. Pre- and post-contrast three-dimensional double inversion-recovery MRI in human glioblastoma

    PubMed Central

    Harris, Robert J.; Cloughesy, Timothy F.; Pope, Whitney B.; Godinez, Sergio; Natsuaki, Yutaka; Nghiemphu, Phioanh L.; Meyer, Heiko; Paul, Dominik; Behbahanian, Yalda; Lai, Albert

    2013-01-01

    Fluid attenuated inversion recovery (FLAIR) MRI sequences have become an indispensible tool for defining the malignant boundary in patients with brain tumors by nulling the signal contribution from cerebro-spinal fluid allowing both regions of edema and regions of non-enhancing, infiltrating tumor to become hyperintense on resulting images. In the current study we examined the utility of a three-dimensional double inversion recovery (DIR) sequence that additionally nulls the MR signal associated with white matter, implemented either pre-contrast or post-contrast, in order to determine whether this sequence allows for better differentiation between tumor and normal brain tissue. T1- and T2-weighted, FLAIR, dynamic susceptibility contrast (DSC)-MRI estimates of cerebral blood volume (rCBV), contrast-enhanced T1-weighted images (T1+C), and DIR data (pre- or post-contrast) were acquired in 22 patients with glioblastoma. Contrast-to-noise (CNR) and tumor volumes were compared between DIR and FLAIR sequences. Line profiles across regions of tumor were generated to evaluate similarities between image contrasts. Additionally, voxel-wise associations between DIR and other sequences were examined. Results suggested post-contrast DIR images were hyperintense (bright) in regions spatially similar those having FLAIR hyperintensity and hypointense (dark) in regions with contrast-enhancement or elevated rCBV due to the high sensitivity of 3D turbo spin echo sequences to susceptibility differences between different tissues. DIR tumor volumes were statistically smaller than tumor volumes as defined by FLAIR (Paired t test, P = 0.0084), averaging a difference of approximately 14 mL or 24 %. DIR images had approximately 1.5× higher lesion CNR compared with FLAIR images (Paired t test, P = 0.0048). Line profiles across tumor regions and scatter plots of voxel-wise coherence between different contrasts confirmed a positive correlation between DIR and FLAIR signal intensity and a

  13. Pre- and post-contrast three-dimensional double inversion-recovery MRI in human glioblastoma.

    PubMed

    Harris, Robert J; Cloughesy, Timothy F; Pope, Whitney B; Godinez, Sergio; Natsuaki, Yutaka; Nghiemphu, Phioanh L; Meyer, Heiko; Paul, Dominik; Behbahanian, Yalda; Lai, Albert; Ellingson, Benjamin M

    2013-04-01

    Fluid attenuated inversion recovery (FLAIR) MRI sequences have become an indispensible tool for defining the malignant boundary in patients with brain tumors by nulling the signal contribution from cerebrospinal fluid allowing both regions of edema and regions of non-enhancing, infiltrating tumor to become hyperintense on resulting images. In the current study we examined the utility of a three-dimensional double inversion recovery (DIR) sequence that additionally nulls the MR signal associated with white matter, implemented either pre-contrast or post-contrast, in order to determine whether this sequence allows for better differentiation between tumor and normal brain tissue. T1- and T2-weighted, FLAIR, dynamic susceptibility contrast (DSC)-MRI estimates of cerebral blood volume (rCBV), contrast-enhanced T1-weighted images (T1+C), and DIR data (pre- or post-contrast) were acquired in 22 patients with glioblastoma. Contrast-to-noise (CNR) and tumor volumes were compared between DIR and FLAIR sequences. Line profiles across regions of tumor were generated to evaluate similarities between image contrasts. Additionally, voxel-wise associations between DIR and other sequences were examined. Results suggested post-contrast DIR images were hyperintense (bright) in regions spatially similar those having FLAIR hyperintensity and hypointense (dark) in regions with contrast-enhancement or elevated rCBV due to the high sensitivity of 3D turbo spin echo sequences to susceptibility differences between different tissues. DIR tumor volumes were statistically smaller than tumor volumes as defined by FLAIR (Paired t test, P = 0.0084), averaging a difference of approximately 14 mL or 24 %. DIR images had approximately 1.5× higher lesion CNR compared with FLAIR images (Paired t test, P = 0.0048). Line profiles across tumor regions and scatter plots of voxel-wise coherence between different contrasts confirmed a positive correlation between DIR and FLAIR signal intensity and a

  14. Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

    SciTech Connect

    Gross, C.L.; Innace, J.K.; Hovatter, R.C.; Meier, H.L.; Smith, W.J.

    1993-12-31

    Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 AM HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NA C), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 LAIHD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.

  15. Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells

    PubMed Central

    Hwang, Jae Youn; Yoon, Chi Woo; Lim, Hae Gyun; Park, Jin Man; Yoon, Sangpil; Lee, Jungwoo; Shung, K. Kirk

    2016-01-01

    Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities. PMID:26150401

  16. Acoustic tweezers for studying intracellular calcium signaling in SKBR-3 human breast cancer cells.

    PubMed

    Hwang, Jae Youn; Yoon, Chi Woo; Lim, Hae Gyun; Park, Jin Man; Yoon, Sangpil; Lee, Jungwoo; Shung, K Kirk

    2015-12-01

    Extracellular matrix proteins such as fibronectin (FNT) play crucial roles in cell proliferation, adhesion, and migration. For better understanding of these associated cellular activities, various microscopic manipulation tools have been used to study their intracellular signaling pathways. Recently, it has appeared that acoustic tweezers may possess similar capabilities in the study. Therefore, we here demonstrate that our newly developed acoustic tweezers with a high-frequency lithium niobate ultrasonic transducer have potentials to study intracellular calcium signaling by FNT-binding to human breast cancer cells (SKBR-3). It is found that intracellular calcium elevations in SKBR-3 cells, initially occurring on the microbead-contacted spot and then eventually spreading over the entire cell, are elicited by attaching an acoustically trapped FNT-coated microbead. Interestingly, they are suppressed by either extracellular calcium elimination or phospholipase C (PLC) inhibition. Hence, this suggests that our acoustic tweezers may serve as an alternative tool in the study of intracellular signaling by FNT-binding activities.

  17. Inhibition of the Autophagy Pathway Synergistically Potentiates the Cytotoxic Activity of Givinostat (ITF2357) on Human Glioblastoma Cancer Stem Cells

    PubMed Central

    Angeletti, Francesca; Fossati, Gianluca; Pattarozzi, Alessandra; Würth, Roberto; Solari, Agnese; Daga, Antonio; Masiello, Irene; Barbieri, Federica; Florio, Tullio; Comincini, Sergio

    2016-01-01

    Increasing evidence highlighted the role of cancer stem cells (CSCs) in the development of tumor resistance to therapy, particularly in glioblastoma (GBM). Therefore, the development of new therapies, specifically directed against GBM CSCs, constitutes an important research avenue. Considering the extended range of cancer-related pathways modulated by histone acetylation/deacetylation processes, we studied the anti-proliferative and pro-apoptotic efficacy of givinostat (GVS), a pan-histone deacetylase inhibitor, on cell cultures enriched in CSCs, isolated from nine human GBMs. We report that GVS induced a significant reduction of viability and self-renewal ability in all GBM CSC cultures; conversely, GVS exposure did not cause a significant cytotoxic activity toward differentiated GBM cells and normal mesenchymal human stem cells. Analyzing the cellular and molecular mechanisms involved, we demonstrated that GVS affected CSC viability through the activation of programmed cell death pathways. In particular, a marked stimulation of macroautophagy was observed after GVS treatment. To understand the functional link between GVS treatment and autophagy activation, different genetic and pharmacological interfering strategies were used. We show that the up-regulation of the autophagy process, obtained by deprivation of growth factors, induced a reduction of CSC sensitivity to GVS, while the pharmacological inhibition of the autophagy pathway and the silencing of the key autophagy gene ATG7, increased the cell death rate induced by GVS. Altogether these findings suggest that autophagy represents a pro-survival mechanism activated by GBM CSCs to counteract the efficacy of the anti-proliferative activity of GVS. In conclusion, we demonstrate that GVS is a novel pharmacological tool able to target GBM CSC viability and its efficacy can be enhanced by autophagy inhibitory strategies. PMID:27833530

  18. Potent Antiproliferative Cembrenoids Accumulate in Tobacco upon Infection with Rhodococcus fascians and Trigger Unusual Microtubule Dynamics in Human Glioblastoma Cells

    PubMed Central

    Nacoulma, Aminata P.; Megalizzi, Veronique; Pottier, Laurent R.; De Lorenzi, Manuela; Thoret, Sylviane; Dubois, Joëlle; Vandeputte, Olivier M.; Duez, Pierre; Vereecke, Danny; Jaziri, Mondher El

    2013-01-01

    Aims Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians. Methods We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry. Results The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability. PMID:24167576

  19. The interaction of bee products with temozolomide in human diffuse astrocytoma, glioblastoma multiforme and astroglia cell lines.

    PubMed

    Borawska, Maria H; Markiewicz-Żukowska, Renata; Naliwajko, Sylwia K; Moskwa, Justyna; Bartosiuk, Emilia; Socha, Katarzyna; Surażyński, Arkadiusz; Kochanowicz, Jan; Mariak, Zenon

    2014-01-01

    In the present study, we investigated the influence of extracts from Salix spp. honey (ESH), beebread (EBB), and royal jelly (ERJ) with and without temozolomide (TMZ) on cell lines derived from a patient with diffuse astrocytoma (DASC), human glioblastoma multiforme (U87MG), and normal human astroglia (SVGp12). DASC was identified by immunocytochemistry. TMZ (20 μM) in combination with ESH (30 μg/mL), EBB (50 μg/mL), and ERJ (30 μg/mL) has stronger cytotoxic activity on U87MG cells after 72 h (20.0, 26.5, and 29.3% of control, respectively) than TMZ alone (about 6% of control). An increase of the cytotoxic effect and inhibition of DNA synthesis in SVGp12 were detected after administering TMZ with the studied extracts. NF-κB p50 subunit was reduced in U87MG cells after treatment with ESH (70.9%) and ESH + TMZ (74.7%). A significant decline of MMP-9 and MMP-2 secretion in cultured U87MG was detected after incubation with EBB (42.9% and 73.0%, respectively) and EBB + TMZ (38.4% and 68.5%, respectively). In conclusion, the use of bee products may increase the cytotoxic effect of TMZ in U87MG but also in SVGp12 cell line. It is important to note that the U87MG cells were sensitive to natural bee products, although there was no influence of natural bee products on the DASC cells.

  20. Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

    PubMed Central

    Bardet, Sylvia M.; Carr, Lynn; Soueid, Malak; Arnaud-Cormos, Delia; Leveque, Philippe; O’Connor, Rodney P.

    2016-01-01

    Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10 ns, high voltage electric pulse (35–45 kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma. PMID:27698479

  1. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  2. A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2013-05-14

    There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

  3. Sensitivity to PRIMA-1MET is associated with decreased MGMT in human glioblastoma cells and glioblastoma stem cells irrespective of p53 status

    PubMed Central

    Patyka, Mariia; Sharifi, Zeinab; Petrecca, Kevin; Mansure, Jose; Jean-Claude, Bertrand; Sabri, Siham

    2016-01-01

    Alterations of the TP53 tumor suppressor gene occur in ~30% of primary glioblastoma (GBM) with a high frequency of missense mutations associated with the acquisition of oncogenic “gain-of-function” (GOF) mutant (mut)p53 activities. PRIMA-1MET/APR-246, emerged as a promising compound to rescue wild-type (wt)p53 function in different cancer types. Previous studies suggested the role of wtp53 in the negative regulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), a major determinant in resistance to therapy in GBM treatment. The potential role of MGMT in expression of p53 and the efficacy of PRIMA-1MET with respect to TP53 status and expression of MGMT in GBM remain unknown. We investigated response to PRIMA-1MET of wtp53/MGMT-negative (U87MG, A172), mutp53/MGMT-positive U138, LN-18, T98/Empty vector (T98/EV) and its isogenic MGMT/shRNA gene knockdown counterpart (T98/shRNA). We show that MGMT silencing decreased expression of mutp53/GOF in T98/shRNA. PRIMA-1MET further cleared T98/shRNA cells of mutp53, decreased proliferation and clonogenic potential, abrogated the G2 checkpoint control, increased susceptibility to apoptotic cell death, expression of GADD45A and sustained expression of phosphorylated Erk1/2. PRIMA-1MET increased expression of p21 protein in U87MG and A172 and promoted senescence in U87MG cell line. Importantly, PRIMA-1MET decreased relative cell numbers, disrupted the structure of neurospheres of patient-derived GBM stem cells (GSCs) and enabled activation of wtp53 with decreased expression of MGMT in MGMT-positive GSCs or decreased expression of mutp53. Our findings highlight the cell-context dependent effects of PRIMA-1MET irrespective of p53 status and suggest the role of MGMT as a potential molecular target of PRIMA-1MET in MGMT-positive GSCs. PMID:27533246

  4. Sensitivity to PRIMA-1MET is associated with decreased MGMT in human glioblastoma cells and glioblastoma stem cells irrespective of p53 status.

    PubMed

    Patyka, Mariia; Sharifi, Zeinab; Petrecca, Kevin; Mansure, Jose; Jean-Claude, Bertrand; Sabri, Siham

    2016-09-13

    Alterations of the TP53 tumor suppressor gene occur in ~30% of primary glioblastoma (GBM) with a high frequency of missense mutations associated with the acquisition of oncogenic "gain-of-function" (GOF) mutant (mut)p53 activities. PRIMA-1MET/APR-246, emerged as a promising compound to rescue wild-type (wt)p53 function in different cancer types. Previous studies suggested the role of wtp53 in the negative regulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), a major determinant in resistance to therapy in GBM treatment. The potential role of MGMT in expression of p53 and the efficacy of PRIMA-1MET with respect to TP53 status and expression of MGMT in GBM remain unknown. We investigated response to PRIMA-1MET of wtp53/MGMT-negative (U87MG, A172), mutp53/MGMT-positive U138, LN-18, T98/Empty vector (T98/EV) and its isogenic MGMT/shRNA gene knockdown counterpart (T98/shRNA). We show that MGMT silencing decreased expression of mutp53/GOF in T98/shRNA. PRIMA-1MET further cleared T98/shRNA cells of mutp53, decreased proliferation and clonogenic potential, abrogated the G2 checkpoint control, increased susceptibility to apoptotic cell death, expression of GADD45A and sustained expression of phosphorylated Erk1/2. PRIMA-1MET increased expression of p21 protein in U87MG and A172 and promoted senescence in U87MG cell line. Importantly, PRIMA-1MET decreased relative cell numbers, disrupted the structure of neurospheres of patient-derived GBM stem cells (GSCs) and enabled activation of wtp53 with decreased expression of MGMT in MGMT-positive GSCs or decreased expression of mutp53. Our findings highlight the cell-context dependent effects of PRIMA-1MET irrespective of p53 status and suggest the role of MGMT as a potential molecular target of PRIMA-1MET in MGMT-positive GSCs.

  5. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

  6. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  7. Activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

    SciTech Connect

    Fogler, W.E.; Fidler, I.J.

    1986-03-15

    The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-..gamma.. (IFN-..gamma..)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and (/sup 3/H)nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced its encapsulation within multilamellar vesicles (MLV). Studies with (/sup 3/H)nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-..gamma..-primed monocytes. The intracellular interaction of (/sup 3/H)nor-MDP with IFN-..gamma..-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.

  8. Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium.

    PubMed

    Vallabhapurapu, Subrahmanya D; Blanco, Víctor M; Sulaiman, Mahaboob K; Vallabhapurapu, Swarajya Lakshmi; Chu, Zhengtao; Franco, Robert S; Qi, Xiaoyang

    2015-10-27

    Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS.

  9. Intracellular RNA recognition pathway activates strong anti-viral response in human mast cells.

    PubMed

    Lappalainen, J; Rintahaka, J; Kovanen, P T; Matikainen, S; Eklund, K K

    2013-04-01

    Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti-viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double-stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti-viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF-α. In contrast, extracellular Toll-like receptor 3 (TLR)-3-activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti-viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up-regulated the expression of melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene-1 (RIG-1), and TLR-3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti-viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti-viral proteins human myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.

  10. Effect of solute concentration on intracellular water volume and hydraulic conductivity of human blood platelets.

    PubMed Central

    Armitage, W J

    1986-01-01

    The intracellular water volume of human blood platelets was determined using tritiated water. The cells responded as osmometers over an observed range of solute concentration from 0.292 to 2.180 osmol kg-1. Only 87% of intracellular water was apparently osmotically active (i.e. Ponder's R was 0.87). Changes in cell volume induced by small step changes in external osmolality were followed photometrically and the time constant for the exponential approach of cell volume to its new equilibrium value was determined. Hydraulic conductivity (LP) was calculated from the time constant and was 1.41 X 10(-6) cm atm-1 s-1 under isotonic conditions at 37 degrees C. LP was inversely dependent on extracellular solute concentration, but it was independent of the direction of movement of water across the plasma membrane. PMID:3746695

  11. Honokiol exerts an anticancer effect in T98G human glioblastoma cells through the induction of apoptosis and the regulation of adhesion molecules.

    PubMed

    Jeong, Jae Ju; Lee, Jae Heun; Chang, Ki Churl; Kim, Hye Jung

    2012-10-01

    Glioblastoma is one of the most lethal and common malignant human brain tumors, with aggressive proliferation and highly invasive properties. Honokiol derived from Magnolia officinalis is able to cross the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB), suggesting a strong possibility that it could be an effective drug for the treatment of brain tumors, including glioblastoma. Thus, we investigated the effects of honokiol on the expression of adhesion molecules in TNF-α-stimulated endothelial cells, and cancer growth and invasion were determined in T98G human glioblastoma cells. Honokiol dose-dependently inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α for 6 h. Pretreatment with honokiol significantly reduced the adhesion of T98G cells to HUVECs. Moreover, honokiol inhibited the invasion of T98G cells, suggesting that honokiol has an anti-metastatic effect. In addition, honokiol increased the cytotoxicity of T98G cells in a dose- and time-dependent manner as assayed by MTT. TUNEL assay showed that honokiol significantly induced apoptosis in T98G cells at doses of 10 µM or more. The induction of apoptotic cell death was mediated by the downregulation of the anti-apoptotic protein Bcl-2 and the upregulation of the pro-apoptotic protein Bax. Taken together, the results of this study suggest that honokiol exerts an anticancer effect by preventing metastasis and inducing apoptotic cell death of brain tumor cells.

