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Sample records for human glioblastoma intracellular

  1. 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

  2. The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

    PubMed Central

    Song, M; Chen, D; Yu, S P

    2014-01-01

    BACKGROUND AND PURPOSE SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca2+ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored. EXPERIMENTAL APPROACH The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca2+ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence. KEY RESULTS SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca2+ ([Ca2+]i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na+/Ca2+ exchanger (NCX) with an EC50 of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G2 phases and activated p38-MAPK and JNK, which were all prevented by the Ca2+ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells. CONCLUSIONS AND IMPLICATIONS At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca2+]i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca2+ homeostasis and suppress human glioblastoma cells. PMID:24641279

  3. 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 ...

  4. 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. PMID:26948367

  5. Decitabine nanoconjugate sensitizes human glioblastoma cells to temozolomide.

    PubMed

    Cui, Yi; Naz, Asia; Thompson, David H; Irudayaraj, Joseph

    2015-04-01

    In this study, we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) based nanoconjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells than that with the free drug. After synthesis, the highly efficient uptake process and intracellular dynamics of this nanoconjugate were monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nanovector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing positive feedback to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to the excellent internalization and endolysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than that of free drug molecules. Hence, the synthesized nanoconjugate and temozolomide could act in synergy to deliver a more potent and long-term antiproliferative effect against malignant GBM cells.

  6. Decitabine Nano-conjugate Sensitizing Human Glioblastoma Cells to Temozolomide

    PubMed Central

    Cui, Yi; Naz, Asia; Thompson, David H.; Irudayaraj, Joseph

    2015-01-01

    In this study we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based nano-conjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells. After synthesis, the highly efficient uptake process and intracellular dynamics of this nano-conjugate was monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nano-vector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing a “positive feedback” to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to excellent internalization and endo-lysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than free drug molecules. Hence, the synthesized nano-conjugate and temozolomide could act in synergy to deliver a more potent and long-term anti-proliferation effect against malignant GBM cells. PMID:25751281

  7. Voltage-Gated Proton Channel in Human Glioblastoma Multiforme Cells.

    PubMed

    Ribeiro-Silva, Luisa; Queiroz, Fernanda Oliveira; da Silva, Annielle Mendes Brito; Hirata, Aparecida Emiko; Arcisio-Miranda, Manoel

    2016-07-20

    Solid tumors tend to have a more glycolytic metabolism leading to an accumulation of acidic metabolites in their cytosol, and consequently, their intracellular pH (pHi) turns critically lower if the cells do not handle the acid excess. Recently, it was proposed that the voltage gated proton channels (HV1) can regulate the pHi in several cancers. Here we report the functional expression of voltage gated proton channels in a human glioblastoma multiforme (GBM) cell line, the most common and lethal brain tumor. T98G cells presented an outward, slow activating voltage-dependent proton current, which was also ΔpH-dependent and inhibited by ZnCl2, characterizing it as being conducted by HV1 channels. Furthermore, blocking HV1 channels with ZnCl2 significantly reduced the pHi, cell survival, and migration, indicating an important role for HV1 for tumor proliferation and progression in GBM. Overall, our results suggest that HV1 channels can be a new therapeutic target for GBM. PMID:27225904

  8. Tax-interacting protein 1 coordinates the spatiotemporal activation of Rho GTPases and regulates the infiltrative growth of human glioblastoma

    PubMed Central

    Wang, Hailun; Han, Miaojun; Whetsell, William; Wang, Jialiang; Rich, Jeremy; Hallahan, Dennis; Han, Zhaozhong

    2014-01-01

    PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. TIP-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as one novel TIP-1 interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector, and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas. PMID:23563176

  9. Metformin selectively affects human glioblastoma tumor-initiating cell viability: A role for metformin-induced inhibition of Akt.

    PubMed

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirano; 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.

  10. 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. PMID:27334753

  11. 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.

  12. 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

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

    PubMed

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

    2014-11-30

    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.

  14. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

    PubMed

    Rachlin, Kenneth; Moore, Dan H; Yount, Garret

    2013-11-01

    The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting. PMID:23165942

  15. 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

    2015-01-01

    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. PMID:25455450

  16. 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

  17. 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

  18. Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent.

    PubMed

    Leone, Roberta; Giussani, Paola; De Palma, Sara; Fania, Chiara; Capitanio, Daniele; Vasso, Michele; Brioschi, Loredana; Riboni, Laura; Viani, Paola; Gelfi, Cecilia

    2015-06-01

    Glioblastoma multiforme is the most aggressive astrocytoma characterized by the development of resistant cells to various cytotoxic stimuli. Nitric oxide (NO) is able to overcome tumor resistance in PTEN mutated rat C6 glioma cells due to its ability to inhibit cell growth by influencing the intracellular distribution of ceramide. The aim of this study is to monitor the effects of NO donor PAPANONOate on ceramide trafficking in human glioma cell lines, CCF-STTG1 (PTEN-mutated, p53-wt) and T98G (PTEN-harboring, p53-mutated), together with the assessment of their differential molecular signature by 2D-DIGE and MALDI mass spectrometry. In the CCF-STTG1 cell line, the results indicate that treatment with PAPANONOate decreased cell proliferation (<50%) and intracellular trafficking of ceramide, assessed by BODIPY-C5Cer, while these events were not observed in the T98G cell line. Proteomic results suggest that CCF-STTG1 cells are characterized by an increased expression of proteins involved in NO-associated ER stress (i.e. protein disulfide-isomerase A3, calreticulin, 78 kDa glucose-regulated protein), which could compromise ceramide delivery from ER to Golgi, leading to ceramide accumulation in ER and partial growth arrest. Conversely, T98G cell lines, resistant to NO exposure, are characterized by increased levels of cytosolic antioxidant proteins (i.e. glutathione-S-transferase P, peroxiredoxin 1), which might buffer intracellular NO. By providing differential ceramide distribution after NO exposure and differential protein expression of two high grade glioma cell lines, this study highlights specific proteins as possible markers for tumor aggressiveness. This study demonstrates that, in two different high grade glioma cell lines, NO exposure results in a different ceramide distribution and protein expression. Furthermore, this study highlights specific proteins as possible markers for tumor aggressiveness. PMID:25797839

  19. Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

    PubMed

    Chen, Jong-Hang; Chou, Chin-Cheng

    2015-08-01

    This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling.

  20. 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.

  1. 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.

  2. In-vitro suppression of metabolic activity in malignant human glioblastomas due to pulsed - low frequency electric potential exposures

    NASA Astrophysics Data System (ADS)

    Schlichting, Abby; Waynant, Ronald W.; Tata, Darrell B.

    2010-02-01

    The role of pulsed - low repetition frequency electric potential was investigated in suppressing the metabolic activities of aggressive human brain cancer cells. Twenty four hours post exposure the glioblastomas were found to be significantly inhibited in their metabolic activity. The findings herein reveal a near complete inhibition of glioblastoma's metabolic activity through selective applications of low frequency pulsed electric potentials.

  3. A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide

    PubMed Central

    Pojo, Marta; Gonçalves, Céline S.; Xavier-Magalhães, Ana; Oliveira, Ana Isabel; Gonçalves, Tiago; Correia, Sara; Rodrigues, Ana J.; Costa, Sandra; Pinto, Luísa; Pinto, Afonso A.; Lopes, José M.; Reis, Rui M.; Rocha, Miguel; Sousa, Nuno; Costa, Bruno M.

    2015-01-01

    Glioblastoma is the most malignant brain tumor, exhibiting remarkable resistance to treatment. Here we investigated the oncogenic potential of HOXA9 in gliomagenesis, the molecular and cellular mechanisms by which HOXA9 renders glioblastoma more aggressive, and how HOXA9 affects response to chemotherapy and survival. The prognostic value of HOXA9 in glioblastoma patients was validated in two large datasets from TCGA and Rembrandt, where high HOXA9 levels were associated with shorter survival. Transcriptomic analyses identified novel HOXA9-target genes with key roles in cancer-related processes, including cell proliferation, DNA repair, and stem cell maintenance. Functional studies with HOXA9-overexpressing and HOXA9-silenced glioblastoma cell models revealed that HOXA9 promotes cell viability, stemness and invasion, and inhibits apoptosis. Additionally, HOXA9 promoted the malignant transformation of human immortalized astrocytes in an orthotopic in vivo model, and caused tumor-associated death. HOXA9 also mediated resistance to temozolomide treatment in vitro and in vivo via upregulation of BCL2. Importantly, the pharmacological inhibition of BCL2 with the BH3 mimetic ABT-737 reverted temozolomide resistance in HOXA9-positive cells. These data establish HOXA9 as a driver of glioma initiation, aggressiveness and resistance to therapy. In the future, the combination of BH3 mimetics with temozolomide should be further explored as an alternative treatment for glioblastoma. PMID:25762636

  4. New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line.

    PubMed

    Giacomelli, Chiara; Natali, Letizia; Trincavelli, Maria Letizia; Daniele, Simona; Bertoli, Alessandra; Flamini, Guido; Braca, Alessandra; Martini, Claudia

    2016-05-01

    Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule. PMID:26939786

  5. A CDC20-APC/SOX2 Signaling Axis Regulates Human Glioblastoma Stem-like Cells.

    PubMed

    Mao, Diane D; Gujar, Amit D; Mahlokozera, Tatenda; Chen, Ishita; Pan, Yanchun; Luo, Jingqin; Brost, Taylor; Thompson, Elizabeth A; Turski, Alice; Leuthardt, Eric C; Dunn, Gavin P; Chicoine, Michael R; Rich, Keith M; Dowling, Joshua L; Zipfel, Gregory J; Dacey, Ralph G; Achilefu, Samuel; Tran, David D; Yano, Hiroko; Kim, Albert H

    2015-06-23

    Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-anaphase-promoting complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma.

  6. Identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (CFSE).

    PubMed

    Deleyrolle, Loic P; Rohaus, Mark R; Fortin, Jeff M; Reynolds, Brent A; Azari, Hassan

    2012-01-01

    Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia(2-8). The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma(2,7,8). The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds(9). The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division(10). CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity(2). This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry(2). The technique has served to identify

  7. M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells.

    PubMed

    Ferretti, Michela; Fabbiano, Cinzia; Di Bari, Maria; Conte, Claudia; Castigli, Emilia; Sciaccaluga, Miriam; Ponti, Donatella; Ruggieri, Paola; Raco, Antonino; Ricordy, Ruggero; Calogero, Antonella; Tata, Ada Maria

    2013-04-01

    Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

  8. Intracellular accumulation and cytotoxicity of doxorubicin with different pharmaceutical formulations in human cancer cell lines.

    PubMed

    Serpe, Loredana; Guido, Marilena; Canaparo, Roberto; Muntoni, Elisabetta; Cavalli, Roberta; Panzanelli, Patrizia; Della Pepal, Carlo; Bargoni, Alessandro; Mauro, Alessandro; Gasco, Maria Rosa; Eandi, Mario; Zara, Gian Paolo

    2006-01-01

    The structure of both carrier and anticancer drug affects the intracellular fate of a transported drug. The study investigated in vitro intracellular accumulation and cytotoxic activity of doxorubicin-loaded solid lipid nanoparticles (SLN), doxorubicin in pegylated liposomes (Caelyx) and free doxorubicin. Intracellular doxorubicin levels and cytotoxic activity were determined by high performance liquid chromatography with fluorescence detection, and by the trypan blue dye exclusion assay, respectively. Doxorubicin-loaded SLN inhibited cell growth more strongly than either free or liposomal doxorubicin, in human colorectal adenocarcinoma, HT-29, retinoblastoma Y79, and glioblastoma U373 cell lines. The IC50 values for doxorubicin-loaded SLN were significantly lower after 24 h exposure than those for free doxorubicin in all cell lines; after 48 h exposure they were lower than those for liposomal doxorubicin in HT-29 and Y79 cells. The enhanced cytotoxic activity of doxorubicin-loaded SLN was associated with increased drug incorporation in cells: intracellular doxorubicin levels were significantly enhanced after exposure to drug-loaded SLN versus either free or liposomal drug. Rate of intracellular accumulation and cytotoxic activity also differed among different cell lines; in particular, cells of epithelial origin were found to be more sensitive to doxorubicin-loaded SLN. In conclusion, the greater sensitivity of HT-29, Y79, and U373 cells to doxorubicin-loaded SLN than to the other drug formulations may be due to the capability of the delivery system to enhance drug action, through a marked uptake and accumulation of SLN within the cell. PMID:17048519

  9. 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

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

    PubMed

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

    2004-10-01

    Serotonin 5-HT(7) 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-HT(7) receptors and 5-HT(7) 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-HT(7) receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89-1.13) and pA(2) values of 8.69-9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT(7) receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT(7) receptor (5-HT(7(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-HT(7) receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT(7) receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines. PMID:15339860

  11. Novel synthetic chalcones induces apoptosis in human glioblastoma cells.

    PubMed

    Bittencourt, Lucas Felipe Fernandes; Oliveira, Karen Andrinéia de; Cardoso, Carine Bropp; Lopes, Flávia Garcia; Dal-Cim, Tharine; Chiaradia-Delatorre, Louise Domeneghini; Mascarello, Alessandra; Maluf, Sharbel Weidner; Yunes, Rosendo Augusto; Garcez, Ricardo Castilho; Tasca, Carla Inês; Nedel, Cláudia Beatriz

    2016-05-25

    Glioblastoma multiforme is the main and most frequent tumor in adults' central nervous system. With a survival average of 5% two years after diagnosis, this type of cancer is a main health problem. Substances like the chalcones have been tested in order to develop new treatments. Here, we studied the effects of three synthetic chalcones (A23, C31 and J11) on A172 and surgery obtained-glioma cells. All chalcones showed a decrease in cell viability, mainly C31. An increase in apoptosis levels with no further increase of necrosis was observed. This augmentation may be linked to the high oxidative effect found, caused by the increased presence of reactive oxygen species and nitric oxide production. Cell cycle distribution showed an arrest at G0/G1 and S phases, suggesting that C31 interferes in cell cycle control. Our results shall aid in directing future research with this substance and its antitumor effect.

  12. Association of epidermal growth factor receptor gene amplification with loss of chromosome 10 in human glioblastoma multiforme.

    PubMed

    von Deimling, A; Louis, D N; von Ammon, K; Petersen, I; Hoell, T; Chung, R Y; Martuza, R L; Schoenfeld, D A; Yaşargil, M G; Wiestler, O D

    1992-08-01

    Although the loss of tumor suppressor genes and the activation of oncogenes have been established as two of the fundamental mechanisms of tumorigenesis in human cancer, little is known about the possible interactions between these two mechanisms. Loss of genetic material on chromosome 10 and amplification of the epidermal growth factor receptor (EGFR) gene are the most frequently reported genetic abnormalities in glioblastoma multiforme. In order to examine a possible correlation between these two genetic aberrations, the authors studied 106 gliomas (58 glioblastomas, 14 anaplastic astrocytomas, five astrocytomas, nine pilocytic astrocytomas, seven mixed gliomas, six oligodendrogliomas, two ependymomas, one subependymoma, one subependymal giant-cell astrocytoma, and three gangliogliomas) with Southern blot analysis for loss of heterozygosity on both arms of chromosome 10 and for amplification of the EGFR gene. Both the loss of genetic material on chromosome 10 and EGFR gene amplification were restricted to the glioblastomas. Of the 58 glioblastoma patients, 72% showed loss of chromosome 10 and 38% showed EGFR gene amplification. The remaining 28% had neither loss of chromosome 10 nor EGFR gene amplification. Without exception, the glioblastomas that exhibited EGFR gene amplification had also lost genetic material on chromosome 10 (p less than 0.001). This invariable association suggests a relationship between the two genetic events. Moreover, the presence of 15 cases of glioblastoma with loss of chromosome 10 but without EGFR gene amplification may further imply that the loss of a tumor suppressor gene (or genes) on chromosome 10 precedes EGFR gene amplification in glioblastoma tumorigenesis.

  13. Intracellular localization of VAMP-1 protein in human neutrophils.

    PubMed

    Nabokina, S M

    2001-02-01

    We studied the intracellular localization of vesicle-associated membrane protein VAMP-1 in human neutrophils. VAMP-1 was associated with membranes of gelatinase and specific secretory granules rapidly mobilized during exocytosis. VAMP-1 probably acts as a component of the SNARE complex during exocytosis of gelatinase and specific granules in human neutrophils.

  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. The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

    PubMed

    Xie, Yuan; Bergström, Tobias; Jiang, Yiwen; Johansson, Patrik; Marinescu, Voichita Dana; Lindberg, Nanna; Segerman, Anna; Wicher, Grzegorz; Niklasson, Mia; Baskaran, Sathishkumar; Sreedharan, Smitha; Everlien, Isabelle; Kastemar, Marianne; Hermansson, Annika; Elfineh, Lioudmila; Libard, Sylwia; Holland, Eric Charles; Hesselager, Göran; Alafuzoff, Irina; Westermark, Bengt; Nelander, Sven; Forsberg-Nilsson, Karin; Uhrbom, Lene

    2015-10-01

    Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research. PMID:26629530

  16. 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.

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

    PubMed

    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; Izpisua Belmonte, Juan Carlos; 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

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

    PubMed

    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; Izpisua Belmonte, Juan Carlos; Chen, Zhiguo; Wang, Xiaomin; Jiang, Tao; Qu, Jing; Tang, Fuchou; Liu, Guang-Hui

    2015-12-03

    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.

  19. Expression of Tax-interacting protein 1 (TIP-1) facilitates angiogenesis and tumor formation of human glioblastoma cells in nude mice

    PubMed Central

    Han, Miaojun; Wang, Hailun; Zhang, Hua-Tang; Han, Zhaozhong

    2012-01-01

    Glioblastoma is the most common and fatal type of primary brain tumors featured with hyperplastic blood vessels. Here, we performed meta-analyses of published data and established a correlation between high TIP-1 expression levels and the poor prognosis of glioblastoma patients. Next, we explored the biological relevance of TIP-1 expression in the pathogenesis of glioblastoma. By using orthotopic and heterotopic mouse models of human glioblastomas, this study has characterized TIP-1 as one contributing factor to the tumor-driven angiogenesis. In vitro and in vivo functional assays, along with biochemical analyses with microarrays and antibody arrays, have demonstrated that TIP-1 utilizes multiple pathways including modulating fibronectin gene expression and uPA protein secretion, to establish or maintain a pro-angiogenic microenvironment within human glioblastoma. In conclusion, this work supports the hypothesis that TIP-1 represents a novel prognostic biomarker and a therapeutic target of human glioblastoma. PMID:23010083

  20. 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.

  1. 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

  2. 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.

  3. Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

    PubMed

    Halamoda Kenzaoui, Blanka; Angeloni, Silvia; Overstolz, Thomas; Niedermann, Philippe; Chapuis Bernasconi, Catherine; Liley, Martha; Juillerat-Jeanneret, Lucienne

    2013-05-01

    Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer.

  4. 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.

  5. Glioblastoma cells induce differential glutamatergic gene expressions in human tumor-associated microglia/macrophages and monocyte-derived macrophages.

    PubMed

    Choi, Judy; Stradmann-Bellinghausen, Beate; Yakubov, Eduard; Savaskan, Nicolai E; Régnier-Vigouroux, Anne

    2015-01-01

    Glioblastoma cells produce and release high amounts of glutamate into the extracellular milieu and subsequently can trigger seizure in patients. Tumor-associated microglia/macrophages (TAMs), consisting of both parenchymal microglia and monocytes-derived macrophages (MDMs) recruited from the blood, are known to populate up to 1/3 of the glioblastoma tumor environment and exhibit an alternative, tumor-promoting and supporting phenotype. However, it is unknown how TAMs respond to the excess extracellular glutamate in the glioblastoma microenvironment. We investigated the expressions of genes related to glutamate transport and metabolism in human TAMs freshly isolated from glioblastoma resections. Quantitative real-time PCR analysis showed (i) significant increases in the expressions of GRIA2 (GluA2 or AMPA receptor 2), SLC1A2 (EAAT2), SLC1A3 (EAAT1), (ii) a near-significant decrease in the expression of SLC7A11 (cystine-glutamate antiporter xCT) and (iii) a remarkable increase in GLUL expression (glutamine synthetase) in these cells compared to adult primary human microglia. TAMs co-cultured with glioblastoma cells also exhibited a similar glutamatergic profile as freshly isolated TAMs except for a slight increase in SLC7A11 expression. We next analyzed these genes expressions in cultured human MDMs derived from peripheral blood monocytes for comparison. In contrast, MDMs co-cultured with glioblastoma cells compared to MDMs co-cultured with normal astrocytes exhibited decreased expressions in the tested genes except for GLUL. This is the first study to demonstrate transcriptional changes in glutamatergic signaling of TAMs in a glioblastoma microenvironment, and the findings here suggest that TAMs and MDMs might potentially elicit different cellular responses in the presence of excess extracellular glutamate. PMID:26047211

  6. Flavonoids suppress human glioblastoma cell growth by inhibiting cell metabolism, migration, and by regulating extracellular matrix proteins and metalloproteinases expression.

    PubMed

    Santos, Balbino L; Oliveira, Mona N; Coelho, Paulo L C; Pitanga, Bruno P S; da Silva, Alessandra B; Adelita, Taís; Silva, Victor Diógenes A; Costa, Maria de F D; El-Bachá, Ramon S; Tardy, Marcienne; Chneiweiss, Hervé; Junier, Marie-Pierre; Moura-Neto, Vivaldo; Costa, Silvia L

    2015-12-01

    The malignant gliomas are very common primary brain tumors with poor prognosis, which require more effective therapies than the current used, such as with chemotherapy drugs. In this work, we investigated the effects of several polyhydroxylated flavonoids namely, rutin, quercetin (F7), apigenin (F32), chrysin (F11), kaempferol (F12), and 3',4'-dihydroxyflavone (F2) in human GL-15 glioblastoma cells. We observed that all flavonoids decreased the number of viable cells and the mitochondrial metabolism. Furthermore, they damaged mitochondria and rough endoplasmic reticulum, inducing apoptosis. Flavonoids also induced a delay in cell migration, related to a reduction in filopodia-like structures on the cell surface, reduction on metalloproteinase (MMP-2) expression and activity, as well as an increase in intra- and extracellular expression of fibronectin, and intracellular expression of laminin. Morphological changes were also evident in adherent cells characterized by the presence of a condensed cell body with thin and long cellular processes, expressing glial fibrillary acidic protein (GFAP). Therefore, these flavonoids should be tested as potential antitumor agents in vitro and in vivo in other malignant glioma models.

  7. Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization.

    PubMed

    Faria, Jane; Romão, Luciana; Martins, Sheila; Alves, Tércia; Mendes, Fabio A; de Faria, Giselle Pinto; Hollanda, Rosenilde; Takiya, Christina; Chimelli, Leila; Morandi, Veronica; de Souza, Jorge Marcondes; Abreu, Jose Garcia; Moura Neto, Vivaldo

    2006-12-01

    The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.

  8. 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

  9. Cytotoxic constituents of Abutilon indicum leaves against U87MG human glioblastoma cells.

    PubMed

    Khan, Rukaiyya Sirajuddin; Senthi, Mahibalan; Rao, Poorna Chandra; Basha, Ameer; Alvala, Mallika; Tummuri, Dinesh; Masubuti, Hironori; Fujimoto, Yoshinori; Begum, Ahil Sajeli

    2015-01-01

    The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for treating glioblastoma. The petroleum ether extract, methanol extract (AIM), chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) prepared from AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extracts exhibited considerable activity (IC50 values of 42.6-64.5 μg/mL). The most active AIM-C fraction was repeatedly chromatographed to yield four known compounds, methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid (3) and pinellic acid (4). Cell viability assay of 1-4 against U87MG cells indicated 2 as most active (IC50 value of 8.2 μg/mL), whereas the other three compounds were much less active. Interestingly, compounds 1-4 were non-toxic towards normal human cells (HEK-293). The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of some more active substances besides methyl caffeate (2) in AIM-C is anticipated.

  10. Cytotoxic constituents of Abutilon indicum leaves against U87MG human glioblastoma cells.

    PubMed

    Khan, Rukaiyya Sirajuddin; Senthi, Mahibalan; Rao, Poorna Chandra; Basha, Ameer; Alvala, Mallika; Tummuri, Dinesh; Masubuti, Hironori; Fujimoto, Yoshinori; Begum, Ahil Sajeli

    2015-01-01

    The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for treating glioblastoma. The petroleum ether extract, methanol extract (AIM), chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) prepared from AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extracts exhibited considerable activity (IC50 values of 42.6-64.5 μg/mL). The most active AIM-C fraction was repeatedly chromatographed to yield four known compounds, methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid (3) and pinellic acid (4). Cell viability assay of 1-4 against U87MG cells indicated 2 as most active (IC50 value of 8.2 μg/mL), whereas the other three compounds were much less active. Interestingly, compounds 1-4 were non-toxic towards normal human cells (HEK-293). The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of some more active substances besides methyl caffeate (2) in AIM-C is anticipated. PMID:25422029

  11. Silencing of phosphoglucose isomerase/autocrine motility factor decreases U87 human glioblastoma cell migration.

    PubMed

    Li, Yang; Wei, Zhenqing; Dong, Bin; Lian, Zhigang; Xu, Yinghui

    2016-04-01

    Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is secreted by tumors and influences tumor growth and metastasis. In order to investigate the effects of silencing PGI/AMF on the migration and the sphere forming abilities of human glioblastoma U87 cells, as well as on the side population cells (SPCs), PGI/AMF was silenced using siRNA. Western blot analysis and RT-qPCR were used to assess the expression of PGI/AMF, Akt and SRY (sex determining region Y)-box 2 (SOX2). Wound healing, migration and tumorsphere formation assays were performed to assess invasion and metastatic potential. The proportion of SPCs was determined using Hoechst 33342 dye and flow cytometric analysis. PGI/AMF silencing inhibited the wound healing capacity and migration ability of U87 cells by 52.6 and 80.4%, respectively, compared with the scrambled siRNA (both P<0.001). Silencing of PGI/AMF decreased the proportion of SPCs in the U87 cells by 80.9% (P<0.01). The silencing of PGI/AMF decreased the number and size of tumorspheres by 53.1 and 39.9%, respectively, compared with the scrambled siRNA (both P<0.01). The silencing of PGI/AMF decreased the levels of phosphorylated Akt (-71.9%, P<0.001) compared with the scrambled siRNA, as well as the levels of the stemness marker, SOX2 (-61.7%, P<0.01). Taken together, these findings suggest that PGI/AMF silencing decreases migration, tumorsphere formation as well as the proportion of SPCs in glioblastoma U87 cells. We suggest that the Akt pathway is involved, and our results provide a potential new target for the treatment of glioblastoma.

  12. 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

  13. 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

  14. Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells.

    PubMed

    Péntek, Adrienn; Pászty, Katalin; Apáti, Ágota

    2016-01-01

    Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well.

  15. Binding of Mycobacterium avium-Mycobacterium intracellulare to human leukocytes.

    PubMed Central

    Catanzaro, A; Wright, S D

    1990-01-01

    We examined nonopsonic binding of Mycobacterium avium-Mycobacterium intracellulare (MAI) by human leukocytes. Macrophages (M phi) avidly bound fluorescently labeled MAI in the absence of serum proteins. Binding appeared to be mediated by a lineage-specific, proteinaceous receptor on M phi, since (i) binding of labeled bacteria could be competitively inhibited by unlabeled MAI, (ii) treatment of M phi with trypsin ablated the ability of M phi to bind MAI, and (iii) the capacity to bind MAI was observed on monocytes, M phi, and stimulated polymorphonuclear cells but not on lymphocytes or unstimulated polymorphonuclear cells. The receptor for MAI appeared mobile in the plane of the membrane, since spreading of M phi on a carpet of immobilized, unlabeled MAI down modulated binding of labeled MAI added in suspension. The receptor required neither calcium nor magnesium for activity and appeared different from other known receptors for intracellular pathogens. Images PMID:2387629

  16. Identification of Survival Genes in Human Glioblastoma Cells by Small Interfering RNA Screening

    PubMed Central

    Thaker, Nikhil G.; Zhang, Fang; McDonald, Peter R.; Shun, Tong Ying; Lewen, Michael D.; Pollack, Ian F.

    2009-01-01

    Target identification and validation remain difficult steps in the drug discovery process, and uncovering the core genes and pathways that are fundamental for cancer cell survival may facilitate this process. Glioblastoma represents a challenging form of cancer for chemotherapy. Therefore, we assayed 16,560 short interfering RNA (siRNA) aimed at identifying which of the 5520 unique therapeutically targetable gene products were important for the survival of human glioblastoma. We analyzed the viability of T98G glioma cells 96 h after siRNA transfection with two orthogonal statistical methods and identified 55 survival genes that encoded proteases, kinases, and transferases. It is noteworthy that 22% (12/55) of the survival genes were constituents of the 20S and 26S proteasome subunits. An expression survey of a panel of glioma cell lines demonstrated expression of the proteasome component PSMB4, and the validity of the proteasome complex as a target for survival inhibition was confirmed in a series of glioma and nonglioma cell lines by pharmacological inhibition and RNA interference. Biological networks were built with the other survival genes using a protein-protein interaction network, which identified clusters of cellular processes, including protein ubiquitination, purine and pyrimidine metabolism, nucleotide excision repair, and NF-κB signaling. The results of this study should broaden our understanding of the core genes and pathways that regulate cell survival; through either small molecule inhibition or RNA interference, we highlight the potential significance of proteasome inhibition. PMID:19783622

  17. Caffeine suppresses the progression of human glioblastoma via cathepsin B and MAPK signaling pathway.

    PubMed

    Cheng, Yu-Chen; Ding, You-Ming; Hueng, Dueng-Yuan; Chen, Jang-Yi; Chen, Ying

    2016-07-01

    Glioblastoma has aggressive proliferative and invasive properties. We investigated the effect of caffeine on the invasion and the anti-cancer effect in human glioblastomas. Caffeine reduced the invasion in U-87MG, GBM8401 and LN229 cells. Caffeine decreased mRNA, protein expression, and activity of cathepsin B. Besides, mRNA and protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) was upregulated by caffeine treatment, whereas matrix metalloproteinase-2 (MMP-2) was downregulated. The expression of Ki67, p-p38, phospforylated extracellular regulated protein kinases (p-ERK), and membranous integrin β1 and β3 was decreased by caffeine. The Rho-associated protein kinase (ROCK) inhibitor, Y27632, blocked the caffeine-mediated reduction of cathepsin B, phosphorylated focal adhesion kinase (p-FAK), and p-ERK, and invasion. Moreover, caffeine decreased the tumor size, cathepsin B and Ki67 expression in animal model. Caffeine reduced the invasion of glioma cells through ROCK-cathepsin B/FAK/ERK signaling pathway and tumor growth in orthotopic xenograft animal model, supporting the anti-cancer potential in glioma therapy.

  18. Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown

    PubMed Central

    2009-01-01

    Background Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. Results Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. Conclusion Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition. PMID:20038293

  19. Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells

    PubMed Central

    Kucharzewska, Paulina; Christianson, Helena C.; Belting, Mattias

    2015-01-01

    Oncogenetic events and unique phenomena of the tumor microenvironment together induce adaptive metabolic responses that may offer new diagnostic tools and therapeutic targets of cancer. Hypoxia, or low oxygen tension, represents a well-established and universal feature of the tumor microenvironment and has been linked to increased tumor aggressiveness as well as resistance to conventional oncological treatments. Previous studies have provided important insights into hypoxia induced changes of the transcriptome and proteome; however, how this translates into changes at the metabolite level remains to be defined. Here, we have investigated dynamic, time-dependent effects of hypoxia on the cancer cell metabolome across all families of macromolecules, i.e., carbohydrate, protein, lipid and nucleic acid, in human glioblastoma cells. Using GC/MS and LC/MS/MS, 345 and 126 metabolites were identified and quantified in cells and corresponding media, respectively, at short (6 h), intermediate (24 h), and prolonged (48 h) incubation at normoxic or hypoxic (1% O2) conditions. In conjunction, we performed gene array studies with hypoxic and normoxic cells following short and prolonged incubation. We found that levels of several key metabolites varied with the duration of hypoxic stress. In some cases, metabolic changes corresponded with hypoxic regulation of key pathways at the transcriptional level. Our results provide new insights into the metabolic response of glioblastoma cells to hypoxia, which should stimulate further work aimed at targeting cancer cell adaptive mechanisms to microenvironmental stress. PMID:25633823

  20. Investigation of imatinib loaded surface decorated biodegradable nanocarriers against glioblastoma cell lines: Intracellular uptake and cytotoxicity studies.