  12. Cell proliferation and drug sensitivity of human glioblastoma cells are altered by the stable modulation of cytosolic 5'-nucleotidase II.

    PubMed

    Cividini, F; Cros-Perrial, E; Pesi, R; Machon, C; Allegrini, S; Camici, M; Dumontet, C; Jordheim, L P; Tozzi, M G

    2015-08-01

    Cytosolic 5'-nucleotidase II (cN-II) has been reported to be involved in cell survival, nucleotide metabolism and in the cellular response to anticancer drugs. With the aim to further evaluate the role of this enzyme in cell biology, we stably modulated its expression the human glioblastoma cell ADF in which the transient inhibition of cN-II has been shown to induce cell death. Stable cell lines were obtained both with inhibition, obtained with plasmids coding cN-II-targeting short hairpin RNA, and stimulation, obtained with plasmids coding Green Fluorescence Protein (GFP)-fused wild type cN-II or a GFP-fused hyperactive mutant (GFP-cN-II-R367Q), of cN-II expression. Silenced cells displayed a decreased proliferation rate while the over expressing cell lines displayed an increased proliferation rate as evidenced by impedance measurement using the xCELLigence device. The expression of nucleotide metabolism relevant genes was only slightly different between cell lines, suggesting a compensatory mechanism in transfected cells. Cells with decreased cN-II expression were resistant to the nucleoside analog fludarabine confirming the involvement of cN-II in the metabolism of this drug. Finally, we observed sensitivity to cisplatin in cN-II silenced cells and resistance to this same drug in cN-II over-expressing cells indicating an involvement of cN-II in the mechanism of action of platinum derivatives, and most probably in DNA repair. In summary, our findings confirm some previous data on the role of cN-II in the sensitivity of cancer cells to cancer drugs, and suggest its involvement in other cellular phenomenon such as cell proliferation.

  13. Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells.

    PubMed

    Ross, Heather H; Rahman, Maryam; Levkoff, Lindsay H; Millette, Sebastien; Martin-Carreras, Teresa; Dunbar, Erin M; Reynolds, Brent A; Laywell, Eric D

    2011-12-01

    Thymidine analogs (TAs) are synthetic nucleosides that incorporate into newly synthesized DNA. Halogenated pyrimidines (HPs), such as bromodeoxyuridine (BrdU), are a class of TAs that can be detected with antibodies and are commonly used for birthdating individual cells and for assessing the proliferative index of cell populations. It is well established that HPs can act as radiosensitizers when incorporated into DNA chains, but they are generally believed not to impair normal cell function in the absence of secondary stressors. However, we and others have shown that HP incorporation leads to a sustained suppression of cell cycle progression in mammalian cells, resulting in cellular senescence in somatic cells. In addition, we have shown that HP incorporation results in delayed tumor progression in a syngeneic rat model of glioma. Here we examine ethynyldeoxyuridine (EdU), a newly developed and alkylated TA, for its anti-cancer activity, both in vitro and in vivo. We show that EdU, like HPs, leads to a severe reduction in the proliferation rate of normal and transformed cells in vitro. Unlike HPs, however, EdU incorporation also causes DNA damage resulting in the death of a substantial subset of treated cells. When administered over an extended time as a monotherapy to mice bearing subcutaneous xenografts of human glioblastoma multiforme tumors, EdU significantly reduces tumor volume and increases survival without apparent significant toxicity. These results, combined with the fact that EdU readily crosses the blood-brain barrier, support the continued investigation of EdU as a potential therapy for malignant brain tumors.

  14. Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells.

    PubMed

    Rao, V Koneti; Wangsa, Darawalee; Robey, Robert W; Huff, Lyn; Honjo, Yasumasa; Hung, Jeffrey; Knutsen, Turid; Ried, Thomas; Bates, Susan E

    2005-07-15

    The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.

  15. Histone deacetylase inhibitors interact with melanoma differentiation associated-7/interleukin-24 to kill primary human glioblastoma cells.

    PubMed

    Hamed, Hossein A; Yacoub, Adly; Park, Margaret A; Archer, Kellie; Das, Swadesh K; Sarkar, Devanand; Grant, Steven; Fisher, Paul B; Dent, Paul

    2013-08-01

    We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca(2+). Quenching of reactive oxygen species and Ca(2+) blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic.

  16. Glioblastoma Multiforme: Novel Therapeutic Approaches

    PubMed Central

    Fialho, Arsenio M.; Salunkhe, Prabhakar; Manna, Sunil; Mahali, Sidharth; Chakrabarty, Ananda M.

    2012-01-01

    The current therapy for glioblastoma multiforme involves total surgical resection followed by combination of radiation therapy and temozolomide. Unfortunately, the efficacy for such current therapy is limited, and newer approaches are sorely needed to treat this deadly disease. We have recently described the isolation of bacterial proteins and peptides with anticancer activity. In phase I human clinical trials, one such peptide, p28, derived from a bacterial protein azurin, showed partial and complete regression of tumors in several patients among 15 advanced-stage cancer patients with refractory metastatic tumors where the tumors were no longer responsive to current conventional drugs. An azurin-like protein called Laz derived from Neisseria meningitides demonstrates efficient entry and high cytotoxicity towards glioblastoma cells. Laz differs from azurin in having an additional 39-amino-acid peptide called an H.8 epitope, which allows entry and high cytotoxicity towards glioblastoma cells. Since p28 has been shown to have very little toxicity and high anti-tumor activity in advanced-stage cancer patients, it will be worthwhile to explore the use of H.8-p28, H.8-azurin, and Laz in toxicity studies and glioblastoma therapy in preclinical and human clinical trials. PMID:22462021

  17. A Cancer Research UK First Time in Human Phase I Trial of IMA950 (Novel Multipeptide Therapeutic Vaccine) in Patients with Newly Diagnosed Glioblastoma.

    PubMed

    Rampling, Roy; Peoples, Sharon; Mulholland, Paul J; James, Allan; Al-Salihi, Omar; Twelves, Christopher J; McBain, Catherine; Jefferies, Sarah; Jackson, Alan; Stewart, Willie; Lindner, Juha; Kutscher, Sarah; Hilf, Norbert; McGuigan, Lesley; Peters, Jane; Hill, Karen; Schoor, Oliver; Singh-Jasuja, Harpreet; Halford, Sarah E; Ritchie, James W A

    2016-10-01

    To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue. Patients were HLA-A*02-positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (Treg) levels, and the effect of steroids on T-cell responses. Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months. IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776-85. ©2016 AACRSee related commentary by Lowenstein and Castro, p. 4760. ©2016 American Association for Cancer Research.

  18. Intracellular calcium mobilization and phospholipid degradation in sphingosylphosphorylcholine-stimulated human airway epithelial cells.

    PubMed Central

    Orlati, S; Porcelli, A M; Hrelia, S; Lorenzini, A; Rugolo, M

    1998-01-01

    Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores. PMID:9729473

  19. Effect of alkali-treated lipopolysaccharide on the intracellular cations of human erythrocytes.

    PubMed Central

    Warren, J R; Kowalski, M M; Wallas, C H

    1977-01-01

    The adsorption to human erythrocytes of Escherichia coli lipopolysaccharide treated by mild alkaline hydrolysis (h-LPS) stimulated an increase in the intracellular Na+ concentration and a decrease in the intracellular K+ concentration of the erythrocytes. Erythrocytes treated by h-LPS remained responsive to the membrane adenosine triphosphatase inhibitors ouabain and ethacrynic acid, indicating that hLPS did not alter erythrocyte cations be depleting energy intermediates or uncoupling energy metabolism from active cation transport. The h-LPS-treated erythrocytes became non-agglutinable by the lectin concanavalin A prior to the development of changes in intracellular cations. In addition, h-LPS-treated erythrocytes demonstrated a three-fold greater cation response to ethacrynic acid than the untreated erythrocytes; this greater response was probably due to local membrane effects by h-LPS on the ethacrynic acid-sensitive adenosine triphosphatase. It is suggested that the h-LPS-induced alteration of erythrocyte cation content was secondary to an increase in ion permeability localized to the concanavalin A receptor regions of the erythrocyte membrane, possibly combined with indirect effects of membrane-bound h-LPS on ethacrynic acid-sensitive adenosine triphosphatase. Images PMID:330408

  20. Effect of alkali-treated lipopolysaccharide on the intracellular cations of human erythrocytes.

    PubMed

    Warren, J R; Kowalski, M M; Wallas, C H

    1977-08-01

    The adsorption to human erythrocytes of Escherichia coli lipopolysaccharide treated by mild alkaline hydrolysis (h-LPS) stimulated an increase in the intracellular Na+ concentration and a decrease in the intracellular K+ concentration of the erythrocytes. Erythrocytes treated by h-LPS remained responsive to the membrane adenosine triphosphatase inhibitors ouabain and ethacrynic acid, indicating that hLPS did not alter erythrocyte cations be depleting energy intermediates or uncoupling energy metabolism from active cation transport. The h-LPS-treated erythrocytes became non-agglutinable by the lectin concanavalin A prior to the development of changes in intracellular cations. In addition, h-LPS-treated erythrocytes demonstrated a three-fold greater cation response to ethacrynic acid than the untreated erythrocytes; this greater response was probably due to local membrane effects by h-LPS on the ethacrynic acid-sensitive adenosine triphosphatase. It is suggested that the h-LPS-induced alteration of erythrocyte cation content was secondary to an increase in ion permeability localized to the concanavalin A receptor regions of the erythrocyte membrane, possibly combined with indirect effects of membrane-bound h-LPS on ethacrynic acid-sensitive adenosine triphosphatase.

  1. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.

  2. Intracellular Na(+) modulates large conductance Ca(2+)-activated K (+) currents in human umbilical vein endothelial cells.

    PubMed

    Liang, Guo Hua; Kim, Moon Young; Park, Seonghee; Kim, Ji Aee; Choi, Shinkyu; Suh, Suk Hyo

    2008-10-01

    We studied the effects of Na(+) influx on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na(+) replacement by NMDG(+) or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na(+) concentration ([Na(+)](i)) and vice versa. In addition, extracellular Na(+) depletion decreased [Na(+)](i). In voltage-clamped cells, BK(Ca) currents were markedly increased by extracellular Na(+) depletion. In inside-out patches, increasing [Na(+)](i) from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK(Ca) channels and decreasing intracellular K(+) concentration ([K(+)](i)) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na(+)](i) gradually from 0 to 70 mM, by replacing K(+), markedly reduced single channel conductance and NPo. The Na(+)-Ca(2+) exchange blocker Ni(2+) or KB-R7943 decreased [Na(+)](i) and increased BK(Ca) currents simultaneously, and the Na(+) ionophore monensin completely inhibited BK(Ca) currents. BK(Ca) currents were significantly augmented by increasing extracellular K(+) concentration ([K(+)](o)) from 6 to 12 mM and significantly reduced by decreasing [K(+)](o) from 12 or 6 to 0 mM or applying the Na(+)-K(+) pump inhibitor ouabain. These results suggest that intracellular Na(+) inhibit single channel conductance of BK(Ca) channels and that intracellular K(+) increases single channel conductance and NPo.

  3. Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells.

    PubMed

    Berti, Denise A; Morano, Cain; Russo, Lilian C; Castro, Leandro M; Cunha, Fernanda M; Zhang, Xin; Sironi, Juan; Klitzke, Clécio F; Ferro, Emer S; Fricker, Lloyd D

    2009-05-22

    Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

  4. Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes

    PubMed Central

    Rittig, Michael G.; Alvarez-Martinez, Maria-Teresa; Porte, Françoise; Liautard, Jean-Pierre; Rouot, Bruno

    2001-01-01

    Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall. PMID:11349069

  5. F NMR measurement of intracellular free calcium in human red blood cells

    SciTech Connect

    Gupta, R.K.; Schanne, F.A.X.

    1986-03-01

    Optical techniques for the measurement of intracellular Ca are not readily applicable to the human red cell because of the intense absorption of hemoglobin. The authors have therefore examined the use of /sup 19/F NMR of 5,5'-difluoro-1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (5FBAPTA) introduced non-disruptively by intracellular hydrolysis of the membrane-permeant acetoxymethyl ester derivative. /sup 19/F NMR spectra of 5FBAPTA-containing erythrocytes at 188 MHz displayed two well resolved resonances corresponding to the free and Ca-bound forms of the chelator, the resonance of the free form being ten-fold larger than that of the Ca-bound form. Addition of the ionophore A23187 resulted in the disappearance of the resonance of the free anion and a quantitative increase in the intensity of the resonance of the Ca-complex. From these data, and a K/sub D/ of 708 nM for the Ca-5FBAPTA complex, the authors estimate red cell free Ca to be 70 nM, which is in the range of values obtained for other cells, despite the fact that the human red cell, which lacks intracellular organelles for storing Ca, possesses only 1 ..mu..mol total Ca/1. cells in comparison to mmols of total Ca found in other cells. The authors ability to use /sup 19/F NMR to measure free Ca in the red blood cell paves the way for future NMR studies of red cell free Ca concentrations in human essential hypertension as well as in other diseases states in which alterations in cellular Ca homeostasis may be involved.

  6. The presence of gonadotropin binding sites in the intracellular organelles of human ovaries.

    PubMed

    Rao, C V; Mitra, S; Sanfilippo, J; Carman, F R

    1981-03-15

    The nuclei (N), plasma membranes (PM), mitochondria-lysosomes, rough endoplasmic reticulum, and combined (light, medium, and heavy) Golgi (G) fractions were isolated from human ovaries. The purities of these fractions were evaluated by assays of appropriate marker enzymes, which revealed that some fractions were very pure but that others had minor contamination. When tested, all of the fractions exhibited 125I-labeled human chorionic gonadotropin (125I-hCG)-specific binding. This intracellular 125I-hCG binding was not due to PM contamination because: (1) N, which had no detectable 5'-nucleotidase (5'-NE) activity, a marker for PM, exhibited 125I-hCG-specific binding; (2) the G, which had only a fraction of the 5'-NE activity of PM, exhibited as much binding as PM; and (3) the ratios between specific 125I-hCG binding and 5'-NE activity in other fractions were not the same as for PM. They should have been the same if PM contamination was responsible for the 125I-hCG binding observed in other organelles. In conclusion, our results demonstrate that gonadotropin-binding sites are present in various intracellular organelles as well as in PM of human ovaries.

  7. Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins.

    PubMed

    Palmgren, Madelene; Hernebring, Malin; Eriksson, Stefanie; Elbing, Karin; Geijer, Cecilia; Lasič, Samo; Dahl, Peter; Hansen, Jesper S; Topgaard, Daniel; Lindkvist-Petersson, Karin

    2017-09-15

    Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.

  8. Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

    PubMed Central

    Chandra, S.; Ahmad, T.; Barth, R. F.; Kabalka, G. W.

    2014-01-01

    Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 (10B) atoms to individual tumor cells. Cell killing results from the 10B (n, α)7Li neutron capture and fission reactions that occur if a sufficient number of 10B atoms are localized in the tumor cells. Intranuclear 10B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of 10B atoms reflects both bound and free pools of boron in individual tumor cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular scale resolution by clinically applicable techniques such as PET and MRI. In this study, secondary ion mass spectrometry (SIMS) based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high grade gliomas, recurrent tumors of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumor cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a

  9. Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular-scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

    PubMed

    Chandra, S; Ahmad, T; Barth, R F; Kabalka, G W

    2014-06-01

    Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron-10 ((10)B) atoms to individual tumour cells. Cell killing results from the (10)B (n, α)(7) Li neutron capture and fission reactions that occur if a sufficient number of (10)B atoms are localized in the tumour cells. Intranuclear (10)B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of (10)B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular-scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L-p-boronophenylalanine (BPA), has been used clinically for BNCT of high-grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron-free nutrient medium. Both BPA and cis-ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This

  10. The expression of human papillomavirus type 16 (HPV16 E7) induces cell cycle arrest and apoptosis in radiation and hypoxia resistant glioblastoma cells.

    PubMed

    Moon, Sung-Ung; Choi, Soo Kyoung; Kim, Han Jo; Kumar Bokara, Kiran; Park, Kyung Ah; Lee, Won Taek; Lee, Jong-Eun

    2011-01-01

    p53 is a widely known tumor-suppressor gene product that plays a key role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents. Human glioma cells with functional p53 are known to be more resistant to γ-radiation. The aim of this study was to investigate whether the mutant glioblastoma cells (U87MG) transfected with human papilloma virus-type 16 E7 (HPV16 E7) genes were capable of increasing sensitivity towards irradiation and hypoxia-induced cell death. The pLXSN retroviral vector expressed HPV-16E7 genes and was infected into the p53 mutated U87MG cell line. A specific amplification band of HPV16 E7 genes was detected in E7 genes and transfected in the U87MG cell line using a reverse transcriptase polymerase chain reaction. The experimental groups included the mutant glioblastoma cell line (U87MG), empty vector (pLXSN) transfected to mutant glioblastoma cell line (U87MG-LXSN), and retrovirus carrying HPV16 E7 genes transfected to the mutant glioblastoma cell line (U87MG-E7). Hypoxic conditions were optimized using LDH assay and the subjects were exposed to hypoxia (16 and 20 h) and irradiation (9 h). Hoechst-propidium iodide (PI) staining results showed that hypoxia and irradiation increased the number of dead cells in the U87MG-E7 cells compared to U87MG and U87MG-LXSN cells. Results of the FACS analysis showed a similar pattern and recorded 80.44 and 58.94% of apoptotic cells in U87MG-E7 and U87MG cells, respectively. Cell cycle analysis by FACS revealed that, following irradiation and hypoxia, cells showed G2-M arrest. Additionally, the Western blot analysis results showed altered expression of E2F-1, Rb and p53 in the irradiation- and hypoxia-induced U87MG-E7 cells compared to U87MG and U87MG LXSN cells. In conclusion, the over-expression of HPV16 E7 genes in U87MG cell lines increasd cell apoptosis and E2F1 expression compared to the HPV non-infected U87MG cells following irradiation and hypoxia. These results indicate that tumor

  11. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells

    PubMed Central

    Podergajs, Neža; Motaln, Helena; Rajčević, Uroš; Verbovšek, Urška; Koršič, Marjan; Obad, Nina; Espedal, Heidi; Vittori, Miloš; Herold-Mende, Christel; Miletic, Hrvoje; Bjerkvig, Rolf; Turnšek, Tamara Lah

    2016-01-01

    The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment. PMID:26573230

  12. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells.