    PubMed

    Khan, Abrar M; Ahmad, Farhan Jalees; Panda, Amulya K; Talegaonkar, Sushama

    2016-06-30

    Overexpression of P-glycoprotein (P-gp) efflux transporter in glioma cells thwarts the build-up of therapeutic concentration of drugs usually resulting into poor therapeutic outcome. To surmount aforesaid challenge, Imatinib (IMM) loaded Poly-lactide-co-glycolic acid nanoparticles (IMM-PLGA-NPs) were developed and optimized by Box Behnken Design as a new treatment stratagem in malignant glioma. Optimized NPs were functionalized with Pluronic(®) P84, P-gp inhibitor (IMM-PLGA-P84-NPs) which showed size, PDI, zeta potential, drug loading, 182.63±13.56nm, 0.196±0.021, -15.2±1.49mV, 40.63±2.04μg/mg, respectively. Intracellular uptake study conducted on A172, U251MG and C6 glioma cells demonstrated significantly high uptake of IMM through NPs when compared with IMM solution (IMM-S), p<0.001. IMM-PLGA-P84-NPs showed better uptake in P-gp expressing cell line (U251MG and C6) while uncoated NPs showed higher uptake in non-P-gp expressing cell line (A-172). Cytotoxicity studies demonstrated significantly low IC50 for both IMM-PLGA-NPs and IMM-PLGA-P84-NPs when compared with IC50 of IMM-S. IMM-PLGA-P84-NPs showed a significantly low IC50 against P-gp overexpressing cell lines when compared with IC50 of IMM-PLGA-NPs. In contrary, IMM-PLGA-NPs showed lower IC50 against non P-gp expressing cell line. This study demonstrated the feasibility of targeting surface decorated NPs to multidrug resistant gliomas. However, to address its clinical utility extensive in vivo studies are required. PMID:27154254

  1. Mechanisms of Defense against Intracellular Pathogens Mediated by Human Macrophages.

    PubMed

    Bloom, Barry R; Modlin, Robert L

    2016-06-01

    The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the human host?" The origins of this work derive from a long-standing interest in the mechanisms of protection against two such paradigmatic intracellular pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, that have brilliantly adapted to the human host. It was obvious that these pathogens, which cause chronic diseases and persist in macrophages, must have acquired subtle strategies to resist host microbicidal mechanisms, yet since the vast majority of individuals infected with M. tuberculosis do not develop disease, there must be some potent human antimicrobial mechanisms. What follows is not a comprehensive review of the vast literature on the role of human macrophages in protection against infectious disease, but a summary of the research in our two laboratories with collaborators that we hope has contributed to some understanding of mechanisms of resistance and pathogenesis. While mouse models revealed some necessary conditions for protection, e.g., innate immunity, Th1 cells and their cytokines, and major histocompatibility complex class I-restricted T cells, here we emphasize multiple antimicrobial mechanisms that exist in human macrophages that differ from those of most experimental animals. Prominent here is the vitamin D-dependent antimicrobial pathway common to human macrophages activated by innate and acquired immune responses, mediated by antimicrobial peptides, e.g., cathelicidin, through an interleukin-15- and interleukin-32-dependent common pathway that is necessary for macrophage killing of M. tuberculosis in vitro. PMID:27337485

  2. Genome-wide transcriptional profiling of human glioblastoma cells in response to ITE treatment.

    PubMed

    Kang, Bo; Zhou, Yanwen; Zheng, Min; Wang, Ying-Jie

    2015-09-01

    A ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is recently revealed to play a key role in embryogenesis and tumorigenesis (Feng et al. [1], Safe et al. [2]) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) (Song et al. [3]) is an endogenous AhR ligand that possesses anti-tumor activity. In order to gain insights into how ITE acts via the AhR in embryogenesis and tumorigenesis, we analyzed the genome-wide transcriptional profiles of the following three groups of cells: the human glioblastoma U87 parental cells, U87 tumor sphere cells treated with vehicle (DMSO) and U87 tumor sphere cells treated with ITE. Here, we provide the details of the sample gathering strategy and show the quality controls and the analyses associated with our gene array data deposited into the Gene Expression Omnibus (GEO) under the accession code of GSE67986.

  3. Effect of thymol on Ca2+ homeostasis and viability in human glioblastoma cells.

    PubMed

    Hsu, Shu-Shong; Lin, Ko-Long; Chou, Chiang-Ting; Chiang, An-Jen; Liang, Wei-Zhe; Chang, Hong-Tai; Tsai, Jeng-Yu; Liao, Wei-Chuan; Huang, Fong-Dee; Huang, Jong Khing; Chen, I-Shu; Liu, Shuih-Inn; Kuo, Chun-Chi; Jan, Chung-Ren

    2011-11-16

    The effect of the natural essential oil thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in human glioblastoma cells was examined. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Thymol 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+). Thymol-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 thymol-induced [Ca(2+)](i) rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca(2+)](i) rise. At concentrations of 200-800 μM, thymol 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 thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis. PMID:21914442

  4. Ubiquitin-specific protease 28 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity by regulating MYC expression

    PubMed Central

    Wang, Zengwu; Song, Qimin; Xue, Jian; Zhao, Yumei

    2015-01-01

    The transcription factor MYC, which is dysregulated in the majority of gliomas, is difficult to target directly. Deubiquitinase ubiquitin-specific protease 28 (USP28) stabilizes oncogenic factors, including MYC. However, the contribution of USP28 in tumorigenesis, particularly in glioma, is unknown. Here, we determined the expression of USP28 and assessed its clinical significance in human glioma. We found that USP28 is overexpressed in human glioma but not in normal brain tissue. The level of USP28 protein expression in human glioma tissues was directly correlated with glioma grade. Meanwhile, the level of USP28 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced USP28 expression promotes SW1783 glioma cell proliferation. Moreover, gliomas that arose from USP28-transfected SW1783 cells displayed tumorigenicity in nude mouse model systems. Inhibition of USP28 expression in glioblastoma U373 cells suppressed anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, USP28 regulates the expression of MYC protein, which is essential in USP28-induced cell growth in glioma cells. These results showed that USP28 is overexpressed in human glioblastomas and it contributes to glioma tumorigenicity. Therefore, USP28 could be a new target of therapy for human malignant glioma. PMID:26209720

  5. 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.

  6. 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.

  7. 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.

  8. 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. PMID:25860932

  9. Analysis of the c-src gene product structure, abundance, and protein kinase activity in human neuroblastoma and glioblastoma cells.

    PubMed

    O'Shaughnessy, J; Deseau, V; Amini, S; Rosen, N; Bolen, J B

    1987-01-01

    We have compared in different human neuroblastoma cell lines and human glioblastoma cells the expression level, structure, and tyrosine-specific protein kinase activity of pp60c-src. Our results show that not all human neuroblastoma cell lines express pp60c-src molecules with amino-terminal structural alterations. In neuroblastoma cells which possess pp60c-src with altered gel migration, the diminished polyacrylamide gel mobility of pp60c-src was found not to be dependent upon amino-terminal phosphorylations since extensive treatment of these molecules with phosphatase did not significantly change their gel migration properties. Similar differences in gel migration were observed when RNA from the various neuroblastoma and glioblastoma cells was translated in vitro using either rabbit reticulocyte or wheat germ lysates. White the level of c-src mRNA in the different cells analyzed was found to be similar, the abundance of pp60c-src in these same cells was found to vary by as much as 12-fold. This suggests that the abundance of pp60c-src in human neuroendocrine tumors is regulated through post-transcriptional and/or post-translational events which may be related to the stage of neuronal differentiation of the cells. Based upon determination of pp60c-src abundance by immunoblot analysis, we demonstrate that pp60c-src molecules derived from human neuroblastoma and glioblastoma cells have very similar in vitro protein kinase activities.

  10. The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells

    PubMed Central

    Atif, Fahim; Patel, Neil R.; Yousuf, Seema; Stein, Donald G.

    2015-01-01

    Glioblastoma multiforme (GBM) is the most common and most aggressive malignant brain tumor. Despite optimal treatment and evolving standard of care, the median survival of patients diagnosed with GBM is only 12–15 months. In this study, we combined progesterone (PROG) and temozolomide (TMZ), a standard chemotherapeutic agent for human GBM, to test whether PROG enhances the antitumor effects of TMZ and reduces its side effects. Two WHO grade IV human GBM cells lines (U87MG and U118MG) and primary human dermal fibroblasts (HDFs) were repeatedly exposed to PROG and TMZ either alone or in combination for 3 and 6 days. Cell death was measured by MTT reduction assay. PROG and TMZ individually induced tumor cell death in a dose-dependent manner. PROG at high doses produced more cell death than TMZ alone. When combined, PROG enhanced the cell death-inducing effect of TMZ. In HDFs, PROG did not reduce viability even at the same high cytotoxic doses, but TMZ did so in a dose-dependent manner. In combination, PROG reduced TMZ toxicity in HDFs. PROG alone and in combination with TMZ suppressed the EGFR/PI3K/Akt/mTOR signaling pathway and MGMT expression in U87MG cells, thus suppressing cell proliferation. PROG and TMZ individually reduced cell migration in U87MG cells but did so more effectively in combination. PROG enhances the cytotoxic effects of TMZ in GBM cells and reduces its toxic side effects in healthy primary cells. PMID:26110872

  11. Combined inhibition of HER1/EGFR and RAC1 results in a synergistic antiproliferative effect on established and primary cultured human glioblastoma cells.

    PubMed

    Karpel-Massler, Georg; Westhoff, M-Andrew; Zhou, Shaoxia; Nonnenmacher, Lisa; Dwucet, Annika; Kast, Richard E; Bachem, Max G; Wirtz, Christian R; Debatin, Klaus-Michael; Halatsch, Marc-Eric

    2013-09-01

    Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR-targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma.

  12. Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

    PubMed Central

    Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

    2014-01-01

    Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

  13. Induction of microRNA-146a is involved in curcumin-mediated enhancement of temozolomide cytotoxicity against human glioblastoma.

    PubMed

    Wu, Hao; Liu, Qiang; Cai, Tao; Chen, Yu-Dan; Wang, Zhi-Fei

    2015-10-01

    MicroRNA (miR)-146a is a negative regulator of nuclear factor-κB (NF-κB) signaling that affects tumor growth and survival. The present study was undertaken to determine whether the cytotoxicity of curcumin (diferuloylmethane), a natural polyphenolic compound isolated from turmeric (Curcuma longa Linn), in glioblastoma cells is mediated through upregulation of miR‑146a. Human U‑87 MG glioblastoma cells were treated with curcumin and temozolomide (TMZ) alone or in combination, and cell proliferation and apoptosis were assessed. The involvement of miR‑146a and NF‑κB signaling in curcumin‑mediated chemosensitization was explored. Curcumin exposure led to upregulation of miR‑146a in U‑87 MG cells. Combined curcumin and TMZ treatment significantly (P<0.05) inhibited U‑87 MG cell proliferation and induced apoptotic death, compared with each alone. Notably, curcumin‑mediated enhancement of TMZ‑induced apoptosis was blocked by depletion of miR‑146a. By contrast, miR‑146a overexpression enhanced apoptosis and suppressed NF‑κB activation in TMZ‑treated cells. Additionally, pharmacological inhibition of NF‑κB signaling significantly increased TMZ‑induced apoptosis. To the best of our knowledge, the present study provides the first evidence that upregulation of miR‑146a and inactivation of NF‑κB signaling mediates the sensitization of human glioblastoma cells to TMZ-induced apoptosis by curcumin. PMID:26239619

  14. Accessibilome of human glioblastoma: collagen-VI-alpha-1 is a new target and a marker of poor outcome.

    PubMed

    Turtoi, Andrei; Blomme, Arnaud; Bianchi, Elettra; Maris, Pamela; Vannozzi, Riccardo; Naccarato, Antonio Giuseppe; Delvenne, Philippe; De Pauw, Edwin; Bevilacqua, Generoso; Castronovo, Vincent

    2014-12-01

    Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody-drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the bloodstream remain scarce. In the current work, we have identified and characterized novel and accessible proteins using an innovative proteomic approach on six human glioblastomas; the corresponding data have been deposited in the PRIDE database identifier PXD001398. Among several clusters of uniquely expressed proteins, we highlight collagen-VI-alpha-1 (COL6A1) as a highly expressed tumor biomarker with low levels in most normal tissues. Immunohistochemical analysis of glioma samples from 61 patients demonstrated that COL6A1 is a significant and consistent feature of high-grade glioma. Deposits of COL6A1 were evidenced in the perivascular regions of the tumor-associated vasculature and in glioma cells found in pseudopalisade structures. Retrospective analysis of public gene-expression data sets from over 300 glioma patients demonstrated a significant correlation of poor patient outcome and high COL6A1 expression. In a proof-of-concept study, we use chicken chorioallantoic membrane in vivo model to show that COL6A1 is a reachable target for IV-injected antibodies. The present data warrant further development of human COL6A1 antibodies for assessing the quantitative biodistribution in the preclinical tumor models. PMID:25325876

  15. 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

  16. Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

    PubMed Central

    Assandri, Roberta; Egger, Marcel; Gassmann, Max; Niggli, Ernst; Bauer, Christian; Forster, Ian; Görlach, Agnes

    1999-01-01

    Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels. PMID:10087335

  17. 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. PMID:22701651

  18. Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death

    PubMed Central

    Daniele, Simona; Giacomelli, Chiara; Zappelli, Elisa; Granchi, Carlotta; Trincavelli, Maria Letizia; Minutolo, Filippo; Martini, Claudia

    2015-01-01

    Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type. PMID:26494310

  19. 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

  20. Silver nanoparticles impregnated alginate-chitosan-blended nanocarrier induces apoptosis in human glioblastoma cells.

    PubMed

    Sharma, Shilpa; Chockalingam, S; Sanpui, Pallab; Chattopadhyay, Arun; Ghosh, Siddhartha Sankar

    2014-01-01

    Herein, a green method for the development of a novel biodegradable silver nanoparticles (NPs) impregnated alginate-chitosan-blended nanocarrier (Ag NPs-Alg-Chi NC) is reported. The synthesis of Ag NPs-Alg-Chi NC is based on the polyelectrolyte complex formation between alginate and chitosan. The composite NC is characterized by ultraviolet-visible spectroscopy, transmission electron microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. The Ag NPs in the NC are found to elicit anticell proliferative effect on refractory U87MG (human glioblastoma) cells at IC50 of 2.4 μg mL(-1) for Ag NPs. The cell cycle analysis shows extensive DNA damage. Elevation in reactive oxygen species level indicates induction of oxidative stress in treated cells. Mitochondrial dysfunction in cell death is evident from the depolarization of mitochondrial membrane potential (ΔΨm ). Fluorescence and SEM images of the treated cells reveal nuclear and morphological changes characteristic of apoptosis, which is further confirmed by TUNEL assay. The induction of apoptosis at low concentration of Ag NPs present in Ag NPs-Alg-Chi NC in comparison with free Ag NPs makes it a promising tool for cancer therapy.

  1. Green tea compounds inhibit tyrosine phosphorylation of PDGF beta-receptor and transformation of A172 human glioblastoma.

    PubMed

    Sachinidis, A; Seul, C; Seewald, S; Ahn, H; Ko, Y; Vetter, H

    2000-04-01

    The effect of the green tea compounds 2-(3,4-dihydroxyphenyl)-3, 4-dihydro-2H-1-benzopyran-3,5,7-triol (catechin), epicathechin (EC), epigallocathechin-3 gallate (EGCG), epicathechin-3 gallate (ECG) and catechin-3 gallate (CG) on the tyrosine phosphorylation of PDGF beta-receptor (PDGF-Rbeta) and on the anchorage-independent growth of A172 glioblastoma cells in semisolid agar has been investigated. Treatment of A172 glioblastoma with 50 microM CG, ECG, EGCG and 25 microM Tyrphostin 1296 resulted in an 82+/-17%, 77+/-21%, 75+/-8% and 55+/-11%, respectively (mean+/-S.D., n=3) inhibition of the PDGF-BB-induced tyrosine phosphorylation of PDGF-Rbeta. The PDGF-Rbeta downstream intracellular transduction pathway including tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) and phosphatidylinositol 3'-kinase (PI 3'-K) was also inhibited. Spheroid formation was completely inhibited by 50 microM ECG, CG, EGCG and by 25 microM Tyrphostin 1296. We conclude that catechins of the green tea possessing the gallate group in their chemical structure act as anticancer agents probably partly via their ability to suppress the tyrosine kinase activity of the PDGF-Rbeta. PMID:10760511

  2. 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.

  3. Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH.

    PubMed

    Gilbert, B; Perfetti, L; Fauchoux, O; Redondo, J; Baudat, P A; Andres, R; Neumann, M; Steen, S; Gabel, D; Mercanti, D; Ciotti, M T; Perfetti, P; Margaritondo, G; De Stasio, G

    2000-07-01

    Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

  4. Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH

    NASA Astrophysics Data System (ADS)

    Gilbert, B.; Perfetti, L.; Fauchoux, O.; Redondo, J.; Baudat, P.-A.; Andres, R.; Neumann, M.; Steen, S.; Gabel, D.; Mercanti, Delio; Ciotti, M. Teresa; Perfetti, P.; Margaritondo, G.; de Stasio, Gelsomina

    2000-07-01

    Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 μm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

  5. 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

  6. RT-21Mre11-Rad50-Nbs1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    PubMed Central

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Fukada, Junichi; Nishikawa, Ryo; Kawata, Tetsuya

    2014-01-01

    PURPOSE: Radiation therapy plays a central part in the treatment of glioblastoma, however, it is not curative due to the high tumor radioresistance. Therefore, increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma. The Mre11, Rad 50 and Nbs1 proteins form a complex (MRN) that has a critical role in DNA damage detection and signaling. Because defects in MRN enhance radiosensitivity, it has been proposed that small molecule inhibitors targeted to these proteins might be used as radiosensitizers. Here, we investigated the effects of the MRN complex inhibitor, Mirin, on radiation response of human glioma cells. MATERIALS AND METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylation (γH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSION: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells.

  7. LRIG1 enhances the radiosensitivity of radioresistant human glioblastoma U251 cells via attenuation of the EGFR/Akt signaling pathway

    PubMed Central

    Yang, Ji-An; Liu, Bao-Hui; Shao, Ling-Min; Guo, Zhen-Tao; Yang, Qian; Wu, Li-Quan; Ji, Bao-Wei; Zhu, Xiao-Nan; Zhang, Shen-Qi; Li, Cheng-Jun; Chen, Qian-Xue

    2015-01-01

    The radiotherapy as a local and regional modality is widely applied in treatment of glioma, but most glioblastomas are commonly resistant to irradiation treatment. It remains challengeable to seek out efficient strategies to conquer the resistance of human glioblastoma cells to radiotherapy. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) is a newly discovered tumor suppressor which involved in regulation of chemosensitivity in various human cancer cells. In the present study, we established a radioresistant U251 cell line (U251R) to investigate the role of LRIG1 in regulation of radiosensitivity in human glioblastoma cells. Significantly decreased expression level of LRIG1 and enhanced expression of EGFR and phosphorylated Akt were detected in U251R cells compared with the parental U251 cells. U251R cells exhibited an advantage in colony formation ability, which accompanied by remarkably reduced X-ray-induced γ-H2AX foci formation and cell apoptosis. LRIG1 overexpression significantly inhibited the colony formation ability of U251R cells and obviously enhanced X-ray-inducedγ-H2AX foci formation and cell apoptosis. In addition, up-regulated expression of LRIG1 suppressed the expression level of EGFR and phosphorylated Akt protein. Our results demonstrated that LRIG1 expression was related to the radiosensitivity of human glioblastoma cells and may play an important role in the regulation of cellular radiosensitivity of human glioblastoma cells through the EGFR/Akt signaling pathway. PMID:26097540

  8. Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

    PubMed Central

    Aldaz, Beatriz; Sagardoy, Ainara; Nogueira, Lorena; Guruceaga, Elizabeth; Grande, Lara; Huse, Jason T.; Aznar, Maria A.; Díez-Valle, Ricardo; Tejada-Solís, Sonia; Alonso, Marta M.; Fernandez-Luna, Jose L.

    2013-01-01

    Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process. PMID:24155920

  9. Significant Association of Multiple Human Cytomegalovirus Genomic Loci with Glioblastoma Multiforme Samples

    PubMed Central

    Ranganathan, Padhma; Clark, Paul A.; Kuo, John S.; Salamat, M. Shahriar

    2012-01-01

    Viruses are appreciated as etiological agents of certain human tumors, but the number of different cancer types induced or exacerbated by viral infections is unknown. Glioblastoma multiforme (GBM)/astrocytoma grade IV is a malignant and lethal brain cancer of unknown origin. Over the past decade, several studies have searched for the presence of a prominent herpesvirus, human cytomegalovirus (HCMV), in GBM samples. While some have detected HCMV DNA, RNA, and proteins in GBM tissues, others have not. Therefore, any purported association of HCMV with GBM remains controversial. In most of the previous studies, only one or a select few viral targets were analyzed. Thus, it remains unclear the extent to which the entire viral genome was present when detected. Here we report the results of a survey of GBM specimens for as many as 20 different regions of the HCMV genome. Our findings indicate that multiple HCMV loci are statistically more likely to be found in GBM samples than in other brain tumors or epileptic brain specimens and that the viral genome was more often detected in frozen samples than in paraffin-embedded archival tissue samples. Finally, our experimental results indicate that cellular genomes substantially outnumber viral genomes in HCMV-positive GBM specimens, likely indicating that only a minority of the cells found in such samples harbor viral DNA. These data argue for the association of HCMV with GBM, defining the virus as oncoaccessory. Furthermore, they imply that, were HCMV to enhance the growth or survival of a tumor (i.e., if it is oncomodulatory), it would likely do so through mechanisms distinct from classic tumor viruses that express transforming viral oncoproteins in the overwhelming majority of tumor cells. PMID:22090104

  10. First in human nanotechnology doxorubicin delivery system to target epidermal growth factor receptors in recurrent glioblastoma.

    PubMed

    Whittle, James R; Lickliter, Jason D; Gan, Hui K; Scott, Andrew M; Simes, John; Solomon, Benjamin J; MacDiarmid, Jennifer A; Brahmbhatt, Himanshu; Rosenthal, Mark A

    2015-12-01

    There are limited treatment options for patients with recurrent glioblastoma (GBM). The EnGeneIC delivery vehicle (EDV) is a novel nanocellular (minicell) compound which packages theoretically effective concentrations of chemotherapeutic drugs that are designed to target tumors via minicell-surface attached bispecific proteins (EnGeneIC, Lane Cove West, NSW, Australia). Epidermal growth factor receptor (EGFR) is overexpressed in 40-50% of patients with GBM and is a promising target for new therapeutics. (V)EDVDox contains doxorubicin (Dox) within the minicells and targets EGFR through Vectibix (V; Amgen Biologicals, Thousand Oaks, CA, USA). We conducted a first in human Phase I study of (V)EDVDox in adults with recurrent GBM expressing EGFR on immunohistochemistry, following standard therapy including radiation and temozolomide, to establish a safe maximum tolerated dose and determine a recommended Phase II dose (RPTD). (V)EDVDox was administered weekly in an 8week cycle, with dose escalation in successive cohorts of patients using a standard 3+3 design. In total, 14 patients were treated at three dose levels, and the RPTD was identified as 5×10(9)(V)EDVDox. Overall (V)EDVDox was well tolerated, with no dose limiting toxicity and no withdrawals from the study due to adverse events. The most common adverse events were nausea, fever, and chills or rigors, experienced in seven, five and five patients, respectively. Transient uncomplicated hypophosphatemia was seen in seven patients and was not dose-related. Our results demonstrate that (V)EDVDox, up to a dose of 5×10(9)(V)EDVDox weekly, is well tolerated in patients with recurrent GBM.

  11. 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.

  12. Human mesenchymal stem cells exploit the immune response mediating chemokines to impact the phenotype of glioblastoma.

    PubMed

    Motaln, Helena; Gruden, Kristina; Hren, Matjaz; Schichor, Christian; Primon, Monika; Rotter, Ana; Lah, Tamara T

    2012-01-01

    In contrast to the application of human mesenchymal stem cells (hMSCs) in regenerative medicine, only a limited number of studies are addressing their use in anticancer therapy. As the latter may represent a new hope to improve the survival of patients with glioblastoma multiformae (GBM), the most common and malignant form of the brain tumors, we aimed to investigate the interactions of hMSCs and GBM cells under in vitro conditions. Four hMSC clones and three different GBM cell lines were used to study their mutual paracrine interactions in cocultures compared to their monocultures, where cells were grown under the same experimental conditions. The effects on cell growth, proliferation, and invasion in Matrigel were quantified. Further, bioinformatics tools were used to relate these results to the data obtained from cytokine macroarrays and cDNA microarrays that revealed proteins and genes significantly involved in cellular cross-talk. We showed that hMSCs are responsible for the impairment of GBM cell invasion and growth, possibly via induction of their senescence. On the other hand, GBM cells inversely affected some of these characteristics in hMSCs. We found CCL2/MCP-1 to be the most significantly regulated chemokine during hMSC and U87-MG paracrine signaling in addition to several chemokines that may account for changed cocultured cells' phenotype by affecting genes associated with proliferation (Pmepa-1, NF-κB, IL-6, IL-1b), invasion (EphB2, Sod2, Pcdh18, Col7A1, Gja1, Mmp1/2), and senescence (Kiaa1199, SerpinB2). As we functionally confirmed the role of CCL2/MCP-1 in GBM cell invasion we thereby propose a novel mechanism of CCL2/MCP-1 antimigratory effects on GBM cells, distinct from its immunomodulatory role. Significant alterations of GBM phenotype in the presence of hMSCs should encourage the studies on the naive hMSC use for GBM treatment.

  13. Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

    PubMed Central

    LeBlanc, Isabelle; Blot, Vincent; Bouchaert, Isabelle; Salamero, Jean; Goud, Bruno; Rosenberg, Arielle R.; Dokhélar, Marie-Christine

    2002-01-01

    Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system. PMID:11752179

  14. 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

  15. 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.

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

    PubMed

    Chen, Chien-Min; Syu, Jhih-Pu; Way, Tzong-Der; Huang, Li-Jiau; Kuo, Sheng-Chu; Lin, Chung-Tien; Lin, Chih-Li

    2015-11-01

    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.

  17. 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

  18. 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

  19. Epithelial-to-mesenchymal transition in paired human primary and recurrent glioblastomas.

    PubMed

    Kubelt, Carolin; Hattermann, Kirsten; Sebens, Susanne; Mehdorn, H Maximilian; Held-Feindt, Janka

    2015-01-01

    Patients with highly malignant glioblastomas have a short median survival time mainly due to aggressive relapses after therapeutic treatment. Beside others, they achieve their progressive character via epithelial-to-mesenchymal transition (EMT). However, comprehensive investigations on EMT in paired primary-recurrent glioblastoma pairs are presently not available. Thus, in our present study we examined the expression profile of different EMT-markers in 17 matched primary and recurrent glioblastomas by qPCR and double-immunofluorescence stainings to identify EMT marker expressing cell types. Additionally, we analyzed the influence of temozolomide on EMT marker expression in vitro. In comparison to primary tumors, expression of β-catenin (p<0.05), Snail1 (p<0.05), Snail2/Slug (p<0.05), biglycan (p<0.05) and Twist1 (p<0.01) was downregulated in recurrence whereas L1CAM showed upregulation (p<0.05; qPCR). Expression of desmoplakin, vimentin, fibronectin and TGF-β1 with its receptors TGF-βR1 and TGF-βR2 was almost unchanged. Comparing each individual pair, five different 'EMT groups' within our glioblastoma collective were identified according to the regulation of mRNA expression of GFAP, desmoplakin, Snail1, Snail2, Twist1 and vimentin. Additionally, double-stainings of EMT markers in combination with cell specific markers (glial fibrillary acidic protein, CD11b, von Willebrand factor) revealed that EMT markers were expressed in a complex pattern with all three cellular types as possible sources. Temozolomide treatment significantly induced mRNA expression of nearly all investigated EMT markers in T98G glioma cells. Thus, EMT seems to be involved in glioma progression in a complex way requiring an individualized analysis, and is influenced by commonly used therapeutic options in glioma therapy. PMID:25845427

  20. Cluster and Principal Component Analysis of Human Glioblastoma Multiforme (GBM) Tumor Proteome

    PubMed Central

    Pooladi, Mehdi; Rezaei-Tavirani, Mostafa; Hashemi, Mehrdad; Hesami-Tackallou, Saeed; Khaghani-Razi-Abad, Solmaz; Moradi, Afshin; Zali, Ali Reza; Mousavi, Masoumeh; Firozi-Dalvand, Leila; Rakhshan, Azadeh; Zamanian Azodi, Mona

    2014-01-01

    Background Glioblastoma Multiforme (GBM) or grade IV astrocytoma is the most common and lethal adult malignant brain tumor. Several of the molecular alterations detected in gliomas may have diagnostic and/or prognostic implications. Proteomics has been widely applied in various areas of science, ranging from the deciphering of molecular pathogen nests of discuses. Methods In this study proteins were extracted from the tumor and normal brain tissues and then the protein purity was evaluated by Bradford test and spectrophotometry. In this study, proteins were separated by 2-Dimensional Gel (2DG) electrophoresis method and the spots were then analyzed and compared using statistical data and specific software. Protein clustering analysis was performed on the list of proteins deemed significantly altered in glioblastoma tumors (t-test and one-way ANOVA; P< 0.05). Results The 2D gel showed totally 876 spots. We reported, 172 spots were exhibited differently in expression level (fold > 2) for glioblastoma. On each analytical 2D gel, an average of 876 spots was observed. In this study, 188 spots exhibited up regulation of expression level, whereas the remaining 232 spots were decreased in glioblastoma tumor relative to normal tissue. Results demonstrate that functional clustering (up and down regulated) and Principal Component Analysis (PCA) has considerable merits in aiding the interpretation of proteomic data. Conclusion 2D gel electrophoresis is the core of proteomics which permitted the separation of thousands of proteins. High resolution 2DE can resolve up to 5,000 proteins simultaneously. Using cluster analysis, we can also form groups of related variables, similar to what is practiced in factor analysis. PMID:25250155

  1. 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

  2. Subcellular SIMS imaging of gadolinium isotopes in human glioblastoma cells treated with a gadolinium containing MRI agent

    NASA Astrophysics Data System (ADS)

    Smith, Duane R.; Lorey, Daniel R.; Chandra, Subhash

    2004-06-01

    Neutron capture therapy is an experimental binary radiotherapeutic modality for the treatment of brain tumors such as glioblastoma multiforme. Recently, neutron capture therapy with gadolinium-157 has gained attention, and techniques for studying the subcellular distribution of gadolinium-157 are needed. In this preliminary study, we have been able to image the subcellular distribution of gadolinium-157, as well as the other six naturally abundant isotopes of gadolinium, with SIMS ion microscopy. T98G human glioblastoma cells were treated for 24 h with 25 mg/ml of the metal ion complex diethylenetriaminepentaacetic acid Gd(III) dihydrogen salt hydrate (Gd-DTPA). Gd-DTPA is a contrast enhancing agent used for MRI of brain tumors, blood-brain barrier impairment, diseases of the central nervous system, etc. A highly heterogeneous subcellular distribution was observed for gadolinium-157. The nuclei in each cell were distinctly lower in gadolinium-157 than in the cytoplasm. Even within the cytoplasm the gadolinium-157 was heterogeneously distributed. The other six naturally abundant isotopes of gadolinium were imaged from the same cells and exhibited a subcellular distribution consistent with that observed for gadolinium-157. These observations indicate that SIMS ion microscopy may be a viable approach for subcellular studies of gadolinium containing neutron capture therapy drugs and may even play a major role in the development and validation of new gadolinium contrast enhancing agents for diagnostic MRI applications.

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

    PubMed

    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

    2015-11-06

    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.

  4. 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

  5. High expression of N-myc (and STAT) interactor predicts poor prognosis and promotes tumor growth in human glioblastoma

    PubMed Central

    Yun, Dapeng; Zhao, Yingjie; Wang, Jingkun; Xu, Tao; Li, Xiaoying; Wang, Yuqi; Yuan, Li; Sun, Ruochuan; Song, Xiao; Huai, Cong; Hu, Lingna; Yang, Song; Min, Taishan; Chen, Juxiang; Chen, Hongyan; Lu, Daru

    2015-01-01

    Glioma is the most malignant brain tumor and glioblastoma (GBM) is the most aggressive type. The involvement of N-myc (and STAT) interactor (NMI) in tumorigenesis was sporadically reported but far from elucidation. This study aims to investigate roles of NMI in human glioma. Three independent cohorts, the Chinese tissue microarray (TMA) cohort (N = 209), the Repository for Molecular Brain Neoplasia Data (Rembrandt) cohort (N = 371) and The Cancer Genome Atlas (TCGA) cohort (N = 528 or 396) were employed. Transcriptional or protein levels of NMI expression were significantly increased according to tumor grade in all three cohorts. High expression of NMI predicted significantly unfavorable clinical outcome for GBM patients, which was further determined as an independent prognostic factor. Additionally, expression and prognostic value of NMI were associated with molecular features of GBM including PTEN deletion and EGFR amplification in TCGA cohort. Furthermore, overexpression or depletion of NMI revealed its regulation on G1/S progression and cell proliferation (both in vitro and in vivo), and this effect was partially dependent on STAT1, which interacted with and was regulated by NMI. These data demonstrate that NMI may serve as a novel prognostic biomarker and a potential therapeutic target for glioblastoma. PMID:25669971

  6. 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

  7. A novel bispecific ligand-directed toxin designed to simultaneously target EGFR on human glioblastoma cells and uPAR on tumor neovasculature

    PubMed Central

    Tsai, Alexander K.; Oh, Seunguk; Chen, Hua; Shu, Yanqun; Ohlfest, John R.

    2011-01-01

    A bispecific ligand-directed toxin (BLT), called EGFATFKDEL, consisting of human epidermal growth factor, a fragment of urokinase, and truncated pseudomonas exotoxin (PE38) was assembled in order to target human glioblastoma. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGFATFKDEL 7mut. In vitro, the drug selectively killed several human glioblastoma cell lines. EGFATFKDEL is our first BLT designed to simultaneously target EGFR on solid tumors and uPAR on the tumor neovasculature. In vitro assays revealed that the agent is effective against glioblastoma cell lines as well as human umbilical vein endothelial cells (HUVEC). Additionally, the bispecific drug displayed enhanced binding to overexpressed epidermal growth factor receptor and urokinase receptor when compared to similar monospecific drugs, EGFKDEL and ATFKDEL. In vivo, an aggressive human glioblastoma cell line was genetically marked with a firefly luciferase reporter gene and administered to the flanks of nude mice. Treatment with intratumoral injections of EGFATFKDEL 7mut eradicated small tumors in over half of the treated mice, which survived with tumor free status at least 100 days post tumor inoculation. ATFKDEL, which primarily targets the tumor neovasculature, prevented tumor growth but did not result in tumor-free mice in most cases. Specificity was shown by treating with an irrelevant BLT control which did not protect mice. Finally, immunization experiments in immunocompetent mice revealed significantly reduced anti-toxin production in EGFATFKDEL 7mut treated groups. Thus, EGFATFKDEL 7mut is an effective drug for glioblastoma therapy in this murine model and warrants further study. PMID:20830604

  8. 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.

  9. [Role of defective intracellular proteolysis in human degenerative diseases].