    PubMed

    Podergajs, Neža; Motaln, Helena; Rajčević, Uroš; Verbovšek, Urška; Koršič, Marjan; Obad, Nina; Espedal, Heidi; Vittori, Miloš; Herold-Mende, Christel; Miletic, Hrvoje; Bjerkvig, Rolf; Turnšek, Tamara Lah

    2016-01-05

    The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.

  13. Human glioblastoma stem-like cells accumulate protoporphyrin IX when subjected to exogenous 5-aminolaevulinic acid, rendering them sensitive to photodynamic treatment.

    PubMed

    Schimanski, Adrian; Ebbert, Lara; Sabel, Michael C; Finocchiaro, Gaetano; Lamszus, Katrin; Ewelt, Christian; Etminan, Nima; Fischer, Johannes C; Sorg, Rüdiger V

    2016-10-01

    Glioblastoma (GBM) is the most frequent and lethal primary brain tumor in adults. Despite multimodal therapy combining resection, radio- and alkylating chemotherapy, disease recurrence is universal and prognosis of patients is poor. Glioblastoma stem-like cells (GSC), which can be grown as neurospheres from primary tumors in vitro, appear to be resistant to the established therapies and are suspected to be the driving force for disease recurrence. Thus, efficacy of emerging therapies may depend on targeting GSC. 5-aminolaevulinic acid-mediated photodynamic therapy (5-ALA/PDT) is a promising therapeutic approach in GBM. It utilizes the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in GBM cells after application of 5-ALA. When exposed to laser light of 635nm wavelength, PPIX initiates a photochemical reaction resulting in the generation of reactive oxygen species, which kill the tumor cells. Whether GSC accumulate PPIX and are sensitive to 5-ALA/PDT is currently unknown. Therefore, human GSC were derived from primary tumors and grown as neurospheres under serum free conditions. When subjected to exogenous 5-ALA, a dose- and time-dependent accumulation of PPIX in GSC was observed by flow cytometry, which varied between individual GSC preparations. Subsequent exposure to laser light of 635nm wavelength substantially killed GSC, whereas treatment with 5-ALA or exposure to laser light only had no effect. LD50 values differed between GSC preparations, but were negatively correlated with PPIX accumulation in GSC. In summary, we report for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to 5-aminolaevulinic acid and are sensitive to 5-aminolaevulinc acid based photodynamic therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Down-regulation of Connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion.

    PubMed

    Strale, Pierre-Olivier; Clarhaut, Jonathan; Lamiche, Coralie; Cronier, Laurent; Mesnil, Marc; Defamie, Norah

    2012-11-01

    Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1.

  15. Sorafenib induces growth arrest and apoptosis of human glioblastoma cells through the dephosphorylation of signal transducers and activators of transcription 3.

    PubMed

    Yang, Fan; Brown, Christine; Buettner, Ralf; Hedvat, Michael; Starr, Renate; Scuto, Anna; Schroeder, Anne; Jensen, Michael; Jove, Richard

    2010-04-01

    Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953-62. (c)2010 AACR.

  16. Branched multipeptide immunotherapy for glioblastoma using human leukocyte antigen-A*0201-restricted cytotoxic T-lymphocyte epitopes from ERBB2, BIRC5 and CD99

    PubMed Central

    Kim, Young-Hee; Tran, Thi-Anh-Thuy; Lee, Hyun-Ju; Jung, Sook-In; Lee, Je-Jung; Jang, Wool-Youl; Moon, Kyung-Sub; Kim, In-Young; Jung, Shin; Jung, Tae-Young

    2016-01-01

    We investigated the use of cytotoxic T-lymphocyte (CTL) epitopes in peptide immunotherapy for glioblastoma. Three peptides (ERBB2, BIRC5 and CD99) were selected based on their peptide-T2 cell binding affinities and combined in a multipeptide cocktail or a branched multipeptide synthesized with mini-polyethylene glycol spacers. Dendritic cells (DCs) pulsed with the multipeptide cocktail or branched multipeptide were compared based on their immunophenotype and cytokine secretion. FACS analysis of alpha-type 1 polarized dendritic cells (αDC1s) revealed that both groups highly expressed CD80, CD83 and CD86, indicating that both treatments efficiently generated mature αDC1s with the expected phenotype. Production of IL-12p70, IL-12p40 and IL-10 also increased upon αDC1 maturation in both groups. CTLs stimulated by either αDC1 group (“DC-CTLs”) included numerous IFN-γ-secreting cells against T2 cells loaded with the corresponding multipeptides. Large numbers of IFN-γ-secreting cells were observed when human glioblastoma cell lines and primary cells were treated with multipeptide-pulsed DC-CTLs. Both multipeptide-pulsed DC-CTL groups exhibited cytotoxic activity of 40-60% against the U251 cell line and 60-80% against primary cells. Branched multipeptide from ERBB2, BIRC5 and CD99 stably bound with T2 cells, and its cytotoxicity toward target cells was similar to that of the multipeptide cocktail. Thus, branched multipeptides could be promising candidates for immunotherapeutic glioblastoma treatment. PMID:27409668

  17. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

    PubMed

    Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

    2004-03-15

    Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

  18. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection

    PubMed Central

    Li, Yuan; Shah-Simpson, Sheena; Okrah, Kwame; Belew, A. Trey; Choi, Jungmin; Caradonna, Kacey L.; Padmanabhan, Prasad; Ndegwa, David M.; Temanni, M. Ramzi; Corrada Bravo, Héctor; El-Sayed, Najib M.; Burleigh, Barbara A.

    2016-01-01

    Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our

  19. Sulforhodamine 101 selectively labels human astrocytoma cells in an animal model of glioblastoma.

    PubMed

    Georges, Joseph F; Martirosyan, Nikolay L; Eschbacher, Jennifer; Nichols, Joshua; Tissot, Maya; Preul, Mark C; Feuerstein, Burt; Anderson, Trent; Spetzler, Robert F; Nakaji, Peter

    2014-05-01

    Sulforhodamine 101 (SR101) is a useful tool for immediate staining of astrocytes. We hypothesized that if the selectivity of SR101was maintained in astrocytoma cells, it could prove useful for glioma research. Cultured astrocytoma cells and acute slices from orthotopic human glioma (n=9) and lymphoma (n=6) xenografts were incubated with SR101 and imaged with confocal microscopy. A subset of slices (n=18) were counter-immunostained with glial fibrillary acidic protein and CD20 for stereological assessment of SR101 co-localization. SR101 differentiated astrocytic tumor cells from lymphoma cells. In acute slices, SR101 labeled 86.50% (±1.86; p<0.0001) of astrocytoma cells and 2.19% (±0.47; p<0.0001) of lymphoma cells. SR101-labeled astrocytoma cells had a distinct morphology when compared with in vivo astrocytes. Immediate imaging of human astrocytoma cells in vitro and in ex vivo rodent xenograft tissue labeled with SR101 can identify astrocytic tumor cells and help visualize the tumor margin. These features are useful in studying astrocytoma in the laboratory and may have clinical applications. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study

    PubMed Central

    2012-01-01

    Background Glioblastoma multiforme (GBM) represent the most aggressive brain tumor with a median overall survival of about 12-15 months. Over 90% of GBM tumors have recently been shown to be infected with human cytomegalovirus (HCMV). In this case-control study, we evaluated whether there was an association between the grade of HCMV infection and long-term survival (> 18 months) in GBM patients. Material and methods Brain tumor tissue sections from consecutive GBMs patients who survived more than 18 months (n = 40), and an equal number of GBM patients, matched to date of diagnosis and surgery, operated at Karolinska University Hospital in 2000-2005 were selected. HCMV infection grade was determined by estimation of the number of HCMV positive cells (scored negative or grade 1-4) in tumor tissue specimens. Using Chi-Square test and logistic regression analysis, we analyzed whether there was an association between long-term survival and HCMV low-grade infection or other clinical parameters known to be associated with prolonged survival of GBM patients; age under 50 years, radical surgery or low recursive partition analysis (RPA) subclass. Results HCMV infection was detected in tumor samples from 79 of 80 patients (99%). Among patients surviving > 18 months, HCMV infection grade 1 in the GBM tumor was predominant. A low grade HCMV infection was found in 19 patients, of these 16 survived > 18 months. Thus, 16 of 40 (40%) GBM patients who lived > 18 months had low-grade HCMV infection while only 3 of 40 (8%) GBM patients who lived < 18 months did (p .0006, Chi-Square test). Multiple logistic regression analyses yielded an odds ratio estimate of 6.604 with 95% confidence interval (1.36-32.1) (p .019) for low grade HCMV after adjustment for RPA class III and IV, radical surgery, age and gamma knife treatment. Conclusion In conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients. PMID:22424569

  1. Immunosuppressive mechanisms in glioblastoma.

    PubMed

    Nduom, Edjah K; Weller, Michael; Heimberger, Amy B

    2015-11-01

    Despite maximal surgical and medical therapy, the treatment of glioblastoma remains a seriously vexing problem, with median survival well under 2 years and few long-term survivors. Targeted therapy has yet to produce significant advances in treatment of these lesions in spite of advanced molecular characterization of glioblastoma and glioblastoma cancer stem cells. Recently, immunotherapy has emerged as a promising mode for some of the hardest to treat tumors, including metastatic melanoma. Although immunotherapy has been evaluated in glioblastoma in the past with limited success, better understanding of the failures of these therapies could lead to more successful treatments in the future. Furthermore, there is a persistent challenge for the use of immune therapy to treat glioblastoma secondary to the existence of redundant mechanisms of tumor-mediated immune suppression. Here we will address these mechanisms of immunosuppression in glioblastoma and therapeutic approaches.

  2. Site-1 protease, a novel metabolic target for glioblastoma.

    PubMed

    Caruana, Beth T; Skoric, Aleksandra; Brown, Andrew J; Lutze-Mann, Louise H

    2017-08-26

    Sterol regulatory element binding proteins (SREBPs) are transcriptional regulators of lipids which promote glioblastoma growth. Here, we investigate the effect of inhibiting expression of SREBP target genes in human glioblastoma cells. This was achieved by using PF-429242 to inhibit site-1 protease (S1P), an enzyme required for SREBP activation. Treatment with PF-429242 decreased glioblastoma cell viability, induced apoptosis and downregulated steroid, isoprenoid and unsaturated fatty acid biosynthetic pathways. Several pro-inflammatory genes were upregulated. Collectively, these results demonstrate the potential of S1P as a target for glioblastoma therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Analysis of the Cytotoxicity of Carbon-Based Nanoparticles, Diamond and Graphite, in Human Glioblastoma and Hepatoma Cell Lines

    PubMed Central

    Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

    2015-01-01

    Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests. PMID:25816103

  4. Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines.

    PubMed

    Zakrzewska, Karolina Ewa; Samluk, Anna; Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Sawosz, Ewa; Chwalibog, André; Pijanowska, Dorota Genowefa; Pluta, Krzysztof Dariusz

    2015-01-01

    Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells are suitable for drug cytotoxicity tests.

  5. Carbon-ion beams effectively induce growth inhibition and apoptosis in human neural stem cells compared with glioblastoma A172 cells.

    PubMed

    Isono, Mayu; Yoshida, Yukari; Takahashi, Akihisa; Oike, Takahiro; Shibata, Atsushi; Kubota, Yoshiki; Kanai, Tatsuaki; Ohno, Tatsuya; Nakano, Takashi

    2015-09-01

    Carbon-ion radiotherapy (CIRT) holds promise in the treatment of glioblastoma, an aggressive X-ray-resistant brain tumor. However, since glioblastoma cells show a highly invasive nature, carbon-ion (C-ion) irradiation of normal tissues surrounding the tumor is inevitable. Recent studies have revealed the existence of neural stem cells in the adult brain. Therefore, the damaging effect of C-ion beams on the neural stem cells has to be carefully considered in the treatment planning of CIRT. Here, we investigated the growth and death mode of human neural stem cells (hNSCs) and glioblastoma A172 cells after X-ray or C-ion beam irradiation. The X-ray dose resulting in a 50% growth rate (D(50)) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D(50) for C-ion beams was 0.4 Gy in hNSCs and 1.6 Gy in A172 cells; the relative biological effectiveness value of C-ion beams was 2.0 in hNSCs and 1.9 in A172 cells. Importantly, both X-rays and C-ion beams preferentially induced apoptosis, not necrosis, in hNSCs; however, radiation-induced apoptosis was less evident in A172 cells. The apoptosis-susceptible nature of the irradiated hNSCs was associated with prolonged upregulation of phosphorylated p53, whereas the apoptosis-resistant nature of A172 cells was associated with a high basal level of nuclear factor kappa B expression. Taken together, these data indicate that apoptosis is the major cell death pathway in hNSCs after irradiation. The high sensitivity of hNSCs to C-ion beams underscores the importance of careful target volume delineation in the treatment planning of CIRT for glioblastoma. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  6. Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells.

    PubMed Central

    Pascual, A; García, I; Ballesta, S; Perea, E J

    1997-01-01

    The penetration of trovafloxacin into human polymorphonuclear leukocytes (PMNs), human peritoneal macrophages, and tissue-cultured epithelial cells (McCoy cells) was evaluated. The cellular concentration to extracellular concentration (C/E) ratios of trovafloxacin were greater than 9 for extracellular concentrations ranging from 0.5 to 25 micrograms/ml. The uptake of trovafloxacin by PMNs was rapid, reversible, nonsaturable, not energy dependent, and significantly increased at 4 degrees C. Ingestion of opsonized zymosan, but not opsonized Staphylococcus aureus, significantly increased the amount of PMN-associated trovafloxacin. This agent at concentrations of 0.5 and 1 microgram/ml induced a greater reduction in the survival of intracellular S. aureus in PMNs than ciprofloxacin and ofloxacin. It was concluded that trovafloxacin reaches concentrations within phagocytic and nonphagocytic cells several times higher than the extracellular ones, while it remains active in PMNs. PMID:9021179

  7. Computational Identification of Tumor Anatomic Location Associated with Survival in 2 Large Cohorts of Human Primary Glioblastomas.

    PubMed

    Liu, T T; Achrol, A S; Mitchell, L A; Du, W A; Loya, J J; Rodriguez, S A; Feroze, A; Westbroek, E M; Yeom, K W; Stuart, J M; Chang, S D; Harsh, G R; Rubin, D L

    2016-04-01

    Tumor location has been shown to be a significant prognostic factor in patients with glioblastoma. The purpose of this study was to characterize glioblastoma lesions by identifying MR imaging voxel-based tumor location features that are associated with tumor molecular profiles, patient characteristics, and clinical outcomes. Preoperative T1 anatomic MR images of 384 patients with glioblastomas were obtained from 2 independent cohorts (n = 253 from the Stanford University Medical Center for training and n = 131 from The Cancer Genome Atlas for validation). An automated computational image-analysis pipeline was developed to determine the anatomic locations of tumor in each patient. Voxel-based differences in tumor location between good (overall survival of >17 months) and poor (overall survival of <11 months) survival groups identified in the training cohort were used to classify patients in The Cancer Genome Atlas cohort into 2 brain-location groups, for which clinical features, messenger RNA expression, and copy number changes were compared to elucidate the biologic basis of tumors located in different brain regions. Tumors in the right occipitotemporal periventricular white matter were significantly associated with poor survival in both training and test cohorts (both, log-rank P < .05) and had larger tumor volume compared with tumors in other locations. Tumors in the right periatrial location were associated with hypoxia pathway enrichment and PDGFRA amplification, making them potential targets for subgroup-specific therapies. Voxel-based location in glioblastoma is associated with patient outcome and may have a potential role for guiding personalized treatment. © 2016 by American Journal of Neuroradiology.

  8. Olanzapine inhibits proliferation, migration and anchorage-independent growth in human glioblastoma cell lines and enhances temozolomide's antiproliferative effect.

    PubMed

    Karpel-Massler, Georg; Kast, Richard Eric; Westhoff, Mike-Andrew; Dwucet, Annika; Welscher, Nathalie; Nonnenmacher, Lisa; Hlavac, Michal; Siegelin, Markus David; Wirtz, Christian Rainer; Debatin, Klaus-Michael; Halatsch, Marc-Eric

    2015-03-01

    The poor prognosis of patients with glioblastoma fuels the search for more effective therapeutic compounds. We previously hypothesised that the neuroleptic olanzapine may enhance antineoplastic effects of temozolomide the standard chemotherapeutic agent used in this disease. This study tested this hypothesis. The anti-proliferative effect of olanzapine was examined by MTT assays and cell count analysis. Soft-agar assays were performed to examine colony-forming ability. In addition, the inhibitory effect of olanzapine on the migratory capacity of U87MG and A172 cells was analyzed by Transwell(®) assays. Moreover, staining for annexin V/propidium iodide or carboxyfluorescein succinimidyl ester was performed prior to flow cytometric analysis in order to better understand the subjacent cellular mechanism. Our initial hypothesis that olanzapine may enhance temozolomide's anti-tumor activity could be confirmed in U87MG and A172 glioblastoma cell lines. Moreover, treatment with olanzapine alone resulted in a marked anti-proliferative effect on U87MG, A172 and two glioma stem-like cells with IC50 values ranging from 25 to 79.9 µM. In U87MG cells, anchorage-independent growth was dose-dependently inhibited. In A172 cells, migration was also shown to be inhibited in a dose-dependent manner. In addition, olanzapine was shown to exert a cell line-dependent pleomorphism with respect to the induction of apoptosis, necrosis and/or cytostasis. Our data show that the neuroleptic olanzapine enhances the anti-tumor activity of temozolomide against glioblastoma cell lines. Moreover, this is the first study to show that olanzapine provides on its own anti-cancer activity in glioblastoma and thus may have potential for repurposing.

  9. Nociceptin/orphanin FQ antagonizes lipopolysaccharide-stimulated proliferation, migration and inflammatory signaling in human glioblastoma U87 cells.