    PubMed

    Nezelof, Christian

    2012-11-01

    Although intracellular protein synthesis has been studied extensively, protein degradation and disposal, know as proteolysis, has been relatively neglected. Modern studies which led two Nobel prizes (de Duve in 1950 and Herschko, Rose and Ciechanover in 1980) established that proteolysis is ensured by two separate but complementary mechanisms: lysosomes responsible for auto and heterophagy and the Ubiquitin-Proteasome System (UPS). The UPS involves ubiquitin, a small molecule consisting of 76 amino acids found in all eukaryotic cells that ensures the identification of the protein to be degraded and its transport to the proteasome, an intracellular complex with enzymes which degrade unneeded or damaged proteins. The proteasome, acting as a composting agent, ensures the enzymatic dissociation of the protein. In this degradation process, as infinite screw, ubiquitin, peptides and amino acids are released and made available for a new cycle. Knowledge of the UPS and its related disorders is continually expanding. Concurrent with lysosomes which work in acidic environment, it is currently known that the UPS provides 80% to 90% of the proteolysis of the short-life proteins and ensures, as chaperon-molecules, the right conformation and hence the correct function of the proteins. The proteolytic activity generates abnormal residues (tau protein, amyloid and related proteins) and various soluble and insoluble wastes. Some are precipitated as inclusion-bodies or aggregosomes, identified years ago by pathologists. These aggregosomes affect almost exclusively long-lived cells (nervous and muscular, macophages). Pigment deposits, such as lipofuscines made by the peroxydation of cell membranes, are the most abundant. Due to their diverse chemical composition, they cannot be empoyed for a scientific classification. Failures of these systems are numerous. They vary not according to the chemical nature of the abnormal protein and wastes but the life span of the targeted cells and

  10. Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

    PubMed

    Strik, Merel C; Bladergroen, Bellinda A; Wouters, Dorine; Kisiel, Walter; Hooijberg, Jan Hendrik; Verlaan, Angelique R; Hordijk, Peter L; Schneider, Pascal; Hack, C Erik; Kummer, J Alain

    2002-11-01

    Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.

  11. Activation of the recombinant human alpha 7 nicotinic acetylcholine receptor significantly raises intracellular free calcium.

    PubMed

    Delbono, O; Gopalakrishnan, M; Renganathan, M; Monteggia, L M; Messi, M L; Sullivan, J P

    1997-01-01

    The alpha 7 nicotinic acetylcholine receptor (nAChR) subtype, unlike other neuronal nicotinic receptors, exhibits a relatively high permeability to Ca++ ions. Although Ca++ entry through this receptor subtype has been implicated in various Ca(++)-dependent processes in the central nervous system, little is known about how this receptor modulates mammalian intracellular Ca++ dynamics. Intracellular Ca++ responses evoked by activation of the human alpha 7 nAChRs stably expressed in HEK-293 (human embryonic kidney) cells were studied. Inward current and intracellular Ca++ transients were recorded simultaneously in response to a fast drug application system. Current recordings under whole-cell voltage-clamp and fast ratiometric intracellular Ca++ imaging acquisition were synchronized to drug pulses. The mean peak [Ca++]i observed with 100 microM (-)-nicotine was 356 +/- 48 nM (n = 8). The magnitude of the intracellular Ca++ elevation corresponds to a 20% fractional current carried by Ca++ ions. The EC50 of the intracellular Ca++ responses for (-)-nicotine, (+/-)-epibatidine, 1,1 dimethyl-4-phenyl-piperazinium and acetylcholine were 51, 3.5, 75 and 108 microM, respectively. These EC50 values strongly correlate with those recorded for the cationic inward current through alpha 7 nAChR. alpha-Bungarotoxin, methyllcaconitine or extracellular Ca++ chelation ablated (-)-nicotine-evoked increase in intracellular Ca++ concentration. This study provides evidence that cation influx through the human alpha 7 nAChR is sufficient to mediate a significant, transient, rise in intracellular Ca++ concentration.

  12. Synthesis of metal nanoparticles inside living human cells based on the intracellular formation process.

    PubMed

    El-Said, Waleed A; Cho, Hyeon-Yeol; Yea, Cheol-Heon; Choi, Jeong-Woo

    2014-02-12

    Intracellular and extracellular formation of Au and Ag NPs with different sizes and shapes using human cells has been developed as green method, which does not require the use of any reducing agents. Also, the cell lysis is used for production of different metal NPs. Our results demonstrate that treatment of human cells with various metal ions cause cell fixation.

  13. 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.

  14. 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. PMID:27060209

  15. 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

  16. 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.

  17. 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. PMID:26270679

  18. 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

  19. Photodynamic therapy using talaporfin sodium induces concentration-dependent programmed necroptosis in human glioblastoma T98G cells.

    PubMed

    Miki, Yuichi; Akimoto, Jiro; Moritake, Keiko; Hironaka, Chihiro; Fujiwara, Yasuyuki

    2015-08-01

    Photodynamic therapy (PDT) using photosensitizer induces several types of cell death, such as apoptosis, necrosis, and autophagy, depending on the PDT procedure, photosensitizer type, and cell type. We previously demonstrated that PDT using the photosensitizer talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6; NPe6-PDT) induces both mitochondrial apoptotic and necrotic cell death in human glioblastoma T98G cells. However, details regarding the mechanism of necrosis caused by NPe6-PDT are unclear. Here, we investigated whether or not necroptosis, a recently suggested form of programmed necrosis, is involved in the necrotic cell death of NPe6-PDT-treated T98G cells. Leakage of lactate dehydrogenase (LDH) from the cell layer into conditioned medium was significantly increased by NPe6 (25 and 50 μg/ml)-PDT, indicating that NPe6-PDT induces necrosis in these cells. NPe6 (25 μg/ml)-PDT treatment also induced conversion of microtubule-associated protein 1 light-chain 3 (LC3)-I into phosphatidylethanolamine-conjugated LC3-II accompanying autophagosome formation, indicators of autophagy; however, of note, NPe6 (50 μg/ml)-PDT did not induce such autophagic changes. In addition, both necrostatin-1 (a necroptosis inhibitor) and knockdown of necroptotic pathway-related proteins [e.g., receptor interacting serine-threonine kinase (RIP)-1, RIP-3, and mixed lineage kinase domain-like protein (MLKL)] inhibited leakage of LDH caused by NPe6 (25 μg/ml)-PDT. Taken together, the present findings revealed that NPe6-PDT-induced necrotic cell death is mediated in part by the necroptosis pathway in glioblastoma T98G cells. PMID:26109138

  20. Hematoporphyrin derivative binding and photosensitization in human glioblastoma cells: comparison of exponential and plateau phase cells.

    PubMed

    Sreenivasan, R; Joshi, P G; Joshi, N B

    1994-11-01

    Plateau phase glioblastoma (U 87MG) cells were found more photosensitive than the exponentially growing cells. In both phases of growth, the photosensitivity showed further enhancement on incubating the cells with HpD for longer duration. Plateau phase cells accumulated more HpD than exponential phase cells for shorter duration of incubation with HpD, however, for longer duration of incubation, the amount of drug uptake was almost the same in both phases of growth. Fluorescence spectra of cell bound HpD showed a difference in spectral intensity distribution in exponential and plateau phase cells. In exponential phase cells, the fluorescence maximum of cell bound HpD was at 615 nm whereas in plateau phase cells the same was at 636 nm. PMID:7896304

  1. A visual intracellular classification strategy for uncharacterized human proteins.

    PubMed

    Hoja, M R; Wahlestedt, C; Höög, C

    2000-08-25

    The human cDNA and genomic sequencing projects will result in the identification and isolation of some 140,000 genes, the majority of which lack predicted functions and for which the cellular localizations are not known. The identification and characterization of protein components of specific cell structures and machineries are essential steps not only toward defining functions of genes but also toward understanding cell function and regulation. We describe here a new approach, termed PROLOC, which uses full-length cDNAs for systematic classification of novel proteins as a functional pointer. We have PCR-amplified 25 uncharacterized human genes and expressed the encoded proteins as GFP fusions in a human cell line. This pilot project has identified novel proteins associated with the nucleolus, mitochondria, the ER, the ER-Golgi-intermediate compartment (ERGIC), the GC, the plasma membrane, and cytoplasmic foci. This visual classification approach may be scaled up to handle a large number of novel genes and permit the generation of a global cellular protein localization map. Such information should be valuable for many aspects of functional genomics and cell biology. PMID:10942595

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

    PubMed Central

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

    2016-01-01

    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. PMID:27074557

  3. Haloperidol, but not olanzapine, may affect expression of PER1 and CRY1 genes in human glioblastoma cell line

    PubMed Central

    Mokros, Łukasz; Karbownik, Michał Seweryn; Nowakowska-Domagała, Katarzyna; Szemraj, Janusz; Wieteska, Łukasz; Woźniak, Karol; Witusik, Andrzej; Antczak, Adam; Pietras, Tadeusz

    2016-01-01

    Abstract Background: There is barely any evidence of antipsychotic drugs affecting the molecular clockwork in human, yet it is suggested that clock genes are associated with dopaminergic transmission, i.e. the main target of this therapeutics. We decided to verify if haloperidol and olanzapine affect expression of CLOCK, BMAL1, PER1 and CRY1 in a human central nervous system cell line model. Methods: U-87MG human glioblastoma cell line was used as an experimental model. The cells were incubated with or without haloperidol and olanzapine in the concentration of 5 and 20 μM for 24 h. Real-time quantitative polymerase chain reaction with the ΔC T analysis was used to examine the effect of haloperidol and olanzapine on the mRNA expression of the genes. Results: At 5 μM, haloperidol decreased expression of CRY1 almost 20-fold. There was nearly a 1.5-fold increase in expression of PER1. Considering the 20 μM haloperidol concentration and both olanzapine concentrations, no other statistically significant effect was observed. Conclusions: At certain concentration, haloperidol seems to affect expression of particular clock genes in a human central nervous system cell line model, yet mechanism underlying this phenomenon remains elusive.

  4. Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells.

    PubMed Central

    Ueba, T; Nosaka, T; Takahashi, J A; Shibata, F; Florkiewicz, R Z; Vogelstein, B; Oda, Y; Kikuchi, H; Hatanaka, M

    1994-01-01

    Mutations of the p53 gene are found in various human cancers. The frequency of its mutation is reported to increase during tumor progression in most tumors. In human gliomas, mutations of the p53 gene are found in about one-third of the malignant forms and in few of the benign ones, indicating their possible involvement in tumor progression. On the other hand, we have recently shown that basic fibroblast growth factor (basic FGF) plays a crucial role in tumor progression as an autocrine growth factor in tissues of human gliomas. Therefore, we hypothesized that p53 might regulate the promoter activity of the basic FGF gene, which has several GC boxes and no typical TATA box. In this study, cotransfection assays using human glioblastoma and hepatocellular carcinoma cells and establishment of stable cell lines expressing mutant-type p53 were performed. The basic FGF gene promoter was demonstrated to be regulated by p53 at the transcriptional level and its basal core promoter was found to be responsive to p53. Expression of endogenous basic FGF was also demonstrated to be activated by mutant type p53. Wild-type p53 repressed gene expression of the basic FGF and its mutant activated it in vitro, implying one of the possible pathways in tumor progression. Images PMID:8090761

  5. Relaxation-compensated CEST-MRI of the human brain at 7T: Unbiased insight into NOE and amide signal changes in human glioblastoma.

    PubMed

    Zaiss, Moritz; Windschuh, Johannes; Paech, Daniel; Meissner, Jan-Eric; Burth, Sina; Schmitt, Benjamin; Kickingereder, Philip; Wiestler, Benedikt; Wick, Wolfgang; Bendszus, Martin; Schlemmer, Heinz-Peter; Ladd, Mark E; Bachert, Peter; Radbruch, Alexander

    2015-05-15

    Endogenous chemical exchange saturation transfer (CEST) effects of protons resonating near to water protons are always diluted by competing effects such as direct water saturation and semi-solid magnetization transfer (MT). This leads to unwanted T2 and MT signal contributions that contaminate the observed CEST signal. Furthermore, all CEST effects appear to be scaled by the T1 relaxation time of the mediating water pool. As MT, T1 and T2 are also altered in tumor regions, a recently published correction algorithm yielding the apparent exchange-dependent relaxation AREX, is used to evaluate in vivo CEST effects. This study focuses on CEST effects of amides (3.5ppm) and Nuclear-Overhauser-mediated saturation transfer (NOE, -3.5ppm) that can be properly isolated at 7T. These were obtained in 10 glioblastoma patients, and this is the first comprehensive study where AREX is applied in human brain as well as in human glioblastoma. The correction of CEST effects alters the contrast significantly: after correction, the CEST effect of amides does not show significant contrast between contrast enhancing tumor regions and normal tissue, whereas NOE drops significantly in the tumor area. In addition, new features in the AREX contrasts are visible. This suggests that previous CEST approaches might not have shown pure CEST effects, but rather water relaxation shine-through effects. Our insights help to improve understanding of the CEST effect changes in tumors and correlations on a cellular and molecular level. PMID:25727379

  6. Intracellular Streptococcus pyogenes in Human Macrophages Display an Altered Gene Expression Profile

    PubMed Central

    Hertzén, Erika; Johansson, Linda; Kansal, Rita; Hecht, Alexander; Dahesh, Samira; Janos, Marton; Nizet, Victor; Kotb, Malak; Norrby-Teglund, Anna

    2012-01-01

    Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems. PMID:22511985

  7. Intracellular Streptococcus pyogenes in human macrophages display an altered gene expression profile.

    PubMed

    Hertzén, Erika; Johansson, Linda; Kansal, Rita; Hecht, Alexander; Dahesh, Samira; Janos, Marton; Nizet, Victor; Kotb, Malak; Norrby-Teglund, Anna

    2012-01-01

    Streptococcus pyogenes is an important human pathogen, which has recently gained recognition as an intracellular microorganism during the course of severe invasive infections such as necrotizing fasciitis. Although the surface anchored M protein has been identified as a pivotal factor affecting phagosomal maturation and S. pyogenes survival within macrophages, the overall transcriptional profile required for the pathogen to adapt and persist intracellularly is as of yet unknown. To address this, the gene expression profile of S. pyogenes within human macrophages was determined and compared to that of extracellular bacteria using customized microarrays and real-time qRT-PCR. In order to model the early phase of infection involving adaptation to the intracellular compartment, samples were collected 2h post-infection. Microarray analysis revealed that the expression of 145 streptococcal genes was significantly altered in the intracellular environment. The majority of differentially regulated genes were associated with metabolic and energy-dependent processes. Key up-regulated genes in early phase intracellular bacteria were ihk and irr, encoding a two-component gene regulatory system (TCS). Comparison of gene expression of selected genes at 2h and 6h post-infection revealed a dramatic shift in response regulators over time with a down-regulation of ihk/irr genes concurring with an up-regulation of the covR/S TCS. In re-infection assays, intracellular bacteria from the 6h time point exhibited significantly greater survival within macrophages than did bacteria collected at the 2h time point. An isogenic S. pyogenes mutant deficient in ihk/irr displayed significantly reduced bacterial counts when compared to wild-type bacteria following infection of macrophages. The findings illustrate how gene expression of S. pyogenes during the intracellular life cycle is fine-tuned by temporal expression of specific two-component systems.

  8. 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

  9. The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers.

    PubMed

    Veeriah, Selvaraju; Brennan, Cameron; Meng, Shasha; Singh, Bhuvanesh; Fagin, James A; Solit, David B; Paty, Philip B; Rohle, Dan; Vivanco, Igor; Chmielecki, Juliann; Pao, William; Ladanyi, Marc; Gerald, William L; Liau, Linda; Cloughesy, Timothy C; Mischel, Paul S; Sander, Chris; Taylor, Barry; Schultz, Nikolaus; Major, John; Heguy, Adriana; Fang, Fang; Mellinghoff, Ingo K; Chan, Timothy A

    2009-06-01

    Tyrosine phosphorylation plays a critical role in regulating cellular function and is a central feature in signaling cascades involved in oncogenesis. The regulation of tyrosine phosphorylation is coordinately controlled by kinases and phosphatases (PTPs). Whereas activation of tyrosine kinases has been shown to play vital roles in tumor development, the role of PTPs is much less well defined. Here, we show that the receptor protein tyrosine phosphatase delta (PTPRD) is frequently inactivated in glioblastoma multiforme (GBM), a deadly primary neoplasm of the brain. PTPRD is a target of deletion in GBM, often via focal intragenic loss. In GBM tumors that do not possess deletions in PTPRD, the gene is frequently subject to cancer-specific epigenetic silencing via promoter CpG island hypermethylation (37%). Sequencing of the PTPRD gene in GBM and other primary human tumors revealed that the gene is mutated in 6% of GBMs, 13% of head and neck squamous cell carcinomas, and in 9% of lung cancers. These mutations were deleterious. In total, PTPRD inactivation occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the growth of GBM and other tumor cells, an effect not observed with PTPRD alleles harboring cancer-specific mutations. Human astrocytes lacking PTPRD exhibited increased growth. PTPRD was found to dephosphorylate the oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor on chromosome 9p that is involved in the development of GBMs and multiple human cancers.

  10. Carnosic acid, a component of rosemary (Rosmarinus officinalis L.), promotes synthesis of nerve growth factor in T98G human glioblastoma cells.

    PubMed

    Kosaka, Kunio; Yokoi, Toshio

    2003-11-01

    Nerve growth factor (NGF) is a factor vital for the growth and functional maintenance of nerve tissue. The authors found that a rosemary (Rosmarinus officinalis L.) extract enhanced the production of NGF in T98G human glioblastoma cells. Furthermore, the results indicated that carnosic acid and carnosol, which are major components of the rosemary extract, were able to promote markedly enhanced synthesis of NGF. PMID:14600414

  11. 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

  12. 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

  13. 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. PMID:26220980

  14. MRE11-RAD50-NBS1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    PubMed Central

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Kawata, Tetsuya; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Nishikawa, Ryo; Shigematsu, Naoyuki

    2014-01-01

    BACKGROUND: (blind field) METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylation (γH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSIONS: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells. SECONDARY CATEGORY: n/a.

  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. An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells

    PubMed Central

    Gil-Ranedo, Jon; Mendiburu-Eliçabe, Marina; García-Villanueva, Mercedes; Medina, Diego; del Álamo, Marta; Izquierdo, Marta

    2011-01-01

    Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor. PMID:22174890

  17. 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. PMID:27133062

  18. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53

    SciTech Connect

    Mercer, W.E.; Shields, M.T.; Amin, M.; Sauve, G.J. ); Appella, E.; Romano, J.W.; Ullrich, S.J. )

    1990-08-01

    To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation the authors constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastomas multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. The authors show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G{sub 0}/G{sub 1} progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G{sub 0}/G{sub 1} phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

  19. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells

    PubMed Central

    Li, Da-Ming; Sun, Hong

    1998-01-01

    PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1. PMID:9860981

  20. 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.

  1. LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages.

    PubMed

    Stephan, Alexander; Batinica, Marina; Steiger, Julia; Hartmann, Pia; Zaucke, Frank; Bloch, Wilhelm; Fabri, Mario

    2016-08-01

    As part of the innate host response neutrophils release neutrophil extracellular traps (NETs), protein:DNA complexes that contain a number of antimicrobial peptides (AMPs), such as cathelicidin. Human cathelicidin in its active form, LL37, has potent antimicrobial activity against bacteria. However, whether LL37 derived from NETs contributes to antimicrobial activity against intracellular pathogens remains unclear. Here, we report that NETs induced by mycobacteria contain cathelicidin. Human macrophages internalized NET-bound cathelicidin, which is transported to lysosomal compartments. Furthermore, using a model of in vitro-generated LL37:DNA complexes we found that LL37 derived from such complexes attacks mycobacteria in macrophage phagolysosomes resulting in antimicrobial activity. Taken together, our results suggest a mechanism by which LL37 in complex with DNA contributes to host defence against intracellular bacteria in human macrophages.

  2. Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

    PubMed

    Wang, Feng; Yang, Lijun; Shi, Lin; Li, Qian; Zhang, Gengshen; Wu, Jianliang; Zheng, Jun; Jiao, Baohua

    2015-08-28

    Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments. PMID:26056081

  3. Nuclear translocation of fibroblast growth factor-2 (FGF2) is regulated by Karyopherin-β2 and Ran GTPase in human glioblastoma cells.

    PubMed

    Wang, Feng; Yang, Lijun; Shi, Lin; Li, Qian; Zhang, Gengshen; Wu, Jianliang; Zheng, Jun; Jiao, Baohua

    2015-08-28

    Human glioblastoma multiforme (GBM) is the most malignant tumor of the central nervous system (CNS). Fibroblast growth factor-2 (FGF2) belongs to the FGF superfamily and functions as a potential oncoprotein in GBM. FGF2 has low molecular weight (18K) and high molecular weight (HMW) isoforms. Nuclear accumulation of HMW-FGF2 strongly promotes glioblastoma cell proliferation, yet mechanism governing such cellular distribution remains unexplored. We investigated the mechanisms regulating FGF2 cellular localization in T98G human brain glioblastoma cells. We found HMW-FGF2, but not 18K-FGF2, is primarily located in the nucleus and interacts with nuclear transport protein Karyopherin-β2/Transportin (Kapβ2). SiRNA-directed Kapβ2 knockdown significantly reduced HMW-FGF2's nuclear translocation. Moreover, inhibiting Ran GTPase activity also resulted in decreased HMW-FGF2 nuclear accumulation. Proliferation of T98G cells is greatly enhanced with transfections HMW-FGF2. Decreased PTEN expression and activated Akt signaling were observed upon HMW-FGF2 overexpression and might mediate pro-survival effect of FGF2. Interestingly, addition of nuclear localization signal (NLS) to 18K-FGF2 forced its nuclear import and dramatically increased cell proliferation and Akt activation. These findings demonstrated for the first time the molecular mechanisms for FGF2's nuclear import, which promotes GBM cell proliferation and survival, providing novel insights to the development of GBM treatments.

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

    PubMed

    Benedetti, Elisabetta; Antonosante, Andrea; 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

    2015-12-01

    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.

  5. 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

  6. Hexane extract from Sargassum serratifolium inhibits the cell proliferation and metastatic ability of human glioblastoma U87MG cells.

    PubMed

    Kang, Chang-Won; Park, Min-Seok; Kim, Nan-Hee; Lee, Ji-Hyun; Oh, Chul-Woong; Kim, Hyeung-Rak; Kim, Gun-Do

    2015-11-01

    The present study is the first to demonstrate the anticancer effects of a hexane extract from the brown algae Sargassum serratifolium (HES) on human cancer cell lines, including glioblastoma U87MG, cervical cancer HeLa and gastric cancer MKN-28 cells, as well as liver cancer SK-HEP 1 cells. Among these cancer cell lines, U87MG cells were most sensitive to the cell death induced by HES. HES exhibited a cytotoxic effect on U87MG cells at concentrations of 14-16 µg/ml, yet an effect was not observed in human embryonic kidney HEK293 cells. The antiproliferative effects of HES were regulated by inhibition of the MAPK/ERK signaling pathway which plays a pivotal role in the proliferation of glioblastoma U87MG cells. In addition, treatment with HES led to cell morphological changes and cell cytoskeleton degradation through regulation of actin dynamic signaling. Furthermore, migration and invasion of the U87MG cells were inhibited by HES via suppression of matrix metalloproteinase (MMP)-2 and -9 expression. Thus, our results suggest that HES is a potential therapeutic agent which has anticancer effects on glioblastoma. PMID:26323587

  7. PTEN mutation and epidermal growth factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression in human glioblastoma cells by transactivating the proximal VEGF promoter.

    PubMed

    Pore, Nabendu; Liu, Shuang; Haas-Kogan, Daphne A; O'Rourke, Donald M; Maity, Amit

    2003-01-01

    Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.

  8. TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance.

    PubMed

    Costa, Barbara; Da Pozzo, Eleonora; Giacomelli, Chiara; Taliani, Sabrina; Bendinelli, Sara; Barresi, Elisabetta; Da Settimo, Federico; Martini, Claudia

    2015-03-01

    Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside.

  9. 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

  10. Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain

    PubMed Central

    Breznik, Barbara; Gredar, Tajda; Hrovat, Katja; Bizjak Mali, Lilijana; Lah, Tamara T

    2016-01-01

    Abstract Background An attractive approach in the study of human cancers is the use of transparent zebrafish (Danio rerio) embryos, which enable the visualization of cancer progression in a living animal. Materials and methods We implanted mixtures of fluorescently labeled glioblastoma (GBM) cells and bonemarrow-derived mesenchymal stem cells (MSCs) into zebrafish embryos to study the cellular pathways of their invasion and the interactions between these cells in vivo. Results By developing and applying a carbocyanine-dye-compatible clearing protocol for observation of cells in deep tissues, we showed that U87 and U373 GBM cells rapidly aggregated into tumor masses in the ventricles and midbrain hemispheres of the zebrafish embryo brain, and invaded the central nervous system, often using the ventricular system and the central canal of the spinal cord. However, the GBM cells did not leave the central nervous system. With co-injection of differentially labeled cultured GBM cells and MSCs, the implanted cells formed mixed tumor masses in the brain. We observed tight associations between GBM cells and MSCs, and possible cell-fusion events. GBM cells and MSCs used similar invasion routes in the central nervous system. Conclusions This simple model can be used to study the molecular pathways of cellular processes in GBM cell invasion, and their interactions with various types of stromal cells in double or triple cell co-cultures, to design anti-GBM cell therapies that use MSCs as vectors. PMID:27247548

  11. 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

    2016-11-01

    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.

  12. Intracellular and extracellular pH dynamics in the human placenta from diabetes mellitus.

    PubMed

    Araos, Joaquín; Silva, Luis; Salsoso, Rocío; Sáez, Tamara; Barros, Eric; Toledo, Fernando; Gutiérrez, Jaime; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Sobrevia, Luis

    2016-07-01

    The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.

  13. 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. PMID:25307056

  14. 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

  15. 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.

  16. Carbon-ion beams effectively induce growth inhibition and apoptosis in human neural stem cells compared with glioblastoma A172 cells

    PubMed Central

    Isono, Mayu; Yoshida, Yukari; Takahashi, Akihisa; Oike, Takahiro; Shibata, Atsushi; Kubota, Yoshiki; Kanai, Tatsuaki; Ohno, Tatsuya; Nakano, Takashi

    2015-01-01

    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 (D50) was 0.8 Gy in hNSCs and 3.0 Gy in A172 cells, while the D50 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. PMID:26070322

  17. AKT2-knockdown suppressed viability with enhanced apoptosis, and attenuated chemoresistance to temozolomide of human glioblastoma cells in vitro and in vivo

    PubMed Central

    Cui, Yong; Lin, Jing; Zuo, Jianling; Zhang, Lei; Dong, Yan; Hu, Guohan; Luo, Chun; Chen, Juxiang; Lu, Yicheng

    2015-01-01

    The AKT2 kinase (protein kinase Bβ) is overexpressed in high-grade gliomas. Upregulation of the AKT2 gene has been previously observed in glioblastoma patients suffering from chemotherapy failure and tumor progress. In this study, we aimed to evaluate the effect of AKT2 on viability and chemoresistance in the human glioblastoma cell line U251. The U251 cell line was stably transfected with short hairpin RNA (shRNA) targeting AKT2. U251 cells underexpressing AKT2 were then examined for viability with temozolomide (TMZ) treatment, and tested for cell apoptosis both in vitro and in tumor-implanted mice. Next, expressions of several chemoresistance-related molecules were measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blot analysis. The results showed that the 50% inhibitory concentration (IC50) of AKT2 shRNA-transfected cells was significantly lower compared with Lenti-GFP-transfected and nontransfected controls and that the tumor growth of the AKT2-shRNA and TMZ combined-treated mice was obviously suppressed in either mass or volume. Concomitantly, the apoptosis of TMZ-treated tumor cells was significantly enhanced after knockdown of AKT2, as measured by flow cytometry and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. Furthermore, AKT2-inhibition in TMZ-treated glioblastoma U251 cells upregulated apoptotic effector caspase-3, whereas it downregulated antiapoptotic protein Bcl-2, DNA repairing protein MGMT, and drug efflux pump protein MRP1. Our study identified AKT2 as an important gene in presenting chemoresistance in glioblastoma, and a potential target to potentiate the clinical effect of chemotherapy in glioma treatment. PMID:26185456

  18. Function of carbonic anhydrase IX in glioblastoma multiforme.

    PubMed

    Proescholdt, Martin A; Merrill, Marsha J; Stoerr, Eva-Maria; Lohmeier, Annette; Pohl, Fabian; Brawanski, Alexander

    2012-11-01

    Carbonic anhydrase (CA) IX is over-expressed in glioblastoma; however, its functions in this context are unknown. Metabolically, glioblastomas are highly glycolytic, leading to a significant lactic acid load. Paradoxically, the intracellular pH is alkaline. We hypothesized that CAIX contributes to the extrusion of hydrogen ions into the extracellular space, thereby moderating intra- and extracellular pH and creating an environment conductive to enhanced invasion. We investigated the role of CAIX as a prognostic marker in patients with glioblastoma and its biological function in vitro. CAIX expression was analyzed in 59 patients with glioblastoma by immunohistochemistry. The expression levels were correlated to overall survival. In vitro, U251 and Ln 18 glioblastoma cells were incubated under hypoxia to induce CAIX expression, and RNA interference (RNAi) was used to examine the function of CAIX on cell attachment, invasion, intracellular energy transfer, and susceptibility to adjuvant treatment. High CAIX expression was identified as an independent factor for poor survival in patients with glioblastoma. In vitro, cell attachment and invasion were strongly reduced after knockdown of CAIX. Finally, the effects of radiation and chemotherapy were strongly augmented after CAIX interference and were accompanied by a higher rate of apoptotic cell death. CAIX is an independent prognostic factor for poor outcome in patients with glioblastoma. Cell attachment, invasion, and survival during adjuvant treatment are significantly influenced by high CAIX expression. These results indicate that inhibition of CAIX is a potential metabolic target for the treatment of patients with glioblastoma. PMID:23074198

  19. 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.

  20. Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells.

    PubMed

    Ho, L H; Ratnaike, R N; Zalewski, P D

    2000-02-01

    Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.

  1. 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.

  2. Lipopolysaccharide Compromises Human Sperm Function by Reducing Intracellular cAMP.

    PubMed

    Li, Zhongyuan; Zhang, Dahu; He, Yuanqiao; Ding, Zhiyong; Mao, Fei; Luo, Tao; Zhang, Xiaoping

    2016-01-01

    A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP. PMID:26782775

  3. Lipopolysaccharide Compromises Human Sperm Function by Reducing Intracellular cAMP.

    PubMed

    Li, Zhongyuan; Zhang, Dahu; He, Yuanqiao; Ding, Zhiyong; Mao, Fei; Luo, Tao; Zhang, Xiaoping

    2016-01-01

    A worldwide decline in the quality of human semen is currently occurring. In mammals, sperm are produced from diploid stem-cell spermatogonia by spermatogenesis in testes and become mature in epididymis. Nevertheless, these biological processes can be affected by Gram-negative bacterial infection mediated by lipopolysaccharide (LPS), the major endotoxin of Gram-negative bacteria. It is well known that LPS can disturb spermatogenesis and affect sperm maturation and quality in vivo. However, the effect of LPS on the ejaculated mature sperm in vitro remains unclear. Thus, this study aimed to assess the in vitro toxicity of LPS on human sperm function and to elucidate the underlying mechanism. Human sperm were incubated with LPS (0.1-100 μg/ml) for 1-12 h in vitro and, subsequently, sperm viability, motility and capacitation, and the acrosome reaction were examined. LPS dose-dependently inhibited total and progressive motility and the ability to move through a viscous medium of the sperm but did not affect sperm viability, capacitation, and the acrosome reaction. To explore the underlying mechanism of LPS's actions, we examined the effects of LPS on the intracellular concentrations of cyclic adenosine monophosphate (cAMP) and calcium ([Ca(2+)]i) and protein-tyrosine phosphorylation of human sperm, which are key regulators of human sperm function. LPS decreased intracellular cAMP dose-dependently but had no effect on [Ca(2+)]i and protein-tyrosine phosphorylation of human sperm. These findings suggest that LPS inhibits human sperm motility by decreasing intracellular cAMP.

  4. MicroRNA-377 inhibited proliferation and invasion of human glioblastoma cells by directly targeting specificity protein 1

    PubMed Central

    Zhang, Rui; Luo, Hui; Wang, Shuai; Chen, Wanghao; Chen, Zhengxin; Wang, Hong-Wei; Chen, Yuanyuan; Yang, Jingmin; Zhang, Xiaotian; Wu, Wenting; Zhang, Shu-Yu; Shen, Shuying; Dong, Qingsheng; Zhang, Yaxuan; Jiang, Tao; Lu, Daru; Zhao, Shiguang; You, Yongping; Liu, Ning; Wang, Huibo

    2014-01-01

    Background Increasing evidence has indicated that microRNAs (miRNAs) are strongly implicated in the initiation and progression of glioblastoma multiforme (GBM). Here, we identified a novel tumor suppressive miRNA, miR-377, and investigated its role and therapeutic effect for GBM. Methods MiRNA global screening was performed on GBM patient samples and adjacent nontumor brain tissues. The expression of miR-377 was detected by real-time reverse-transcription PCR. The effects of miR-377 on GBM cell proliferation, cell cycle progression, invasion, and orthotopic tumorigenicity were investigated The therapeutic effect of miR-377 mimic was explored in a subcutaneous GBM model. Western blot and luciferase reporter assay were used to identify the direct and functional target of miR-377. Results MiR-377 was markedly downregulated in human GBM tissues and cell lines. Overexpression of miR-377 dramatically inhibited cell growth both in culture and in orthotopic xenograft tumor models, blocked G1/S transition, and suppressed cell invasion in GBM cells. Importantly, introduction of miR-377 could strongly inhibit tumor growth in a subcutaneous GBM model. Subsequent investigation revealed that specificity protein 1 (Sp1) was a direct and functional target of miR-377 in GBM cells. Silencing of Sp1 recapitulated the antiproliferative and anti-invasive effects of miR-377, whereas restoring the Sp1 expression antagonized the tumor-suppressive function of miR-377. Finally, analysis of miR-377 and Sp1 levels in human GBM tissues revealed that miR-377 is inversely correlated with Sp1 expression. Conclusion These findings reveal that miR-377/Sp1 signaling that may be required for GBM development and may consequently serve as a therapeutic target for the treatment of GBM. PMID:24951112

  5. Anti-tumor effects of progesterone in human glioblastoma multiforme: role of PI3K/Akt/mTOR signaling.

    PubMed

    Atif, Fahim; Yousuf, Seema; Stein, Donald G

    2015-02-01

    Glioblastoma multiforme (GBM) is an aggressive primary brain tumor with a mean patient survival of 13-15 months despite surgical resection, radiation therapy and standard-of-care chemotherapy. We investigated the chemotherapeutic effects of the hormone progesterone (P4) on the growth of human GBM in four genetically different cell lines (U87MG, U87dEGFR, U118MG, LN-229) in vitro and in a U87MG subcutaneous xenograft mouse model. At high concentrations (20, 40, and 80 μM), P4 significantly (P<0.05) decreased tumor cell viability in all cell lines except LN-229. This effect was not blocked by the P4 receptor antagonist RU468. Conversely, at low physiological concentrations (0.1, 1, and 5 μM) P4 showed a proliferative effect in all cell lines which was blocked by RU486. In nude mice, P4 (100 and 200 mg/kg) inhibited tumor growth significantly (P<0.05) over 5 weeks of treatment and extended survival time of tumor-bearing mice by 60% without signs of systemic toxicity. P4 suppressed tumor vascularization as indicated by the expression of CD31, vascular endothelial growth factor and matrix metalloproteinase-9. Apoptosis in tumor tissue was detected by the expression of cleaved caspase-3, BCl-2, BAD and p53 proteins and confirmed by TUNEL assay. P4 treatment also suppressed PI3K/Akt/mTOR signaling, which regulates tumor growth, as demonstrated by the suppression of proliferating cell nuclear antigen. Our data can be interpreted to suggest that P4 suppresses the growth of human GBM cells both in vitro and in vivo and enhances survival time in mice without any demonstrable side effects. This article is part of a Special Issue entitled 'Sex steroids and brain disorders'.

  6. 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

  7. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26462157

  9. 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. PMID:27610031

  10. 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

  11. 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.

  12. Intracellular trafficking pathway of BK virus in human renal proximal tubular epithelial cells

    PubMed Central

    Moriyama, Takahito; Sorokin, Andrey

    2009-01-01

    Intracellular trafficking of BK Virus (BKV) in human renal proximal tubular epithelial cells (HRPTEC) is critical for BKV nephritis. However, the major trafficking components utilized by BKV remain unknown. Co-incubation of HRPTEC with BKV and microtubule disrupting agents prevented BKV infection as detected by immunofluorescence and western blot analysis with antibodies which recognize BKV large T antigen. However, inhibition of a dynein, cellular motor protein, did not interfere with BKV infection in HRPTEC. A colocalization study of BKV with the markers of the endoplasmic reticulum (ER) and the Golgi apparatus (GA), indicated that BKV reached the ER from 6 to 10 hours, while bypassing the GA or passing through the GA too transiently to be detected. This study contributes to the understanding of mechanisms of intracellular trafficking used by BKV in the infection of HRPTEC. PMID:17976677

  13. Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1.

    PubMed

    Tinkelenberg, A H; Liu, Y; Alcantara, F; Khan, S; Guo, Z; Bard, M; Sturley, S L

    2000-12-29

    Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.

  14. Histone Deacetylase Inhibitors Interact with Melanoma Differentiation Associated-7/Interleukin-24 to Kill Primary Human Glioblastoma Cells

    PubMed Central

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

    2013-01-01

    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 Ca2+. Quenching of reactive oxygen species and Ca2+ 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. PMID:23661648

  15. Hypoxia Modulates the Swelling-Activated Cl Current in Human Glioblastoma Cells: Role in Volume Regulation and Cell Survival.

    PubMed

    Sforna, Luigi; Cenciarini, Marta; Belia, Silvia; Michelucci, Antonio; Pessia, Mauro; Franciolini, Fabio; Catacuzzeno, Luigi

    2017-01-01

    The malignancy of glioblastoma multiforme (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non-tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling-activated Cl current (ICl,swell ). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia-activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably, the hypoxia-induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell . Since, a hypoxia-induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia-activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia-induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. J. Cell. Physiol. 232: 91-100, 2017. © 2016 Wiley Periodicals, Inc.

  16. (C-11)-thymidine PET imaging as a measure of DNA synthesis rate: A preliminary quantitative study of human brain glioblastoma

    SciTech Connect

    Wong, C.Y.O.; Yung, B.C.Y.; Conti, P.

    1994-05-01

    (C-11)-Thymidine (TdR) PET imaging can potentially be used to measure the tumor proliferation in-vivo and monitor treatment. Twenty-four stereotactic brain biopsies (SBB) following in-vivo bromodeoxyuridine (BUDR) under MRI guidance were obtained to correlate with TdR PET imaging of primary glioblastoma in human brain. Following data acquisition, standard 4 by 4 pixel (2mm/pixel) regions of interest (ROIs) were placed over the tumor site based on SBB and the corresponding homologous region of contralateral normal cortices. After correcting input function for major metabolites and subtracting TdR activity in the normal side from the tumor side of the brain, 2- and 3- compartmental analysis was performed for all the ROIs. Akaike :(AIC) and Bayes (BIC) information criteria was calculated to compare these 2 kinetic models for differentiating pure blood pool effects from TdR incorporation into DNA. Of 24 SBB regions, 20 non-overlapping and corresponding ROIs in PET were identified and quantified. Eight ROIs were selected based on the AIC, BIC and root-mean-square errors (RMSE < 0.1) (4 couldn`t be modelled and 8 most likely represented blood flow effects). The percentage (%) of BUDR per high power field area %BUDR labelling. The k3, the forward phosphorylation rate (hence an index of DNA synthesis), was categorized into 2 groups according to a threshold value of %BUDR/hpfa - 5%. The tumor regions with low proliferative index (%BUDR/hpfa<5%) have significantly lower k3 than those with high proliferative index (p<0.005). We also find that k4 is at least an order less than k3, suggesting minimal effects of dephosphorylation and efflux of metabolites. We conclude that 3-compartmental, 4-parameter modeling is adequate for TdR PET studies and k3 correlates with DNA synthesis rate.

  17. Immunomodulatory Effects of Streptococcus suis Capsule Type on Human Dendritic Cell Responses, Phagocytosis and Intracellular Survival

    PubMed Central

    Meijerink, Marjolein; Ferrando, Maria Laura; Lammers, Geraldine; Taverne, Nico; Smith, Hilde E.; Wells, Jerry M.

    2012-01-01

    Streptococcus suis is a major porcine pathogen of significant commercial importance worldwide and an emerging zoonotic pathogen of humans. Given the important sentinel role of mucosal dendritic cells and their importance in induction of T cell responses we investigated the effect of different S. suis serotype strains and an isogenic capsule mutant of serotype 2 on the maturation, activation and expression of IL-10, IL-12p70 and TNF-α in human monocyte-derived dendritic cells. Additionally, we compared phagocytosis levels and bacterial survival after internalization. The capsule of serotype 2, the most common serotype associated with infection in humans and pigs, was highly anti-phagocytic and modulated the IL-10/IL-12 and IL-10/TNF-α cytokine production in favor of a more anti-inflammatory profile compared to other serotypes. This may have consequences for the induction of effective immunity to S. suis serotype 2 in humans. A shielding effect of the capsule on innate Toll-like receptor signaling was also demonstrated. Furthermore, we showed that 24 h after phagocytosis, significant numbers of viable intracellular S. suis were still present intracellularly. This may contribute to the dissemination of S. suis in the body. PMID:22558240

  18. Citrus bergamia Risso Elevates Intracellular Ca2+ in Human Vascular Endothelial Cells due to Release of Ca2+ from Primary Intracellular Stores

    PubMed Central

    Kang, Purum; Han, Seung Ho; Moon, Hea Kyung; Lee, Jeong-Min; Kim, Hyo-Keun; Min, Sun Seek; Seol, Geun Hee

    2013-01-01

    The purpose of the present study is to examine the effects of essential oil of Citrus bergamia Risso (bergamot, BEO) on intracellular Ca2+ in human umbilical vein endothelial cells. Fura-2 fluorescence was used to examine changes in intracellular Ca2+ concentration [Ca2+]i . In the presence of extracellular Ca2+, BEO increased [Ca2+]i , which was partially inhibited by a nonselective Ca2+ channel blocker La3+. In Ca2+-free extracellular solutions, BEO increased [Ca2+]i in a concentration-dependent manner, suggesting that BEO mobilizes intracellular Ca2+. BEO-induced [Ca2+]i increase was partially inhibited by a Ca2+-induced Ca2+ release inhibitor dantrolene, a phospholipase C inhibitor U73122, and an inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker, 2-aminoethoxydiphenyl borane (2-APB). BEO also increased [Ca2+]i in the presence of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+ uptake. In addition, store-operated Ca2+ entry (SOC) was potentiated by BEO. These results suggest that BEO mobilizes Ca2+ from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+ release and affect promotion of Ca2+ influx, likely via an SOC mechanism. PMID:24348719

  19. Maintenance of Large Numbers of Virus Genomes in Human Cytomegalovirus-Infected T98G Glioblastoma Cells

    PubMed Central

    Duan, Ying-Liang; Ye, Han-Qing; Zavala, Anamaria G.; Yang, Cui-Qing; Miao, Ling-Feng; Fu, Bi-Shi; Seo, Keun Seok; Davrinche, Christian

    2014-01-01

    ABSTRACT After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (1). HCMV infection has been further characterized in T98Gs, emphasizing the presence of HCMV DNA over an extended time frame. T98Gs were infected with either HCMV Towne or AD169-IE2-enhanced green fluorescent protein (eGFP) strains. Towne infections yielded mixed IE1 antigen-positive and -negative (Ag+/Ag−) populations. AD169-IE2-eGFP infections also yielded mixed populations, which were sorted to obtain an IE2− (Ag−) population. Viral gene expression over the course of infection was determined by immunofluorescent analysis (IFA) and reverse transcription-PCR (RT-PCR). The presence of HCMV genomes was determined by PCR, nested PCR (n-PCR), and fluorescence in situ hybridization (FISH). Compared to the HCMV latency model, THP-1, Towne-infected T98Gs expressed IE1 and latency-associated transcripts for longer periods, contained many more HCMV genomes during early passages, and carried genomes for a greatly extended period of passaging. Large numbers of HCMV genomes were also found in purified Ag− AD169-infected cells for the first several passages. Interestingly, latency transcripts were observed from very early times in the Towne-infected cells, even when IE1 was expressed at low levels. Although AD169-infected Ag− cells expressed no detectable levels of either IE1 or latency transcripts, they also maintained large numbers of genomes within the cell nuclei for several passages. These results identify HCMV-infected T98Gs as an attractive new model in the study of the long-term maintenance of virus genomes in the context of neural cell types. IMPORTANCE Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus

  20. Release of plasminogen activator inhibitor-1 from human astrocytes is regulated by intracellular ceramide.

    PubMed

    Kimura, M; Soeda, S; Oda, M; Ochiai, T; Kihara, T; Ono, N; Shimeno, H

    2000-12-15

    The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

  1. 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

  2. Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles.

    PubMed

    Lavado, Andrea S; Chauhan, Veeren M; Zen, Amer Alhaj; Giuntini, Francesca; Jones, D Rhodri E; Boyle, Ross W; Beeby, Andrew; Chan, Weng C; Aylott, Jonathan W

    2015-09-14

    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.

  3. Intracellular free calcium concentration and calcium transport in human erythrocytes of lead-exposed workers

    SciTech Connect

    Quintanar-Escorza, M.A.; Gonzalez-Martinez, M.T.; Navarro, L.; Maldonado, M.; Arevalo, B.; Calderon-Salinas, J.V. . E-mail: jcalder@cinvestav.mx

    2007-04-01

    Erythrocytes are the route of lead distribution to organs and tissues. The effect of lead on calcium homeostasis in human erythrocytes and other excitable cells is not known. In the present work we studied the effect of lead intoxication on the uptake and efflux (measured as (Ca{sup 2+}-Mg{sup 2+})-ATPase activity) of calcium were studied in erythrocytes obtained from lead-exposed workers. Blood samples were taken from 15 workers exposed to lead (blood lead concentration 74.4 {+-} 21.9 {mu}g/dl) and 15 non-exposed workers (9.9 {+-} 2 {mu}g/dl). In erythrocytes of lead-exposed workers, the intracellular free calcium was 79 {+-} 13 nM, a significantly higher concentration (ANOVA, P < 0.01) than the one detected in control (30 {+-} 9 nM). The enhanced intracellular free calcium was associated with a higher osmotic fragility and with important modifications in erythrocytes shape. The high intracellular free calcium in lead-exposed workers was also related to a 100% increase in calcium incorporation and to 50% reduction of (Ca{sup 2+}-Mg{sup 2+})-ATPase activity. Lipid peroxidation was 1.7-fold higher in erythrocytes of lead-exposed workers as compared with control. The alteration on calcium equilibrium in erythrocytes is discussed in light of the toxicological effects in lead-exposed workers.

  4. Intracellular distribution of Fe3O4 nanoparticles in both human and mouse cells

    NASA Astrophysics Data System (ADS)

    Palihawadana Arachchige, Maheshika; Laha, Suvra; Rajagopal, Amulya; Kulkarni, Sanjana; Wang, Shuo; Flack, Amanda; Li, Chunying; Jena, Bhanu; Lawes, Gavin

    2014-03-01

    In recent years there has been an increasing interest in developing Fe3O4 nanoparticles for biomedical applications including targeted drug delivery and magnetic resonance imaging. Understanding of the intracellular distribution of these nanoparticles is crucial when considering these nanoparticles for specific applications. We have synthesized Fe3O4 nanoparticles having average size of 14 nm using a co-precipitation technique, which were coated with dextran. We studied the structural and morphological characteristics of the nanoparticles using x-ray diffraction, electron microscopy, dynamic light scattering, and zeta potential measurements. We also characterized the magnetic properties of the nanoparticles. In order to investigate the intracellular distribution of these Fe3O4 nanoparticles, we functionalized the dextran coated Fe3O4 nanoparticles with a fluorescent dye, Fluorescein isothiocyanate (FITC), and cultured them with both mouse insulinoma MIN 6 cells and human pancreatic MIA PaCa 2 cells. Using optical microscope we investigated the intracellular distribution of the nanoparticles and the effects on cell growth.

  5. Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages.

    PubMed

    Rekha, Rokeya Sultana; Rao Muvva, S S V Jagadeeswara; Wan, Min; Raqib, Rubhana; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta

    2015-01-01

    LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca(2+), and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages.

  6. Fangchinoline suppresses the growth and invasion of human glioblastoma cells by inhibiting the kinase activity of Akt and Akt-mediated signaling cascades.

    PubMed

    Guo, Bingyu; Xie, Peng; Su, Jingyuan; Zhang, Tingting; Li, Xiaoming; Liang, Guobiao

    2016-02-01

    Glioblastoma multiforme (GBM) is one of the most palindromic and malignant central nervous system neoplasms, and the current treatment is not effectual for GBM. Research of specific medicine for GBM is significant. Fangchinoline possesses a wide range of pharmacological activities and attracts more attentions due to its anti-tumor effects. In this study, two WHO grade IV human GBM cell lines (U87 MG and U118 MG) were exposed to fangchinoline, and we found that fangchinoline specifically inhibits the kinase activity of Akt and markedly suppresses the phosphorylation of Thr308 and Ser473 of Akt in human GBM cells. We also observed that fangchinoline inhibits tumor cell proliferation and invasiveness and induces apoptosis through suppressing the Akt-mediated signaling cascades, including Akt/p21, Akt/Bad, and Akt/matrix metalloproteinases (MMPs). These data demonstrated that fangchinoline exerts its anti-tumor effects in human glioblastoma cells, at least partly by inhibiting the kinase activity of Akt and suppressing Akt-mediated signaling cascades. PMID:26408176

  7. Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction

    PubMed Central

    Chen, Tingting; Blanc, Caroline; Eder, Anke Z.; Prados-Rosales, Rafael; Souza, Ana Camila Oliveira; Kim, Ryung S.; Glatman-Freedman, Aharona; Joe, Maju; Bai, Yu; Lowary, Todd L.; Tanner, Rachel; Brennan, Michael J.; Fletcher, Helen A.; McShane, Helen; Casadevall, Arturo; Achkar, Jacqueline M.

    2016-01-01

    Background. The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. Methods. Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. Results. Immunoglobulin G (IgG) responses to AM increased significantly 4–8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. Conclusions. Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans. PMID:27056953

  8. Enrichment and characterization of human dermal stem/progenitor cells by intracellular granularity.

    PubMed

    Shim, Joong Hyun; Lee, Tae Ryong; Shin, Dong Wook

    2013-04-15

    Adult stem cells from the dermis would be an attractive cell source for therapeutic purposes as well as studying the process of skin aging. Several studies have reported that human dermal stem/progenitor cells (hDSPCs) with multipotent properties exist within the dermis of adult human skin. However, these cells have not been well characterized, because methods for their isolation or enrichment have not yet been optimized. In the present study, we enriched high side scatter (SSC(high))-hDSPCs from normal human dermal fibroblasts using a structural characteristic, intracellular granularity, as a sorting parameter. The SSC(high)-hDSPCs had high in vitro proliferation properties and expressed high levels of SOX2 and S100B, similar to previously identified mouse SOX2+ hair follicle dermal stem cells. The SSC(high)-hDSPCs could differentiate into not only mesodermal cell types, for example, adipocytes, chondrocytes, and osteoblasts, but also neuroectodermal cell types, such as neural cells. In addition, the SSC(high)-hDSPCs exhibited no significant differences in the expression of nestin, vimentin, SNAI2, TWIST1, versican, and CORIN compared with non-hDSPCs. These cells are therefore different from the previously identified multipotent fibroblasts and skin-derived progenitors. In this study, we suggest that hDSPCs can be enriched by using characteristic of their high intracellular granularity, and these SSC(high)-hDSPCs exhibit high in vitro proliferation and differentiation potentials.

  9. [Intracellular localization of transcription factor PROX1 in the human retina in ontogeny].

    PubMed

    Markitantova, Iu V; Zinov'eva, R D

    2014-01-01

    The spatiotemporal intracellular localization of the transcription factor PROX1 in the human retina during prenatal development (fetal weeks 9.5 to 31) and in the adult human retina was studied for the first time. The PROX1 protein was identified in the cell nuclei of the neuroblast retinal layers at the stage of active cell proliferation (fetal week 9.5) as well as in the nuclei of differentiating neurons of the inner nuclear retinal layer (horizontal, amacrine, and bipolar cells) from weeks 13 to 31 of prenatal development. The PROX1 protein localization in the adult retina was the same as at the late stage of prenatal development. Our results indicate the involvement of the transcription factor PROX1 in the regulation of proliferation of progenitor cells and differentiation of the inner nuclear layer cells of the human retina. These results confirm the conservative functions of Prox1/PROX1 in the vertebrate retina.

  10. 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

  11. 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

  12. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection.

    PubMed

    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

    2016-04-01

    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

  13. 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

  14. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection.

    PubMed

    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

    2016-04-01

    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

  15. Laser microspectrofluorometry for measuring dynamic changes of intracellular free Ca2+ in human airway gland cells

    NASA Astrophysics Data System (ADS)

    Millot, Jean-Marc; Merten, M.; Sharonov, S.; Figarella, C.; Jacquot, J.; Manfait, Michel

    1996-01-01

    Intracellular Ca2+ is a ubiquitous second messenger that regulates a wide variety of cellular functions including secretion, transepithelial solute and fluid transport. Laser confocal microspectrofluorometry (DILOR, Lille, France) was applied to visualize fluorescence emission spectra of the Indo-1 for measuring the intracellular free Ca2+ levels ([Ca2+]i) in a human tracheal gland immortalized cell line (MM39 cell line). Under a 351 nm laser excitation (0.5 (mu) W), the intracellular spectrum was analyzed as a ratio of the emission intensities at 420 and 500 nm. Previously, the intracellular Ca2+ calibration has been performed to define the relation between the intensity ratio and [Ca2+]i. Dynamic changes of single-cell [Ca2+]i were measured either from one substrate-attached cell or from different adjacent cells in monolayer culture. Measurements of [Ca2+]i are taken successively in different subcellular locations (up to 10 measurement points). Each measurement cycle was repeated 60 times. To do so, an (X,Y) motorized stage coupled with a computer allowed us to store the (X,Y) positions of several chosen points for the laser radiation. Cells were monitored for about 10 min. After agonist stimulation. Upon stimulating with calcium ionophore, 4BrA23187 (1 (mu) M), [Ca2+]i increased immediately up to 10 fold from a resting value of 31 plus or minus 6 nM (n equals 36). Histamine (1 to 100 (mu) M) increased [Ca2+]i in a concentration dependent manner with levels of up to 88 nM and 140 nM for 1 (mu) M and 100 (mu) M concentration, respectively, followed by a smooth decay back to baseline. Removal of extracellular Ca2+ did not abolish the histamine-stimulation [Ca2+]i rise, suggesting that a part of Ca2+ mobilization comes from intracellular Ca2+ stores. These results show that the combined use of the UV microspectrofluorometry and Indo-1 is well adapted and straight forward for the measurement of rapid responses of substrate-attached cells during experiments of long

  16. New perspectives in glioblastoma antiangiogenic therapy

    PubMed Central

    Popescu, Alisa Madalina; Purcaru, Stefana Oana; Alexandru, Oana

    2015-01-01

    Glioblastoma (GB) is highly vascularised tumour, known to exhibit enhanced infiltrative potential. One of the characteristics of glioblastoma is microvascular proliferation surrounding necrotic areas, as a response to a hypoxic environment, which in turn increases the expression of angiogenic factors and their signalling pathways (RAS/RAF/ERK/MAPK pathway, PI3K/Akt signalling pathway and WTN signalling cascade). Currently, a small number of anti-angiogenic drugs, extending glioblastoma patients survival, are available for clinical use. Most medications are ineffective in clinical therapy of glioblastoma due to acquired malignant cells or intrinsic resistance, angiogenic receptors cross-activation and redundant intracellular signalling, or the inability of the drug to cross the blood-brain barrier and to reach its target in vivo. Researchers have also observed that GB tumours are different in many aspects, even when they derive from the same tissue, which is the reason for personalised therapy. An understanding of the molecular mechanisms regulating glioblastoma angiogenesis and invasion may be important in the future development of curative therapeutic approaches for the treatment of this devastating disease. PMID:27358588

  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. 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. PMID:27588717

  19. 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.

  20. Some Attenuated Variants of Vesicular Stomatitis Virus Show Enhanced Oncolytic Activity against Human Glioblastoma Cells relative to Normal Brain Cells▿

    PubMed Central

    Wollmann, Guido; Rogulin, Vitaliy; Simon, Ian; Rose, John K.; van den Pol, Anthony N.

    2010-01-01

    Vesicular stomatitis virus (VSV) has been shown in laboratory studies to be effective against a variety of tumors, including malignant brain tumors. However, attenuation of VSV may be necessary to balance the potential toxicity toward normal cells, particularly when targeting brain tumors. Here we compared 10 recombinant VSV variants resulting from different attenuation strategies. Attenuations included gene shifting (VSV-p1-GFP/RFP), M protein mutation (VSV-M51), G protein cytoplasmic tail truncations (VSV-CT1/CT9), G protein deletions (VSV-dG-GFP/RFP), and combinations thereof (VSV-CT9-M51). Using in vitro viability and replication assays, the VSV variants were grouped into three categories, based on their antitumor activity and non-tumor-cell attenuation. In the first group, wild-type-based VSV-G/GFP, tumor-adapted VSV-rp30, and VSV-CT9 showed a strong antitumor profile but also retained some toxicity toward noncancer control cells. The second group, VSV-CT1, VSV-dG-GFP, and VSV-dG-RFP, had significantly diminished toxicity toward normal cells but showed little oncolytic action. The third group displayed a desired combination of diminished general toxicity and effective antitumor action; this group included VSV-M51, VSV-CT9-M51, VSV-p1-GFP, and VSV-p1-RFP. A member of the last group, VSV-p1-GFP, was then compared in vivo against wild-type-based VSV-G/GFP. Intranasal inoculation of young, postnatal day 16 mice with VSV-p1-GFP showed no adverse neurological effects, whereas VSV-G/GFP was associated with high lethality (80%). Using an intracranial tumor xenograft model, we further demonstrated that attenuated VSV-p1-GFP targets and kills human U87 glioblastoma cells after systemic application. We concluded that some, but not all, attenuated VSV mutants display a favorable oncolytic profile and merit further investigation. PMID:19906910

  1. 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.

  2. 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. PMID:25816103

  3. 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

  4. 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.

  5. 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.

  6. Positive interaction of thyme (red) essential oil with human polymorphonuclear granulocytes in eradicating intracellular Candida albicans.

    PubMed

    Tullio, Vivian; Mandras, Narcisa; Allizond, Valeria; Nostro, Antonia; Roana, Janira; Merlino, Chiara; Banche, Giuliana; Scalas, Daniela; Cuffini, Anna Maria

    2012-10-01

    The essential oils have started to be recognized for their potential antimicrobial role only in recent years. Clinical experience showed that the efficacy of antimicrobial agents depends not only on their direct effect on a given microorganism but also on the functional activity of the host immune system. Since data on the effects of essential oils on the innate immune system are scanty and fragmentary, the aim of this study was to evaluate the influence of thyme (red) essential oil (EO), at subinhibitory/inhibitory concentrations, on intracellular killing activity by human polymorphonuclear granulocytes (PMNs) against Candida albicans. In order to provide a frame of reference for the activity of this EO, its in vitro killing activity in the absence of PMNs was also evaluated.Results showed that EO at subminimal inhibitory (subMIC)/minimal inhibitory (MIC) concentrations significantly enhanced intracellular killing of C. albicans in comparison with EO-free controls and was comparable to the positive control (fluconazole). In in vitro killing assays without PMNs, we observed progressive growth of the yeast cells in the presence of EO subMIC/MIC concentrations. A positive antifungal interaction with phagocytes could explain why this EO, which appeared to be only fungistatic in time-kill assays, had efficacy in killing yeast cells once incubated with PMNs. PMID:22872591

  7. 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

  8. M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.

    PubMed

    Buchacher, Tanja; Ohradanova-Repic, Anna; Stockinger, Hannes; Fischer, Michael B; Weber, Viktoria

    2015-01-01

    Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence

  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 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

  11. Inhibition of intracellular growth of Histoplasma capsulatum yeast cells by cytokine-activated human monocytes and macrophages.

    PubMed Central

    Newman, S L; Gootee, L; Bucher, C; Bullock, W E

    1991-01-01

    Human monocytes/macrophages (M psi) were infected with Histoplasma capsulatum yeast cells, and intracellular growth was quantified after 24 h of incubation in medium alone or in medium containing cytokines. Yeast cells multiplied within freshly isolated monocytes, cultured M psi, and alveolar M psi with intracellular generation times of 14.2 +/- 1.4, 18.5 +/- 2.1, and 19.9 +/- 1.9 h (mean +/- standard error of the mean), respectively. Monocytes and M psi inhibited the intracellular growth of yeast cells in response to cytokine supernatant; maximum inhibition was obtained when cytokines were added to cell monolayers immediately after infection. Opsonization of yeast cells in normal serum or in H. capsulatum-immune serum did not affect the intracellular generation time of yeast cells in either control M psi or cytokine-activated M psi. PMID:1898916

  12. 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

  13. Saponin 6 derived from Anemone taipaiensis induces U87 human malignant glioblastoma cell apoptosis via regulation of Fas and Bcl‑2 family proteins.

    PubMed

    Ji, Chen-Chen; Tang, Hai-Feng; Hu, Yi-Yang; Zhang, Yun; Zheng, Min-Hua; Qin, Hong-Yan; Li, San-Zhong; Wang, Xiao-Yang; Fei, Zhou; Cheng, Guang

    2016-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor, and is associated with a poor prognosis. Saponin 6, derived from Anemone taipaiensis, exerts potent cytotoxic effects against the human hepatocellular carcinoma HepG2 cell line and the human promyelocytic leukemia HL‑60 cell line; however, the effects of saponin 6 on glioblastoma remain unknown. The present study aimed to evaluate the effects of saponin 6 on human U87 malignant glioblastoma (U87 MG) cells. The current study revealed that saponin 6 induced U87 MG cell death in a dose‑ and time‑dependent manner, with a half maximal inhibitory concentration (IC50) value of 2.83 µM after treatment for 48 h. However, saponin 6 was needed to be used at a lesser potency in HT‑22 cells, with an IC50 value of 6.24 µM. Cell apoptosis was assessed by flow cytometry using Annexin V‑fluorescein isothiocyanate/propidium iodide double staining. DNA fragmentation and alterations in nuclear morphology were examined by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling and transmission electron microscopy, respectively. The present study demonstrated that treatment with saponin 6 induced cell apoptosis in U87 MG cells, and resulted in DNA fragmentation and nuclear morphological alterations typical of apoptosis. In addition, flow cytometric analysis revealed that saponin 6 was able to induce cell cycle arrest. The present study also demonstrated that saponin 6‑induced apoptosis of U87 MG cells was attributed to increases in the protein expression levels of Fas, Fas ligand, and cleaved caspase‑3, ‑8 and ‑9, and decreases in the levels of B‑cell lymphoma 2. The current study indicated that saponin 6 may exhibit selective cytotoxicity toward U87 MG cells by activating apoptosis via the extrinsic and intrinsic pathways. Therefore, saponin 6 derived from A. taipaiensis may possess therapeutic potential for the treatment of GBM. PMID

  14. 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

  15. Immunocytochemical studies on the effect of 405-nm low-power laser irradiation on human-derived A-172 glioblastoma cells.

    PubMed

    Ang, Foong Yee; Fukuzaki, Yumi; Yamanoha, Banri; Kogure, Shinichi

    2012-09-01

    The application of low-power laser irradiation (LLI) affects the cell cycle and cell proliferation in various kinds of cells. LLI at a wavelength of 808 nm and a power of 30 mW has been found to significantly decrease the proliferation rate of cells of the human-derived glioblastoma cell line A-172. To determine if this effect of LLI is specific to 808-nm LLI, the present study was designed to reveal the effects of 405-nm LLI under the same experimental conditions. A-172 glioblastoma cells were cultured in 96-well plates according to the conventional protocol. Two different schedules of 405-nm LLI (27 mW) were tested: longer periods of 20, 40 and 60 min and shorter periods of 1, 2, 3, 5, 10 and 15 min. Cells on a digital image displayed on a computer monitor were counted and the proliferation ratio was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) staining. Annexin-V-FLUOS staining and acridine-orange/ethidium-bromide staining were in an immunocytochemical assay to determine if cells were viable or dead (due to apoptosis or necrosis). Cell counting and MTT staining showed that longer 405-nm LLI significantly suppressed the proliferation of A-172 cells at 48 h after LLI (p < 0.05 or p < 0.01) and that the effect of LLI tended to be dose-dependent with morphological changes including cell death. At 90 min after LLI, shorter 405-nm LLI caused necrotic as well as apoptotic cell death, and these effects depended on irradiation time, power and energy density. Detailed analysis revealed that this lethal effect occurred after LLI and was not sustainable. It is concluded that 405-nm LLI has a lethal effect on human-derived glioblastoma A-172 cells, that is different from the suppressive effect without morphological changes induced by 808-nm LLI.

  16. Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells

    PubMed Central

    Jafarnejad, M.; Cromer, W. E.; Kaunas, R. R.; Zhang, S. L.; Zawieja, D. C.

    2015-01-01

    The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca2+]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca2+]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca2+]i. Step changes in shear stress resulted in a rapid increase in [Ca2+]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca2+]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm2 stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm2 stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca2+]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry. PMID:25617358

  17. β2-Integrin-Mediated Adhesion and Intracellular Ca2+ Release in Human Eosinophils

    PubMed Central

    Bankers-Fulbright, Jennifer L.; Bartemes, Kathleen R.; Kephart, Gail M.; Kita, Hirohito

    2009-01-01

    Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO3-buffered HybriCare medium maintained in a humidified 5% CO2 incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca2+] that precedes adhesion with BAPTA-AM (10 µM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca2+] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β2-integrins, or jasplakinolide (2 µM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 µM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca2+] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion. PMID:19290459

  18. Nickel Mobilizes Intracellular Zinc to Induce Metallothionein in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Leikauf, George D.; Pitt, Bruce R.; Wasserloos, Karla J.; Barchowsky, Aaron

    2009-01-01

    We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release. PMID:19097988

  19. Intracellular induction of the Bartonella henselae virB operon by human endothelial cells.

    PubMed

    Schmiederer, M; Arcenas, R; Widen, R; Valkov, N; Anderson, B

    2001-10-01

    One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2 gene revealed a region with sequence homology to the vir box of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virB genes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A. tumefaciens. PMID:11553594

  20. 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

  1. 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.

  2. Reversing HOXA9 Oncogene Activation by PI3K Inhibition: Epigenetic Mechanism and Prognostic Significance in Human Glioblastoma

    PubMed Central

    Costa, Bruno M.; Smith, Justin S.; Chen, Ying; Chen, Justin; Phillips, Heidi S.; Aldape, Kenneth D.; Zardo, Giuseppe; Nigro, Janice; James, C. David; Fridlyand, Jane; Reis, Rui M.; Costello, Joseph F.

    2010-01-01

    HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using RT-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrantly activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a PI3K inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with TRAIL resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets, and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies. PMID:20068170

  3. Activating transcription factor 3 is overexpressed in human glioma and its knockdown in glioblastoma cells causes growth inhibition both in vitro and in vivo.