    PubMed

    Bedini, Andrea; Baiula, Monica; Vincelli, Gabriele; Formaggio, Francesco; Lombardi, Sara; Caprini, Marco; Spampinato, Santi

    2017-09-15

    Glioblastoma is among the most aggressive brain tumors and has an exceedingly poor prognosis. Recently, the importance of the tumor microenvironment in glioblastoma cell growth and progression has been emphasized. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and endogenous ligands originating from dying cells or the extracellular matrix involved in host defense and in inflammation. G-protein coupled receptors (GPCRs) have gained interest in anti-tumor drug discovery due to the role that they directly or indirectly play by transactivating other receptors, causing cell migration and proliferation. A proteomic analysis showed that the nociceptin receptor (NOPr) is among the GPCRs significantly expressed in glioblastoma cells, including U87 cells. We describe a novel role of the peptide nociceptin (N/OFQ), the endogenous ligand of the NOPr that counteracts cell migration, proliferation and increase in IL-1β mRNA elicited by LPS via TLR4 in U87 glioblastoma cells. Signaling pathways through which N/OFQ inhibits LPS-mediated cell migration and elevation of [Ca(2+)]i require β-arrestin 2 and are sensitive to TNFR-associated factor 6, c-Src and protein kinase C (PKC). LPS-induced cell proliferation and increase in IL-1β mRNA are counteracted by N/OFQ via β-arrestin 2, PKC and extracellular signal-regulated kinase 1/2; furthermore, the contributions of the transcription factors NF-kB and AP-1 were investigated. Independent of LPS, N/OFQ induces a significant increase in cell apoptosis. Contrary to what was observed in other cell models, a prolonged exposure to this endotoxin did not promote any tolerance of the cellular effects above described, including NOPr down-regulation while N/OFQ loses its inhibitory role. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Changes in Intracellular Na+ following Enhancement of Late Na+ Current in Virtual Human Ventricular Myocytes

    PubMed Central

    Giles, Wayne R.

    2016-01-01

    The slowly inactivating or late Na+ current, INa-L, can contribute to the initiation of both atrial and ventricular rhythm disturbances in the human heart. However, the cellular and molecular mechanisms that underlie these pro-arrhythmic influences are not fully understood. At present, the major working hypothesis is that the Na+ influx corresponding to INa-L significantly increases intracellular Na+, [Na+]i; and the resulting reduction in the electrochemical driving force for Na+ reduces and (may reverse) Na+/Ca2+ exchange. These changes increase intracellular Ca2+, [Ca2+]i; which may further enhance INa-L due to calmodulin-dependent phosphorylation of the Na+ channels. This paper is based on mathematical simulations using the O’Hara et al (2011) model of baseline or healthy human ventricular action potential waveforms(s) and its [Ca2+]i homeostasis mechanisms. Somewhat surprisingly, our results reveal only very small changes (≤ 1.5 mM) in [Na+]i even when INa-L is increased 5-fold and steady-state stimulation rate is approximately 2 times the normal human heart rate (i.e. 2 Hz). Previous work done using well-established models of the rabbit and human ventricular action potential in heart failure settings also reported little or no change in [Na+]i when INa-L was increased. Based on our simulations, the major short-term effect of markedly augmenting INa-L is a significant prolongation of the action potential and an associated increase in the likelihood of reactivation of the L-type Ca2+ current, ICa-L. Furthermore, this action potential prolongation does not contribute to [Na+]i increase. PMID:27875582

  11. Changes in Intracellular Na+ following Enhancement of Late Na+ Current in Virtual Human Ventricular Myocytes.

    PubMed

    Cardona, Karen; Trenor, Beatriz; Giles, Wayne R

    2016-01-01

    The slowly inactivating or late Na+ current, INa-L, can contribute to the initiation of both atrial and ventricular rhythm disturbances in the human heart. However, the cellular and molecular mechanisms that underlie these pro-arrhythmic influences are not fully understood. At present, the major working hypothesis is that the Na+ influx corresponding to INa-L significantly increases intracellular Na+, [Na+]i; and the resulting reduction in the electrochemical driving force for Na+ reduces and (may reverse) Na+/Ca2+ exchange. These changes increase intracellular Ca2+, [Ca2+]i; which may further enhance INa-L due to calmodulin-dependent phosphorylation of the Na+ channels. This paper is based on mathematical simulations using the O'Hara et al (2011) model of baseline or healthy human ventricular action potential waveforms(s) and its [Ca2+]i homeostasis mechanisms. Somewhat surprisingly, our results reveal only very small changes (≤ 1.5 mM) in [Na+]i even when INa-L is increased 5-fold and steady-state stimulation rate is approximately 2 times the normal human heart rate (i.e. 2 Hz). Previous work done using well-established models of the rabbit and human ventricular action potential in heart failure settings also reported little or no change in [Na+]i when INa-L was increased. Based on our simulations, the major short-term effect of markedly augmenting INa-L is a significant prolongation of the action potential and an associated increase in the likelihood of reactivation of the L-type Ca2+ current, ICa-L. Furthermore, this action potential prolongation does not contribute to [Na+]i increase.

  12. Establishment and genetic characterization of ANGM-CSS, a novel, immortal cell line derived from a human glioblastoma multiforme.

    PubMed

    Notarangelo, Angelantonio; Trombetta, Domenico; D'Angelo, Vincenzo; Parrella, Paola; Palumbo, Orazio; Storlazzi, Clelia Tiziana; Impera, Luciana; Muscarella, Lucia Anna; La Torre, Antonella; Affuso, Andrea; Fazio, Vito Michele; Carella, Massimo; Zelante, Leopoldo

    2014-03-01

    Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.

  13. Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration.

    PubMed

    Patten, Daniel A; Wilson, Garrick K; Bailey, Dalan; Shaw, Robert K; Jalkanen, Sirpa; Salmi, Marko; Rot, Antal; Weston, Chris J; Adams, David H; Shetty, Shishir

    2017-01-01

    The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs.

  14. Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles

    NASA Astrophysics Data System (ADS)

    Lavado, Andrea S.; Chauhan, Veeren M.; Alhaj Zen, Amer; Giuntini, Francesca; Jones, D. Rhodri E.; Boyle, Ross W.; Beeby, Andrew; Chan, Weng C.; Aylott, Jonathan W.

    2015-08-01

    Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn

  15. Infected T98G glioblastoma cells support human cytomegalovirus reactivation from latency.

    PubMed

    Cheng, Shuang; Jiang, Xuan; Yang, Bo; Wen, Le; Zhao, Fei; Zeng, Wen-Bo; Liu, Xi-Juan; Dong, Xiao; Sun, Jin-Yan; Ming, Ying-Zi; Zhu, Hua; Rayner, Simon; Tang, Qiyi; Fortunato, Elizabeth; Luo, Min-Hua

    2017-10-01

    T98G cells have been shown to support long-term human cytomegalovirus (HCMV) genome maintenance without infectious virus release. However, it remains unclear whether these viral genomes could be reactivated. To address this question, a recombinant HCMV (rHCMV) containing a GFP gene was used to infect T98G cells, and the infected cells absent of infectious virus production were designated T98G-LrV. Upon dibutyryl cAMP plus IBMX (cAMP/IBMX) treatment, a serial of phenomena were observed, including GFP signal increase, viral genome replication, lytic genes expression and infectious viruses release, indicating the reactivation of HCMV in T98G-LrV cells from a latent status. Mechanistically, HCMV reactivation in the T98G-LrV cells induced by cAMP/IBMX was associated with the PKA-CREB signaling pathway. These results demonstrate that HCMV was latent in T98G-LrV cells and could be reactivated. The T98G-LrV cells represent an effective model for investigating the mechanisms of HCMV reactivation from latency in the context of neural cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. A New Approach in Gene Therapy of Glioblastoma Multiforme: Human Olfactory Ensheathing Cells as a Novel Carrier for Suicide Gene Delivery.

    PubMed

    Hashemi, Mansoureh; Fallah, Ali; Aghayan, Hamid Reza; Arjmand, Babak; Yazdani, Nasrin; Verdi, Javad; Ghodsi, Seyed Mohammad; Miri, Seyed Mojtaba; Hadjighassem, Mahmoudreza

    2016-10-01

    Olfactory ensheathing cells (OECs) of human olfactory mucosa are a type of glial-like cells that possess good migratory and tropism properties. We believe that neuronal-derived vehicle may have better capability to receive to the site of injury. In addition to, obtaining of such vehicle from the patient reduces risk of unwanted complications. So, in this study, we investigate whether human olfactory ensheathing cells can be used as a cell source for the first time in gene delivery to assay the tumoricidal effect of herpes simplex virus thymidine kinase gene (HSV-tk) on glioblastoma multiforme (GBM). We obtained OECs from superior turbinate of human nasal cavity mucosa, and cell phenotype was confirmed by the expression of cell-specific antigens including low-affinity nerve growth factor receptor (p75 neurotrophin receptor), microtubule-associated protein-2 (MAP2), and S100 calcium binding protein B (S100-beta) using immunocytochemistry. Then, these cells were transduced by lentiviral vector for transient and stable expression of the herpes simplex virus thymidine kinase gene (OEC-tk). The migratory capacity of OEC-tk, their potency to convert prodrug ganciclovir to toxic form, and cytotoxic effect on astrocyte cells were assayed in vitro. The OECs showed fibroblast-like morphology and expressed specific antigens such as p75 neurotrophin receptor, S100-beta, and MAP2. Our results indicated that OECs-tk were able to migrate toward primary cultured human glioblastoma multiforme and affected survival rate of tumor cells according to exposure time and concentration of ganciclovir. Also, OECs-HSV-tk was capable of inducing apoptosis in tumor cells. Our findings suggest that human OECs could employ as a possible tool to transfer anticancer agent in gene therapy of brain tumor.

  17. Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

    PubMed

    Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N; Melo, Rómulo; Oyarzún, Carlos; San Martín, Rody; Quezada, Claudia

    2016-10-11

    MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

  18. Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells

    PubMed Central

    Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N.; Melo, Rómulo; Oyarzún, Carlos; Martín, Rody San; Quezada, Claudia

    2016-01-01

    MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs. PMID:27634913

  19. Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

    SciTech Connect

    Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra; Zupanska, Agata; Chouaib, Salem; Kaminska, Bozena . E-mail: bozenakk@nencki.gov.pl

    2007-03-23

    Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2 and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.

  20. Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS).

    PubMed

    Chandra, S; Tjarks, W; Lorey, D R; Barth, R F

    2008-01-01

    Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron-10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p-boronophenylalanine (BPA) and 3-[4-(o-carboran-1-yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3-carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase-mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS-3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze-fractured, freeze-dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria-rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na-K-ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA-treated interphase

  1. Intracellular localization of human cytidine deaminase. Identification of a functional nuclear localization signal.

    PubMed

    Somasekaram, A; Jarmuz, A; How, A; Scott, J; Navaratnam, N

    1999-10-01

    The cytidine deaminases belong to the family of multisubunit enzymes that catalyze the hydrolytic deamination of their substrate to a corresponding uracil product. They play a major role in pyrimidine nucleoside and nucleotide salvage. The intracellular distribution of cytidine deaminase and related enzymes has previously been considered to be cytosolic. Here we show that human cytidine deaminase (HCDA) is present in the nucleus. A highly specific, affinity purified polyclonal antibody against HCDA was used to analyze the intracellular localization of native HCDA in a variety of mammalian cells by in situ immunochemistry. Native HCDA was found to be present in the nucleus as well as the cytoplasm in several cell types. Indirect immunofluorescence microscopy indicated a predominantly nuclear localization of FLAG-tagged HCDA overexpressed in these cells. We have identified an amino-terminal bipartite nuclear localization signal that is both necessary and sufficient to direct HCDA and a non-nuclear reporter protein to the nucleus. We also show HCDA binding to the nuclear import receptor, importin alpha. Similar putative bipartite nuclear localization sequences are found in other cytidine/deoxycytidylate deaminases. The results presented here suggest that the pyrimidine nucleotide salvage pathway may operate in the nucleus. This localization may have implications in the regulation of nucleoside and nucleotide metabolism and nucleic acid biosynthesis.

  2. Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes.

    PubMed

    Neumeister, B; Reiff, G; Faigle, M; Dietz, K; Northoff, H; Lang, F

    2000-03-01

    Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449-2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.

  3. Rapid intracellular release of calcium in human platelets by stimulation of 5-HT2-receptors.

    PubMed Central

    Erne, P.; Pletscher, A.

    1985-01-01

    The concentration of intracellular free Ca2+ ( [Ca2+]i) in human blood platelets was measured by use of the fluorescent probe quin-2. 5-Hydroxytryptamine (5-HT) caused a rapid increase of [Ca2+]i in the presence or absence of Ca2+ in the medium. The [Ca2+]i-rise was less marked in the absence of Ca2+ and could be antagonized by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate-hydrochloride (TMB-8), an inhibitor of calcium release from internal stores. 5-HT induced a shape change reaction in the presence or absence of extracellular Ca2+, but the pEC50 of 5-HT was slightly higher in the presence of the cation. Shape change reaction and [Ca2+]i-rise showed similar time courses. Various 5-HT-agonists caused a rise of [Ca2+]i, whereas 5-HT-antagonists, but not the 5-HT-uptake inhibitor desmethylimipramine and the alpha 2-adrenoceptor antagonist yohimbine, counteracted the 5-HT-induced rise of the cation in a stereospecific manner. The antagonists were more potent than the agonists. The orders of potencies of the drugs affecting [Ca2+]i and platelet shape were similar. It is concluded that stimulation of 5-HT2-receptors of platelets causes a rapid release of intracellular calcium which, by activation of the contractile system, mediates the shape change reaction. PMID:3156650

  4. Oxidation of intracellular glutathione after exposure of human red blood cells to hypochlorous acid.

    PubMed Central

    Vissers, M C; Winterbourn, C C

    1995-01-01

    Exposure of human red blood cells to low doses of hypochlorous acid (HOCl) resulted in the loss of intracellular GSH. Oxidation occurred less than 2 min after the addition of HOCl, and required approx. 2.5 mol of HOCl per mol of GSH lost. Loss of GSH preceded oxidation of membrane thiols, the formation of chloramines and haemoglobin oxidation. The susceptibility of intracellular GSH to oxidation by HOCl was two-thirds that of GSH in cell lysates. These results indicate that HOCl can penetrate the red cell membrane, which provides little barrier protection for cytoplasmic components, and that GSH oxidation by HOCl may be a highly selective process. Virtually all of the GSH lost was converted into GSSG. If glucose was added to the medium, most of the GSH oxidized by low doses of HOCl was rapidly regenerated. At higher doses, recovery was less efficient. However, when HOCl was added as a slow infusion rather than in a single bolus, there was increased recovery at higher doses. This indicates that in metabolically active cells regeneration is rapid and GSH may protect cell components from damage by HOCl. HOCl-induced lysis was only slightly delayed by adding glucose to the medium, indicating that lytic injury is not ameliorated by GSH. Images Figure 5 PMID:7717994

  5. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

    PubMed Central

    Giengkam, Suparat; Blakes, Alex; Utsahajit, Peemdej; Chaemchuen, Suwittra; Atwal, Sharanjeet; Blacksell, Stuart D.; Paris, Daniel H.; Day, Nicholas P. J.; Salje, Jeanne

    2015-01-01

    Background Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families. Methodology A clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells. Key results Development of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including

  6. Regulation of human airway ciliary beat frequency by intracellular pH

    PubMed Central

    Sutto, Zoltan; Conner, Gregory E; Salathe, Matthias

    2004-01-01

    pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate-free medium were 7.2 ± 0.2 Hz and 7.49 ± 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pHi 7.78 ± 0.02 resulted in a 2.2 ± 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 ± 0.02 and CBF to 5.8 ± 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 μm forskolin), broad inhibition of protein kinases (100 μm H-7), inhibition of PKA (10 μm H-89), nor inhibition of phosphatases (10 μm cyclosporin + 1.5 μm okadaic acid) changed pHi-mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re-differentiated at the air–liquid interface, was 3.9 ± 0.3, 5.7 ± 0.4, 7.0 ± 0.3 and 7.3 ± 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery. PMID:15308676

  7. Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma

    PubMed Central

    Ma, Jun; Meng, Fanjie; Li, Shuai; Liu, Libo; Zhao, Lini; Liu, Yunhui; Hu, Yi; Li, Zhen; Yao, Yilong; Xi, Zhuo; Teng, Hao; Xue, Yixue

    2015-01-01

    This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo. PMID:26648842

  8. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

    PubMed Central

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T.

    2016-01-01

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects. PMID:26907261

  9. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    PubMed

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  10. Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

    PubMed Central

    1987-01-01

    Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types. PMID:3694176

  11. Actions of serum and plasma albumin on intracellular Ca2+ in human endothelial cells.

    PubMed Central

    Fuentes, E; Nadal, A; Jacob, R; McNaughton, P

    1997-01-01

    1. The effects of serum and plasma albumin on [Ca2+]i in human endothelial cells were examined using single-cell Ca2+ imaging. Two types of endothelial cell were used: human umbilical vein endothelial cells (HUVEC) in primary culture, and the endothelial-derived cell line ECV304. 2. Serum albumin caused a large and transient rise in [Ca2+]i, due to Ca2+ release from an IP3-sensitive internal store, followed by a maintained elevation in [Ca2+]i attributable to Ca2+ influx from the external medium. A half-maximal rise in [Ca2+]i was produced by a concentration of serum albumin of about 1 microgram ml-1. 3. The Ca(2+)-releasing action of serum albumin is abolished by methanol extraction and is therefore attributable to an attached polar lipid. A possible candidate is lysophosphatidic acid, known to be released from platelets during blood coagulation, which produced similar effects to those of serum albumin. 4. In HUVEC, plasma albumin caused a sustained decrease in [Ca2+]i from the mean resting level of 114 nM to 58 nM. No effect of plasma albumin was observed in ECV304 cells. 5. The decrease in [Ca2+]i caused by plasma albumin is due to an uptake into intracellular stores. The store loading substantially potentiates the action of Ca(2+)-releasing agonists such as histamine. 6. The results show that normal plasma albumin, which carries few lipids, lowers [Ca2+]i and potentiates the actions of Ca(2+)-releasing agonists by promoting Ca2+ uptake into intracellular stores. When converted to the serum form, by binding lysophosphatidic acid released during blood coagulation, albumin has a potent effect in elevating [Ca2+]i. Blood coagulation may therefore play a role in regulating vascular tone and capillary permeability. PMID:9365906

  12. Concurrent therapy to enhance radiotherapeutic outcomes in glioblastoma

    PubMed Central

    2016-01-01

    Glioblastoma is one of the most fatal and incurable human cancers characterized by nuclear atypia, mitotic activity, intense microvascular proliferation and necrosis. The current standard of care includes maximal safe surgical resection followed by radiation therapy (RT) with concurrent and adjuvant temozolomide (TMZ). The prognosis remains poor with median survival of 14.6 months with RT plus TMZ. Majority will have a recurrence within 2 years from diagnosis despite adequate treatment. Radiosensitizers, radiotherapy dose escalation and altered fractionation have failed to improve outcome. The molecular biology of glioblastoma is complex and poses treatment challenges. High rate of mutation, genotypic and phenotypic heterogeneity, rapid development of resistance, existence of blood-brain barrier (BBB), multiple intracellular and intercellular signalling pathways, over-expression of growth factor receptors, angiogenesis and antigenic diversity renders the tumor cells differentially susceptible to various treatment modalities. Thus, the treatment strategies require personalised or individualized approach based on the characteristics of tumor. Several targeted agents have been evaluated in clinical trials but the results have been modest despite these advancements. This review summarizes the current standard of care, results of concurrent chemoradiation trials, evolving innovative treatments that use targeted therapy with standard chemoradiation or RT alone, outcome of various recent trials and future outlook. PMID:26904576

  13. Enhanced susceptibility of Escherichia coli to intracellular killing by human polymorphonuclear leukocytes after in vitro incubation with chloramphenicol.