    PubMed

    Ma, Siqi; Pang, Changhe; Song, Laijun; Guo, Fuyou; Sun, Hongwei

    2015-06-01

    Glioblastomas are highly malignant gliomas that are extremely invasive with high rates of recurrence and mortality. It has been reported that activating transcription factor 3 (ATF3) is expressed in elevated levels in multiple malignant tumors. The purpose of this study was to investigate the function of ATF3 in the development of glioma and its clinical significance. Immunohistochemical staining, western blot analysis and RT-qPCR revealed that the mRNA and protein levels of ATF3 and matrix metalloproteinase 2 (MMP2) were higher in the glioma than in the normal human brain tissues, and that their levels were proportional to the pathological grades. By contrast, the mRNA and protein levels of mammary serine protease inhibitor (maspin; SERPINB5) were significantly lower in the glioma than in the normal brain tissue, and maspin expression was inversely proportional to the glioma pathological grade. The transfection of U373MG glioblastoma cells with ATF3-siRNA induced a number of changes in cell behavior; the cell proliferative activity was decreased and flow cytometry revealed an increased proportion of cells arrested in the G0/G1 phase of the cell cycle. In addition, TUNEL staining indicated an increased proportion of cells undergoing apoptosis and Transwell assays revealed impaired cell mobility. The sizes of the tumors grown as xenografts in nude mice were also significantly reduced by treatment of host mice with ATF3-siRNA. Taken together, these results suggest that ATF3 promotes the progression of human gliomas. PMID:25872784

  4. Activating transcription factor 3 is overexpressed in human glioma and its knockdown in glioblastoma cells causes growth inhibition both in vitro and in vivo

    PubMed Central

    MA, SIQI; PANG, CHANGHE; SONG, LAIJUN; GUO, FUYOU; SUN, HONGWEI

    2015-01-01

    Glioblastomas are highly malignant gliomas that are extremely invasive with high rates of recurrence and mortality. It has been reported that activating transcription factor 3 (ATF3) is expressed in elevated levels in multiple malignant tumors. The purpose of this study was to investigate the function of ATF3 in the development of glioma and its clinical significance. Immunohistochemical staining, western blot analysis and RT-qPCR revealed that the mRNA and protein levels of ATF3 and matrix metalloproteinase 2 (MMP2) were higher in the glioma than in the normal human brain tissues, and that their levels were proportional to the pathological grades. By contrast, the mRNA and protein levels of mammary serine protease inhibitor (maspin; SERPINB5) were significantly lower in the glioma than in the normal brain tissue, and maspin expression was inversely proportional to the glioma pathological grade. The transfection of U373MG glioblastoma cells with ATF3-siRNA induced a number of changes in cell behavior; the cell proliferative activity was decreased and flow cytometry revealed an increased proportion of cells arrested in the G0/G1 phase of the cell cycle. In addition, TUNEL staining indicated an increased proportion of cells undergoing apoptosis and Transwell assays revealed impaired cell mobility. The sizes of the tumors grown as xenografts in nude mice were also significantly reduced by treatment of host mice with ATF3-siRNA. Taken together, these results suggest that ATF3 promotes the progression of human gliomas. PMID:25872784

  5. Autocrine VEGFR1 and VEGFR2 signaling promotes survival in human glioblastoma models in vitro and in vivo

    PubMed Central

    Szabo, Emese; Schneider, Hannah; Seystahl, Katharina; Rushing, Elisabeth Jane; Herting, Frank; Weidner, K. Michael

    2016-01-01

    Background Although the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has become a prime target for antiangiogenic treatment, its biological role in glioblastoma beyond angiogenesis has remained controversial. Methods Using neutralizing antibodies to VEGF or placental growth factor (PlGF) or the tyrosine kinase inhibitor, cediranib, or lentiviral gene silencing, we delineated autocrine signaling in glioma cell lines. The in vivo effects of VEGFR1 and VEGFR2 depletion were evaluated in orthotopic glioma xenograft models. Results VEGFR1 and VEGFR2 modulated glioma cell clonogenicity, viability, and invasiveness in vitro in an autocrine, cell–line-specific manner. VEGFR1 silencing promoted mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling, whereas VEGFR2 silencing resulted in cell-type dependent activation of the protein kinase B (PKB)/AKT and MAPK/ERK pathways. These responses may represent specific escape mechanisms from VEGFR inhibition. The survival of orthotopic glioma-bearing mice was prolonged upon VEGFR1 silencing in the LNT-229, LN-308, and U87MG models and upon VEGFR2 silencing in LN-308 and U87MG. Disruption of VEGFR1 and VEGFR2 signaling was associated with decreased tumor size, increased tumor necrosis, or loss of matrix metalloproteinase 9 (MMP9) immunoreactivity. Neutralizing VEGF and PlGF by specific antibodies was superior to either antibody treatment alone in the VEGFR1-dependent LNT-229 model. Conclusions Differential dependence on autocrine signaling through VEGFR1 and VEGFR2 suggests a need for biomarker–stratified VEGF(R)-based therapeutic approaches to glioblastoma. PMID:27009237

  6. Heparin modulates intracellular cyclic AMP in human trabecular bone cells and adherent rheumatoid synovial cells.

    PubMed Central

    Crisp, A J; Roelke, M S; Goldring, S R; Krane, S M

    1984-01-01

    Cells were cultured from explants of human trabecular bone excised from eight patients and incubated usually for 20 minutes with bovine parathyroid hormone, salmon calcitonin, prostaglandin E2, or heparin. The intracellular content of cyclic AMP was measured by radioimmunoassay and was significantly increased by parathyroid hormone in four, by calcitonin in two, by prostaglandin E2 in eight, and by heparin in seven out of eight cultures. In the two cultures containing calcitonin-responsive cells heparin inhibited the cyclic AMP response induced by calcitonin. Heparin did not affect the cyclic AMP response to parathyroid hormone or prostaglandin E2. Heparin also increased the cyclic AMP content of cultured adherent rheumatoid synovial cells. It is proposed that, in certain situations of focal pathological bone resorption, although concentrations of circulating hormones may be normal, the local release of products such as heparin may modify the effect of hormones which regulate connective tissue homoeostasis. local changes in hormone responses could contribute to the enhanced bone resorption associated with inflammatory processes such as rheumatoid arthritis. Images PMID:6089675

  7. Novel robust biomarkers for human bladder cancer based on activation of intracellular signaling pathways.

    PubMed

    Lezhnina, Ksenia; Kovalchuk, Olga; Zhavoronkov, Alexander A; Korzinkin, Mikhail B; Zabolotneva, Anastasia A; Shegay, Peter V; Sokov, Dmitry G; Gaifullin, Nurshat M; Rusakov, Igor G; Aliper, Alexander M; Roumiantsev, Sergey A; Alekseev, Boris Y; Borisov, Nikolay M; Buzdin, Anton A

    2014-10-15

    We recently proposed a new bioinformatic algorithm called OncoFinder for quantifying the activation of intracellular signaling pathways. It was proved advantageous for minimizing errors of high-throughput gene expression analyses and showed strong potential for identifying new biomarkers. Here, for the first time, we applied OncoFinder for normal and cancerous tissues of the human bladder to identify biomarkers of bladder cancer. Using Illumina HT12v4 microarrays, we profiled gene expression in 17 cancer and seven non-cancerous bladder tissue samples. These experiments were done in two independent laboratories located in Russia and Canada. We calculated pathway activation strength values for the investigated transcriptomes and identified signaling pathways that were regulated differently in bladder cancer (BC) tissues compared with normal controls. We found, for both experimental datasets, 44 signaling pathways that serve as excellent new biomarkers of BC, supported by high area under the curve (AUC) values. We conclude that the OncoFinder approach is highly efficient in finding new biomarkers for cancer. These markers are mathematical functions involving multiple gene products, which distinguishes them from "traditional" expression biomarkers that only assess concentrations of single genes. PMID:25296972

  8. 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

  9. 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. PMID:24079969

  10. 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.

  11. cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium.

    PubMed Central

    Haskill, S; Martin, G; Van Le, L; Morris, J; Peace, A; Bigler, C F; Jaffe, G J; Hammerberg, C; Sporn, S A; Fong, S

    1991-01-01

    A cDNA encoding a receptor antagonist of interleukin 1 (IL-1ra), secreted from human monocytes, has recently been isolated and sequenced [Eisenberg, S. P., Evans, R. J., Arend, W. P., Verderber, E., Brewer, M. T., Hannum, C. H. & Thompson, R. C. (1990) Nature (London) 343, 341-346]. We have identified another version of this IL-1ra, which is predominantly expressed in epithelial cells. This IL-1ra lacks a leader sequence and, thus, is probably intracellular. Both proteins are derived from the same gene through use of an alternative transcriptional start site and internal splice-acceptor site. Expression of intracellular IL-1ra cDNA in COS cells demonstrated that the intracellular product specifically inhibited exogenous interleukin 1-dependent responses. Keratinocytes were shown to contain significant amounts of nonsecreted IL-1ra protein. Constitutive expression of the intracellular IL-1ra may be an intracellular defensive mechanism in exposed epithelial cells and/or may serve to regulate autocrine interleukin 1-mediated pathways of differentiation. Images PMID:1827201

  12. 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.

  13. Advances in treating glioblastoma

    PubMed Central

    Weathers, Shiao-Pei

    2014-01-01

    Glioblastoma is the most common and most aggressive primary brain tumor in adults. Optimized standard treatment only confers a modest improvement in progression and overall survival, underscoring the pressing need for the development of novel therapies. Our understanding of glioblastoma (a molecularly heterogeneous disorder) has been accelerated in the setting of large scale genomic analyses, lending insight into potential actionable targets. Antiangiogenic therapies have been used in the treatment of glioblastoma, and our understanding of the means to optimize the role of these agents is continuing to evolve. Recently, immunotherapy has garnered increasing attention as a therapeutic approach in the treatment of gliomas. Promising novel approaches are under active development in the treatment of glioblastoma. PMID:24991423

  14. Bevacizumab for glioblastoma

    PubMed Central

    Narita, Yoshitaka

    2015-01-01

    Individuals with glioblastoma are often characterized by older age, advanced neurologic manifestations at the primary stage, and unresectable tumors, and these factors are associated with poor treatment outcomes. Administration of bevacizumab (BV, Avastin®) promotes tumor regression and improves cerebral edema, and is expected to improve neurologic findings in many patients with malignant gliomas, including glioblastoma. Although the addition of BV to the conventional standard therapy (chemoradiotherapy with temozolomide) for newly diagnosed glioblastoma prolonged the progression-free survival time and the performance status of patients, it failed to extend overall survival time. However, more than 50% of glioblastoma patients show Karnofsky performance status ≤70 at initial presentation; therefore, BV should be used to improve or maintain their performance status as an initial treatment. Most of the adverse events of BV, except hypertension and proteinuria, occur as complications of glioblastoma, and explanation of the advantages and disadvantages of BV administration to patients is important. Herein, the efficacy, safety, and challenges of using BV for treating glioblastoma were reviewed. PMID:26664126

  15. Nucleolipids of Canonical Purine ß‐d‐Ribo‐Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells

    PubMed Central

    Knies, Christine; Hammerbacher, Katharina; Kinscherf, Ralf

    2015-01-01

    Abstract We report on the synthesis of two series of canonical purine ß‐d‐ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by 1H and 13C NMR, and pH‐dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS‐3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS‐3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3‐{4‐hydroxymethyl‐2‐methyl‐6‐[6‐oxo‐1‐(3,7,11‐trimethyl‐dodeca‐2,6,10‐trienyl)‐1,6‐dihydro‐purin‐9‐yl]‐tetrahydro‐furo[3,4‐d][1,3]dioxol‐2‐yl}propionate (8 c) toward both human and rat glioblastoma cell lines in vitro. PMID:27308225

  16. Targeting delivery of etoposide to inhibit the growth of human glioblastoma multiforme using lactoferrin- and folic acid-grafted poly(lactide-co-glycolide) nanoparticles.

    PubMed

    Kuo, Yung-Chih; Chen, Yu-Chun

    2015-02-01

    Lactoferrin (Lf) and folic acid (FA) were crosslinked on poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for transporting etoposide across the blood-brain barrier (BBB) and treating human brain malignant glioblastoma. Lf- and FA-grafted PLGA NPs (Lf/FA/PLGA NPs) were employed to permeate the monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes and to inhibit the multiplication of U87MG cells. Lf/FA/PLGA NPs showed a satisfactory entrapment efficiency of etoposide and characteristics of sustained drug release. When compared with PLGA NPs, the permeability coefficient for etoposide across the BBB using Lf/FA/PLGA NPs increased about twofold. The antiproliferative efficacy against the growth of U87MG cells was in the following order: Lf/FA/PLGA NPs>FA/PLGA NPs>PLGA NPs>free etoposide solution. In addition, the targeting ability of Lf/FA/PLGA NPs was evidenced by immunostaining of Lf receptor on HBMECs and folate receptor on U87MG cells during endocytosis. Lf/FA/PLGA NPs with loaded etoposide can be a promising anticancer pharmacotherapy to enhance the delivery of etoposide to malignant brain tumors for preclinical trials.

  17. Nucleolipids of Canonical Purine ß-d-Ribo-Nucleosides: Synthesis and Cytostatic/Cytotoxic Activities Toward Human and Rat Glioblastoma Cells.

    PubMed

    Knies, Christine; Hammerbacher, Katharina; Bonaterra, Gabriel A; Kinscherf, Ralf; Rosemeyer, Helmut

    2016-04-01

    We report on the synthesis of two series of canonical purine ß-d-ribonucleoside nucleolipids derived from inosine and adenosine, which have been characterized by elemental analyses, electrospray ionization mass spectrometry (ESI MS) as well as by (1)H and (13)C NMR, and pH-dependent UV/Vis spectroscopy. A selection of the novel nucleolipids with different lipophilic moieties were first tested on their cytotoxic effect toward human macrophages. Compounds without a significant inhibitory effect on the viability of the macrophages were tested on their cytostatic/cytotoxic effect toward human astrocytoma/oligodendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. In order to additionally investigate the potential molecular mechanisms involved in the cytotoxic effects of the derivatives on GOS-3 or BT4Ca cells, we evaluated the induction of apoptosis and observed the particular activity of the nucleolipid ethyl 3-{4-hydroxymethyl-2-methyl-6-[6-oxo-1-(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-1,6-dihydro-purin-9-yl]-tetrahydro-furo[3,4-d][1,3]dioxol-2-yl}propionate (8 c) toward both human and rat glioblastoma cell lines in vitro. PMID:27308225

  18. 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.

  19. 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.

  20. MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production

    PubMed Central

    Tong, Zhongsheng; Wang, Qinning; Liu, Weihuang; Wang, Yan; Liu, Wei; Chen, Jinbo; Xu, Li; Chen, Liuqing; Duan, Yiqun

    2012-01-01

    Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production. PMID:23239982

  1. 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.

  2. 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

  3. Synthesis, intracellular processing and secretion of thrombospondin in human endothelial cells.

    PubMed

    Vischer, P; Beeck, H; Voss, B

    1985-12-16

    The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from 'high-mannose' to 'complex' structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A-Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition. PMID:3935437

  4. Molecular therapy for glioblastoma.

    PubMed

    Karpati, G; Li, H; Nalbantoglu, J

    1999-10-01

    Glioblastoma (GB), the relatively frequent and most malignant form of primary brain tumor, is fatal within 1 to 2 years of onset of symptoms, despite conventional therapy. Molecular therapy promises to be an effective and possibly curative treatment. Several molecular strategies have been tested, either in animal models or clinical trials. These include: prodrug activating systems, introduction of tumor suppressor or cell-cycle-related genes, inhibition of growth factors and/or their receptors, inhibition of neovascularization, immunomodulatory maneuvers, oncolytic viruses and inhibition of matrix metalloproteinases. Of special interest for the development of optimal molecular therapy of GB, is the choice of the most efficient and least toxic gene vectors (adenovirus, retrovirus, herpes simplex virus), the route of administration of the therapeutic agent (intratumoral with or without debulking and intracarotid), avoidance of collateral damage to the perineoplastic neuropil and adequate preclinical studies. The ultimate molecular therapy will probably involve the application of multiple simultaneous (combinatorial) therapeutic modalities. The safety and efficiency of these in humans can only be judged by properly controlled therapeutic trials. PMID:11249660

  5. Stereotactic Radiosurgery for Glioblastoma.

    PubMed

    Redmond, Kristin J; 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

  6. 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

  7. Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells

    PubMed Central

    Zappelli, Elisa; Marinelli, Luciana; Novellino, Ettore; Da Settimo, Federico; Taliani, Sabrina; Trincavelli, Maria L.; Martini, Claudia

    2016-01-01

    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. PMID:26761214

  8. Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

    PubMed Central

    Faletto, M B; Miller, W H; Garvey, E P; St Clair, M H; Daluge, S M; Good, S S

    1997-01-01

    The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity. PMID:9145876

  9. Human β-Cell Proliferation and Intracellular Signaling Part 2: Still Driving in the Dark Without a Road Map

    PubMed Central

    Bernal-Mizrachi, Ernesto; Kulkarni, Rohit N.; Scott, Donald K.; Mauvais-Jarvis, Franck; Stewart, Andrew F.; Garcia-Ocaña, Adolfo

    2014-01-01

    Enhancing β-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of β-cell intracellular signaling pathways that promote β-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent β-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human β-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human β-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for β-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human β-cell replication. This is a critical aspect in the long-term goal of expanding human β-cells for the prevention and/or cure of type 1 and type 2 diabetes. PMID:24556859

  10. Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages

    PubMed Central

    Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso

    2015-01-01

    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. PMID:26503663

  11. 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

    2016-01-01

    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. PMID:26503663

  12. An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages.

    PubMed

    Siqueira-Neto, Jair L; Moon, Seunghyun; Jang, Jiyeon; 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

  13. 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.

  14. 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

    2015-03-01

    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.

  15. 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.

  16. 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.

  17. 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. PMID:24582893

  18. Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

    PubMed Central

    Schwan, W R; Kopecko, D J

    1997-01-01

    A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive. PMID:9353061

  19. Anti-vascular endothelial growth factor antibody and nimustine as combined therapy: effects on tumour growth and angiogenesis in human glioblastoma xenografts.

    PubMed Central

    Takano, Shingo; Tsuboi, Koji; Matsumura, Akira; Nose, Tadao

    2003-01-01

    We evaluated the effectiveness of vascular endothelial growth factor (VEGF) blockade alone and in combination with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU, nimustine), a cytotoxic agent commonly used in the treatment of malignant gliomas, to eradicate tumors of human glioblastoma cell lines implanted in SCID (severe combined immunodeficiency) mice. ACNU, but not cisplatin and etoposide, elevated VEGF expression in a glioma cell line in vitro. VEGF antibody alone inhibited glioma growth in vivo as a result of angiogenesis inhibition. The combination with ACNU resulted in an additive effect for inhibition of glioma growth. ACNU also induced VEGF up-regulation in glioma tissues, which was decreased with VEGF antibody treatment. One of the mechanisms of the additive effect of the VEGF antibody and ACNU combination is the blockade of VEGF up-regulation induced by ACNU. As such, the combination of antiangiogenic therapy with conventional therapy is promising for glioma treatment in the future. PMID:12626127

  20. Telomere targeting with a novel G-quadruplex-interactive ligand BRACO-19 induces T-loop disassembly and telomerase displacement in human glioblastoma cells.

    PubMed

    Zhou, Guangtong; Liu, Xinrui; Li, Yunqian; Xu, Songbai; Ma, Chengyuan; Wu, Xinmin; Cheng, Ye; Yu, Zhiyun; Zhao, Gang; Chen, Yong

    2016-03-22

    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

  1. Molecular recognition force spectroscopy study of the dynamic interaction between aptamer GBI-10 and extracellular matrix protein tenascin-C on human glioblastoma cell.

    PubMed

    Li, Yongjun; Qiao, Haiyan; Yan, Wei; Zhang, Jing; Xing, Chunyan; Wang, Hongda; Zhang, Bailin; Tang, Jilin

    2013-01-01

    Molecular recognition force spectroscopy (MR-FS) was applied to investigate the dynamic interaction between aptamer GBI-10 and tenascin-C (TN-C) on human glioblastoma cell surface at single-molecule level. The unbinding force between aptamer GBI-10 and TN-C was 39 pN at the loading rate of 0.3 nN sec⁻¹. A series of kinetic parameters concerning interaction process such as the unbinding force f(u) , the association rate constant k(on) , dissociation rate constant at zero force k(off) , and dissociation constant K(D) for aptamer GBI-10/TN-C complexes were acquired. In addition, the interaction of aptamer GBI-10 with TN-C depended on the presence of Mg²⁺. This work demonstrates that MR-FS can be used as an attractive tool for exploring the interaction forces and dynamic process of aptamer and ligand at the single-molecule level. As a future perspective, MR-FS may be used as a potential diagnostic and therapeutic tool by combining with other techniques.

  2. Intratumoral hemorrhage-related differences in the expression of vascular endothelial growth factor, basic fibroblast growth factor and thioredoxin reductase 1 in human glioblastoma

    PubMed Central

    Kaya, Bulent; Çiçek, Onur; Erdi, Fatih; Findik, Siddika; Karatas, Yasar; Esen, Hasan; Keskin, Fatih; Kalkan, Erdal

    2016-01-01

    The present study was designed to evaluate the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and thioredoxin reductase 1 (TrxR1) in glioblastoma multiforme (GBM) with and without intratumoral hemorrhage. Surgically resected human GBM samples from 20 patients who underwent surgery at our institute were extracted from the histopathological specimens and divided into two groups. A total of 10 samples from each type of GBM (World Health Organization grade IV, intratumoral hemorrhage-positive or -negative) were included in each group. VEGF, bFGF and TrxR1 expression was analyzed using immunohistochemistry and the results were compared between groups. VEGF and bFGF immunoreactivity was significantly higher in GBMs containing intratumoral hemorrhage. Furthermore, VEGF, bFGF and TrxR1 immunointensity was significantly higher in GBMs containing intratumoral hemorrhage. Thus, the present study demonstrated a higher VEGF, bFGF and TrxR1 expression in GBMs contain intratumoral hemorrhage, indicatiogn a role of VEGF, bFGF and TrxR1 expression in the promotion of tumoral angiogenesis and tumoral growth by complex mechanisms that require further elucidation.

  3. Involvement of intracellular oxidative stress-sensitive pathway in phloxine B-induced photocytotoxicity in human T lymphocytic leukemia cells.

    PubMed

    Qi, Hang; Zhu, Beiwei; Abe, Naomi; Shin, Yuko; Murata, Yoshiyuki; Nakamura, Yoshimasa

    2012-06-01

    We investigated the molecular mechanisms underlying phloxine B (PhB)-induced photocytotoxicity in human T lymphocytic leukemia Jurkat cells. In addition to apoptosis-related biochemical events, photo-irradiated PhB generated intracellular reactive oxygen species (ROS), induced phosphorylation of c-Jun-N-terminal kinase (JNK) in an oxidative stress-dependent manner and up-regulated the gene expression of interferon (IFN)-γ, an inducer of diverse apoptosis-related molecules in activated T cells. PhB-induced apoptosis was significantly inhibited by N-acetyl-l-cysteine, but not by catalase, indicating that ROS generation occurred intracellularly, and by SP600125 and AG490, specific inhibitors of JNK and IFN-γ signaling, respectively, confirming their roles in the apoptotic pathway. IFN-γ up-regulation was also inhibited by SP600125, indicating that it was downstream of JNK activation. These results suggest that PhB-induced apoptosis in Jurkat cells partially involves the intracellular oxidative stress-sensitive and T cell-specific IFN-γ pathway. These data present a novel insight into the mechanisms of photocytotoxicity induced by artificial food colorants in human T lymphocytic leukemia cells. PMID:22440610

  4. Heatshock protein vaccines against Glioblastoma: From bench to bedside

    PubMed Central

    Ampie, Leonel; Choy, Winward; Lamano, Jonathan B; Fakurnejad, Shayan; Bloch, Orin; Parsa, Andrew T.

    2015-01-01

    Current adjuvant treatment regimens available for the treatment of glioblastoma are widely ineffective and offer a dismal prognosis. Advancements in conventional treatment strategies have only yielded modest improvements in overall survival. Immunotherapy remains a promising adjuvant in the treatment of GBM through eliciting tumor specific immune responses capable of producing sustained antitumor response while minimizing systemic toxicity. Heat Shock Proteins (HSP) function as intracellular chaperones and have been implicated in the activation of both innate and adaptive immune systems. Vaccines formulated from HSP-peptide complexes, derived from autologous tumor, have been applied to the field of immunotherapy for glioblastoma. The results from the phase I and II clinical trials have been promising. Here we review the role of HSP in cellular function and immunity, and its application in the treatment of glioblastoma. PMID:26093618

  5. The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane.

    PubMed

    Re, Michelle; Pampillo, Macarena; Savard, Martin; Dubuc, Céléna; McArdle, Craig A; Millar, Robert P; Conn, P Michael; Gobeil, Fernand; Bhattacharya, Moshmi; Babwah, Andy V

    2010-07-08

    The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

  6. Entry of Sanfetrinem into Human Polymorphonuclear Granulocytes and Its Cell-Associated Activity against Intracellular, Penicillin-Resistant Streptococcus pneumoniae

    PubMed Central

    Cuffini, Anna Maria; Tullio, Vivian; Bonino, Alessandro; Allocco, Alessandra; Palarchio, Angela Ianni; Carlone, Nicola A.

    1998-01-01

    The entry of antibiotics into phagocytes is necessary for activity against intracellular pathogens. The ability of sanfetrinem, the first member of a new class of antibiotics, to penetrate human polymorphonuclear granulocytes and its consequences upon subsequent phagocytosis and killing of ingested penicillin-resistant Streptococcus pneumoniae have been evaluated. Sanfetrinem penetrated into human polymorphonuclear leukocytes (PMNs) at all concentrations tested, with cellular concentration/extracellular concentration ratios of 6.6 to 5.03 and 4.21 when sanfetrinem was used at 0.25 to 0.5 and 1 μg/ml, respectively, within 30 min of incubation. The uptake was complete within 5 min and was not energy dependent, since it was not affected by cell viability, environmental temperature, or the addition of a metabolic inhibitor. At a concentration of one-half the MIC, sanfetrinem significantly enhanced human PMN phagocytosis and increased intracellular bactericidal activity against penicillin-resistant S. pneumoniae. Following preexposure of PMNs to a concentration of one-half the MIC of sanfetrinem, there was a significant increase in both phagocytosis and killing compared with that for the controls, indicating the ability of sanfetrinem to interact with biological membranes and remain active within PMNs. Preexposure of streptococci to sanfetrinem made penicillin-resistant S. pneumoniae more susceptible to the bactericidal mechanisms of human PMNs than untreated organisms. PMID:9661015

  7. Intracellular transport of nanodiamond particles in human endothelial and epithelial cells.

    PubMed

    Solarska-Ściuk, Katarzyna; Gajewska, Agnieszka; Glińska, Sława; Studzian, Maciej; Michlewska, Sylwia; Balcerzak, Łucja; Skolimowski, Janusz; Kolago, Bogumiła; Bartosz, Grzegorz

    2014-08-01

    During the recent years nanodiamonds have been the subject of interest as possible means of targeted delivery of anticancer substances. Detonation nanodiamonds are attractive candidates for intracellular studies due to their synthesis methods, low cost, good biocompatibility and facile surface functionalizability. Our previous study, in which we used nanoparticles obtained by different methods showed the significance of size and way of production of nanodiamonds in their cellular effects. The aim of this study was to check the ability of surface-modified detonation nanodiamonds to reach intracellular compartments without degradation of the surface-conjugated drug or fluorescent marker. In this study we examined the penetration HUVEC-ST and A549 cells by detonation nanodiamonds (grain size <20 nm) modified by adding to, employing four pharmacological inhibitors of endocytosis, using optical, confocal and transmission electron microscopy We discuss the possibilities, the challenges of studying the endocytic pathways involved in cellular uptake of nanoparticles. Our results suggest that fluorescent nanomaterials are very promising for monitoring the intracellular fate of nanodiamonds. PMID:24882084

  8. Space Flight Effects on Intracellular Ions in Sublingual Cells of Non-Human Primates

    NASA Technical Reports Server (NTRS)

    Arnaud, Sara B.; Dotsenko, R.; Fung, P.; Navidi, M.; Silver, B.; Wade, Charles E. (Technical Monitor)

    1994-01-01

    We have used a novel technique that quantifies minerals and electrolytes from smears of sublingual cells by x-ray microanalysis to monitor metabolic changes in bed rest subjects. Increases in intracellular calcium (Ca), phosphorus (P), and potassium (K) were characteristic of subjects whose exercise regimen was inadequate to maintain calcium metabolism. To test the effects of space flight on intracellular ions, we analyzed cells from 2-4 kg Rhesus monkeys before and after 2 weeks in space or chair restraint (CR). There were increases in sublingual cell Ca, P and K after space flight which paralleled the clinical estimates of metabolic status of the animals and exceeded the levels found during CR on R+11. Increases after 2 weeks CR were 26% in Ca, 6% in P and 29% in K. Species similarity ill responses of intracellular ions to inactivity imposed by bed rest, restraint or microgravity suggest that this innovative non-invasive technique would be a useful in-flight monitor of exercise countermeasures directed toward maintaining calcium balance.

  9. Analysis of intracellular reducing levels in human hepatocytes on three-dimensional focusing microchip.

    PubMed

    Xu, Chunxiu; Cai, Longfei

    2014-02-01

    A novel three-dimensional hydrodynamic focusing microfluidic device integrated with high-throughput cell sampling and detection of intracellular contents is presented. It has a pivotal role in maintaining the reducing environment in cells. Intracellular reducing species such as vitamin C and glutathione in normal and tumor cells were labeled by a newly synthesized 2,2,6,6-tetramethyl-piperidine-1-oxyl-based fluorescent probe. Hepatocytes are adherent cells, which are prone to attaching to the channel surface. To avoid the attachment of cells on the channel surface, a single channel microchip with three sheath-flow channels located on both sides of and below the sampling channel was developed. Hydrostatic pressure generated by emptying the sample waste reservoir was used as driving force of fluid on the microchip. Owing to the difference between the liquid levels of the reservoirs, the labeled cells were three-dimensional hydrodynamically focused and transported from the sample reservoir to the sample waste reservoir. Hydrostatic pressure takes advantage of its ease of generation on a microfluidic chip without any external pressure pump, which drives three sheath-flow streams to constrain a sample flow stream into a narrow stream to avoid blockage of the sampling channel by adhered cells. The intracellular reducing levels of HepG2 cells and L02 cells were detected by home-built laser-induced fluorescence detector. The analysis throughput achieved in this microfluidic system was about 59-68 cells/min.

  10. Pine (Pinus morrisonicola Hayata) needle extracts sensitize GBM8901 human glioblastoma cells to temozolomide by downregulating autophagy and O(6)-methylguanine-DNA methyltransferase expression.

    PubMed

    Liao, Chia-Leng; Chen, Chien-Min; Chang, Yan-Zin; Liu, Guang-Yaw; Hung, Hui-Chih; Hsieh, Tung-Ying; Lin, Chih-Li

    2014-10-29

    Pine needle extracts of Pinus morrisonicola (Hayata) are commonly used as a functional health beverage. However, it remains unclear what the mechanism is underlying the antitumor activity of pine needle extract. The aims of present study were to investigate the anti-glioblastoma effects of pine needle extracts as well as its bioactive compounds. From three different solvent extracts of pine needles, the water extract displayed the strongest cytotoxicity effects on GBM8901 glioblastoma cells. The isolated compounds were identified as pinocembrin, chrysin, and tiliroside. Chrysin was the most active ingredient of pine needle extract for the induction of apoptosis and suppression of migration and invasion. It also markedly inhibited temozolomide (TMZ)-induced autophagy and O(6)-methylguanine-DNA methyltransferase (MGMT) expression. Because both autophagy and MGMT overexpression have been implicated to TMZ-induced drug resistance in glioblastoma, our results showed that pine needle extract and chrysin may serve as a potential anticancer agent against glioblastoma, especially with regard to sensitizing glioblastoma cells resistant to TMZ.

  11. RNF135, RING finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the ERK pathway

    PubMed Central

    Liu, Yongjian; Wang, Feng; Liu, Yongsheng; Yao, Yiqun; Lv, Xiupeng; Dong, Bin; Li, Jun; Ren, Siyang; Yao, Yiwen; Xu, Yinghui

    2016-01-01

    Ring finger protein 135 (RNF135), located on chromosome 17q11.2, is a RING finger domain-containing E3 ubiquitin ligase that was identified as a bio-marker and therapy target of glioblastoma. In our study, we confirmed that RNF135 was up-regulated in glioblastoma tissues compared with normal brain (NB) tissues, and that RNF135 knockdown inhibited proliferation and migration and led to cell cycle arrest in the G0/G1 phase in vivo. By lowering RNF135 expression, phosphorylated Erk and cell cycle protein CDK4 were down-regulated, while p27Kip1 and p21Waf1/Cip1 were up-regulated in U87 and U251 cells in vitro. In addition, using the immunofluorescence double labelling method, we found that RNF135 and P-Erk were co-localized in the cytoplasm and were highly expressed in glioblastoma samples compared with NB tissues. Moreover, the growth of U87 cell-transplanted tumours in nude mice was inhibited while transduced with Lv-shRNF135. Taken together, our findings demonstrate the biological effects of RNF135 in glioblastoma cell proliferation, migration and cell cycle, and its role in the progression of glioblastoma may be associated with the ERK signal transduction pathway. PMID:26856755

  12. Differential expression in glioblastoma multiforme and cerebral hemangioblastoma of cytoplasmic proteins that bind two different domains within the 3'-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.

    PubMed Central

    Tsukamoto, H; Boado, R J; Pardridge, W M

    1996-01-01

    The glucose transporter 1 (GLUT1) protein is underexpressed in human glioblastoma multiforme and is overexpressed in human cerebral hemangioblastoma. To gain in-sight into possible posttranscriptional mechanisms regulating the expression of the GLUT1 protein in human brain tumors, cytosolic proteins were prepared from these two tumors and used in RNase T1 protection assays that employed [32P]human GLUT1 synthetic RNA prepared from transcription plasmids. Gel shift mobility assays and ultra-violet light cross-linking studies demonstrated the formation of specific RNA/protein complexes that migrated with a mol mass of 120, 44, and 41 kD. RNase T1 mapping and oligodeoxynucleotide competition studies showed that the 120 kD complex was comprised of an RNA fragment that localized to nucleotides 2186-2203 of the GLUT1 mRNA. The 44 kD complex contained an adenosine-uridine-rich RNA fragment that localized to nucleotides 1885-1906 of the human GLUT1 mRNA, and the formation of this complex was inhibited by synthetic RNA enriched in adenosine-uridine sequences. The 44 kD complex was selectively downregulated in hemangioblastoma as compared to glioblastoma multiforme. These studies demonstrate that human brain tumors have differential regulation of cytosolic proteins that specifically interact with two different domains in the 3'-untranslated region of the GLUT1 mRNA, which may serve to mediate the posttranscriptional regulation of GLUT1 gene expression in these tumors. PMID:8675694

  13. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases.

    PubMed

    Lizarte Neto, F S; Tirapelli, D P C; Ambrosio, S R; Tirapelli, C R; Oliveira, F M; Novais, P C; Peria, F M; Oliveira, H F; Carlotti Junior, C G; Tirapelli, L F

    2013-01-01

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  14. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    PubMed Central

    Lizarte, F.S.; Tirapelli, D.P.C.; Ambrosio, S.R.; Tirapelli, C.R.; Oliveira, F.M.; Novais, P.C.; Peria, F.M.; Oliveira, H.F.; Carlotti, C.G.; Tirapelli, L.F.

    2013-01-01

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors. PMID:23314342

  15. SI113, a SGK1 inhibitor, potentiates the effects of radiotherapy, modulates the response to oxidative stress and induces cytotoxic autophagy in human glioblastoma multiforme cells

    PubMed Central

    Talarico, Cristina; Dattilo, Vincenzo; D'Antona, Lucia; Barone, Agnese; Amodio, Nicola; Belviso, Stefania; Musumeci, Francesca; Abbruzzese, Claudia; Bianco, Cataldo; Trapasso, Francesco; Schenone, Silvia; Alcaro, Stefano; Ortuso, Francesco; Florio, Tullio; Paggi, Marco G.; Perrotti, Nicola; Amato, Rosario

    2016-01-01

    Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy. PMID:26908461

  16. Effects of paeonol on intracellular calcium concentration and expression of RUNX3 in LoVo human colon cancer cells.

    PubMed

    Li, Ming; Tan, Shi-Yun; Zhang, Jun; You, Hong-Xia

    2013-05-01

    Paeonol, a major phenolic component of the root bark of Paeonia moutan, is known to exhibit antitumor effects. However, the underlying mechanisms remain unknown. In the present study, the effects of paeonol on cell viability, intracellular calcium concentration and the expression of runt‑related transcription factor 3 (RUNX3) were analyzed in LoVo human colon cancer cells. Results revealed that paeonol markedly reduced LoVo cell viability in a time‑ and dose‑dependent manner. Flow cytometry assays demonstrated that paeonol blocked the cell cycle at the G1 to S transition and significantly induced apoptosis in LoVo cells. Intracellular calcium accumulation occurred following a 48 h treatment with paeonol. Furthermore, RUNX3 gene expression was increased in paeonol‑treated cells. These observations indicate that paeonol possesses antiproliferative properties and apoptosis‑inducing activity. One of the antitumor mechanisms of paeonol may be its apoptosis‑inducing activity through an increased intracellular calcium concentration and the upregulation of RUNX3 expression. Paeonol may be a promising antitumor agent for colon carcinoma treatment.

  17. Intracellular multiplication of Legionella species and the influence of amoebae on their intracellular growth in human monocytes: mono mac 6 cells and Acanthamoeba castellanii as suitable in vitro models.

    PubMed

    Neumeister, Birgid

    2004-01-01

    Legionellae are important etiological agents of pneumonia. Legionella pneumophila (predominantly serogroup 1) is detected in most cases of legionellosis; other species only occasionally cause infections, predominantly in immunocompromized patients. Aquiferous technical systems are the primary source of infection (air-conditioning systems, refrigerators, showers, whirlpools, springs, taps, moisturizing equipment, medical nebulizers, and swimming pools). Legionellae are present in the water in these systems, within the amoebae, flagellates, and ciliates in which they replicate. After inhalation of contaminated aerosols, the bacteria multiply intracellularly within alveolar macrophages. The ability to multiply within monocytic host cells is usually considered to correspond to pathogenicity. The mechanisms of intracellular replication have been only partially characterized. Analysis of the molecular pathogenesis of Legionella infection, both in the pathogen itself and in the host cell, is the subject of current research and may lead to new options in prophylaxis and treatment. We have established the human Mono Mac 6 cell line (MM6) instead of the previously used histiocytic lymphoma cell line U 937 or the promyelocytic leukemia cell line HL-60 to investigate the intracellular replication of legionellae and the molecular pathogenesis of Legionella infection within human monocytic host cells. MM6 cells represent a more mature macrophage-like cell line that expresses phenotypic and functional properties of mature monocytes and that does not need to be stimulated by phorbol esters or 1,25-dihydroxyvitamin D3. A good correlation between the prevalence of a given Legionella species and its intracellular multiplication in MM6 cells could be demonstrated.In addition to Legionella, MM6 cells were found to support the intracellular growth of Mycobacterium tuberculosis and Chlamydia pneumoniae, two other important bacterial agents involved in induction of pneumonia. Therefore

  18. Intracellular Bacteria in Protozoa

    NASA Astrophysics Data System (ADS)

    Görtz, Hans-Dieter; Brigge, Theo

    Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.

  19. Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Crawford, N

    1984-08-15

    By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

  20. β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages

    PubMed Central

    Morris, Jessica D.; Rajaram, Murugesan V.S.; Schlesinger, Larry S.

    2014-01-01

    The yeast polysaccharide, β-glucan, has been shown to promote both anti-microbial and anti-tumor activities through its interaction with macrophages. Here we analyzed the effects of an insoluble whole glucan particle (WGP), a 1,3/1,6-β-glucan from Saccharomyces cerevisiae, and a soluble poly-1-6-β-d-glucopyranosyl-1-3-β-d-glucopyranose (PGG), a hydrolytic product of WGP, on the anti-microbial response of human macrophages against mycobacterial infection. Treatment of macrophages with WGP and PGG significantly decreased cell association and intracellular growth of Mycobacterium bovis BCG, but not Mycobacterium tuberculosis (M.tb) when compared to untreated controls. We characterized the influence of β-glucans on the generation of macrophage oxidative products and pro-inflammatory cytokines, two important anti-microbial defense mechanisms. WGP but not PGG treatment enhanced the oxidative response of macrophages as determined by the 2′,7′-dichlorofluorescin (DCF) assay. WGP treatment also induced macrophages to produce pro-inflammatory cytokines. The β-glucan receptor, Dectin-1, was found to be involved in the WGP-induced macrophage oxidative burst and intracellular growth inhibition of M. bovis BCG. This report indicates that although some forms of β-glucan are able to stimulate the respiratory burst and cytokine production in human macrophages, and exhibit antimicrobial properties against M. bovis BCG, the β-glucans tested here did not inhibit growth of M.tb within human macrophages. PMID:21762773

  1. The parvoviral capsid controls an intracellular phase of infection essential for efficient killing of stepwise-transformed human fibroblasts.

    PubMed

    Paglino, Justin; Tattersall, Peter

    2011-07-20

    Members of the rodent subgroup of the genus Parvovirus exhibit lytic replication and spread in many human tumor cells and are therefore attractive candidates for oncolytic virotherapy. However, the significant variation in tumor tropism observed for these viruses remains largely unexplained. We report here that LuIII kills BJ-ELR 'stepwise-transformed' human fibroblasts efficiently, while MVM does not. Using viral chimeras, we mapped this property to the LuIII capsid gene, VP2, which is necessary and sufficient to confer the killer phenotype on MVM. LuIII VP2 facilitates a post-entry, pre-DNA-amplification step early in the life cycle, suggesting the existence of an intracellular moiety whose efficient interaction with the incoming capsid shell is critical to infection. Thus targeting of human cancers of different tissue-type origins will require use of parvoviruses with capsids that effectively make this critical interaction. PMID:21600623

  2. [Roles of intracellular calcium and monomeric G-proteins in regulating exocytosis of human neutrophils].

    PubMed

    Zhu, Ying; Wang, Jun-Han; Wu, Jian-Min; Xu, Tao; Zhang, Chun-Guang

    2003-12-25

    Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level. PMID:14695488

  3. Changes in force and intracellular metabolites during fatigue of human skeletal muscle.

    PubMed Central

    Cady, E B; Jones, D A; Lynn, J; Newham, D J

    1989-01-01

    1. The relationship between intracellular metabolites and the generation of force during fatigue has been examined in the first dorsal interosseous muscle of the hand. With the arm made ischaemic, the muscle was fatigued by three bouts of maximal voluntary contraction, leaving approximately three minutes ischaemic rest between contractions. During one series of experiments intracellular phosphorus metabolites were measured by nuclear magnetic resonance during the intervals between the fatiguing contractions: in the second series contractile properties were tested with brief electrical stimulation during the rest intervals. 2. The relationships between loss of force and change in metabolite concentrations obtained with four normal subjects were compared with those from one subject with myophosphorylase deficiency (MPD) who could not utilize muscle glycogen and therefore produced no hydrogen ion from glycolysis during exercise. 3. For both the MPD and normal subjects the relationship between relative force loss and inorganic phosphate (Pi) concentration was curvilinear, force changing little in the early stages of the contraction when the intracellular Pi was accumulating rapidly but falling faster when the Pi was above 25 mM and increasing relatively slowly. 4. In the normal subjects intracellular pH fell from a mean of 7.03 +/- 0.01 (mean +/- S.E. of mean, n = 19) in the fresh muscle to 6.51 +/- 0.02 at the end of the fatiguing exercise; force, as a percentage of the initial value, fell in proportion to the increase in H+ concentration. In the MPD subject pH did not change and force loss was therefore independent of H+ accumulation. In the normal subjects the force of the fatiguing muscle showed an approximately linear relationship with the concentration of the monobasic form of inorganic phosphate. However, the MPD subject showed a quite different relationship, with force loss being much greater for a given concentration of monobasic phosphate. This result

  4. Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

    PubMed Central

    Xie, J; Drumm, M L; Zhao, J; Ma, J; Davis, P B

    1996-01-01

    The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0

  5. Targeting Bone Marrow to Potentiate the Anti-Tumor Effect of Tyrosine Kinase Inhibitor in Preclinical Rat Model of Human Glioblastoma

    PubMed Central

    Shaaban, S.; Alsulami, M.; Arbab, S.A.; Ara, R.; Shankar, A.; Iskander, A.; Angara, K.; Jain, M.; Bagher-Ebadian, H.; Achyut, B.R.; Arbab, A.S.

    2016-01-01

    Antiangiogenic agents caused paradoxical increase in pro-growth and pro-angiogenic factors and caused tumor growth in glioblastoma (GBM). It is hypothesized that paradoxical increase in pro-angiogenic factors would mobilize Bone Marrow Derived Cells (BMDCs) to the treated tumor and cause refractory tumor growth. The purposes of the studies were to determine whether whole body irradiation (WBIR) or a CXCR4 antagonist (AMD3100) will potentiate the effect of vatalanib (a VEGFR2 tyrosine kinase inhibitor) and prevent the refractory growth of GBM. Human GBM were grown orthotopically in three groups of rats (control, pretreated with WBIR and AMD3100) and randomly selected for vehicle or vatalanib treatments for 2 weeks. Then all animals underwent Magnetic Resonance Imaging (MRI) followed by euthanasia and histochemical analysis. Tumor volume and different vascular parameters (plasma volume (vp), forward transfer constant (Ktrans), back flow constant (kep), extravascular extracellular space volume (ve) were determined from MRI. In control group, vatalanib treatment increased the tumor growth significantly compared to that of vehicle treatment but by preventing the mobilization of BMDCs and interaction of CXCR4-SDF-1 using WBIR and ADM3100, respectively, paradoxical growth of tumor was controlled. Pretreatment with WBIR or AMD3100 also decreased tumor cell migration, despite the fact that ADM3100 increased the accumulation of M1 and M2 macrophages in the tumors. Vatalanib also increased Ktrans and ve in control animals but both of the vascular parameters were decreased when the animals were pretreated with WBIR and AMD3100. In conclusion, depleting bone marrow cells or CXCR4 interaction can potentiate the effect of vatalanib. PMID:27429653

  6. MicroPET/CT Imaging of an Orthotopic Model of Human Glioblastoma Multiforme and Evaluation of Pulsed Low-Dose Irradiation

    SciTech Connect

    Park, Sean S.; Chunta, John L.; Robertson, John M.; Martinez, Alvaro A.; Oliver Wong, Ching-Yee; Amin, Mitual; Wilson, George D.; Marples, Brian

    2011-07-01

    Purpose: Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Methods: Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Results: Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 x 10{sup 6} cells produced a 50- to 70-mm{sup 3} tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). Conclusion: This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage.

  7. Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities.

    PubMed

    Norkin, L C; Steinberg, V I; Kosz-Vnenchak, M

    1985-02-01

    A stable, persistent infection of A172 human glioblastoma cells with simian virus 40 (SV40) was readily established after infection at an input of 450 PFU per cell. Only 11% of the cells were initially susceptible to SV40, as shown by indirect immunofluorescent staining for the SV40 T antigen at 48 h. However, all cells produced T antigen by week 11. In contrast, viral capsid proteins were made in only about 1% of the cells in the established carrier system. Weekly viral yields ranged between 10(4) and 10(6) PFU/ml. Most of the capsid protein-producing cells contained enormous aberrant (lobulated or multiple) nuclei. Persistent viral DNA appeared in an episomal or "free" state exclusively in Southern blots and was indistinguishable from standard SV40 DNA by restriction analysis. Viral autointerference activity was not detected, and yield reduction assays did not indicate defective interfering particle activity, further implying that variant viruses were not a factor in this carrier system. Interferon was also not a factor in the system, as shown by direct challenge with vesicular stomatitis virus. Persistent infection resulted in cellular growth changes (enhanced saturation density and plating efficiency) characteristic of SV40 transformation. Persistent infection also led to an increased frequency of cytogenetic effects. These included sister chromatid exchanges, a variety of chromosomal abnormalities (ring chromosomes, acentric fragments, breaks, and gaps), and an increase in the chromosome number. Nevertheless, the persistently infected cells continued to display a bipolar glial cell-like morphology with extensive process extension and intercellular contacts.

  8. miR-29b attenuates tumorigenicity and stemness maintenance in human glioblastoma multiforme by directly targeting BCL2L2

    PubMed Central

    Chung, Hyun Joo; Choi, Young Eun; Kim, Eun Sook; Han, Young-Hoon; Park, Myung-Jin; Bae, In Hwa

    2015-01-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor and exhibits aggressive and invasive behavior. We previously identified four miRNAs—miR-29b, 494, 193a-3p, and 30e—with enhanced expression in GBM following treatment of ionizing radiation by miRNA microarray analysis. In this study, we found that only miR-29b inhibited tumor cell migration and invasion by reducing MMP-2 activity via phospho-AKT/β-catenin signaling, and stimulated a more epithelial-like morphology. Moreover, miR-29b inhibits angiogenesis by attenuating tube formation and the expression of VEGF and Ang-2, and stemness maintenance in GBM cells, as demonstrated by decreasing neurosphere formation and cancer stem cell marker protein expression. These findings support the anti-tumor properties of miR-29b in human GBM cells. Furthermore, miR-29b expression was inversely proportional to that of BCL2L2 mRNA or protein in various cancer cell types. Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. To further elucidate the relationship between miR-29b and BCL2L2 in GBM, we performed co-transfection reporter assays and determined that miR-29b downregulates BCL2L2 expression by directly binding its 3′UTR. Finally, we confirmed that BCL2L2 repression is of central importance to miR-29b anti-tumor activity using functional assays to examine cell migration, invasion, angiogenesis, and stemness. From these data, we propose that miR-29b may be a useful therapeutic agent in GBM. PMID:26155940

  9. Use of in Vivo Two-dimensional MR Spectroscopy to Compare the Biochemistry of the Human Brain to That of Glioblastoma

    PubMed Central

    Ramadan, Saadallah; Andronesi, Ovidiu C.; Stanwell, Peter; Lin, Alexander P.; Sorensen, A. Gregory

    2011-01-01

    Purpose: To develop an in vivo two-dimensional localized correlation spectroscopy technique with which to monitor the biochemistry of the human brain and the pathologic characteristics of diseases in a clinically applicable time, including ascertainment of appropriate postprocessing parameters with which to allow diagnostic and prognostic molecules to be measured, and to investigate how much of the chemical information, known to be available from malignant cultured cells, could be recorded in vivo from human brain. Materials and Methods: The study was approved by the institutional review board and was compliant with HIPAA. With use of a 3.0-T clinical magnetic resonance (MR) unit and a 32-channel head coil, localized correlation spectroscopy was performed in six healthy control subjects and six patients with glioblastoma multiforme (GBM) with an acquisition time of 11 minutes. Two-dimensional spectra were processed and analyzed and peak volume ratios were tabulated. The data used were proved to be normally distributed by passing the Shapiro-Wilk normality test. The first row of the spectra was extracted to examine diagnostic features. The pathologic characteristics and grade of each GBM were determined after biopsy or surgery. Statistically significant differences were assessed by using a t test. Results: The localized correlation spectroscopy method assigned biochemical species from the healthy human brain. The correlation spectra of GBM were of sufficiently high quality that many of the cross peaks, recorded previously from malignant cell models in vitro, were observed, demonstrating a statistically significant difference (P < .05) between the cross peak volumes measured for healthy subjects and those with GBM (which include lipid, alanine, N-acetylaspartate, γ-aminobutyric acid, glutamine and glutamate, glutathione, aspartate, lysine, threonine, total choline, glycerophosphorylcholine, myo-inositol, imidazole, uridine diphosphate glucose, isocitrate, lactate

  10. Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Croset, M; Crawford, N

    1986-02-01

    Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar

  11. Role of LIMP-2 in the intracellular trafficking of β-glucosidase in different human cellular models.

    PubMed

    Malini, Erika; Zampieri, Stefania; Deganuto, Marta; Romanello, Milena; Sechi, Annalisa; Bembi, Bruno; Dardis, Andrea

    2015-09-01

    Acid β-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.

  12. Differential localization of glioblastoma subtype: implications on glioblastoma pathogenesis

    PubMed Central

    Steed, Tyler C.; Treiber, Jeffrey M.; Patel, Kunal; Ramakrishnan, Valya; Merk, Alexander; Smith, Amanda R.; Carter, Bob S.; Dale, Anders M.; Chow, Lionel M. L.; Chen, Clark C.

    2016-01-01

    Introduction The subventricular zone (SVZ) has been implicated in the pathogenesis of glioblastoma. Whether molecular subtypes of glioblastoma arise from unique niches of the brain relative to the SVZ remains largely unknown. Here, we tested whether these subtypes of glioblastoma occupy distinct regions of the cerebrum and examined glioblastoma localization in relation to the SVZ. Methods Pre-operative MR images from 217 glioblastoma patients from The Cancer Imaging Archive were segmented automatically into contrast enhancing (CE) tumor volumes using Iterative Probabilistic Voxel Labeling (IPVL). Probabilistic maps of tumor location were generated for each subtype and distances were calculated from the centroid of CE tumor volumes to the SVZ. Glioblastomas that arose in a Genetically Modified Murine Model (GEMM) model were also analyzed with regard to SVZ distance and molecular subtype. Results Classical and mesenchymal glioblastomas were more diffusely distributed and located farther from the SVZ. In contrast, proneural and neural glioblastomas were more likely to be located in closer proximity to the SVZ. Moreover, in a GFAP-CreER; PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1−/− GEMM model of glioblastoma where tumor can spontaneously arise in different regions of the cerebrum, tumors that arose near the SVZ were more likely to be of proneural subtype (p < 0.0001). Conclusions Glioblastoma subtypes occupy different regions of the brain and vary in proximity to the SVZ. These findings harbor implications pertaining to the pathogenesis of glioblastoma subtypes. PMID:27056901

  13. Modeling of progesterone-induced intracellular calcium signaling in human spermatozoa.

    PubMed

    Li, Long-Fei; Xiang, Cheng; Zhu, Ya-Bing; Qin, Kai-Rong

    2014-06-21

    Calcium ion is a secondary messenger of mammalian spermatozoa. The dynamic change of its concentration plays a vital role in the process of sperm motility, capacitation, acrosome and fertilization. Progesterone released by the cumulus cells, as a potent stimulator of fertilization, can activate the calcium channels on the plasma membrane, which in turn triggers the dynamic change of intracellular calcium concentration. In this paper, a mathematical model of calcium dynamic response in mammalian spermatozoa induced by progesterone is proposed and numerical simulation of the dynamic model is conducted. The results show that the dynamic response of calcium concentration predicted by the model is in accordance with experimental evidence. The proposed dynamic model can be used to explain the phenomena observed in the experiments and predict new phenomena to be revealed by experimental investigations, which will provide the basis to quantitatively investigate the fluid mechanics and biochemistry for the sperm motility induced by progesterone.

  14. Quantitative non-invasive intracellular imaging of Plasmodium falciparum infected human erythrocytes

    NASA Astrophysics Data System (ADS)

    Edward, Kert; Farahi, Faramarz

    2014-05-01

    Malaria is a virulent pathological condition which results in over a million annual deaths. The parasitic agent Plasmodium falciparum has been extensively studied in connection with this epidemic but much remains unknown about its development inside the red blood cell host. Optical and fluorescence imaging are among the two most common procedures for investigating infected erythrocytes but both require the introduction of exogenous contrast agents. In this letter, we present a procedure for the non-invasive in situ imaging of malaria infected red blood cells. The procedure is based on the utilization of simultaneously acquired quantitative phase and independent topography data to extract intracellular information. Our method allows for the identification of the developmental stages of the parasite and facilitates in situ analysis of the morphological changes associated with the progression of this disease. This information may assist in the development of efficacious treatment therapies for this condition.

  15. Intracellular mechanisms of hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells.

    PubMed

    Okayama, N; Coe, L; Oshima, T; Itoh, M; Alexander, J S

    1999-03-01

    We examined which endothelial second messengers are involved in peroxide-mediated endothelial-neutrophil adhesion with respect to endothelial P-selectin expression and platelet-activating factor (PAF). Peroxide (0.5 mM)-mediated adhesion was blocked by a protein kinase C (PKC) inhibitor, Gö6976 (10 nM); an intracellular calcium chelator, TMB-8 (0.1 mM); and a protein kinase G (PKG) inhibitor, KT5823 (0.5 microM); but not by a tyrosine kinase inhibitor, genistein (1 microM), or a protein kinase A inhibitor, H-89 (0.1 microM). These data were consistent with the proadhesive effects of PMA (0.1 microM), a PKC activator; a calcium ionophore, A23187 (1 microM); and dibutyryl cGMP (0.5 and 1 mM); but not phenylarsine oxide (0.1 mM), a tyrosine phosphatase inhibitor, or dibutyryl cAMP (1 mM). Conversely, peroxide-mediated P-selectin expression was blocked by Gö6976 and KT5823, but not by TMB-8. These data are strengthened by the observation that PMA and dibutyryl cGMP, but not A23187, increased P-selectin expression. WEB 2086 (10 microM), a PAF-receptor antagonist, blocked peroxide-, PMA-, and A23187-mediated adhesion, but not peroxide-mediated P-selectin expression. PAF itself (10 nM) stimulated adhesion, but not P-selectin expression. These data indicate that PKC and PKG are involved in peroxide-mediated neutrophil adhesion via P-selectin mobilization and PAF synthesis; however, intracellular calcium appears to mediate adhesion only through PAF synthesis.

  16. Immunological Evasion in Glioblastoma

    PubMed Central

    Magaña-Maldonado, Roxana; Chávez-Cortez, Elda Georgina; Olascoaga-Arellano, Nora Karen; López-Mejía, Mariana; Maldonado-Leal, Fernando Manuel; Sotelo, Julio

    2016-01-01

    Glioblastoma is the most aggressive tumor in Central Nervous System in adults. Among its features, modulation of immune system stands out. Although immune system is capable of detecting and eliminating tumor cells mainly by cytotoxic T and NK cells, tumor microenvironment suppresses an effective response through recruitment of modulator cells such as regulatory T cells, monocyte-derived suppressor cells, M2 macrophages, and microglia as well as secretion of immunomodulators including IL-6, IL-10, CSF-1, TGF-β, and CCL2. Other mechanisms that induce immunosuppression include enzymes as indolamine 2,3-dioxygenase. For this reason it is important to develop new therapies that avoid this immune evasion to promote an effective response against glioblastoma. PMID:27294132

  17. Microarray Analysis in Glioblastomas

    PubMed Central

    Bhawe, Kaumudi M.; Aghi, Manish K.

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930–2942, 2012)To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013)To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59–70, 2013; Verhaak et al., Cancer Cell 17(1):98–110, 2010) While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  18. Microarray Analysis in Glioblastomas.

    PubMed

    Bhawe, Kaumudi M; Aghi, Manish K

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: i. To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930-2942, 2012). ii. To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013). iii. To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59-70, 2013; Verhaak et al., Cancer Cell 17(1):98-110, 2010). While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  19. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    PubMed

    Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L; Chae, Jae Jin; Shelhamer, James H

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.

  20. Prostaglandin E2 inhibits NLRP3 inflammasome activation through EP4 receptor and intracellular cAMP in human macrophages

    PubMed Central

    Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L.; Chae, Jae Jin; Shelhamer, James H.

    2015-01-01

    Prostaglandin E2 (PGE2) is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. NLRP3 inflammasome plays an important role in host defense. Uncontrolled activation of NLRP3 inflammasome, due to mutations in the NLRP3 gene causes cryopyrin-associated periodic syndromes (CAPS). Here, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through prostaglandin E receptor 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac). A specific agonist of EP4 mimicked, while its antagonist or EP4 knockdown reversed PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. PKA or Epac agonists did not mimic and their antagonists did not reverse PGE2-mediated NLRP3 inhibition. In addition, constitutive IL-1β secretion from LPS-primed PBMCs of CAPS patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or siRNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  1. Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models

    PubMed Central

    Katayanagi, Mishina; Hashimoto, Fumie

    2015-01-01

    Background Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. Objective This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax, respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Methods Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Results Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. Conclusion LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis. PMID:26082583

  2. β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages.

    PubMed

    Betz, Bret E; Azad, Abul K; Morris, Jessica D; Rajaram, Murugesan V S; Schlesinger, Larry S

    2011-10-01

    The yeast polysaccharide, β-glucan, has been shown to promote both anti-microbial and anti-tumor activities through its interaction with macrophages. Here we analyzed the effects of an insoluble whole glucan particle (WGP), a 1,3/1,6-β-glucan from Saccharomyces cerevisiae, and a soluble poly-1-6-β-d-glucopyranosyl-1-3-β-d-glucopyranose (PGG), a hydrolytic product of WGP, on the anti-microbial response of human macrophages against mycobacterial infection. Treatment of macrophages with WGP and PGG significantly decreased cell association and intracellular growth of Mycobacterium bovis BCG, but not Mycobacterium tuberculosis (M.tb) when compared to untreated controls. We characterized the influence of β-glucans on the generation of macrophage oxidative products and pro-inflammatory cytokines, two important anti-microbial defense mechanisms. WGP but not PGG treatment enhanced the oxidative response of macrophages as determined by the 2',7'-dichlorofluorescin (DCF) assay. WGP treatment also induced macrophages to produce pro-inflammatory cytokines. The β-glucan receptor, Dectin-1, was found to be involved in the WGP-induced macrophage oxidative burst and intracellular growth inhibition of M. bovis BCG. This report indicates that although some forms of β-glucan are able to stimulate the respiratory burst and cytokine production in human macrophages, and exhibit anti-microbial properties against M. bovis BCG, the β-glucans tested here did not inhibit growth of M.tb within human macrophages.

  3. Nanoparticle-Mediated Intracellular Delivery Enables Cryopreservation of Human Adipose-Derived Stem Cells Using Trehalose as the Sole Cryoprotectant

    PubMed Central

    Rao, Wei; Huang, Haishui; Wang, Hai; Zhao, Shuting; Dumbleton, Jenna; Zhao, Gang; He, Xiaoming

    2016-01-01

    In this study, pH responsive genipin-crosslinked Pluronic F127-chitosan nanoparticles (GNPs) was synthesized to encapsulate trehalose for intracellular delivery to cryopreserve primary human adipose-derived stem cells (hADSCs). Trehalose is a disaccharide of glucose used by lower-organisms to survive extreme cold in nature and has been used to cryopreserve various biomacromolecules. However, it does not enter mammalian cells due to its highly hydrophilic nature and has only been used in combination with other cell-penetrating cryoprotectants such as DMSO to cryopreserve mammalian cells. Our data show that trehalose can be efficiently encapsulated in our GNPs for intracellular delivery, which enables cryopreservation of primary hADSCs using the nontoxic sugar as the sole cryoprotectant. This capability is important because the conventional approach of cryopreserving mammalian cells using highly toxic (at body temperature) cell-penetrating cryoprotectants requires multi-step washing of the cryopreserved cells to remove the toxic cryoprotectant for further use, which is time-consuming and associated with significant cell loss (~10% during each washing step). By contrast, the trehalose-cryopreserved cells can be used without washing, which should significantly facilitate the wide application of the burgeoning cell-based medicine. PMID:25679454

  4. P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages1

    PubMed Central

    Lees, Michael P.; Fuller, Stephen J.; McLeod, Rima; Boulter, Nicola R.; Miller, Catherine M.; Zakrzewski, Alana M.; Mui, Ernest J.; Witola, William H.; Coyne, Jessica J.; Hargrave, Aubrey C.; Jamieson, Sarra E.; Blackwell, Jenefer M.; Wiley, James S.; Smith, Nicholas C.

    2010-01-01

    The P2X7 receptor (P2X7R)4 is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms (SNPs) that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this paper we show that macrophages from people with the 1513C (rs3751143) loss-of-function P2X7R SNP are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knock-out mice (P2X7R−/−) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production. PMID:20488797

  5. Genetics and Epigenetics of Glioblastoma: Applications and Overall Incidence of IDH1 Mutation

    PubMed Central

    Liu, Aizhen; Hou, Chunfeng; Chen, Hongfang; Zong, Xuan; Zong, Peijun

    2016-01-01

    Glioblastoma is the most fatal brain cancer found in humans. Patients suffering from glioblastoma have a dismal prognosis, with a median survival of 15 months. The tumor may develop rapidly de novo in older patients or through progression from anaplastic astrocytomas in younger patients if glioblastoma is primary or secondary, respectively. During the past decade, significant advances have been made in the understanding of processes leading to glioblastoma, and several important genetic defects that appear to be important for the development and progression of this tumor have been identified. Particularly, the discovery of recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) gene has shed new light on the molecular landscape in glioblastoma. Indeed, emerging research on the consequences of mutant IDH1 protein expression suggests that its neomorphic enzymatic activity catalyzing the production of the oncometabolite 2-hydroxyglutarate influences a range of cellular programs that affect the epigenome and contribute to glioblastoma development. One of the exciting observations is the presence of IDH1 mutation in the vast majority of secondary glioblastoma, while it is almost absent in primary glioblastoma. Growing data indicate that this particular mutation has clinical and prognostic importance and will become a critical early distinction in diagnosis of glioblastoma. PMID:26858939

  6. Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

    PubMed

    Gobin, V; De Bock, M; Broeckx, B J G; Kiselinova, M; De Spiegelaere, W; Vandekerckhove, L; Van Steendam, K; Leybaert, L; Deforce, D

    2015-09-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression.

  7. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    PubMed

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M; Gupta, Goutam; McCleskey, T Mark; Chaudhary, Anu

    2009-12-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1beta secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.

  8. Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

    SciTech Connect

    Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.; Moore, Ronald J.; Clauss, Therese RW; Monroe, Matthew E.; Du, Xiuxia; Adkins, Joshua N.; Wong, Scott; Smith, Richard D.

    2008-03-07

    Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.

  9. Caffeine-induced nuclear translocation of FoxO1 triggers Bim-mediated apoptosis in human glioblastoma cells.

    PubMed

    Sun, Fei; Han, Dong-Feng; Cao, Bo-Qiang; Wang, Bo; Dong, Nan; Jiang, De-Hua

    2016-03-01

    Caffeine is one of the most commonly ingested neuroactive compounds and exhibits anticancer effects through induction of apoptosis and suppression of cell proliferation. However, the mechanisms underlying these effects are currently unknown. In this study, we investigated the mechanisms of caffeine-induced apoptosis in U251 cells (human glioma cell line). We analyzed the inhibitory effects of caffeine on cell proliferation by performing WST-8 and colony formation assays; in addition, cell survival was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and flow cytometric analysis. Western blotting was used to investigate the role played by FoxO1 in the proapoptotic effects of caffeine on glioma cells. Results showed that caffeine inhibited proliferation and survival of human glioma cells, induced apoptosis, and increased the expression of FoxO1 and its proapoptotic target Bim. In addition, we found that FoxO1 enhanced the transcription of its proapoptotic target Bim. In summary, our data indicates that FoxO1-Bim mediates caffeine-induced regression of glioma growth by activating cell apoptosis, thereby providing new mechanistic insight into the possible use of caffeine in treating human cancer.

  10. Tyrphostin AG 1296 induces glioblastoma cell apoptosis in vitro and in vivo

    PubMed Central

    LI, HONGWEI; ZHENG, JUNNING; GUAN, RUIYUN; ZHU, ZIFENG; YUAN, XIANHOU

    2015-01-01

    Glioblastoma is the most common type of malignant human brain tumor. Currently available chemotherapies for glioblastoma focus on targeting tyrosine kinases. However, the existing inhibitors of tyrosine kinases have not produced the therapeutic outcomes that were anticipated. In order to investigate the viability alternative chemotherapeutic agents in this disease, the present study examined the anticancer effects of tyrphostin AG 1296, focusing on its involvement in apoptosis in glioblastoma cells. The study aimed to identify whether tyrphostin AG 1296 affects glioblastoma cell growth by inducing cell apoptosis. To achieve this, cell viability, propidium iodide analysis and cell invasion assay were used to measure cell growth, cell apoptosis and cell migration of human glioblastoma cells. The results showed that tyrphostin AG 1296 treatment reduced cell viability and suppressed migration of human glioblastoma cells. It was also demonstrated that tyrphostin AG 1296 induced cell apoptosis in vitro. Finally, tyrphostin AG 1296 was also shown to significantly inhibit the growth of glioblastoma cells and to increase tumor cell apoptosis in vivo. These findings suggest that tyrphostin AG 1296 induces apoptosis, thereby reducing cell viability and capacity for migration of glioblastoma cells. PMID:26788146

  11. Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β

    PubMed Central

    Paino, Annamari; Tuominen, Heidi; Jääskeläinen, Mari; Alanko, Jonna; Nuutila, Jari; Asikainen, Sirkka E.; Pelliniemi, Lauri J.; Pöllänen, Marja T.; Chen, Casey; Ihalin, Riikka

    2011-01-01

    Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation. PMID:21533109

  12. Locking intracellular helices 2 and 3 together inactivates human P-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2014-01-01

    The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(β,γ-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.