    PubMed Central

    Pruul, H; Wetherall, B L; McDonald, P J

    1981-01-01

    The effect of brief exposure to chloramphenicol of a pathogenic strain of Escherichia coli on susceptibility to normal human leukocytes was examined. Leukocytes killed chloramphenicol-pretreated E. coli more efficiently than they did untreated controls. Phagocytosis of pretreated bacteria, as measured by the uptake of radiolabeled bacteria and by direct visual count of engulfed bacteria, was not significantly increased. The decrease in viability was associated with enhanced intracellular killing of phagocytosed antibiotic-damaged bacteria. Chloramphenicol pretreatment altered the frequency distribution of intracellular bacteria by decreasing the number of leukocytes containing multiple stainable bacteria. Leukocytes failed to kill chloramphenicol-pretreated E. coli in the presence of phenylbutazone, which allowed an accumulation of intracellular bacteria. These results indicate that exposure of E. coli to chloramphenicol renders the bacteria more susceptible to intracellular killing and degradation. PMID:7023384

  14. Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter*

    PubMed Central

    Hasenhuetl, Peter S.; Freissmuth, Michael; Sandtner, Walter

    2016-01-01

    The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na+ concentrations because intracellular Na+ release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K+ and, notably, also H+. In addition, our data suggest that Cl− is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K+ or of H+ to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na+ release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na+ release and K+ (or H+) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form. PMID:27756841

  15. Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.

    PubMed

    Hasenhuetl, Peter S; Freissmuth, Michael; Sandtner, Walter

    2016-12-09

    The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na(+) concentrations because intracellular Na(+) release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K(+) and, notably, also H(+) In addition, our data suggest that Cl(-) is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K(+) or of H(+) to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na(+) release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na(+) release and K(+) (or H(+)) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

    PubMed Central

    2005-01-01

    β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation. PMID:16225456

  17. Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking.

    PubMed

    Wichit, Sineewanlaya; Hamel, Rodolphe; Bernard, Eric; Talignani, Loïc; Diop, Fodé; Ferraris, Pauline; Liegeois, Florian; Ekchariyawat, Peeraya; Luplertlop, Natthanej; Surasombatpattana, Pornapat; Thomas, Frédéric; Merits, Andres; Choumet, Valérie; Roques, Pierre; Yssel, Hans; Briant, Laurence; Missé, Dorothée

    2017-06-09

    Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

  18. Predominant contribution of L-type amino acid transporter to 4-borono-2-(18)F-fluoro-phenylalanine uptake in human glioblastoma cells.

    PubMed

    Yoshimoto, Mitsuyoshi; Kurihara, Hiroaki; Honda, Natsuki; Kawai, Keiichi; Ohe, Kazuyo; Fujii, Hirofumi; Itami, Jun; Arai, Yasuaki

    2013-07-01

    4-Borono-2-(18)F-fluoro-phenylalanine ((18)F-FBPA) has been used to anticipate the therapeutic effects of boron neutron capture therapy (BNCT) with 4-borono-L-phenylalanine (BPA). Similarly, L-[methyl-(11)C]-methionine ((11)C-MET), the most popular amino acid PET tracer, is a possible candidate for this purpose. We investigated the transport mechanism of (18)F-FBPA and compared it with that of (14)C-MET in human glioblastoma cell lines. Uptake of (18)F-FBPA and (14)C-MET was examined in A172, T98G, and U-87MG cells using 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (a system L-specific substrate), 2-(methylamino)-isobutyric acid (a system A-specific substrate), and BPA. Gene expression was analyzed by quantitative real time polymerase chain reaction. System L was mainly involved in the uptake of (18)F-FBPA (74.5%-81.1% of total uptake) and (14)C-MET (48.3%-59.4%). System A and ASC also contributed to the uptake of (14)C-MET. Inhibition experiments revealed that BPA significantly decreased the uptake of (18)F-FBPA, whereas 31%-42% of total (14)C-MET uptake was transported by BPA non-sensitive transporters. In addition, (18)F-FBPA uptake correlated with LAT1 and total LAT expressions. This study demonstrated that (18)F-FBPA was predominantly transported by system L in human glioblastoma cells compared to (14)C-MET. Although further studies are needed to elucidate the correlation between (18)F-FBPA uptake and BPA content in tumor tissues, (18)F-FBPA is suitable for the selection of patients who benefit from BNCT with BPA. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes

    PubMed Central

    Mohebbi, Sara; Erfurth, Florian; Hennersdorf, Philipp; Brakhage, Axel A.; Saluz, Hans Peter

    2016-01-01

    Hyperspectral imaging (HSI) is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell. PMID:27727286

  20. Endotoxic lipid A induces intracellular Ca2+ increase in human platelets.

    PubMed Central

    Romano, M; Molino, M; Cerletti, C

    1991-01-01

    The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (tmax). Maximum [Ca2+]i levels, observed at 30 microM lipid A, were 432 +/- 60 nM. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of tmax, but shortening the time for 50% recovery (t50) from greater than 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1 microM], prolonging the tmax. to 54.7 +/- 5.1 s, but decreasing the t50 to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0.15 microM). These results suggest that platelet activation by lipid A involves an interaction between [Ca2+]i elevation and protein kinase C activation. PMID:1909116

  1. UVB radiation induces an increase in intracellular zinc in human epidermal keratinocytes.

    PubMed

    Stork, Christian J; Martorano, Lisa M; Li, Yang V

    2010-10-01

    Ultraviolet (UV) radiation is known to cause oxidative stress, inflammation, DNA damage and apoptotic cell death; however, many details of these malign mechanism have yet to be elucidated. In this study, the exposure of adult human epidermal keratinocytes (HEKa) with UVB (>100 mJ/cm(2)) resulted in the significant increase of intracellular zinc that was released from its storage and was detected by fluorescent zinc indicators. Toxicity testing revealed that UVB-induced zinc release in HEKa is associated with HEKa cell death. Cells that showed elevated intracellular zinc fluorescence upon UVB exposure were also stained by propidium iodide (PI), a traditional viability indicator whose fluorescent signal is as a result of its intercalating with DNA fragments and is unaffected by zinc concentration, showing significant colocalization [Pearson's correlation coefficients r=0.956 (n=6)]. The cytotoxicity of zinc was also determined by an MTT assay after applying the exogenous zinc (ZnCl2) along with its ionophore pyrithione (20 microM) into HEKa culture medium. A significant reduction in cell viability as a function of both zinc concentration and exposure time was observed. The treatments of 1, 10 and 100 microM ZnCl2 with pyrithione demonstrated 2.3, 60 and 84% cell deaths, respectively (control 0.5%) after 30 min. ZnCl2 (100 microM) was also found to induce complete HEKa death after 1 h. Thus, the present study demonstrates that UVB irradiation-induced increased zinc is detrimental to HEKa viability, and zinc may be a necessary step in UVB-induced cell death signaling pathways.

  2. Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

    SciTech Connect

    Levy, J.R.; Olefsky, J.M.

    1988-05-05

    The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4/sup 0/C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37/sup 0/C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.

  3. Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.

    PubMed Central

    Duan, L; Bagasra, O; Laughlin, M A; Oakes, J W; Pomerantz, R J

    1994-01-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections. Images PMID:8197188

  4. Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

    NASA Astrophysics Data System (ADS)

    Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

    1994-05-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

  5. Role of intracellular Ca2+ signal in the ascorbate-induced apoptosis in a human hepatoma cell line.

    PubMed

    Lee, Yong Soo

    2004-12-01

    Although ascorbate (vitamin C) has been shown to have anti-cancer actions, its effect on human hepatoma cells has not yet been investigated, and thus, the exact mechanism of this action is not fully understood. In this study, the mechanism by which ascorbate induces apoptosis using HepG2 human hepatoblastoma cells is investigated. Ascorbate induced apoptotic cell death in a dose-dependent manner in the cells, was assessed through flow cytometric analysis. Contrary to expectation, ascorbate did not alter the cellular redox status, and treatment with antioxidants (N-acetyl cysteine and N,N-diphenyl-p-phenylenediamine) had no influence on the ascorbate-induced apoptosis. However, ascorbate induced a rapid and sustained increase in intracellular Ca2+ concentration. EGTA, an extracellular Ca2+ chelator did not significantly alter the ascorbate-induced intracellular Ca2+ increase and apoptosis, whereas dantrolene, an intracellular Ca2+ release blocker, completely blocked these actions of ascorbate. In addition, phospholipase C (PLC) inhibitors (U-73122 and manoalide) significantly suppressed the intracellular Ca2+ release and apoptosis induced by ascorbate. Collectively, these results suggest that ascorbate induced apoptosis without changes in the cellular redox status in HepG2 cells, and that the PLC-coupled intracellular Ca2+ release mechanism may mediate ascorbate-induced apoptosis.

  6. The role of alkaline phosphatase in intracellular lipid accumulation in the human hepatocarcinoma cell line, HepG2.

    PubMed

    Chirambo, George M; van Niekerk, Chantal; Crowther, Nigel J

    2017-04-01

    Inhibition of tissue non-specific alkaline phosphatase (TNALP) decreases intracellular lipid accumulation in human preadipocytes and the murine preadipocyte cell line, 3T3-L1. Therefore, the current study was performed to determine if TNALP is required for intracellular lipid deposition in the human hepatocyte cell line, HepG2. Intracellular lipid accumulation, TNALP activity and peroxisome proliferator activated receptor (PPAR) γ gene expression were measured in HepG2 and 3T3-L1 cells in the presence and absence of the TNALP inhibitors levamisole and histidine. Sub-cellular TNALP activity was localized using cytochemical analysis. Both PPARγ gene expression and TNALP activity increased during intracellular lipid accumulation in HepG2 and 3T3-L1 cells. Inhibition of TNALP blocked intracellular lipid accumulation but did not alter expression of the PPARγ gene. In HepG2 cells, TNALP co-localized with adipophilin on the lipid droplet membrane. These data suggest a role for TNALP in lipid droplet formation, possibly downstream from PPARγ, within HepG2 and 3T3-L1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Nanotechnology applications for glioblastoma.

    PubMed

    Nduom, Edjah K; Bouras, Alexandros; Kaluzova, Milota; Hadjipanayis, Costas G

    2012-07-01

    Glioblastoma remains one of the most difficult cancers to treat and represents the most common primary malignancy of the brain. Although conventional treatments have found modest success in reducing the initial tumor burden, infiltrating cancer cells beyond the main mass are responsible for tumor recurrence and ultimate patient demise. Targeting residual infiltrating cancer cells requires the development of new treatment strategies. The emerging field of cancer nanotechnology holds promise in the use of multifunctional nanoparticles for imaging and targeted therapy of glioblastoma. This article examines the current state of nanotechnology in the treatment of glioblastoma and directions of further study.

  8. Dual effects of mastoparan on intracellular free Ca2+ concentrations in human astrocytoma cells.

    PubMed Central

    Nakahata, N.; Ishimoto, H.; Mizuno, K.; Ohizumi, Y.; Nakanishi, H.

    1994-01-01

    1. The effect of mastoparan, a wasp venom toxin, on intracellular free Ca2+ concentration ([Ca2+]i) was examined in human astrocytoma cells. Mastoparan inhibited [Ca2+]i induced by carbachol (100 microM) in a concentration-dependent manner in the absence of extracellular Ca2+, consistent with our previous results showing that mastoparan inhibits phosphoinositide hydrolysis in human astrocytoma cells. 2. In contrast, mastoparan itself increased [Ca2+]i and augmented carbachol-induced increase in the [Ca2+]i in the presence of extracellular Ca2+, suggesting that mastoparan elicited Ca2+ influx from the extracellular medium. The increase appeared to be maximum at extracellular Ca2+ concentrations of 0.1-0.2 mM. The higher concentrations of extracellular Ca2+ depressed the influx. 3. Pertussis toxin did not affect mastoparan-induced inhibition of [Ca2+]i in the absence of extracellular Ca2+, consistent with the previous results that pertussis toxin did not affect mastoparan-induced inhibition of phosphoinositide hydrolysis. 4. Pertussis toxin augmented mastoparan-induced increase in [Ca2+]i in the presence of extracellular Ca2+, suggesting that pertussis toxin substrate(s) seems to be inhibitory for Ca2+ influx induced by mastoparan. 5. Verapamil, nifedipine and diltiazem (each 10 microM), L-type Ca2+ antagonists, did not affect mastoparan-induced Ca2+ influx. However, verapamil (10 microM) slightly inhibited the increase in [Ca2+]i induced by carbachol in the presence of mastoparan. 6. The results obtained in the present study indicate that mastoparan has two opposite effects on [Ca2+]i in human astrocytoma cells and possibly has at least two sites of action. PMID:8032654

  9. Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS.

    PubMed

    Dohmen, Luuk C T; Navas, Adriana; Vargas, Deninson Alejandro; Gregory, David J; Kip, Anke; Dorlo, Thomas P C; Gomez, Maria Adelaida

    2016-04-29

    Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Sodium-23 NMR analysis of human whole blood, erythrocytes, and plasma. Chemical shift, spin relaxation, and intracellular sodium concentration studies

    NASA Astrophysics Data System (ADS)

    Pettegrew, Jay W.; Woessner, Donald E.; Minshew, Nancy J.; Glonek, Thomas

    Sodium-23 NMR analysis was performed on freshly obtained human whole blood, erythrocytes, and plasma. The intracellular and extracellular sodium signals were separated by adding dysprosium: tripolyphosphate to the plasma bathing the erythrocytes. Quantitation of the intracellular sodium content was easily accomplished by sodium NMR and was shown to agree well with the values obtained by flame photometry. T1 and T2 relaxation studies demonstrated that the sodium in human plasma and within human erythrocytes is substantially different in its physical characteristics than sodium in aqueous solution, and that some fraction of the plasma and erythrocyte sodium is relatively immobilized. Sodium NMR would appear therefore to be a useful method for studying sodium biology in inherited and acquired human diseases.

  11. Nutraceutical phycocyanin nanoformulation for efficient drug delivery of paclitaxel in human glioblastoma U87MG cell line

    NASA Astrophysics Data System (ADS)

    Agrawal, Madhunika; Yadav, Sanjeev Kumar; Agrawal, Satyam Kumar; Karmakar, Surajit

    2017-08-01

    To enhance the therapeutic efficacy of chemotherapy on glioblastoma U87MG cell line, paclitaxel-loaded phycocyanin nanoparticles (PTX-PcNPs) were prepared by modified desolvation process. PTX-PcNPs were characterised in terms of size, zeta potential, drug loading efficiency and drug release. Confocal laser scanning microscopy showed PTX-PcNPs could be internalised by U87MG cells. The anti-cancer activity was investigated in vitro by 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with and without photodynamic therapy. It was observed that formulation could significantly inhibit growth of U87MG cells as compared to PTX alone and also induced apoptosis, which was evidenced by presence of apoptotic bodies and nuclear fragmentation in treated cells. The present study suggests that PTX-PcNPs can act as a promising drug delivery system for cancer treatment. [Figure not available: see fulltext.

  12. Intracellular Ca(2+) remodeling during the phenotypic journey of human coronary smooth muscle cells.

    PubMed

    Muñoz, Eva; Hernández-Morales, Miriam; Sobradillo, Diego; Rocher, Asunción; Núñez, Lucía; Villalobos, Carlos

    2013-11-01

    Vascular smooth muscle cells undergo phenotypic switches after damage which may contribute to proliferative disorders of the vessel wall. This process has been related to remodeling of Ca(2+) channels. We have tested the ability of cultured human coronary artery smooth muscle cells (hCASMCs) to return from a proliferative to a quiescent behavior and the contribution of intracellular Ca(2+) remodeling to the process. We found that cultured, early passage hCASMCs showed a high proliferation rate, sustained increases in cytosolic [Ca(2+)] in response to angiotensin II, residual voltage-operated Ca(2+) entry, increased Stim1 and enhanced store-operated currents. Non-steroidal anti-inflammatory drugs inhibited store-operated Ca(2+) entry and abolished cell proliferation in a mitochondria-dependent manner. After a few passages, hCASMCs turned to a quiescent phenotype characterized by lack of proliferation, oscillatory Ca(2+) response to angiotensin II, increased Ca(2+) store content, enhanced voltage-operated Ca(2+) entry and Cav1.2 expression, and decreases in Stim1, store-operated current and store-operated Ca(2+) entry. We conclude that proliferating hCASMCs return to quiescence and this switch is associated to a remodeling of Ca(2+) channels and their control by subcellular organelles, thus providing a window of opportunity for targeting phenotype-specific Ca(2+) channels involved in proliferation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

    PubMed

    López-Torres, Aideé S; González-González, María E; Mata-Martínez, Esperanza; Larrea, Fernando; Treviño, Claudia L; Chirinos, Mayel

    2017-02-05

    In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca(2+) concentrations ([Ca(2+)]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca(2+) oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 μg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Quercetin suppresses intracellular ROS formation, MMP activation, and cell motility in human fibrosarcoma cells.

    PubMed

    Lee, Dong Eun; Chung, Min-Yu; Lim, Tae Gyu; Huh, Won Bum; Lee, Hyong Joo; Lee, Ki Won

    2013-09-01

    Cell metastasis is a major cause of death from cancer and can arise from excessive levels of oxidative stress. The objective of this study was to investigate whether the natural flavonoid quercetin can inhibit matrix metalloproteinase (MMP)-2 and -9 activities through the attenuation of reactive oxygen species (ROS) formation, an event expected to lead to the inhibition of cell motility. To induce sustained ROS formation, cells were treated with phenazine methosulfate (PMS; 1 μM). Noncytotoxic concentrations of quercetin inhibited PMS-induced increases in cell motility in HT1080 human fibrosarcoma (HT1080) cells. While nearly 100% of cells were observed to migrate after 24 h of PMS treatment, quercetin significantly (P < 0.01) suppressed this effect. We also found that quercetin, up to 10 μg/mL, attenuated PMS-induced MMP-2 activation. We then investigated whether the decreased levels of MMP-2 activation could be attributable to lower levels of ROS formation by quercetin. We found that quercetin treatments significantly attenuated PMS-induced ROS formation (P < 0.01) and resulted in decreased cell motility associated with a reduction in MMP-2 and -9 activitiy in HT1080 cells, even in the absence of PMS treatment. Collectively, these results suggest that quercetin inhibits cell motility via the inhibition of MMP activation in HT1080 cells in the presence and absence of PMS. This is likely to be a result of the suppression of intracellular ROS formation by quercetin. © 2013 Institute of Food Technologists®

  15. Involvement of human beta-defensin-2 in intracellular signaling: in vitro study.

    PubMed

    Lytvyn, D; Zhuravel, E; Markeeva, N; Soldatkina, M; Dudchenko, T; Pogrebnoy, P

    2006-09-01

    To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. A431cells were cultured in the presence of 1 microg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. 30 min incubation of A431 cells with 1 mug/ml of hBD-2 didn't influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment - in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment - in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells.

  16. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

    PubMed Central

    Kambhampati, Siva P.; Mishra, Manoj K.; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A.; Kannan, Rangaramanujam M.

    2016-01-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (~21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ~100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  17. Inhibition of Intracellular Triglyceride Lipolysis Suppresses Cold-Induced Brown Adipose Tissue Metabolism and Increases Shivering in Humans.

    PubMed

    Blondin, Denis P; Frisch, Frédérique; Phoenix, Serge; Guérin, Brigitte; Turcotte, Éric E; Haman, François; Richard, Denis; Carpentier, André C

    2017-02-07

    Indirect evidence from human studies suggests that brown adipose tissue (BAT) thermogenesis is fueled predominantly by fatty acids hydrolyzed from intracellular triglycerides (TGs). However, no direct experimental evidence to support this assumption currently exists in humans. The aim of this study was to determine the role of intracellular TG in BAT thermogenesis, in cold-exposed men. Using positron emission tomography with (11)C-acetate and (18)F-fluorodeoxyglucose, we showed that oral nicotinic acid (NiAc) administration, an inhibitor of intracellular TG lipolysis, suppressed the cold-induced increase in BAT oxidative metabolism and glucose uptake, despite no difference in BAT blood flow. There was a commensurate increase in shivering intensity and shift toward a greater reliance on glycolytic muscle fibers without modifying total heat production. Together, these findings show that intracellular TG lipolysis is critical for BAT thermogenesis and provides experimental evidence for a reciprocal role of BAT thermogenesis and shivering in cold-induced thermogenesis in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS

    PubMed Central

    Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

    2013-01-01

    Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (~5 million cells) were collected and lysed in 1 mL 70% methanol containing 1.2 mM tris buffer (pH7.4). The lysate (80 uL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20 µL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100 × 2.1 mm, 3 µm) eluted with 100mM ammonium acetate (pH9.8) and acetonitrile in a gradient mode at a flow rate of 0.4 mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI−) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4 min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52 to 76 nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19 pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not

  19. Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.

    PubMed

    Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

    2013-12-01

    Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI(-)) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed

  20. Lactate modulates the intracellular pH sensitivity of human TREK1 channels.

    PubMed

    Ghatak, Swagata; Sikdar, Sujit Kumar

    2016-05-01

    Tissue acidosis and high lactate concentrations are associated with cerebral ischaemia. The degree of acidosis is dependent on circulating glucose concentration, hyperglycaemia being associated with increased acidosis. Among other agents, lactate and protons have been shown to activate the leak potassium channel; TREK1 (TWIK related potassium channel 1) from the intracellular side and its increased activity is implicated in tolerance towards ischaemic cell damage. In the present study, we show that ischaemic concentrations of lactate (30 mM) at pH 7.0 and 6.5, commonly observed during ischemia, cause robust potentiation of human TREK1 (hTREK1) activity at single-channel level in cell-free inside-out membrane patches, while 30 mM lactate at pH 6.0 to 5.5, commonly observed during hyperglycaemic ischemia, reduces hTREK1 channel activity significantly. The biphasic effect of 30 mM lactate (ischaemic concentrations) on modulation of hTREK1 by varying pH conditions is specific since basal concentrations of lactate (3 mM) and 30 mM pyruvate at pH 7.0 and 5.5 failed to show similar effect as lactate. Experiments with deletion and point mutants of hTREK1 channel suggest that lactate changes the pH modulation of hTREK1 by interacting differently with the histidine residue at 328th position (H328) above and below its pKa (∼6.0) in the intracellular carboxyl-terminal domain of TREK1. This lactate-induced pH modulation of hTREK1 is absent in C-terminal deletion mutant, CTDΔ100, and is similar in E321A-hTREK1 mutant as in wild-type hTREK1 suggesting that it is independent of pH-sensitive glutamate residue at 321st position. Such a differential pH-dependent effect of lactate on an ion channel function has not been reported earlier and has important implications in different stages of ischaemia.

  1. The potential origin of glioblastoma initiating cells

    PubMed Central

    Chesler, David A.; Berger, Mitchell S.; Quinones-Hinojosa, Alfredo

    2013-01-01

    Despite intensive clinical and laboratory research and effort, Glioblastoma remains the most common and invariably lethal primary cancer of the central nervous system. The identification of stem cell and lineage-restricted progenitor cell populations within the adult human brain in conjunction with the discovery of stem-like cells derived from gliomas which are themselves tumorigenic and have been shown to have properties of self-renewal and multipotency, has led to the hypothesis that this population of cells may represent glioma initiating cells. Extensive research characterizing the anatomic distribution and phenotype of neural stem cells in the adult brain, and the genetic underpinnings needed for malignant transformation may ultimately lead to the identification of the cellular origin for glioblastoma. Defining the cellular origin of this lethal disease may ultimately provide new therapeutic targets and modalities finally altering an otherwise bleak outcome for patients with glioblastoma. PMID:22202053

  2. Secondary Lymphoid Organ Homing Phenotype of Human Myeloid Dendritic Cells Disrupted by an Intracellular Oral Pathogen

    PubMed Central

    Miles, Brodie; Zakhary, Ibrahim; El-Awady, Ahmed; Scisci, Elizabeth; Carrion, Julio; O'Neill, John C.; Rawlings, Aaron; Stern, J. Kobi; Susin, Cristiano

    2014-01-01

    Several intracellular pathogens, including a key etiological agent of chronic periodontitis, Porphyromonas gingivalis, infect blood myeloid dendritic cells (mDCs). This infection results in pathogen dissemination to distant inflammatory sites (i.e., pathogen trafficking). The alteration in chemokine-chemokine receptor expression that contributes to this pathogen trafficking function, particularly toward sites of neovascularization in humans, is unclear. To investigate this, we utilized human monocyte-derived DCs (MoDCs) and primary endothelial cells in vitro, combined with ex vivo-isolated blood mDCs and serum from chronic periodontitis subjects and healthy controls. Our results, using conditional fimbria mutants of P. gingivalis, show that P. gingivalis infection of MoDCs induces an angiogenic migratory profile. This profile is enhanced by expression of DC-SIGN on MoDCs and minor mfa-1 fimbriae on P. gingivalis and is evidenced by robust upregulation of CXCR4, but not secondary lymphoid organ (SLO)-homing CCR7. This disruption of SLO-homing capacity in response to respective chemokines closely matches surface expression of CXCR4 and CCR7 and is consistent with directed MoDC migration through an endothelial monolayer. Ex vivo-isolated mDCs from the blood of chronic periodontitis subjects, but not healthy controls, expressed a similar migratory profile; moreover, sera from chronic periodontitis subjects expressed elevated levels of CXCL12. Overall, we conclude that P. gingivalis actively “commandeers” DCs by reprogramming the chemokine receptor profile, thus disrupting SLO homing, while driving migration toward inflammatory vascular sites. PMID:24126519

  3. [Study on the cell apoptosis induced by intracellular hyperthermia in human lung adenocarcinoma SPC-A1 cells].

    PubMed

    Ma, Yongjie; Li, Hong; Yan, Zhubing; Gu, Hongchen

    2007-12-01

    This is a comparative study on the efficacy of differential cell apoptosis induced by three methods (intracellular hyperthermia, with water bath hyperthermia and extracellular hyperthermia) in human lung adenocarcinoma SPC-A1 cells in vitro. The effects of hyperthermia on cell apoptosis were determined by Transmission electron microscopy(TEM), agarose gel electrophoresis and flow cytometry methods, respectively. The intracellular effect of particle heating was compared with that of water bath hyperthermia and extracellular hyperthermia; significant differences between these heating methods were detected, the rate of apoptosis being 36.59%, 5.66%, 7.78% respectively. When treated with intracellular hyperthermia, the SPC-A1 cells manifested typical morphological characters of apoptosis by TEM observation, and the SPC-A1 cell DNA was degraded into large fragments by agarose gel electrophoresis assay. Our results showed that amino-silane Fe3O4 induced intracellular hyperthermia was superior to water bath hyperthermia and extracellular hyperthermia. It is mainly the interaction between intracellular nanoparticles and cell that induced apoptosis. Therefore, the aminosilane-coated Fe3O4 may be used in hyperthermia or chemotherapeutics on cancer cells for further clinical application.

  4. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

    PubMed

    El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

    2015-02-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  5. Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

    PubMed Central

    El-Awady, Ahmed R.; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B.; Palani, Chithra D.; Arce, Roger M.; Waller, Jennifer L.; Genco, Caroline A.; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V.; Cutler, Christopher W.

    2015-01-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs. PMID:25679217

  6. Increased level of H19 long noncoding RNA promotes invasion, angiogenesis, and stemness of glioblastoma cells.

    PubMed

    Jiang, Xiaochun; Yan, Yukui; Hu, Minghua; Chen, Xiande; Wang, Yaxian; Dai, Yi; Wu, Degang; Wang, Yongsheng; Zhuang, Zhixiang; Xia, Hongping

    2016-01-01

    OBJECT Increased levels of H19 long noncoding RNA (lncRNA) have been observed in many cancers, suggesting that overexpression of H19 may be important in the development of carcinogenesis. However, the role of H19 in human glioblastoma is still unclear. The object of this study was to examine the level of H19 in glioblastoma samples and investigate the role of H19 in glioblastoma carcinogenesis. METHODS Glioblastoma and nontumor brain tissue specimens were obtained from tissue obtained during tumor resection in 30 patients with glioblastoma. The level of H19 lncRNA was detected by real-time quantitative reverse transcription polymerase chain reaction. The role of H19 in invasion, angiogenesis, and stemness of glioblastoma cells was then investigated using commercially produced cell lines (U87 and U373). The effects of H19 overexpression on glioblastoma cell invasion and angiogenesis were detected by in vitro Matrigel invasion and endothelial tube formation assay. The effects of H19 on glioblastoma cell stemness and tumorigenicity were investigated by neurosphere formation and an in vivo murine xenograft model. RESULTS The authors found that H19 is significantly overexpressed in glioblastoma tissues, and the level of expression was associated with patient survival. In the subsequent investigations, the authors found that overexpression of H19 promotes glioblastoma cell invasion and angiogenesis in vitro. Interestingly, H19 was also significantly overexpressed in CD133(+) glioblastoma cells, and overexpression of H19 was associated with increased neurosphere formation of glioblastoma cells. Finally, stable overexpression of H19 was associated with increased tumor growth in the murine xenograft model. CONCLUSIONS The results of this study suggest that increased expression of H19 lncRNA promotes invasion, angiogenesis, stemness, and tumorigenicity of glioblastoma cells. Taken together, these findings indicate that H19 plays an important role in tumorigenicity and

  7. TCGA Workshop: Genomics and Biology of Glioblastoma Multiforme (GBM) - TCGA

    Cancer.gov

    The National Cancer Institute (NCI) and National Human Genome Research Institute (NHGRI) held a workshop entitled, “Genomics and Biology of Glioblastoma Multiforme (GBM),” to review the initial GBM data from the TCGA pilot project.

  8. Radiosensitisation by pharmacological ascorbate in glioblastoma multiforme cells, human glial cells, and HUVECs depends on their antioxidant and DNA repair capabilities and is not cancer specific.

    PubMed

    Castro, M Leticia; McConnell, Melanie J; Herst, Patries M

    2014-09-01

    We previously showed that 5 mM ascorbate radiosensitized early passage radioresistant glioblastoma multiforme (GBM) cells derived from one patient tumor. Here we investigate the sensitivity of a panel of cell lines to 5 mM ascorbate and 6 Gy ionizing radiation, made up of three primary human GBM cells, three GBM cell lines, a human glial cell line, and primary human vascular endothelial cells. The response of different cells lines to ascorbate and/or radiation was determined by measuring viability, colony-forming ability, generation and repair of double-stranded DNA breaks (DSBs), cell cycle progression, antioxidant capacity and generation of reactive oxygen species. Individually, radiation and ascorbate both decreased viability and clonogenicity by inducing DNA damage, but had differential effects on cell cycle progression. Radiation led to G2/M arrest in most cells whereas ascorbate caused accumulation in S phase, which was moderately associated with poor DSB repair. While high dose ascorbate radiosensitized all cell lines in clonogenic assays, the sensitivity to radiation, high dose ascorbate, and combined treatment varied between cell lines. Normal glial cells were similar to GBM cells with respect to free radical scavenging potential and effect of treatment on DNA damage and repair, viability, and clonogenicity. Both GBM cells and normal cells coped equally poorly with oxidative stress caused by radiation and/or high dose ascorbate, dependent primarily on their antioxidant and DSB repair capacity.

  9. An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells

    PubMed Central

    Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Martínez-Soler, Fina; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Velasco, Roser; Plans, Gerard; Vidal, Noemi; Tortosa, Avelina; Barcia, Carlos; Bruna, Jordi; Yuste, Victor J.

    2016-01-01

    Background Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. Methods We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. Results We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. Conclusions Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy. PMID:26755073

  10. Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie

    2017-01-01

    Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH. PMID:28255266

  11. Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells.

    PubMed

    Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie

    2017-01-01

    Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH.

  12. Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans

    PubMed Central

    Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Passos, Renata L. Freitas; Fonseca, Michele Atalla; Oliveira, Kenya Paula Moreira; Lima, Milene Rodrigues Malheiros; Guimarães, Juliana Bohen; Ferreira-Júnior, João Batista; Martini, Angelo R. P.; Lima, Nilo R. V.; Soares, Danusa Dias; Oliveira, Edilamar Menezes

    2010-01-01

    In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL−1; p < 0.05). In HST2, there was no change in plasma Hsp72 (REST: 0.94 ± 0.08, POST: 1.20 ± 0.15, 1 h POST: 1.17 ± 0.16 ng mL−1; p > 0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p < 0.05). Exercise-induced increased intracellular Hsp72 expression was observed on HST1 (HST1: REST, 1 ± 0.02 vs. POST, 2.9 ± 0.9 density units, mean ± SE, p < 0.05) but was inhibited on HST2 (HST2: REST, 4.2 ± 1.2 vs. POST, 4.4 ± 1.1 density units, p > 0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = −0

  13. Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans.

    PubMed

    Magalhães, Flávio de Castro; Amorim, Fabiano Trigueiro; Passos, Renata L Freitas; Fonseca, Michele Atalla; Oliveira, Kenya Paula Moreira; Lima, Milene Rodrigues Malheiros; Guimarães, Juliana Bohen; Ferreira-Júnior, João Batista; Martini, Angelo R P; Lima, Nilo R V; Soares, Danusa Dias; Oliveira, Edilamar Menezes; Rodrigues, Luiz Oswaldo Carneiro

    2010-11-01

    In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL(-1); p < 0.05). In HST2, there was no change in plasma Hsp72 (REST: 0.94 ± 0.08, POST: 1.20 ± 0.15, 1 h POST: 1.17 ± 0.16 ng mL(-1); p > 0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p < 0.05). Exercise-induced increased intracellular Hsp72 expression was observed on HST1 (HST1: REST, 1 ± 0.02 vs. POST, 2.9 ± 0.9 density units, mean ± SE, p < 0.05) but was inhibited on HST2 (HST2: REST, 4.2 ± 1.2 vs. POST, 4.4 ± 1.1 density units, p > 0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = -0.85, p

  14. Stereotactic Radiosurgery for Glioblastoma

    PubMed Central

    Mehta, Minesh

    2015-01-01

    Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and one of the most aggressive of all human cancers. GBM tumors are highly infiltrative and relatively resistant to conventional therapies. Aggressive management of GBM using a combination of surgical resection, followed by fractionated radiotherapy and chemotherapy has been shown to improve overall survival; however, GBM tumors recur in the majority of patients and the disease is most often fatal. There is a need to develop new treatment regimens and technological innovations to improve the overall survival of GBM patients. The role of stereotactic radiosurgery (SRS) for the treatment of GBM has been explored and is controversial. SRS utilizes highly precise radiation techniques to allow dose escalation and delivery of ablative radiation doses to the tumor while minimizing dose to the adjacent normal structures. In some studies, SRS with concurrent chemotherapy has shown improved local control with acceptable toxicities in select GBM patients. However, because GBM is a highly infiltrative disease, skeptics argue that local therapies, such as SRS, do not improve overall survival. The purpose of this article is to review the literature regarding SRS in both newly diagnosed and recurrent GBM, to describe SRS techniques, potential eligible SRS candidates, and treatment-related toxicities. In addition, this article will propose promising areas for future research for SRS in the treatment of GBM. PMID:26848407

  15. Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells

    SciTech Connect

    Lagrange, Magali; Boulade-Ladame, Charlotte; Mailly, Laurent; Weiss, Etienne; Orfanoudakis, Georges; Deryckere, Francois . E-mail: francois.deryckere@esbs.u-strasbg.fr

    2007-09-21

    The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies.

  16. Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment- a new step in migration

    PubMed Central

    Patten, Daniel A.; Wilson, Garrick K.; Bailey, Dalan; Shaw, Robert K.; Jalkanen, Sirpa; Salmi, Marko; Rot, Antal; Weston, Christopher J.; Adams, David H.; Shetty, Shishir

    2017-01-01

    The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSEC), a functionally and phenotypically distinct sub-population of endothelial cells. Using flow based adhesion assays to study the migration of lymphocytes across primary human HSEC, we found that lymphocytes enter into HSEC, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon gamma increased intracellular localization of lymphocytes within HSECs. Furthermore using confocal imaging and time-lapse recordings we demonstrated ‘intracellular crawling’ of lymphocytes entering into one endothelial cell from another. This required the expression of ICAM-1 and stabilin-1 and was facilitated by the junctional complexes between HSEC. Conclusion: We demonstrate a new step in lymphocyte migration which is facilitated by the unique structure of HSEC. We believe ‘intracellular crawling’ contributes to optimal lymphocyte positioning in liver tissue during chronic hepatitis. PMID:27770554

  17. Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes.