  13. Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

    PubMed

    Hare, Joanna E; Goodchild, Sophia C; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

    2016-07-12

    Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35. PMID:27299171

  14. Intracellular Analysis of the Interaction between the Human Papillomavirus Type 16 E6 Oncoprotein and Inhibitory Peptides

    PubMed Central

    Stutz, Christina; Reinz, Eileen; Honegger, Anja; Bulkescher, Julia; Schweizer, Johannes; Zanier, Katia; Travé, Gilles; Lohrey, Claudia; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2015-01-01

    Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer and other malignancies in humans. The HPV E6 oncoprotein is considered to be an attractive therapeutic target since its inhibition can lead to the apoptotic cell death of HPV-positive cancer cells. The HPV type 16 (HPV16) E6-binding peptide pep11, and variants thereof, induce cell death specifically in HPV16-positive cancer cells. Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket. Thus, these peptides can serve as prototype E6-inhibitory molecules which target the E6AP pocket. We here analyzed their intracellular interaction with HPV16 E6. By comprehensive intracellular binding studies and GST pull-down assays, we show that E6-binding competent pep11 variants induce the formation of a trimeric complex, consisting of pep11, HPV16 E6 and p53. These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53. The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts. Yet, total cellular p53 amounts were also increased, indicating that the E6 / E6AP-mediated degradation of p53 is blocked. These findings suggest that inhibition of oncogenic activities by targeting the E6AP pocket on HPV16 E6 could be a strategy for therapeutic intervention. PMID:26151636

  15. Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress

    NASA Technical Reports Server (NTRS)

    Stamatas, G. N.; Patrick, C. W. Jr; McIntire, L. V.

    1997-01-01

    The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

  16. The role of intracellular calcium signals in inflammatory responses of polarised cystic fibrosis human airway epithelia.

    PubMed

    Ribeiro, Carla Maria Pedrosa

    2006-01-01

    Hyperinflammatory host responses to bacterial infection have been postulated to be a key step in the pathogenesis of cystic fibrosis (CF) lung disease. Previous studies have indicated that the CF airway epithelium itself contributes to the hyperinflammation of CF airways via an excessive inflammatory response to bacterial infection. However, it has been controversial whether the hyperinflammation of CF epithelia results from mutations in the CF transmembrane conductance regulator (CFTR) and/or is a consequence of persistent airways infection. Recent studies have demonstrated that intracellular calcium (Ca2+i) signals consequent to activation of apical G protein-coupled receptors (GPCRs) by pro-inflammatory mediators are increased in CF airway epithelia. Because of the relationship between Ca2+i mobilisation and inflammatory responses, the mechanism for the increased Ca2+i signals in CF was investigated and found to result from endoplasmic reticulum (ER) Ca2+ store expansion. The ER Ca2+ store expansion imparts a hyperinflammatory phenotype to chronically infected airway epithelia as a result of the larger Ca2+i mobilisation coupled to an excessive inflammatory response following GPCR activation. The ER expansion is not dependent on ER retention of misfolded DeltaF508 CFTR, but reflects an epithelial response acquired following persistent luminal airway infection. With respect to the mechanism of ER expansion in CF, the current view is that chronic airway epithelial infection triggers an unfolded protein response as a result of the increased flux of newly synthesised inflammatory mediators and defensive factors into the ER compartment. This unfolded protein response is coupled to X-box binding protein 1 (XBP-1) mRNA splicing and transcription of genes associated with the expansion of the protein-folding capacity of the ER (e.g. increases in ER chaperones and ER membranes). These studies have revealed a novel adaptive response in chronically infected airway epithelia

  17. Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2.

    PubMed

    Forest, Chantal G; Ferraro, Elyse; Sabbagh, Sébastien C; Daigle, France

    2010-12-01

    For successful infection, Salmonella enterica secretes and injects effector proteins into host cells by two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands (SPIs)-1 and -2. The SPI-2 T3SS is involved in intracellular survival of S. enterica serovar Typhimurium and systemic disease. As little is known regarding the function of the SPI-2 T3SS from S. enterica serovar Typhi, the aetiological agent of typhoid fever, we investigated its role for survival in human macrophages. Mutations in the translocon (sseB), basal secretion apparatus (ssaR) and regulator (ssrB) did not result in any reduction in survival under many of the conditions tested. Similar results were obtained with another S. Typhi strain or by using human primary cells. Results were corroborated based on complete deletion of the SPI-2 T3SS. Surprisingly, the data suggest that the SPI-2 T3SS of S. Typhi is not required for survival in human macrophages.

  18. A novel ultrasensitive LC-MS/MS assay for quantification of intracellular raltegravir in human cell extracts

    PubMed Central

    Robbins, Brian L.; Nelson, Sarah R.; Fletcher, Courtney V.

    2012-01-01

    An assay using ultrahigh pressure liquid chromatography and mass spectrometry detection was developed and validated for measurement of the HIV integrase inhibitor raltegravir (MK-0518) in human cell extracts. The assay is designed to utilize 200 µl of 70% MeOH cell extract derived from human peripheral blood mononuclear cells or human tissue samples. The assay is linear over a range from 0.0023 to 9.2 ng ml−1. The average % CV (SD/Mean)*100 and % Deviation ((observed – target)/target)*100 were less than 20% at the lower limit of quantification and less than 15% over the range of the curve. This assay is an accurate and highly sensitive method for determining raltegravir concentrations in cellular extracts with a lower limit 40 to over 100-fold lower than other methods in the literature. We also present a new processing method where a rapid spin through oil produced a significant increase in apparent intracellular raltegravir concentration compared with conventional processing. PMID:22727807

  19. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  20. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    PubMed Central

    Ramanauskiene, Kristina; Raudonis, Raimondas

    2016-01-01

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies.

  1. Rosmarinic Acid and Melissa officinalis Extracts Differently Affect Glioblastoma Cells

    PubMed Central

    Ramanauskiene, Kristina; Raudonis, Raimondas

    2016-01-01

    Lemon balm (Melissa officinalis L.) has many biological effects but especially important is its neuroprotective activity. The aim of the study is to produce different extracts of Melissa officinalis and analyse their chemical composition and biological properties on rat glioblastoma C6 cells. Results revealed that rosmarinic acid (RA) is the predominant compound of lemon balm extracts. RA has cytotoxic effect on glioblastoma cells (LC50 290.5 μM after the incubation of 24 h and LC50 171.3 μM after 48 h). RA at concentration 80–130 μM suppresses the cell proliferation and has an antioxidant effect. 200 μM and higher concentrations of RA have a prooxidant effect and initiate cell death through necrosis. The aqueous extract of lemon balm is also enriched in phenolic compounds: protocatechuic, caftaric, caffeic, ferulic, and cichoric acids and flavonoid luteolin-7-glucoside. This extract at concentrations 50 μM–200 μM RA has cytotoxic activity and initiates cell death through apoptosis. Extracts prepared with 70% ethanol contain the biggest amount of active compounds. These extracts have the highest cytotoxic activity on glioblastoma cells. They initiate generation of intracellular ROS and cell death through apoptosis and necrosis. Our data suggest that differently prepared lemon balm extracts differently affect glioblastoma cells and can be used as neuroprotective agents in several therapeutic strategies. PMID:27688825

  2. Oncogenic Role of Merlin/NF2 in Glioblastoma

    PubMed Central

    Guerrero, Paola A.; Yin, Wei; Camacho, Laura; Marchetti, Dario

    2014-01-01

    Glioblastoma is the most common and aggressive primary brain tumor in adults, with a poor prognosis because of its resistance to radiotherapy and chemotherapy. Merlin/NF2 (neurofibromatosis type 2) is a tumor suppressor found to be mutated in most nervous system tumors; however, it is not mutated in glioblastomas. Merlin associates with several transmembrane receptors and intracellular proteins serving as an anchoring molecule. Additionally, it acts as a key component of cell motility. By selecting subpopulations of U251 glioblastoma cells, we observed that high expression of phosphorylated Merlin at serine 518 (S518-Merlin), Notch1 and epidermal growth factor receptor (EGFR) correlated with increased cell proliferation and tumorigenesis. These cells were defective in cell-contact inhibition with changes in Merlin phosphorylation directly affecting Notch1, EGFR expression as well as downstream targets Hes1 and Ccnd. Of note, we identified a function for S518-Merlin which is distinct from what has been reported when the expression of Merlin is diminished in relation to EGFR and Notch expression, providing first-time evidence that demonstrates that the phosphorylation of Merlin at S518 in glioblastoma promotes oncogenic properties that are not only the result of inactivation of the tumor suppressor role of Merlin, but also, an independent process implicating a Merlin-driven regulation of Notch1 and EGFR. PMID:25043298

  3. A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

    SciTech Connect

    Vik-Mo, Einar Osland; Sandberg, Cecilie; Joel, Mrinal; Stangeland, Biljana; Watanabe, Yasuhiro; Mackay-Sim, Alan; Moe, Morten Carstens; Murrell, Wayne; Langmoen, Iver Arne

    2011-04-15

    Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2-93%)/SSEA1 (3-15%)/CXCR4 (1-72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells, but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.

  4. Targeting and intracellular trafficking of clinically relevant hTHTR1 mutations in human cell lines.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Said, Hamid M

    2007-07-01

    The micronutrient thiamine is required for normal growth and development of human tissues, and is accumulated into cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in human cell lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Delta383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, cellular targeting and/or protein transport activity.

  5. Modulation of cellular calcium by sigma-2 receptors: release from intracellular stores in human SK-N-SH neuroblastoma cells.

    PubMed

    Vilner, B J; Bowen, W D

    2000-03-01

    Human SK-N-SH neuroblastoma cells expressed sigma-1 and sigma-2 receptors with similar pharmacological profiles to those of rodent-derived tissues, although sigma-2 receptors exhibited some affinity differences that might suggest heterogeneity or species differences. Structurally diverse sigma ligands produced two types of increases in intracellular (cytosolic) Ca(2+) concentration ([Ca(2+)](i)) in these cells. CB-64D, CB-64L, JL-II-147, BD737, LR172, BD1008, haloperidol, reduced haloperidol, and ibogaine all produced an immediate, dose-dependent, and transient rise in [Ca(2+)](i). Sigma-inactive compounds structurally similar to the most active sigma ligands and ligands for several neurotransmitter receptors produced little or no effect. The high activity of CB-64D and ibogaine (sigma-2-selective ligands) compared with the low activity of (+)-pentazocine and other (+)-benzomorphans (sigma-1-selective ligands), in addition to enantioselectivity for CB-64D over CB-64L, strongly indicated mediation by sigma-2 receptors. The effect of CB-64D and BD737 was blocked by the sigma antagonists BD1047 and BD1063, further confirming specificity as a receptor-mediated event. The transient rise in [Ca(2+)](i) occurred in the absence of extracellular Ca(2+) and was completely eliminated by pretreatment of cells with thapsigargin. Thus, sigma-2 receptors stimulate a transient release of Ca(2+) from the endoplasmic reticulum. Prolonged exposure of cells to sigma-receptor ligands resulted in a latent and sustained rise in [Ca(2+)](i), with a pharmacological profile identical to that of the transient rise. This sustained rise in [Ca(2+)](i) was affected by neither the removal of extracellular Ca(2+) nor thapsigargin pretreatment, suggesting latent sigma-2 receptor-induced release from thapsigargin-insensitive intracellular Ca(2+) stores. Sigma-2 receptors may use Ca(2+) signals in producing cellular effects.

  6. Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity

    PubMed Central

    Vicetti Miguel, Rodolfo D.; Harvey, Stephen A. K.; LaFramboise, William A.; Reighard, Seth D.; Matthews, Dean B.; Cherpes, Thomas L.

    2013-01-01

    While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4+ T cells. Also among women with genital tract Chlamydia infection, peripheral CD3+ CD4+ and CD3+ CD4- cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures. PMID:23555586

  7. Confocal microscopy as a tool to reveal the tridimensional organization of intracellular lumens and intercellular cysts in a human colon adenocarcinoma cell line.

    PubMed

    Remy, L; Gorvel, J P; Jacquier, M F; Rigal, A; Davoust, J

    1990-01-01

    Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

  8. A critical intracellular concentration of fully reduced non-methylated folate polyglutamates prevents macrocytosis and diminished growth rate of human cell line K562 in culture.

    PubMed Central

    Watkins, D; Cooper, B A

    1983-01-01

    Growth rate of human leukaemic cell line K562 was independent of intracellular folate concentration when this was greater than 1.5 microM. When intracellular folate concentration was less than 1.5 microM, the rate of growth was proportional to the logarithm of intracellular concentration of non-methylated fully reduced folates, but not to the logarithm of the intracellular concentration of N5-methyltetrahydropteroylglutamate. Intracellular folate concentration sufficient to support an optimal growth rate was maintained by either DL-N5-formyltetrahydropteroylglutamate or DL-N5-methyltetrahydropteroylglutamate at a 100-fold lower concentration than pteroylglutamate. Addition of hypoxanthine to culture medium partially restored growth of folate-depleted cells: thymidine had no effect on growth rate either alone or in combination with thymidine. Folate-depleted cells with diminished growth rate were larger than replete cells, but did not have megaloblastic morphology. The mitotic index was not decreased in cultures with diminished growth rate. The rate of growth and cell size of K562 cells is thus dependent on a critical intracellular concentration of non-methylated tetrahydrofolates, which may be maintained by different concentrations of either reduced folates or pteroylglutamate. PMID:6577860

  9. A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

    PubMed Central

    Scott, G K; Robles, R; Park, J W; Montgomery, P A; Daniel, J; Holmes, W E; Lee, J; Keller, G A; Li, W L; Fendly, B M

    1993-01-01

    Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors. Images PMID:8096058

  10. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

    PubMed Central

    Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, Mauro

    2016-01-01

    The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. PMID:27437406

  11. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria.

    PubMed

    Schiavano, Giuditta Fiorella; Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, Mauro

    2016-01-01

    The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. PMID:27437406

  12. The anti-hypertensive drug prazosin inhibits glioblastoma growth via the PKCδ-dependent inhibition of the AKT pathway.

    PubMed

    Assad Kahn, Suzana; Costa, Silvia Lima; Gholamin, Sharareh; Nitta, Ryan T; Dubois, Luiz Gustavo; Fève, Marie; Zeniou, Maria; Coelho, Paulo Lucas Cerqueira; El-Habr, Elias; Cadusseau, Josette; Varlet, Pascale; Mitra, Siddhartha S; Devaux, Bertrand; Kilhoffer, Marie-Claude; Cheshier, Samuel H; Moura-Neto, Vivaldo; Haiech, Jacques; Junier, Marie-Pierre; Chneiweiss, Hervé

    2016-01-01

    A variety of drugs targeting monoamine receptors are routinely used in human pharmacology. We assessed the effect of these drugs on the viability of tumor-initiating cells isolated from patients with glioblastoma. Among the drugs targeting monoamine receptors, we identified prazosin, an α1- and α2B-adrenergic receptor antagonist, as the most potent inducer of patient-derived glioblastoma-initiating cell death. Prazosin triggered apoptosis of glioblastoma-initiating cells and of their differentiated progeny, inhibited glioblastoma growth in orthotopic xenografts of patient-derived glioblastoma-initiating cells, and increased survival of glioblastoma-bearing mice. We found that prazosin acted in glioblastoma-initiating cells independently from adrenergic receptors. Its off-target activity occurred via a PKCδ-dependent inhibition of the AKT pathway, which resulted in caspase-3 activation. Blockade of PKCδ activation prevented all molecular changes observed in prazosin-treated glioblastoma-initiating cells, as well as prazosin-induced apoptosis. Based on these data, we conclude that prazosin, an FDA-approved drug for the control of hypertension, inhibits glioblastoma growth through a PKCδ-dependent mechanism. These findings open up promising prospects for the use of prazosin as an adjuvant therapy for glioblastoma patients. PMID:27138566

  13. Synergistic anti-tumor actions of luteolin and silibinin prevented cell migration and invasion and induced apoptosis in glioblastoma SNB19 cells and glioblastoma stem cells.

    PubMed

    Chakrabarti, Mrinmay; Ray, Swapan K

    2015-12-10

    Glioblastoma is the most lethal brain tumor. Failure of conventional chemotherapies prompted the search for natural compounds for treatment of glioblastoma. Plant-derived flavonoids could be alternative medicine for inhibiting not only glioblastoma cells but also glioblastoma stem cells (GSC). Two plant-derived flavonoids are luteolin (LUT) and silibinin (SIL). We investigated anti-tumor mechanisms of LUT and SIL in different human glioblastoma cells and GSC and found significant synergistic inhibition of human glioblastoma LN18 and SNB19 cells and GSC following treatment with combination of 20µM LUT and 50µM SIL. Combination of 20µM LUT and 50µM SIL was more effective than a conventional chemotherapeutic agent (BCNU or TMZ). We continued our studies with SNB19 cells and GSC and found dramatic inhibition of cell migration from spheroids and also cell invasion through matrigel following treatment with combination of LUT and SIL. This combination was highly effective to block angiogenesis and survival pathways leading to induction of apoptosis. Inhibition of PKCα, XIAP, and iNOS ultimately caused induction of extrinsic and intrinsic pathways of apoptosis. Collectively, synergistic efficacy of LUT and SIL could be a promising therapy to inhibit cell migration and invasion and induce apoptosis in different glioblastoma cells including GSC.

  14. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

    PubMed

    Lahouassa, Hichem; Daddacha, Waaqo; Hofmann, Henning; Ayinde, Diana; Logue, Eric C; Dragin, Loïc; Bloch, Nicolin; Maudet, Claire; Bertrand, Matthieu; Gramberg, Thomas; Pancino, Gianfranco; Priet, Stéphane; Canard, Bruno; Laguette, Nadine; Benkirane, Monsef; Transy, Catherine; Landau, Nathaniel R; Kim, Baek; Margottin-Goguet, Florence

    2012-03-01

    SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate. PMID:22327569

  15. A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

    PubMed

    Wu, Fang; Christen, Philipp; Gehring, Heinz

    2011-07-01

    Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 μM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest. PMID:21454364

  16. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody.

    PubMed Central

    Marasco, W A; Haseltine, W A; Chen, S Y

    1993-01-01

    A single-chain antibody, derived from a human monoclonal antibody that recognizes the CD4 binding region of the human immunodeficiency virus type 1 (HIV-1) envelope protein, has been designed for intracellular expression in eukaryotic cells. The single-chain antibody is composed of an immunoglobulin heavy-chain leader sequence and heavy- and light-chain variable regions that are joined by an interchain linker. The antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic to the cells. The antibody binds to the envelope protein within the cell and inhibits processing of the envelope precursor and syncytia formation. The infectivity of the HIV-1 particles produced by cells that express the single-chain antibody is substantially reduced. These studies illustrate the feasibility of designing antibodies that bind and inactivate molecules intracellularly. Antibodies that act on target molecules within cells should provide a useful tool for research as well as for control of infectious and other diseases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8356098

  17. Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.

    PubMed

    Simões-Wüst, A P; Grãos, M; Duarte, C B; Brenneisen, R; Hamburger, M; Mennet, M; Ramos, M H; Schnelle, M; Wächter, R; Worel, A M; von Mandach, U

    2010-10-01

    The use of preparations from Bryophyllum pinnatum in tocolysis is supported by both clinical (retrospective comparative studies) and experimental (using uterus strips) evidence. We studied here the effect of B. pinnatum juice on the response of cultured human myometrial cells to stimulation by oxytocin, a hormone known to be involved in the control of uterine contractions by increasing the intracellular free calcium concentration ([Ca2+]i). In this work, [Ca2+]i was measured online during stimulation of human myometrial cells (hTERT-C3 and M11) with oxytocin, which had been pre-incubated in the absence or in the presence of B. pinnatum juice. Since no functional voltage-gated Ca2+ channels could be detected in these myometrial cells, the effect of B. pinnatum juice was as well studied in SH-SY5Y neuroblastoma cells, which are known to have such channels and can be depolarised with KCl. B. pinnatum juice prevented the oxytocin-induced increase in [Ca2+]i in hTERT-C3 human myometrial cells in a dose-dependent manner, achieving a ca. 80% inhibition at a 2% concentration. Comparable results were obtained with M11 human primary myometrial cells. In hTERT-C3 cells, prevention of the oxytocin-induced increase in [Ca2+]i was independent of the extracellular Ca2+ concentration and of voltage-dependent Ca2+-channels. B. pinnatum juice delayed, but did not prevent the depolarization-induced increase in [Ca2+]i in SH-SY5Y cells. Taken together, the data suggest a specific and concentration-dependent effect of B. pinnatum juice on the oxytocin signalling pathway, which seems to corroborate its use in tocolysis. Such a specific mechanism would explain the rare and minor side-effects in tocolysis with B. pinnatum as well as its high therapeutic index. PMID:20381326

  18. GLUT-1-independent infection of the glioblastoma/astroglioma U87 cells by the human T cell leukemia virus type 1

    SciTech Connect

    Jin Qingwen; Agrawal, Lokesh; VanHorn-Ali, Zainab; Alkhatib, Ghalib . E-mail: galkhati@iupui.edu

    2006-09-15

    The human glucose transporter protein 1 (GLUT-1) functions as a receptor for human T cell leukemia virus (HTLV). GLUT-1 is a twelve-transmembrane cell surface receptor with six extracellular (ECL) and seven intracellular domains. To analyze HTLV-1 cytotropism, we utilized polyclonal antibodies to a synthetic peptide corresponding to the large extracellular domain of GLUT-1. The antibodies caused significant blocking of envelope (Env)-mediated fusion and pseudotyped virus infection of HeLa cells but had no significant effect on infection of U87 cells. This differential effect correlated with the detection of high-level surface expression of GLUT-1 on HeLa cells and very weak staining of U87 cells. To investigate this in terms of viral cytotropism, we cloned GLUT-1 cDNA from U87 cells and isolated two different versions of cDNA clones: the wild-type sequence (encoding 492 residues) and a mutant cDNA with a 5-base pair deletion (GLUT-1{delta}5) between nucleotides 1329 and 1333. The deletion, also detected in genomic DNA, resulted in a frame-shift and premature termination producing a truncated protein of 463 residues. Transfection of the wild-type GLUT-1 but not GLUT-1{delta}5 cDNA into CHO cells resulted in efficient surface expression of the human GLUT-1. Co-expression of GLUT-1 with GLUT-1{delta}5 produces a trans-inhibition by GLUT-1{delta}5 of GLUT-1-mediated HTLV-1 envelope (Env)-mediated fusion. Co-immunoprecipitation experiments demonstrated physical interaction of the wild-type and mutant proteins. Northern blot and RT-PCR analyses demonstrated lower GLUT-1 RNA expression in U87 cells. We propose two mechanisms to account for the impaired cell surface expression of GLUT-1 on U87 cells: low GLUT-1 RNA expression and the formation of GLUT-1/GLUT-1{delta}5 heterodimers that are retained intracellularly. Significant RNAi-mediated reduction of endogenous GLUT-1 expression impaired HTLV-1 Env-mediated fusion with HeLa cells but not with U87 cells. We propose a

  19. Immunoperoxidase staining of surface and intracellular immunoglobulin in human neoplastic lymphoid cells.

    PubMed

    Mason, D Y; Leonard, R C; Laurent, G; Gourdin, M F

    1980-07-01

    An immunoperoxidase technique for the optical microscopic detection of cellular immunoglobulin has been used to stain fixed smears of human neoplastic B lymphoid cells. Only four out of 28 cases of chronic lymphatic leukaemia (CLL) showed membrane labelling by this technique. In contrast, when 14 samples from other types of B lymphoproliferative disorder (including hairy cell leukaemia, non-Hodgkin's lymphoma, and prolymphocytic leukaemia) were studied, all samples showed membrane immunoglobulin labelling (confirmed by capping experiments). This discrepancy was attributed to the greater density of surface immunoglobulin present on neoplastic cells in the latter group of disorders compared to CLL. This immunoperoxidase technique is therefore less sensitive than immunofluorescent staining of cells in suspension for the demonstration of neoplastic cell surface immunoglobulin. However, it offers a number of advantages (eg, excellent visualisation of cell morphology, permanence of stained preparations, and applicability to stored samples) which recommend it as the method of choice in certain clinical haematological contexts. PMID:7000833

  20. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.

    PubMed

    Willumsen, N J; Boucher, R C

    1992-09-01

    1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in

  1. Glioblastoma with oligodendroglial components: glioblastoma or anaplastic oligodendroglial tumors.

    PubMed

    Takeuchi, Hiroaki; Hosoda, Tetsuya; Kitai, Ryuhei; Kodera, Toshiaki; Arishima, Hidetaka; Tsunetoshi, Kenzo; Neishi, Hiroyuki; Yamauchi, Takahiro; Sato, Kazufumi; Imamura, Yoshiyuki; Itoh, Hiroshi; Kubota, Toshihiko; Kikuta, Ken-ichiro

    2012-07-01

    There have been some recent reports about glioblastoma with oligodendroglial (OG) components and malignant glioma with primitive neuroectodermal tumor (PNET)-like components. We investigated whether the presence and extent of OG components and PNET-like components influenced the prognosis in patients with glioblastoma. Eighty-six patients with glioblastoma were divided into an OG group (28 %), which revealed areas with a honeycomb appearance, and a non-OG group (72 %) without a honeycomb appearance. Patients with glioblastoma were also divided into a PNET group (27 %), which revealed areas with PNET-like features defined as neoplastic cells with high N/C ratios and hyperchromatic oval-carrot-shaped nuclei, and lacked the typical honeycomb appearance, and a non-PNET group (73 %) without PNET features. There were no significant differences in overall survival among the OG, the non-OG, the PNET, and the non-PNET groups. Two patients who survived longer than 36 months had both OG and PNET components with 1p or 19q loss of heterozygosity. Perinuclear halo, which is a characteristic feature of oligodendrogliomas, is an artifact of tissue fixation. Therefore, we should not readily use the term glioblastoma with OG components. PNET-like components, which are considered rare in malignant gliomas, may be frequently identified in glioblastomas. PMID:22527749

  2. Replacement of Val3 in Human Thymidylate Synthase Affects Its Kinetic Properties and Intracellular Stability

    SciTech Connect

    Huang, Xiao; Gibson, Lydia M.; Bell, Brittnaie J.; Lovelace, Leslie L.; Pea, Maria Marjorette O.; Berger, Franklin G.; Berger, Sondra H.; Lebioda, Lukasz

    2010-11-03

    Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of {approx}27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K{sub m,app} values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F {center_dot} FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K{sub m,app} value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K{sub m,app} values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

  3. Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability .

    PubMed

    Huang, Xiao; Gibson, Lydia M; Bell, Brittnaie J; Lovelace, Leslie L; Peña, Maria Marjorette O; Berger, Franklin G; Berger, Sondra H; Lebioda, Lukasz

    2010-03-23

    Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of approximately 27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F.FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K(m,app) value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K(m,app) values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

  4. Immunoperoxidase staining of surface and intracellular immunoglobulin in human neoplastic lymphoid cells.

    PubMed Central

    Mason, D Y; Leonard, R C; Laurent, G; Gourdin, M F

    1980-01-01

    An immunoperoxidase technique for the optical microscopic detection of cellular immunoglobulin has been used to stain fixed smears of human neoplastic B lymphoid cells. Only four out of 28 cases of chronic lymphatic leukaemia (CLL) showed membrane labelling by this technique. In contrast, when 14 samples from other types of B lymphoproliferative disorder (including hairy cell leukaemia, non-Hodgkin's lymphoma, and prolymphocytic leukaemia) were studied, all samples showed membrane immunoglobulin labelling (confirmed by capping experiments). This discrepancy was attributed to the greater density of surface immunoglobulin present on neoplastic cells in the latter group of disorders compared to CLL. This immunoperoxidase technique is therefore less sensitive than immunofluorescent staining of cells in suspension for the demonstration of neoplastic cell surface immunoglobulin. However, it offers a number of advantages (eg, excellent visualisation of cell morphology, permanence of stained preparations, and applicability to stored samples) which recommend it as the method of choice in certain clinical haematological contexts. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7000833

  5. Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

    PubMed

    Kim, Kihoon; Umamoto, Sean; Trang, Phong; Hai, Rong; Liu, Fenyong

    2004-03-01

    A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

  6. Genomic understanding of glioblastoma expanded

    Cancer.gov

    Glioblastoma multiforme (GBM) was the first cancer type to be systematically studied by TCGA in 2008. In a new, complementary report, TCGA experts examined more than 590 GBM samples--the largest to date utilizing genomic characterization techniques and ne

  7. [The Relevance of MicroRNAs in Glioblastoma Stem Cells].

    PubMed

    Kleinová, R; Slabý, O; Šána, J

    2015-01-01

    Glioblastoma multiforme is the most common intracranial malignity of astrocyte origin in adults. Despite complex therapy consisting of maximal surgical resection, adjuvant concomitant chemoradiotherapy with temozolomide followed by temozolomide in monotherapy, the median of survival ranges between 12 and 15 months from dia-gnosis. This infaust prognosis is very often caused by both impossibility of achieving of sufficient radical surgical resection and tumor resistance to adjuvant therapy, which relates to the presence of glioblastoma stem cells. Similarly to normal stem cells, glioblastoma stem cells are capable of self -renewal, differentiation, and unlimited slow proliferation. Their resistance to conventional therapy is also due to higher expressions of DNA repair enzymes, antiapoptotic factors and multidrug transporters. Therefore, targeting these unique properties could be a novel promising therapeutic approach leading to more effective therapy and better prognosis of glioblastoma multiforme patients. One of the approaches how to successfully regulate above -mentioned properties is targeted regulation of microRNAs (miRNAs). These small noncoding RNA molecules posttranscriptionally regulate expression of more than 2/ 3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers, including glioblastoma multiforme. PMID:26480861

  8. Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells

    PubMed Central

    Duhem-Tonnelle, Véronique; Bièche, Ivan; Vacher, Sophie; Loyens, Anne; Maurage, Claude-Alain; Collier, Francis; Baroncini, Marc; Blond, Serge; Prevot, Vincent; Sharif, Ariane

    2010-01-01

    Glioblastomas are the most common CNS tumors in adults, and they remain resistant to current treatments. ErbB1 signaling is frequently altered in these tumors, which indicates that the erbB receptor family is a promising target for molecular therapy. However, data on erbB signaling in glioblastomas are still sparse. Therefore, we undertook a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of nine glioblastomas that were compared to non-neoplastic cerebral tissue containing neocortex and corresponding portions of subcortical convolutional white matter and we determined the distribution patterns of erbB receptors among the main neural cell types that are present in these tumors, particularly the putative tumoral stem cell population. Using quantitative RT-PCR and western blot analysis, we showed that erbB1 signaling and erbB2 receptors exhibited highly variable deregulation profiles among tumors, ranging from under- to overexpression, while erbB3 and erbB4 were down-regulated. Immunohistochemistry revealed an important inter- and intra-tumoral heterogeneity in all four erbB expression profiles. However, each receptor exhibited a distinct repartition pattern among the GFAP-, Olig2-, NeuN- and CD133-positive populations. Interestingly, while erbB1 immunoreactivity was only detected in small subsets of CD133-positive putative tumoral stem cells, erbB3 immunoreactivity was prominent in this cell population thus suggesting that erbB3 may represent a new potential target for molecular therapy. PMID:20467331

  9. Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells.

    PubMed

    Samse, Kaitlen; Emathinger, Jacqueline; Hariharan, Nirmala; Quijada, Pearl; Ilves, Kelli; Völkers, Mirko; Ormachea, Lucia; De La Torre, Andrea; Orogo, Amabel M; Alvarez, Roberto; Din, Shabana; Mohsin, Sadia; Monsanto, Megan; Fischer, Kimberlee M; Dembitsky, Walter P; Gustafsson, Åsa B; Sussman, Mark A

    2015-05-29

    Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated β-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.

  10. Evidence that leishmania donovani utilizes a mannose receptor on human mononuclear phagocytes to establish intracellular parasitism

    SciTech Connect

    Wilson, M.E.; Pearson, R.D.

    1986-01-01

    The pathogenic protozoan Leishmania donovani must gain entrance into mononuclear phagocytes to successfully parasitize man. The parasite's extracellular promastigote stage is ingested by human peripheral blood monocytes or monocyte-derived macrophages in the absence of serum, in a manner characteristic of receptor-mediated endocytosis. Remarkable similarities have been found between the macrophage receptor(s) for promastigotes and a previously characterized eucaryotic receptor system, the mannose/fucose receptor (MFR), that mediates the binding of zymosan particles and mannose- or fucose-terminal glycoconjugates to macrophages. Ingestion of promastigotes by monocyte-derived macrophages was inhibited by several MFR ligands; that is mannan, mannose-BSA and fucose-BSA. In contrast, promastigote ingestion by monocytes was unaffected by MFR ligands. Furthermore, attachment of promastigotes to macrophages, assessed by using cytochalasin D to prevent phagocytosis, was reduced 49.8% by mannan. Reorientation of the MFR to the ventral surface of the cell was achieved by plating macrophages onto mannan-coated coverslips, reducing MFR activity on the exposed cell surface by 94% as assessed by binding of /sup 125/I-mannose-BSA. Under these conditions, ingestion of promastigotes was inhibited by 71.4%. Internalization of the MFR by exposure of macrophages to zymosan before infection with promastigotes resulted in a 62.3% decrease in parasite ingestion. Additionally, NH/sub 4/Cl decreased macrophage ingestion of promastigotes by 38.2%. Subinhibitory concentration of NH/sub 4/Cl (10 mM) and of mannan (0.25 mg/ml) together inhibited parsite ingestion by 76.4%.