    PubMed

    Dziduszko, Agnieszka; Ozbun, Michelle A

    2013-07-01

    Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca(2+)-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

  18. Annexin A2 and S100A10 Regulate Human Papillomavirus Type 16 Entry and Intracellular Trafficking in Human Keratinocytes

    PubMed Central

    Dziduszko, Agnieszka

    2013-01-01

    Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca2+-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome. PMID:23637395

  19. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  20. Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells.

    PubMed

    Weisburg, J H; Roepe, P D; Dzekunov, S; Scheinberg, D A

    1999-04-16

    In previous work (Weisburg, J. H., Curcio, M., Caron, P. C., Raghi, G., Mechetner, E. B., Roepe, P. D., and Scheinberg, D. A. (1996) J. Exp. Med. 183, 2699-2704), we showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV+) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pHi) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pHi by two independent methods (CO2 "conditioning" or isotonic Cl- substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pHi by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl-/HCO3- exchange upon Cl- substitution is blocked by treatment with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate. Continuous photometric monitoring of 2',7'-bis(carboxyethyl)-5, 6-carboxyfluorescein (BCECF), to assess changes in pHi or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pHi upon CO2 conditioning and Cl- substitution and release of BCECF upon formation of MAC pores. Antibody binding and internalization kinetics are similar in both the parental and resistant cell lines as measured by radioimmunoassay, but flow cytometric data showed that net complement deposition in the cell membrane is both delayed and reduced in

  1. Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N).

    PubMed

    Squires, P E; Amiranoff, B; Dunne, M J

    1994-10-01

    Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 microM) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 microM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 microM acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 microM), verapamil (100 microM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.

  2. Different intracellular signalling properties induced by human and porcine growth hormone.

    PubMed

    Hong, Pan; Lan, Hainan; Li, Yumeng; Fu, Zhiling; Zheng, Xin

    2016-04-01

    Growth hormone (GH) is reportedly species-specific. Primate growth hormone can trigger non-primate growth hormone receptor (GHR), but primates GHR cannot be activated by non-primate GH. However, it is also unclear that why primate GH and non-primate GH have different biological activities. Thus, we analysed primate growth hormone (human growth hormone (hGH)) or non-primate GH (porcine growth hormone (pGH))-induced intracellular signalling in 3T3-F442A cells and rat hepatocytes in a dose- and time-dependent manner to explore the different biological activities between them. The results revealed that both hGH and pGH can activate Janus kinase 2 (JAK2), Signal transducers and activators of transcription 1, 3 and 5 (STATs 1, 3 and 5) and extracellular signal-regulated kinase 1/2 (ERK1/2). There were no significant differences in JAK2 or ERK1/2 tyrosine phosphorylation after hGH and pGH treatment, but there were different between hGH and pGH in STAT/1/3/5 tyrosine phosphorylation, and JAK2, STAT/1/3/5 tyrosine phosphorylation was time-dependent and dose-dependent, whereas ERK1/2 was not. Both hGH and pGH demonstrated similar kinetics for STATs 1, 3 and 5 phosphorylation, but the pGH-mediated tyrosine phosphorylation was weaker than that mediated by hGH. Our observations indicated that the levels of main signalling proteins phosphorylation triggered by hGH or pGH were not exactly the same, which may explain the different biological activities showed by primate GH and non-primate GH. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16

    PubMed Central

    Gottschalk, Elinor Y.

    2015-01-01

    ABSTRACT The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. IMPORTANCE In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study

  4. A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

    PubMed

    Gottschalk, Elinor Y; Meneses, Patricio I

    2015-09-01

    The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

  5. In vitro cytotoxicity of SiO2 or ZnO nanoparticles with different sizes and surface charges on U373MG human glioblastoma cells

    PubMed Central

    Kim, Jung-Eun; Kim, Hyejin; An, Seong Soo A; Maeng, Eun Ho; Kim, Meyoung-Kon; Song, Yoon-Jae

    2014-01-01

    Silicon dioxide (SiO2) and zinc oxide (ZnO) nanoparticles are widely used in various applications, raising issues regarding the possible adverse effects of these metal oxide nanoparticles on human cells. In this study, we determined the cytotoxic effects of differently charged SiO2 and ZnO nanoparticles, with mean sizes of either 100 or 20 nm, on the U373MG human glioblastoma cell line. The overall cytotoxicity of ZnO nanoparticles against U373MG cells was significantly higher than that of SiO2 nanoparticles. Neither the size nor the surface charge of the ZnO nanoparticles affected their cytotoxicity against U373MG cells. The 20 nm SiO2 nanoparticles were more toxic than the 100 nm nanoparticles against U373MG cells, but the surface charge had little or no effect on their cytotoxicity. Both SiO2 and ZnO nanoparticles activated caspase-3 and induced DNA fragmentation in U373MG cells, suggesting the induction of apoptosis. Thus, SiO2 and ZnO nanoparticles appear to exert cytotoxic effects against U373MG cells, possibly via apoptosis. PMID:25565841

  6. Human brain glioblastoma cells do not induce but do respond to the bleomycin-induced bystander response from lung adenocarcinoma cells.

    PubMed

    Basheerudeen, Safa Abdul Syed; Mani, Chinnadurai; Kulkarni, Megha Anil Kumar; Pillai, Karthika; Rajan, Anila; Venkatachalam, Perumal

    2013-10-09

    To determine whether the bleomycin (BLM)-induced bystander response occurs in human brain glioblastoma (BMG-1) cells, the BMG-1 cells were exposed to two different concentrations of BLM. The co-culture methodology was adopted to study the in vitro bystander effects. DNA damage was measured using the micronucleus (MN) and γ-H2AX assays. Cytotoxicity was measured using the trypan blue assay. Cell cycle kinetics was analyzed using flow cytometry. The overall results did not show any significant increase in either genotoxicity or cytotoxicity or a delay in the cell cycle kinetics in BMG-1 bystander cells co-cultured with BLM-exposed cells, suggesting that BLM did not induce a bystander response in the BMG-1 cells. Furthermore, the MN results of the BLM-exposed BMG-1 cells co-cultured with unexposed bystander human lung adenocarcinoma (A549 and NCI-H460) cells and vice versa suggested that the BMG-1 cells do not secrete bystander signals but do respond to those signals. Analyzing the underlying mechanism and pathways involved in preventing the cells from secreting bystander signals will provide new insights that can be applied to inhibit these mechanisms in other cell types, thereby preventing and controlling the bystander response and genomic instability and increasing the therapeutic gain in chemotherapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low-frequency electromagnetic field (100Hz, 100G).

    PubMed

    Akbarnejad, Zeinab; Eskandary, Hossein; Dini, Luciana; Vergallo, Cristian; Nematollahi-Mahani, Seyed Noureddin; Farsinejad, Alireza; Abadi, Maryam Fekri Soofi; Ahmadi, Meysam

    2017-08-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer that causes high mortality in humans. It responds poorly to the most common cancer treatments, such as surgery, chemo- and radiation therapy. Temozolomide (TMZ) is an alkylating agent that has been widely used to treat GBM; resistance to this drug is often found. One unexplored possibility for overcoming this resistance is a treatment based on concomitant exposure to electromagnetic fields (EMF) and TMZ. Indeed, many evidences show that EMF affects cancer cells and drug performance. In this study, we evaluated the potential synergistic effect of 100μM TMZ and EMF (100Hz, 100G) on two human glioma cells line, i.e., U87 and T98G above single treatments, TMZ or EMF. Co-treatment synergistically enhanced apoptosis in U87 and T98G cells, by increasing the expression of P53, Bax, and Caspase-3 and decreasing that of Bcl-2 and Cyclin-D1. We also observed an increase in reactive oxygen species (ROS) production and the overexpression of the heme oxygenase-1 (HO-1) gene in comparison to controls. In conclusion, since EMF enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism, the TMZ/EMF combination may be effective for glioma cancer treating. Further studies are needed to reveal the action mechanism of this possible novel therapeutic approach. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells.

    PubMed

    Daniele, Simona; Barresi, Elisabetta; Zappelli, Elisa; Marinelli, Luciana; Novellino, Ettore; Da Settimo, Federico; Taliani, Sabrina; Trincavelli, Maria L; Martini, Claudia

    2016-02-16

    The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies.

  9. Inflammatory landscape of human brain tumors reveals an NFκB dependent cytokine pathway associated with mesenchymal glioblastoma.

    PubMed

    Zanotto-Filho, Alfeu; Gonçalves, Rosângela Mayer; Klafke, Karina; de Souza, Priscila Oliveira; Dillenburg, Fabiane Cristine; Carro, Luigi; Gelain, Daniel Pens; Moreira, José Cláudio Fonseca

    2017-04-01

    The tumor microenvironment is being increasingly recognized as a key factor in cancer aggressiveness. In this study, we characterized the inflammatory gene signatures altered in glioma cell lines and tumor specimens of differing histological and molecular subtypes. The results showed that glioblastoma multiforme (GBM) shows upregulation of a subset of inflammatory genes when compared to astrocytomas and oligodendrogliomas. With molecular subtypes of GBM, the expression of inflammatory genes is heterogeneous, being enriched in mesenchymal and downregulated in Proneural/GCIMP. Other inflammation-associated processes such as tumor-associated macrophage (TAM) signatures are upregulated in mesenchymal, and a subset of 33 mesenchymal-enriched inflammatory and TAM markers showed correlation with poor survival. We found that various GBM tumor-upregulated genes such as IL6, IL8 and CCL2 are also actively expressed in glioma cell lines, playing differential and cooperative roles in promoting proliferation, invasion, angiogenesis and macrophage polarization in vitro. These genes can be stimulated by pathways typically altered in GBM, including the EGFR, PDGFR, MEK1/2-ERK1/2, PI3K/Akt and NFκB cascades. Taken together, the results presented herein depict some inflammatory pathways altered in gliomas and highlight potentially relevant targets to therapy improvement.

  10. Establishment and partial characterization of a human tumor cell line, GBM-HSF, from a glioblastoma multiforme.

    PubMed

    Qu, Jiagui; Rizak, Joshua D; Fan, Yaodong; Guo, Xiaoxuan; Li, Jiejing; Huma, Tanzeel; Ma, Yuanye

    2014-07-01

    This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10% fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5% of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, β-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.

  11. PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells.

    PubMed

    Benedetti, Elisabetta; d'Angelo, Michele; Ammazzalorso, Alessandra; Gravina, Giovanni Luca; Laezza, Chiara; Antonosante, Andrea; Panella, Gloria; Cinque, Benedetta; Cristiano, Loredana; Dhez, Anne Chloè; Astarita, Carlo; Galzio, Renato; Cifone, Maria Grazia; Ippoliti, Rodolfo; Amoroso, Rosa; Di Cesare, Ernesto; Giordano, Antonio; Cimini, Annamaria

    2017-06-01

    Glioblastoma (GB) is the most common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions, and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work, we used a new PPARα antagonist on patient-derived GB primary cells, with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity, and decrease of migration. Therefore, AA452 is proposed as a new adjuvant for the gold standard therapies for GB, opening the possibility for preclinical and clinical trials for this class of compounds. J. Cell. Physiol. 232: 1458-1466, 2017. © 2016 Wiley Periodicals, Inc.

  12. CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells

    PubMed Central

    Drachsler, M; Kleber, S; Mateos, A; Volk, K; Mohr, N; Chen, S; Cirovic, B; Tüttenberg, J; Gieffers, C; Sykora, J; Wirtz, C R; Mueller, W; Synowitz, M; Martin-Villalba, A

    2016-01-01

    Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy. PMID:27124583

  13. Intracellular transport of human lysosomal α-mannosidase and α-mannosidosis-related mutants

    PubMed Central

    2004-01-01

    Human LAMAN (lysosomal α-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease α-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355→Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320). PMID:15035660

  14. Sulforaphane-cysteine induces apoptosis by sustained activation of ERK1/2 and caspase 3 in human glioblastoma U373MG and U87MG cells.

    PubMed

    Wu, Sai; Zhou, Yan; Yang, Gaoxiang; Tian, Hua; Geng, Yang; Hu, Yabin; Lin, Kai; Wu, Wei

    2017-05-01

    We previously demonstrated that sulforaphane (SFN) inhibited invasion via sustained activation of ERK1/2 in human glioblastoma cells. However, sulforaphane-cysteine (SFN-Cys), an analog of SFN, enriched in plasma with longer half-life, had more potentiality to induce apoptosis. Here we investigated the molecular mechanisms of SFN-Cys-induced apoptosis in human glioblastoma U373MG and U87MG cells. Cell viability assay showed that SFN-Cys inhibited cell viability in a dose-dependent manner. Cell morphology observation also showed SFN-Cys increased the phenotype of cell death in a dose-dependent manner. Furthermore, flow cytometry assay showed that SFN-Cys induced apoptosis significantly in a dose-dependent manner in both cell lines. Furthermore, western blot analysis demonstrated that SFN-Cys induced activation of ERK1/2 in a sustained manner and the activation contributed to upregulation of Bax/Bcl-2 ratio and cleaved caspase 3, and these results can be reversed by the ERK1/2 blocker PD98059. Our results showed that SFN-Cys induced cell apoptosis via sustained activation of ERK1/2 and the ERK1/2 mediated signaling pathways such as activation of caspase 3 and apoptosis-related proteins, thus indicating that SFN-Cys might be a more promising therapeutic agent versus SFN to resist glioblastoma cells, especially in Taxol-resistant cancer cells.

  15. Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

    PubMed

    Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso; Av-Gay, Yossef

    2015-10-26

    Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

  16. P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance.

    PubMed

    Tang, Xiao; Basavarajappa, Devaraj; Haeggström, Jesper Z; Wan, Min

    2015-08-01

    Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence-dependent endocytotic process. Both clathrin- and caveolae/lipid raft-mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

  17. An Image-Based High-Content Screening Assay for Compounds Targeting Intracellular Leishmania donovani Amastigotes in Human Macrophages

    PubMed Central

    Yang, Gyongseon; Lee, Changbok; Moon, Hong Kee; Chatelain, Eric; Genovesio, Auguste; Cechetto, Jonathan; Freitas-Junior, Lucio H.

    2012-01-01

    Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania. PMID:22720099

  18. Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes.

    PubMed

    Baltch, Aldona L; Bopp, Lawrence H; Smith, Raymond P; Michelsen, Phyllis B; Ritz, William J

    2005-07-01

    The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin. For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h. The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC. All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

  19. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

    PubMed

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-03-03

    NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

  20. Sodium/proton exchanger isoform 1 regulates intracellular pH and cell proliferation in human ovarian cancer.

    PubMed

    Sanhueza, Carlos; Araos, Joaquín; Naranjo, Luciano; Toledo, Fernando; Beltrán, Ana R; Ramírez, Marco A; Gutiérrez, Jaime; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2017-01-01

    Cancer cells generate protons (H(+)) that are extruded to the extracellular medium mainly via the Na(+)/H(+) exchanger 1 (NHE1), which regulates intracellular pH (pHi) and cell proliferation. In primary cultures of human ascites-derived ovarian cancer cells (haOC) we assayed whether NHE1 was required for pHi modulation and cell proliferation. Human ovary expresses NHE1, which is higher in haOC and A2780 (ovarian cancer cells) compared with HOSE cells (normal ovarian cells). Basal pHi and pHi recovery (following a NH4Cl pulse) was higher in haOC and A2780, compared with HOSE cells. Zoniporide (NHE1 inhibitor) caused intracellular acidification and pHi recovery was independent of intracellular buffer capacity, but reduced in NHE1 knockdown A2780 cells. Zoniporide reduced the maximal proliferation capacity, cell number, thymidine incorporation, and ki67 (marker of proliferation) fluorescence in haOC cells. SLC9A1 (for NHE1) amplification associated with lower overall patient survival. In conclusion, NHE1 is expressed in human ovarian cancer where it has a pro-proliferative role. Increased NHE1 expression and activity constitute an unfavourable prognostic factor in these patients. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance.

    PubMed

    Lutsenko, Svetlana; Gupta, Arnab; Burkhead, Jason L; Zuzel, Vesna

    2008-08-01

    The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

  2. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

    PubMed Central

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-01-01

    NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

  3. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  4. Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

    PubMed

    Rizaner, Nahit; Onkal, Rustem; Fraser, Scott P; Pristerá, Alessandro; Okuse, Kenji; Djamgoz, Mustafa B A

    2016-10-01

    The possible association of intracellular Ca(2+) with metastasis in human cancer cells is poorly understood. We have studied Ca(2+) signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca(2+) was measured using a membrane-permeant fluorescent Ca(2+)-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca(2+) oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca(2+) level could be induced by applying a high-K(+) solution. Various parameters of the oscillations depended on extracellular Ca(2+) and voltage-gated Na(+) channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na(+) channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca(2+) oscillations. It is concluded that the functional voltage-gated Na(+) channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca(2+) activity. Possible mechanisms and consequences of the Ca(2+) oscillations are discussed.

  5. In vitro antitumor activity of stellettin B, a triterpene from marine sponge Jaspis stellifera, on human glioblastoma cancer SF295 cells.

    PubMed

    Tang, Sheng-An; Zhou, Qianxiang; Guo, Wen-Zhi; Qiu, Yuling; Wang, Ran; Jin, Meihua; Zhang, Wenjing; Li, Ke; Yamori, Takao; Dan, Shingo; Kong, Dexin

    2014-07-15

    Stellettin B was isolated from marine sponge Jaspis stellifera. In vitro antitumor activities were investigated on 39 human cancer cell lines. Stellettin B exhibited highly potent inhibition against the growth of a human glioblastoma cell line SF295, with a GI50 of 0.01 μM. In contrast, stellettin B showed very weak inhibitory activity on normal cell lines including HMEC, RPTEC, NHBE and PrEC, with GI50s higher than 10 μM, suggesting its relatively selective cytotoxicity against human cancer cells compared to normal human cell lines. We then focused on the antitumor activity of this compound on SF295 cells. Flow cytometric analysis indicated that stellettin B induced apoptosis in SF295 cells in a concentration-dependent manner. Further study indicated that stellettin B increased the production of ROS, the activity of caspase 3/7, as well as the cleavage of PARP, each of which is known to be involved in apoptosis. To investigate the molecular mechanism for cell proliferation inhibition and apoptosis induction, effect on the phosphorylation of several signal proteins of PI3K/Akt and RAS/MAPK pathways was examined. Stellettin B inhibited the phosphorylation of Akt potently, with no activity on p-ERK and p-p38, suggesting that inhibition of PI3K/Akt pathway might be involved in the antiproliferative and apoptosis-inducing effect. However, homogenous time-resolved fluorescence (HTRF) assay indicated that stellettin B did not inhibit PI3K activity, suggesting that the direct target might be signal protein upstream of Akt pathway other than PI3K.

  6. In Vitro Antitumor Activity of Stellettin B, a Triterpene from Marine Sponge Jaspis stellifera, on Human Glioblastoma Cancer SF295 Cells

    PubMed Central

    Tang, Sheng-An; Zhou, Qianxiang; Guo, Wen-Zhi; Qiu, Yuling; Wang, Ran; Jin, Meihua; Zhang, Wenjing; Li, Ke; Yamori, Takao; Dan, Shingo; Kong, Dexin

    2014-01-01

    Stellettin B was isolated from marine sponge Jaspis stellifera. In vitro antitumor activities were investigated on 39 human cancer cell lines. Stellettin B exhibited highly potent inhibition against the growth of a human glioblastoma cell line SF295, with a GI50 of 0.01 μM. In contrast, stellettin B showed very weak inhibitory activity on normal cell lines including HMEC, RPTEC, NHBE and PrEC, with GI50s higher than 10 μM, suggesting its relatively selective cytotoxicity against human cancer cells compared to normal human cell lines. We then focused on the antitumor activity of this compound on SF295 cells. Flow cytometric analysis indicated that stellettin B induced apoptosis in SF295 cells in a concentration-dependent manner. Further study indicated that stellettin B increased the production of ROS, the activity of caspase 3/7, as well as the cleavage of PARP, each of which is known to be involved in apoptosis. To investigate the molecular mechanism for cell proliferation inhibition and apoptosis induction, effect on the phosphorylation of several signal proteins of PI3K/Akt and RAS/MAPK pathways was examined. Stellettin B inhibited the phosphorylation of Akt potently, with no activity on p-ERK and p-p38, suggesting that inhibition of PI3K/Akt pathway might be involved in the antiproliferative and apoptosis-inducing effect. However, homogenous time-resolved fluorescence (HTRF) assay indicated that stellettin B did not inhibit PI3K activity, suggesting that the direct target might be signal protein upstream of Akt pathway other than PI3K. PMID:25028795

  7. Association between epidermal growth factor receptor amplification and ADP-ribosylation factor 1 methylation in human glioblastoma.

    PubMed

    López-Ginés, Concha; Navarro, Lara; Muñoz-Hidalgo, Lisandra; Buso, Enrique; Morales, José Manuel; Gil-Benso, Rosario; Gregori-Romero, Mariela; Megías, Javier; Roldán, Pedro; Segura-Sabater, Remedios; Almerich-Silla, José Manuel; Monleón, Daniel; Cerdá-Nicolás, Miguel

    2017-08-01

    Glioblastoma (GB) is the most frequent and most malignant primary brain tumor in adults. Previously, it has been found that both genetic and epigenetic factors may play critical roles in its etiology and prognosis. In addition, it has been found that the epidermal growth factor receptor gene (EGFR) is frequently over-expressed and amplified in primary GBs. Here, we assessed the promoter methylation status of 10 genes relevant to GB and explored associations between these findings and the EGFR gene amplification status. Tumor samples were obtained from 36 patients with primary GBs. In addition, 6 control specimens were included from patients who were operated for diseases other than brain tumors. The amplification status of the EGFR gene, and its deletion mutant EGFRvIII, were evaluated using FISH and MLPA, respectively. The IDH1/2 gene mutation status was verified using Sanger sequencing. A commercial DNA methylation kit was used to assess the promoter methylation status of 10 pre-selected genes. Metabolic profiles were measured using HR-MAS NMR spectroscopy. The EGFR and ARF1 mRNA expression levels were quantified using qRT-PCR. Of the 10 genes analyzed, we found that only ARF1 promoter hypermethylation was significantly associated with EGFR gene amplification. ARF1 is a GTPase that is involved in vesicle trafficking and the Golgi apparatus. Subsequent tumor metabolism measurements revealed a positive association between EGFR amplification and different membrane precursors and methyl-donor metabolites. Finally, we found that EGFR gene amplifications were associated with distinct tumor infiltration patterns, thus representing a putative novel functional association between EGFR gene amplification and ARF1 gene promoter methylation in GB. The results reported here provide a basis for a new hypotheses connecting EGFR gene amplification in GB cells with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and tumor metabolism. The mechanism

  8. Computational identification of tumor anatomic location associated with survival in two large cohorts of human primary glioblastomas

    PubMed Central

    Liu, Tiffany T.; Achrol, Achal S.; Mitchell, Lex A.; Du, William A.; Loya, Joshua J.; Rodriguez, Scott A.; Feroze, Abdullah; Westbroek, Erick M.; Yeom, Kristen W.; Stuart, Joshua M.; Chang, Steven D.; Harsh, Griffith R.; Rubin, Daniel L.

    2015-01-01

    Background and Purpose Tumor location has been shown to be a significant prognostic factor in patients with glioblastoma (GBM). The purpose of this study is to characterize GBM lesions by identifying MRI voxel-based tumor location features that are associated with tumor molecular profiles, patient characteristics and clinical outcomes. Materials and Methods Preoperative T1 anatomic MR images of 384 GBM patients were obtained from two independent cohorts (N=253 from our local (name withheld to preserve anonymity) Medical Center for training and N=131 from the Cancer Genome Atlas (TCGA) for validation). An automated computational image analysis pipeline was developed to determine the anatomic locations of tumor in each patient. Voxel-based differences in tumor location between good (overall survival (OS) > 17 months) and poor (OS < 11 months) survival groups identified in the training cohort were used to classify patients in the TCGA cohort into two brain location groups, for which clinical features, mRNA expression, and copy number changes were compared to elucidate the biological basis of tumors located in different brain regions. Results Tumors in the right occipito-temporal periventricular white matter were significantly associated with poor survival in both training and test cohorts (both log-rank P < 0.05) and had larger tumor volume compared to tumors in other locations. Tumors in the right peri-atrial location were associated with hypoxia pathway enrichment and PDGFRA amplification, making them potential targets for subgroup-specific therapies. Conclusion Voxel-based location in GBM is associated with patient outcome and may have a potential role for guiding personalized treatment. PMID:26744442

  9. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

    PubMed

    Xu, Yunpeng; Zhao, Gang; Zhou, Xiaoming; Ding, Weiping; Shu, Zhiquan; Gao, Dayong

    2014-04-01

    The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Regulation of intracellular pH in Calu-3 human airway cells.

    PubMed

    Inglis, S K; Finlay, L; Ramminger, S J; Richard, K; Ward, M R; Wilson, S M; Olver, R E

    2002-01-15

    The Calu-3 human cell line exhibits features of submucosal gland serous cells and secretes HCO(3)(-). The aim of this study was to identify the HCO(3)(-) transporters present in these cells by studying their role in the regulation of intracellular pH (pH(i)). Calu-3 cells were grown on coverslips, loaded with the pH-sensitive fluorescent dye BCECF, and their fluorescence intensity monitored as an indication of pH(i). Cells were acidified with NH(4)Cl (25 mM, 1 min) and pH(i) recovery recorded. In the absence of HCO(3)(-), initial recovery was 0.208 +/- 0.016 pH units min(-1) (n = 37). This was almost abolished by removal of extracellular Na(+) and by amiloride (1 mM), consistent with the activity of a Na(+)-H(+) exchanger (NHE). In the presence of HCO(3)(-) and CO(2), recovery (0.156 +/- 0.018 pH units min(-1)) was abolished (reduced by 91.8 +/- 6.7 %, n = 7) by removal of Na(+) but only attenuated (by 63.3 +/- 5.8 %, n = 9) by amiloride. 4,4-Dinitrostilbene-2,2-disulfonic acid (DNDS) inhibited recovery by 45.8 +/- 5.0 % (n = 7). The amiloride-insensitive recovery was insensitive to changes in membrane potential, as confirmed by direct microelectrode measurements, brought about by changing extracellular [K(+)] in the presence of either valinomycin or the K(+) channel opener 1-EBIO. In addition, forskolin (10 microM), which activates the cystic fibrosis transmembrane conductance regulator Cl(-) conductance in these cells and depolarises the cell membrane, had no effect on recovery. Removal of extracellular Cl(-) trebled pH(i) recovery rates, suggesting that an electroneutral, DNDS-sensitive, Cl(-)-HCO(3)(-) exchanger together with a NHE may be involved in pH(i) regulation and HCO(3)(-) secretion in these cells. RT-PCR detected the expression of the electrogenic Na(+)-HCO(3)(-) cotransporter NBC1 and the Cl(-)-HCO(3)(-) exchanger (AE2) but not the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1.

  11. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

    PubMed

    Rey, Osvaldo; Young, Steven H; Jacamo, Rodrigo; Moyer, Mary P; Rozengurt, Enrique

    2010-10-01

    The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

  12. Ginsenoside 20(S)-Protopanaxadiol Suppresses Viability of Human Glioblastoma Cells via Down-regulation of Cell Adhesion Proteins and Cell-cycle Arrest.

    PubMed

    Wanderi, Cheryl; Kim, Eunsoo; Chang, Soyoung; Choi, Chulhee; Choi, Kyungsun

    2016-03-01

    Pharmacologically active components of ginseng, particularly protopanaxadiol (PPD)-type ginsenosides, have potent anticancer effects, although their effects on highly malignant glioblastoma multiforme (GBM) have not been systemically evaluated. Identification of effective anticancer ginsenosides and further delineation of their mechanisms of action may provide valuable information that aids in the development of alternative or adjuvant therapy for malignant cancer. We examined the viability of human GBM U251-MG and U87-MG cells treated with structurally related PPD-type ginsenosides, including F2, Rh2, compound K (C-K), and PPD. Incubation with PPD, C-K, and Rh2 significantly reduced the viability of U251-MG and U87-MG cells in a dose- and time-dependent manner. The cytotoxic effect of PPD was accompanied by reduced expression of cell adhesion proteins, including N-cadherin and integrin β1, which led to reduced phosphorylation of focal adhesion kinase. Furthermore, incubation with PPD reduced the expression of cyclin D1 and subsequently induced cell-cycle arrest at the G1 phase. These results collectively indicate that PPD might provide a new strategy for treating malignant GBM, which is quite resistant to conventional anticancer treatment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  13. Hydrogen sulfide generation from l-cysteine in the human glioblastoma-astrocytoma U-87 MG and neuroblastoma SHSY5Y cell lines.

    PubMed

    Bronowicka-Adamska, Patrycja; Bentke, Anna; Wróbel, Maria

    2017-01-01

    Hydrogen sulfide (H2S) is endogenously synthesized from l-cysteine in reactions catalyzed by cystathionine beta-synthase (CBS, EC 4.2.1.22) and gamma-cystathionase (CSE, EC 4.4.1.1). The role of 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) in H2S generation is also considered; it could be important for tissues with low CTH activity, e.g. cells of the nervous system. The expression and activity of CBS, CTH, and MPST were detected in the human glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cell lines. In both cell lines, the expression and activity of MPST were the highest among the investigated enzymes, suggesting its possible role in the generation of H2S. The RP-HPLC method was used to determine the concentration of cystathionine and alpha-ketobutyrate, products of the CBS- and CTH-catalyzed reactions. The difference in cystathionine levels between cell homogenates treated with totally CTH-inhibiting concentrations of dl-propargylglycine and without the inhibitor was used to evaluate the activity of CBS. The higher expression and activity of CBS, CTH and MPST in the neuroblastoma cells were associated with more intensive generation of H2S in the presence of 2 mM cysteine. A threefold higher level of sulfane sulfur, a potential source of hydrogen sulfide, was detected in the astrocytoma cells in comparison to the neuroblastoma cells.

  14. Dichloroacetate induces cell cycle arrest in human glioblastoma cells persistently infected with measles virus: a way for controlling viral persistent infection.

    PubMed

    Takahashi, Megumi; Watari, Eiji; Takahashi, Hidemi

    2015-01-01

    We have previously established a human glioblastoma cell line persistently infected with mutant measles virus (MV), and found increased functions of mitochondria in MV persistently infected cells compared with uninfected or acutely infected cells. Moreover, impairment of mitochondria functions induced a breakdown of persistent infection, which suggested that mitochondria might play an important role in the maintenance of persistent infection and loss or functional alterations of mitochondria might be a candidate for possible intervention in persistent infection. In this study we examined the effect of dichloroacetate (DCA), which is known to increase pyruvate oxidation, on mitochondrial functions in MV persistently infected cells. DCA caused mitochondrial dysfunction and cell cycle arrest in MV persistently infected cells; consequently, the expression level of MV proteins were decreased in a DCA dependent manner. Here, we present a new therapeutic approach for persistent infection targeting mitochondrial respiration. Advantage to targeting mitochondrial respiration is that the impairment of mitochondrial functions by DCA is found in MV persistently infected cells not in uninfected cells, which may offer a promising chemotherapeutic strategy with few adverse effects. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Telomere targeting with a novel G-quadruplex-interactive ligand BRACO-19 induces T-loop disassembly and telomerase displacement in human glioblastoma cells

    PubMed Central

    Zhou, Guangtong; Liu, Xinrui; Li, Yunqian; Xu, Songbai; Ma, Chengyuan; Wu, Xinmin; Cheng, Ye; Yu, Zhiyun; Zhao, Gang; Chen, Yong

    2016-01-01

    Interference with telomerase and telomere maintenance is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA 3′-overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends, making these ligands good candidates for chemotherapeutic purposes. BRACO-19 is one of the most effective and specific ligand for telomeric G4. It is shown here that BRACO-19 suppresses proliferation and reduces telomerase activity in human glioblastoma cells, paralleled by the displacement of telomerase from nuclear to cytoplasm. Meanwhile, BRACO-19 triggers extensive DNA damage response at telomere, which may result from uncapping and disassembly of telomeric T-loop structure, characterized by the formation of anaphase bridge and telomere fusion, as well as the release of telomere-binding protein from telomere. The resulting dysfunctional telomere ultimately provokes p53 and p21-mediated cell cycle arrest, apoptosis and senescence. Notably, normal primary astrocytes do not respond to the treatment of BRACO-19, suggesting the agent's good selectivity for cancer cells. These results reinforce the notion that G-quadruplex binding compounds can act as broad inhibitors of telomere-related processes and have potential as selective antineoplastic drugs for various tumors including malignant gliomas. PMID:26908447

  16. Evaluation of Heterogeneous Metabolic Profile in an Orthotopic Human Glioblastoma Xenograft Model Using Compressed Sensing Hyperpolarized 3D 13C Magnetic Resonance Spectroscopic Imaging

    PubMed Central

    Park, Ilwoo; Hu, Simon; Bok, Robert; Ozawa, Tomoko; Ito, Motokazu; Mukherjee, Joydeep; Phillips, Joanna J.; James, C. David; Pieper, Russell O.; Ronen, Sabrina M.; Vigneron, Daniel B.; Nelson, Sarah J.

    2013-01-01

    High resolution compressed sensing hyperpolarized 13C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in 13C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D 13C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor-bearing rats following injection of hyperpolarized [1-13C]-pyruvate using a 3T scanner. The 13C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high-resolution compressed sensing 13C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct 13C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of 13C metabolic profiles between viable, non-necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue. PMID:22851374

  17. MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells

    PubMed Central

    Guo, Xing; Xue, Hao; Guo, Xiaofan; Gao, Xiao; Xu, Shugang; Yan, Shaofeng; Han, Xiao; Li, Tong; Shen, Jie; Li, Gang

    2015-01-01

    Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells. PMID:26536662

  18. Determination of intracellular free magnesium by nuclear magnetic resonance in human magnesium deficiency.

    PubMed

    Ryzen, E; Servis, K L; DeRusso, P; Kershaw, A; Stephen, T; Rude, R K

    1989-12-01

    Magnesium (Mg) deficiency is a common clinical problem. As Mg is predominantly an intracellular cation and Mg deficiency may exist despite normal serum Mg (sMg) concentrations, we have utilized nuclear magnetic resonance (NMR) techniques in an attempt to measure intracellular free Mg (Mg2+) in red blood cells (RBC). Twenty normal subjects, 22 hypomagnesemic patients, and 17 normomagnesemic alcoholic patients were studied. Mean RBC Mg2+ in normal subjects (178 +/- 6.3 microM) was significantly greater than in hypomagnesemic patients (146 +/- 7.1 microM, p less than 0.002). RBC Mg2+ correlated with sMg concentration (r = 0.54, p less than 0.001). In addition, four normal subjects were given a low Mg diet for 3 weeks. There was a progressive fall in both the sMg concentration and RBC Mg2+ during Mg depletion, with a concomitant rise in retention of a parenterally administered Mg load. These data suggest that the determination of intracellular Mg2+ by NMR may be a useful research tool in assessing the effect of changes in Mg2+ on intracellular processes. Its utility in the clinical evaluation of disorders of Mg deficiency remains to be determined.

  19. Effect of extracellular glucose and K+ on intracellular osmolytes and volume in a human kidney cell line.

    PubMed

    Hubert, Marcy D; Indyk, Elisabeth; Pena-Rasgado, Cecilia; Pierce, Sydney K; Rasgado-Flores, Hector; Garber, Sarah S

    2007-05-01

    The goal of this study was to assess the effect of extracellular glucose and K+ ((K)o) on the intracellular osmolyte content and cell volume maintenance and regulation in a human embryonic kidney cell line (tsA201a). Cell volume maintenance was studied by isotonic (313 +/- 5 mOsm) replacement of culture media by a glucose-free Ringer solution containing (in mM) 0, 3, 6, or 10 K+. Cell volume regulation was studied by exposing cells to hypotonic (250 +/- 5 mOsm) glucose-free Ringer solution containing the various (K)o. The results showed that: 1) intracellular osomlyte content (i.e. Na+, Cl-, Urea and free amino acids (FAA)) and cell volume increased when culture media was replaced with isotonic Ringer at all (K)o; 2) osmolyte content decreased with continuous exposure to isotonic Ringer at all (K)o but cell volume changes depended on (K)o. Volume recovery occurred at 6 and 10 mM K+; 3) exposure to hypotonic Ringer induced swelling at all (K)o followed by a reduction in measured intracellular osmolytes. Regulatory volume decrease occurred in 6 or 10 mM K+ but swelling continued in 0 or 3 mM K+; and 4) addition of ouabain produced swelling without recovery under iso- and hypotonic conditions. These results indicate that the removal of extracellular glucose produced a transient inhibition of the Na+/K+ ATPase resulting in a transient increase in the intracellular content of Urea, FAA and cell volume and (K)o regulated an as yet unidentified