  11. Lactate levels with glioblastoma multiforme

    PubMed Central

    Kahlon, Arunpreet Singh; Alexander, Mariam; Kahlon, Arundeep

    2016-01-01

    A 37-year-old woman with known glioblastoma multiforme was admitted for treatment of new deep vein thrombosis. Anion gap and plasma lactate levels were found to be elevated. Magnetic resonance imaging of the brain showed a stable, advanced glioblastoma multiforme. All causes of lactic acidosis, including infections and medications, were ruled out. Aggressive tumors have been shown to produce lactate levels in minute quantities in their microenvironment, which helps them metastasize and evade immune response and even radiation. PMID:27365883

  12. IgE Mediates Killing of Intracellular Toxoplasma gondii by Human Macrophages through CD23-Dependent, Interleukin-10 Sensitive Pathway

    PubMed Central

    Vouldoukis, Ioannis; Mazier, Dominique; Moynet, Daniel; Thiolat, Denis; Malvy, Denis; Mossalayi, M. Djavad

    2011-01-01

    Background In addition to helminthic infections, elevated serum IgE levels were observed in many protozoal infections, while their contribution during immune response to these pathogens remained unclear. As IgE/antigen immune complexes (IgE-IC) bind to human cells through FcεRI or FcεRII/CD23 surface molecules, the present study aimed to identify which functional receptor may be involved in IgE-IC interaction with human macrophages, the major effector cell during parasite infection. Methodology/Principal Findings Human monocyte-derived macrophages were infected with Toxoplasma gondii before being incubated with IgE-IC. IgE receptors were then identified using appropriate blocking antibodies. The activation of cells and parasiticidal activity were evaluated by mediator quantification and direct counting of infected macrophages. RNAs were extracted and cell supernatants were also collected for their content in tumor necrosis factor (TNF)-α, interleukin-10 (IL-10) and nitrites. Sera from symptomatic infected patients were also tested for their content of IgE, IL-10 and nitrites, and compared to values found in healthy donors. Results showed that IgE-IC induced intracellular elimination of parasites by human macrophages. IgE-mediated effect was FcεRI-independent, but required cross-linking of surface FcεRII/CD23, cell activation and the generation of nitric oxide (NO). Although TNF-α was shown to be produced during cell activation, this cytokine had minor contribution in this phenomenon while endogenous and exogenous IL-10 down-regulated parasite killing. Inverse relationship was found between IL-10 and NO expression by infected human macrophages at both mRNA and mediator levels. The relationship between these in vitro data and in vivo levels of various factors in T. gondii infected patients supports the involvement of CD23 antigen and IL-10 expression in disease control. Conclusion Thus, IgE may be considered as immune mediator during antiprotozoal activity of

  13. BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells

    PubMed Central

    Klumpp, Lukas; Haehl, Erik; Schilbach, Karin; Lukowski, Robert; Kühnle, Matthias; Bernhardt, Günther; Buschauer, Armin; Zips, Daniel; Ruth, Peter; Huber, Stephan M.

    2016-01-01

    Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy. PMID:26893360

  14. BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.

    PubMed

    Edalat, Lena; Stegen, Benjamin; Klumpp, Lukas; Haehl, Erik; Schilbach, Karin; Lukowski, Robert; Kühnle, Matthias; Bernhardt, Günther; Buschauer, Armin; Zips, Daniel; Ruth, Peter; Huber, Stephan M

    2016-03-22

    Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy. PMID:26893360

  15. BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.

    PubMed

    Edalat, Lena; Stegen, Benjamin; Klumpp, Lukas; Haehl, Erik; Schilbach, Karin; Lukowski, Robert; Kühnle, Matthias; Bernhardt, Günther; Buschauer, Armin; Zips, Daniel; Ruth, Peter; Huber, Stephan M

    2016-03-22

    Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

  16. [Palliative care for glioblastoma].

    PubMed

    Dieudonné, Nathalie; De Micheli, Rita; Hottinger, Andreas

    2016-04-27

    Patients with glioblastoma have a limited life expectancy and an impaired quality of life and they should be offered palliative care soon after the diagnosis is established. Still, only a quarter of patients aged over 65 return home or medical institution after completing treatments. Home care must be promoted by coordinating assistance and care, combining disciplines such as physiotherapy and ergotherapy, medical and nursing care and psychosocial support. Patients are at risk of mood, personality and behavioural disorders. Limited awareness of these troubles and their physical limitations alter their capacity of rehabilitation and social relationships. Isolation of relatives, exhaustion and misunderstandings should be prevented. The therapeutic goals should be discussed and determined upstream to anticipate difficulties and questions concerning end of life. PMID:27281945

  17. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1989-01-01

    We have investigated the role of iron in the intracellular biology of Legionella pneumophila in human monocytes and in the effector arm of cell-mediated immune defense against this intracellular bacterial pathogen. To determine if L. pneumophila intracellular multiplication is iron dependent, we studied the effect of the iron chelator deferoxamine on L. pneumophila infection of monocytes. Deferoxamine at 15 microM completely inhibited L. pneumophila intracellular multiplication. The inhibitory effect of deferoxamine was reversed with equimolar iron-saturated transferrin but not apotransferrin. To examine the potential role of iron in monocyte activation, we investigated the influence of iron-saturated transferrin on L. pneumophila multiplication in IFN gamma-activated monocytes. Iron transferrin, but not apotransferrin, neutralized the capacity of activated monocytes to inhibit L. pneumophila multiplication. To explore a potential mechanism by which activated monocytes might limit the availability of intracellular iron, we examined transferrin receptor expression on nonactivated and activated monocytes cultured in vitro for 5 d. By fluorescence-activated flow cytometry, activated monocytes exhibited markedly fewer transferrin receptors than nonactivated monocytes. By Scatchard analysis of 125I-transferrin binding to monocytes, nonactivated monocytes had 38,300 +/- 12,700 (mean +/- SE) transferrin binding sites, whereas activated monocytes had 10,300 +/- 1,600, a reduction of 73%. Activated and nonactivated monocytes had a similar mean Kd (1.8 +/- 0.2 nM). This study demonstrates that (a) L. pneumophila intracellular multiplication is iron dependent; (b) activated monocytes inhibit L. pneumophila multiplication by limiting the availability of intracellular iron; and (c) transferrin receptors are downregulated on IFN gamma-activated monocytes. Images PMID:2496141

  18. Intracellular potassium stabilizes human ether-à-go-go-related gene channels for export from endoplasmic reticulum.

    PubMed

    Wang, Lu; Dennis, Adrienne T; Trieu, Phan; Charron, Francois; Ethier, Natalie; Hebert, Terence E; Wan, Xiaoping; Ficker, Eckhard

    2009-04-01

    Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+) pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K(+)-either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media-is sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K(+)](i)-mediated conformational defect directly in the hERG channel protein. PMID:19139152

  19. RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

    SciTech Connect

    Poch, Enric; Minambres, Rebeca; Mocholi, Enric; Ivorra, Carmen; Perez-Arago, Amparo; Guerri, Consuelo; Perez-Roger, Ignacio . E-mail: iperez@uch.ceu.es; Guasch, Rosa M. . E-mail: guasch@cipf.es

    2007-02-15

    Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.

  20. Extra- and Intracellular Imaging of Human Matrix Metalloprotease 11 (hMMP-11) with a Cell-penetrating FRET Substrate*

    PubMed Central

    Meyer, B. Sina; Rademann, Jörg

    2012-01-01

    Matrix metalloprotease 11 (MMP-11), a protease associated with invasion and aggressiveness of cancerous tissue, was postulated as a prognostic marker for pancreatic, breast, and colon cancer patients. Expression analysis, however, did not reveal localization and regulation of this protease. Thus, cellular tools for the visualization of MMP-11 are highly desirable to monitor presence and activity and to elucidate the functional role of MMP-11. Therefore, fluorescein-Dabcyl-labeled Foerster resonance energy transfer (FRET) substrates were developed. The design focused on enhanced peptide binding to human MMP-11, employing an unusual amino acid for the specificity pocket P1′. The addition of several arginines resulted in a cell-permeable FRET substrate SM-P124 (Ac-GRRRK(Dabcyl)-GGAANC(MeOBn)RMGG-fluorescein). In vitro evaluation of SM-P124 with human MMP-11 showed a 25-fold increase of affinity (kcat/Km = 9.16 × 103 m−1 s−1, Km = 8 μm) compared with previously published substrates. Incubation of pancreatic adenocarcinoma cell line MIA PaCa-2 and mamma adenocarcinoma cell line MCF-7 with the substrate SM-P124 (5 μm) indicated intra- and extracellular MMP-11 activity. A negative control cell line (Jurkat) showed no fluorescent signal either intra- or extracellularly. Negative control FRET substrate SM-P123 produced only insignificant extracellular fluorescence without any intracellular fluorescence. SM-P124 therefore enabled intra- and extracellular tracking of MMP-11-overexpressing cancers such as pancreatic and breast adenocarcinoma and might contribute to the understanding of the activation pathways leading to MMP-11-mediated invasive processes. PMID:22927434

  1. Improbability of Effective Vaccination Against Human Immunodeficiency Virus Because of Its Intracellular Transmission and Rectal Portal of Entry

    NASA Astrophysics Data System (ADS)

    Sabin, Albert B.

    1992-09-01

    The worldwide effort to produce a vaccine against AIDS continues to disregard the fact that even human immunodeficiency virus (HIV)-specific neutralizing antibodies and cell-mediated immunity are ineffective against virus within cells without viral antigens on the cell membrane-and that much of HIV infection is transmitted in this manner. According to a recent report, a simian immunodeficiency virus vaccine that protected monkeys against an intravenous challenge with cell-free virus was, as predicted, ineffective against an intravenous challenge with the same amount of virus in infected cells. Moreover, antibody and HIV have been found to coexist in cell-free plasma from asymptomatic and symptomatic patients. Excluding direct introduction of HIV into the bloodstream, the most common and efficient form of transmission of HIV infection is by receptive anal intercourse, and semen contains large numbers of infected cells per milliliter. Recent reports showing that colorectal cells can be persistently infected by HIV and that HIV RNA and cDNA are present in the cells of the colon of dead AIDS patients indicate that either cell-free or intracellular HIV has the capacity to multiply at the portal of entry in the colorectal area without interference from neutralizing antibodies. The available data provide no basis for testing any HIV vaccine in human beings either before or after infection. The main challenge is to find a way to kill cells with chromosomally integrated HIV cDNA without harming normal cells, perhaps by identifying repressor proteins that might be produced by the cells with integrated HIV cDNA and thus could become specific targets for cell-killing drugs.

  2. Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

    PubMed Central

    Rafiee, Parvaneh; Heidemann, Jan; Ogawa, Hitoshi; Johnson, Nathan A; Fisher, Pamela J; Li, Mona S; Otterson, Mary F; Johnson, Christopher P; Binion, David G

    2004-01-01

    The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection. PMID:15175101

  3. Cytotoxicity, intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells.

    PubMed

    Tlotleng, Nonhlanhla; Vetten, Melissa A; Keter, Frankline K; Skepu, Amanda; Tshikhudo, Robert; Gulumian, Mary

    2016-08-01

    Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs. PMID:27184667

  4. Argon laser phototherapy of human malignancies using rhodamine-123 as a new laser dye: The intracellular role of oxygen

    SciTech Connect

    Castro, D.J.; Saxton, R.E.; Markley, J.; Foote, C.S.; Fetterman, H.R.; Castro, D.J.; Ward, P.H. )

    1990-08-01

    Recent studies demonstrated that the cationic, mitochondrial-specific dye Rhodamine-123 (Rh-123), is an efficient tumor photosensitizer for Argon laser treatment of human cancer cells both in vitro and in tumors grown as xenografts in athymic mice. To demonstrate the photodynamic mechanism of action of this reaction, the intracellular role of oxygen and temperature changes in treated cells have to be defined. In the current study, a large panel of human tumor cell lines of diverse histologic origin were tested for in vitro sensitivity to Rh-123 and the Argon laser (514.5 nm) in oxygen, deuterium oxide (D2O), and nitrogen (N2) environment. Tumor cells in suspension were first sensitized to Rh-123 (1 or 20 micrograms/ml for 1 hour), cooled on ice to 4 degrees C, and then exposed to the Argon laser (delta T = 14 +/- 1 degree C). Cell proliferation measured by (3H)-thymidine uptake 24 hours after sensitization with Rh-123 and laser treatment was significantly decreased in tumor cells kept in oxygen and D2O atmospheres. No decrease in DNA synthesis was seen in Rh-123 and laser treated cells kept in an N2 environment. Control tumor cells treated with Rh-123 or the Argon laser separately did not show any decreased (3H)-thymidine uptake in oxygen, D2O or N2 environment. These results provide evidence of a photodynamic process since Rh-123 sensitization and Argon laser activation occur at nonthermal levels of energy and are oxygen dependent. The high effectiveness of this technique of photodynamic therapy with the Argon laser, and low toxicity of Rh-123 could make its clinical use very attractive for the treatment of superficial malignancies.

  5. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine.

    PubMed

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-10-09

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1).

  6. Shikonin Inhibits the Migration and Invasion of Human Glioblastoma Cells by Targeting Phosphorylated β-Catenin and Phosphorylated PI3K/Akt: A Potential Mechanism for the Anti-Glioma Efficacy of a Traditional Chinese Herbal Medicine

    PubMed Central

    Zhang, Feng-Ying; Hu, Yi; Que, Zhong-You; Wang, Ping; Liu, Yun-Hui; Wang, Zhen-Hua; Xue, Yi-Xue

    2015-01-01

    Shikonin is an anthraquinone derivative extracted from the root of lithospermum. Shikonin is traditionally used in the treatment of inflammatory and infectious diseases such as hepatitis. Shikonin also inhibits proliferation and induces apoptosis in various tumors. However, the effect of shikonin on gliomas has not been fully elucidated. In the present study, we aimed to investigate the effects of shikonin on the migration and invasion of human glioblastoma cells as well as the underlying mechanisms. U87 and U251 human glioblastoma cells were treated with shikonin at 2.5, 5, and 7.5 μmol/L and cell viability, migration and invasiveness were assessed with CCK8, scratch wound healing, in vitro Transwell migration, and invasion assays. The expression and activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the expression of phosphorylated β-catenin (p-β-catenin) and phosphorylated PI3K/Akt were also checked. Results showed that shikonin significantly inhibited the cell proliferation, migration, invasion, and expression of MMP-2 and MMP-9 in U87 and U251 cells. The expression of p-β-catenin showed contrary trends in two cell lines. It was significantly inhibited in U87 cells and promoted in U251 cells. Results in this work indicated that shikonin displayed an inhibitory effect on the migration and invasion of glioma cells by inhibiting the expression and activity of MMP-2 and -9. In addition, shikonin also inhibited the expression of p-PI3K and p-Akt to attenuate cell migration and invasion and MMP-2 and MMP-9 expression in both cell lines, which could be reversed by the PI3K/Akt pathway agonist, insulin-like growth factor-1 (IGF-1). PMID:26473829

  7. Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.

    PubMed

    Spanò, Stefania; Liu, Xiaoyun; Galán, Jorge E

    2011-11-01

    Unlike broad-host Salmonella serovars, which cause self-limiting disease, Salmonella enterica serovar Typhi can infect only humans causing typhoid fever, a life-threatening systemic disease. The molecular bases for these differences are presently unknown. Here we show that the GTPase Rab29 (Rab7L1) distinguishes the intracellular vacuole of human-adapted and broad-host Salmonella serovars. A screen to identify host factors required for the export of typhoid toxin, which is exclusively encoded by the human-specific Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi (S. Paratyphi) identified Rab29. We found that Rab29 is recruited to the S. Typhi-containing vacuole but not to vacuoles containing broad-host Salmonella. We observed that in cells infected with broad-host Salmonella Rab29 is specifically cleaved by the proteolytic activity of GtgE, a unique type III secretion effector protein that is absent from S. Typhi. An S. Typhi strain engineered to express GtgE and therefore able to cleave Rab29 exhibited increased intracellular replication in human macrophages. These findings indicate significant differences in the intracellular biology of human-adapted and broad-host Salmonella and show how subtle differences in the assortment of effector proteins encoded by highly related pathogens can have a major impact in their biology.

  8. Glioblastoma Stem Cells as a New Therapeutic Target for Glioblastoma

    PubMed Central

    Kalkan, Rasime

    2015-01-01

    Primary and secondary glioblastomas (GBMs) are two distinct diseases. The genetic and epigenetic background of these tumors is highly variable. The treatment procedure for these tumors is often unsuccessful because of the cellular heterogeneity and intrinsic ability of the tumor cells to invade healthy tissues. The fatal outcome of these tumors promotes researchers to find out new markers associated with the prognosis and treatment planning. In this communication, the role of glioblastoma stem cells in tumor progression and the malignant behavior of GBMs are summarized with attention to the signaling pathways and molecular regulators that are involved in maintaining the glioblastoma stem cell phenotype. A better understanding of these stem cell-like cells is necessary for designing new effective treatments and developing novel molecular strategies to target glioblastoma stem cells. We discuss hypoxia as a new therapeutic target for GBM. We focus on the inhibition of signaling pathways, which are associated with the hypoxia-mediated maintenance of glioblastoma stem cells, and the knockdown of hypoxia-inducible factors, which could be identified as attractive molecular target approaches for GBM therapeutics. PMID:26617463

  9. Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1-Transfected Human Embryonic Kidney 293 Cells.

    PubMed

    Chien, Huan-Chieh; Zur, Arik A; Maurer, Tristan S; Yee, Sook Wah; Tolsma, John; Jasper, Paul; Scott, Dennis O; Giacomini, Kathleen M

    2016-03-01

    Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/10(6) cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K(+) buffer (140 mM KCl) compared with normal K(+) buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells. PMID:26700958

  10. Mutation-Driven Divergence and Convergence Indicate Adaptive Evolution of the Intracellular Human-Restricted Pathogen, Bartonella bacilliformis

    PubMed Central

    Paul, Sandip; Minnick, Michael F.; Chattopadhyay, Sujay

    2016-01-01

    Among all species of Bartonella, human-restricted Bartonella bacilliformis is the most virulent but harbors one of the most reduced genomes. Carrión’s disease, the infection caused by B. bacilliformis, has been afflicting poor rural populations for centuries in the high-altitude valleys of the South American Andes, where the pathogen’s distribution is probably restricted by its sand fly vector’s range. Importantly, Carrión’s disease satisfies the criteria set by the World Health Organization for a disease amenable to elimination. However, to date, there are no genome-level studies to identify potential footprints of B. bacilliformis (patho)adaptation. Our comparative genomic approach demonstrates that the evolution of this intracellular pathogen is shaped predominantly via mutation. Analysis of strains having publicly-available genomes shows high mutational divergence of core genes leading to multiple sub-species. We infer that the sub-speciation event might have happened recently where a possible adaptive divergence was accelerated by intermediate emergence of a mutator phenotype. Also, within a sub-species the pathogen shows inter-clonal adaptive evolution evidenced by non-neutral accumulation of convergent amino acid mutations. A total of 67 non-recombinant core genes (over-representing functional categories like DNA repair, glucose metabolic process, ATP-binding and ligase) were identified as candidates evolving via adaptive mutational convergence. Such convergence, both at the level of genes and their encoded functions, indicates evolution of B. bacilliformis clones along common adaptive routes, while there was little diversity within a single clone. PMID:27167125

  11. Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y Cells

    SciTech Connect

    Liu, P.-S. . E-mail: psliu@mail.scu.edu.tw; Chiung, Y.-M.; Kao, Y.-Y.

    2006-03-01

    The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca{sup 2+}]{sub c}) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca{sup 2+} mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca{sup 2+}]{sub c} by releasing Ca{sup 2+} from the intracellular stores and extracellular Ca{sup 2+} influx. 500 {mu}M TDI induced a net [Ca{sup 2+}]{sub c} increase of 112 {+-} 8 and 78 {+-} 6 nM in the presence and absence of extracellular Ca{sup 2+}, respectively. In Ca{sup 2+}-free buffer, TDI induced Ca{sup 2+} release from internal stores to reduce their Ca{sup 2+} content and this reduction was evidenced by a suppression occurring on the [Ca{sup 2+}]{sub c} rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca{sup 2+}, simultaneous exposure to TDI and methacholine led a higher level of [Ca{sup 2+}]{sub c} compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca{sup 2+}]{sub c} homeostasis including releasing Ca{sup 2+} from internal stores and inducing extracellular Ca{sup 2+} influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.

  12. Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

    PubMed

    Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

    2015-01-01

    Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

  13. Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis.

    PubMed

    Sucic, Sonja; Bryan-Lluka, Lesley J

    2005-09-01

    The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [3H]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mm MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

  14. Human Leukocyte Antigen-G Is Frequently Expressed in Glioblastoma and May Be Induced in Vitro by Combined 5-Aza-2′-Deoxycytidine and Interferon-γ Treatments

    PubMed Central

    Wastowski, Isabela J.; Simões, Renata T.; Yaghi, Layale; Donadi, Eduardo A.; Pancoto, João T.; Poras, Isabelle; Lechapt-Zalcman, Emmanuèle; Bernaudin, Myriam; Valable, Samuel; Carlotti, Carlos G.; Flajollet, Sébastien; Jensen, Stine S.; Ferrone, Soldano; Carosella, Edgardo D.; Kristensen, Bjarne W.; Moreau, Philippe

    2014-01-01

    Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2′-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2′-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G–negative tumors. PMID:23219427

  15. A Phase 1 trial of intravenous boronophenylalanine-fructose complex in patients with glioblastoma multiforme

    SciTech Connect

    Bergland, R.; Elowitz, E.; Chadha, M.; Coderre, J.A.; Joel, D.

    1996-10-01

    Boron neutron capture therapy (BNCT) of glioblastoma multiforme was initially performed at the Brookhaven National Laboratory in the early 1950`s While this treatment for malignant brain tumors has continued in Japan, new worldwide interest has been stimulated by the development of new and more selective boron compounds. Boronophenylalanine (BPA) is a blood-brain barrier penetrating compound that has been used in BNCT of malignant melanomas. SPA has been employed experimentally in BNCT of rat gliosarcoma and has potential use in the treatment of human glioblastoma. As a preface to clinical BNCT trials, we studied the biodistribution of SPA in patients with glioblastoma.

  16. Salinomycin encapsulated nanoparticles as a targeting vehicle for glioblastoma cells.

    PubMed

    Tığlı Aydın, R Seda; Kaynak, Gökçe; Gümüşderelioğlu, Menemşe

    2016-02-01

    Salinomycin has been introduced as a novel alternative to traditional anti-cancer drugs. The aim of this study was to test a strategy designed to deliver salinomycin to glioblastoma cells in vitro. Salinomycin-encapsulated polysorbate 80-coated poly(lactic-co-glycolic acid) nanoparticles (P80-SAL-PLGA) were prepared and characterized with respect to particle size, morphology, thermal properties, drug encapsulation efficiency and controlled salinomycin-release behaviour. The in vitro cellular uptake of P80-SAL-PLGA (5 and 10 µM) or uncoated nanoparticles was assessed in T98G human glioblastoma cells, and the cell viability was investigated with respect to anti-growth activities. SAL, which was successfully transported to T98G glioblastoma cells via P80 coated nanoparticles (∼14% within 60 min), greatly decreased (p < 0.01) the cellular viability of T98G cells. Substantial morphological changes were observed in the T98G cells with damaged actin cytoskeleton. Thus, P80-SAL-PLGA nanoparticles induced cell death, suggesting a potential therapeutic role for this salinomycin delivery system in the treatment of human glioblastoma. PMID:26476239

  17. Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine.

    PubMed

    de Muys, J M; Gourdeau, H; Nguyen-Ba, N; Taylor, D L; Ahmed, P S; Mansour, T; Locas, C; Richard, N; Wainberg, M A; Rando, R F

    1999-08-01

    The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a

  18. Cyclophilin B supports Myc and mutant p53-dependent survival of glioblastoma multiforme cells.

    PubMed

    Choi, Jae Won; Schroeder, Mark A; Sarkaria, Jann N; Bram, Richard J

    2014-01-15

    Glioblastoma multiforme is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here, we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in glioblastoma multiforme cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human glioblastoma multiforme cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of glioblastoma multiforme cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS-mitogen-activated protein kinase pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1, and Janus-activated kinase/STAT3 signaling. Elevated reactive oxygen species, ER expansion, and abnormal unfolded protein responses in CypB-depleted glioblastoma multiforme cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of glioblastoma multiforme tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for glioblastoma multiforme therapy. PMID:24272483

  19. Coordinate activation of Shh and PI3K signaling in PTEN-deficient glioblastoma: new therapeutic opportunities.

    PubMed

    Filbin, Mariella Gruber; Dabral, Sukriti K; Pazyra-Murphy, Maria F; Ramkissoon, Shakti; Kung, Andrew L; Pak, Ekaterina; Chung, Jarom; Theisen, Matthew A; Sun, Yanping; Franchetti, Yoko; Sun, Yu; Shulman, David S; Redjal, Navid; Tabak, Barbara; Beroukhim, Rameen; Wang, Qi; Zhao, Jean; Dorsch, Marion; Buonamici, Silvia; Ligon, Keith L; Kelleher, Joseph F; Segal, Rosalind A

    2013-11-01

    In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma. PMID:24076665

  20. Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells.

    PubMed

    Wang, Xueqin; Tu, Miaomiao; Tian, Baoming; Yi, Yanjie; Wei, ZhenZhen; Wei, Fang

    2016-11-01

    Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe2O3 nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future.

  1. Synthesis of tumor-targeted folate conjugated fluorescent magnetic albumin nanoparticles for enhanced intracellular dual-modal imaging into human brain tumor cells.

    PubMed

    Wang, Xueqin; Tu, Miaomiao; Tian, Baoming; Yi, Yanjie; Wei, ZhenZhen; Wei, Fang

    2016-11-01

    Superparamagnetic iron oxide nanoparticles (SPIO NPs), utilized as carriers are attractive materials widely applied in biomedical fields, but target-specific SPIO NPs with lower toxicity and excellent biocompatibility are still lacking for intracellular visualization in human brain tumor diagnosis and therapy. Herein, bovine serum albumin (BSA) coated superparamagnetic iron oxide, i.e. γ-Fe2O3 nanoparticles (BSA-SPIO NPs), are synthesized. Tumor-specific ligand folic acid (FA) is then conjugated onto BSA-SPIO NPs to fabricate tumor-targeted NPs, FA-BSA-SPIO NPs as a contrast agent for MRI imaging. The FA-BSA-SPIO NPs are also labeled with fluorescein isothiocyanate (FITC) for intracellular visualization after cellular uptake and internalization by glioma U251 cells. The biological effects of the FA-BSA-SPIO NPs are investigated in human brain tumor U251 cells in detail. These results show that the prepared FA-BSA-SPIO NPs display undetectable cytotoxicity, excellent biocompatibility, and potent cellular uptake. Moreover, the study shows that the made FA-BSA-SPIO NPs are effectively internalized for MRI imaging and intracellular visualization after FITC labeling in the targeted U251 cells. Therefore, the present study demonstrates that the fabricated FITC-FA-BSA-SPIO NPs hold promising perspectives by providing a dual-modal imaging as non-toxic and target-specific vehicles in human brain tumor treatment in future. PMID:27523645

  2. [Glioblastoma multiforme... with multifocal presentation].

    PubMed

    Sousa, Gabriela; Rocha, Armando; Alfaiate, Teresa; Carvalho, Teresa; Veiga e Moura, António; Ferreira, Mário Rui

    2002-01-01

    Glioblastoma multiforme is the most common malignant primary brain tumor in adults (+/- 40% of the Central Nervous System primary tumors). Representing only 2% of all oncologic processes, they are associated with a great deterioration of cerebral functions and a poor prognosis, facts that contribute to their great individual and social impact. The authors report a case of glioblastoma multiforme, with multifocal lesions "ab initio" and show the difficulty to make a correct diagnosis, even with the most modern imagiologic techniques. They also make a brief review of the literature. PMID:12525027

  3. Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

    PubMed

    Kim, Ha Won; Chan, Qilin; Afton, Scott E; Caruso, Joseph A; Lai, Barry; Weintraub, Neal L; Qin, Zhenyu

    2012-02-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

  4. Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds

    PubMed Central

    Kim, Ha Won; Chan, Qilin; Afton, Scott E.; Caruso, Joseph A.; Lai, Barry; Weintraub, Neal L.; Qin, Zhenyu

    2013-01-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound

  5. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  6. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  7. The integrated landscape of driver genomic alterations in glioblastoma

    PubMed Central

    Frattini, Veronique; Trifonov, Vladimir; Chan, Joseph Minhow; Castano, Angelica; Lia, Marie; Abate, Francesco; Keir, Stephen T.; Ji, Alan X.; Zoppoli, Pietro; Niola, Francesco; Danussi, Carla; Dolgalev, Igor; Porrati, Paola; Pellegatta, Serena; Heguy, Adriana; Gupta, Gaurav; Pisapia, David J.; Canoll, Peter; Bruce, Jeffrey N.; McLendon, Roger E.; Yan, Hai; Aldape, Ken; Finocchiaro, Gaetano; Mikkelsen, Tom; Privé, Gilbert G.; Bigner, Darell D.; Lasorella, Anna; Rabadan, Raul; Iavarone, Antonio

    2013-01-01

    Glioblastoma remains one of the most challenging forms of cancer to treat. Here, we develop a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We find mutations with loss of heterozygosity of LZTR-1, an adaptor of Cul3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR-1 function, which restrains self-renewal and growth of glioma spheres retaining stem cell features. Loss-of-function mutations of CTNND2 target a neural-specific gene and are associated with transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 as the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate Stat3 signaling and confer mitogen independency and sensitivity to EGFR inhibition. These results provide important insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention. PMID:23917401

  8. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  9. Corticosteroids compromise survival in glioblastoma.

    PubMed

    Pitter, Kenneth L; Tamagno, Ilaria; Alikhanyan, Kristina; Hosni-Ahmed, Amira; Pattwell, Siobhan S; Donnola, Shannon; Dai, Charles; Ozawa, Tatsuya; Chang, Maria; Chan, Timothy A; Beal, Kathryn; Bishop, Andrew J; Barker, Christopher A; Jones, Terreia S; Hentschel, Bettina; Gorlia, Thierry; Schlegel, Uwe; Stupp, Roger; Weller, Michael; Holland, Eric C; Hambardzumyan, Dolores

    2016-05-01

    Glioblastoma is the most common and most aggressive primary brain tumour. Standard of care consists of surgical resection followed by radiotherapy and concomitant and maintenance temozolomide (temozolomide/radiotherapy→temozolomide). Corticosteroids are commonly used perioperatively to control cerebral oedema and are frequently continued throughout subsequent treatment, notably radiotherapy, for amelioration of side effects. The effects of corticosteroids such as dexamethasone on cell growth in glioma models and on patient survival have remained controversial. We performed a retrospective analysis of glioblastoma patient cohorts to determine the prognostic role of steroid administration. A disease-relevant mouse model of glioblastoma was used to characterize the effects of dexamethasone on tumour cell proliferation and death, and to identify gene signatures associated with these effects. A murine anti-VEGFA antibody was used in parallel as an alternative for oedema control. We applied the dexamethasone-induced gene signature to The Cancer Genome Atlas glioblastoma dataset to explore the association of dexamethasone exposure with outcome. Mouse experiments were used to validate the effects of dexamethasone on survival in vivo Retrospective clinical analyses identified corticosteroid use during radiotherapy as an independent indicator of shorter survival in three independent patient cohorts. A dexamethasone-associated gene expression signature correlated with shorter survival in The Cancer Genome Atlas patient dataset. In glioma-bearing mice, dexamethasone pretreatment decreased tumour cell proliferation without affecting tumour cell viability, but reduced survival when combined with radiotherapy. Conversely, anti-VEGFA antibody decreased proliferation and increased tumour cell death, but did not affect survival when combined with radiotherapy. Clinical and mouse experimental data suggest that corticosteroids may decrease the effectiveness of treatment and shorten

  10. Targeting Oncogenic ALK and MET: A Promising Therapeutic Strategy for Glioblastoma

    PubMed Central

    Wallace, Gerald C; Dixon-Mah, Yaenette N; Vandergrift, W Alex; Ray, Swapan K; Haar, Catherine P; Mittendorf, Amber M; Patel, Sunil J; Banik, Naren L; Giglio, Pierre; Das, Arabinda

    2015-01-01

    Glioblastoma is the most common aggressive, highly glycolytic, and lethal brain tumor. In fact, it is among the most commonly diagnosed lethal malignancies, with thousands of new cases reported in the United States each year. Glioblastoma's lethality is derived from a number of factors including highly active pro-mitotic and pro-metastatic pathways. Two factors increasingly associated with the intracellular signaling and transcriptional machinery required for such changes are anaplastic lymphoma kinase (ALK) and the hepatocyte growth factor receptor (HGFR or, more commonly MET). Both receptors are members of the receptor tyrosine kinase (RTK) family, which has itself gained much attention for its role in modulating mitosis, migration, and survival in cancer cells. ALK was first described as a vital oncogene in lymphoma studies, but it has since been connected to many carcinomas, including non-small cell lung cancer and glioblastoma. As the receptor for HGF, MET has also been highly characterized and regulates numerous developmental and wound healing events which, when upregulated in cancer, can promote tumor progression. The wealth of information gathered over the last 30 years regarding these RTKs suggests three downstream cascades that depend upon activation of STAT3, Ras, and AKT. This review outlines the significance of ALK and MET as they relate to glioblastoma, explores the significance of STAT3, Ras, and AKT downstream of ALK/MET, and touches on the potential for new chemotherapeutics targeting ALK and MET to improve glioblastoma patient prognosis. PMID:23543207

  11. Simulation Predicts IGFBP2-HIF1α Interaction Drives Glioblastoma Growth

    PubMed Central

    Lin, Ka Wai; Liao, Angela; Qutub, Amina A.

    2015-01-01

    Tremendous strides have been made in improving patients’ survival from cancer with one glaring exception: brain cancer. Glioblastoma is the most common, aggressive and highly malignant type of primary brain tumor. The average overall survival remains less than 1 year. Notably, cancer patients with obesity and diabetes have worse outcomes and accelerated progression of glioblastoma. The root cause of this accelerated progression has been hypothesized to involve the insulin signaling pathway. However, while the process of invasive glioblastoma progression has been extensively studied macroscopically, it has not yet been well characterized with regards to intracellular insulin signaling. In this study we connect for the first time microscale insulin signaling activity with macroscale glioblastoma growth through the use of computational modeling. Results of the model suggest a novel observation: feedback from IGFBP2 to HIF1α is integral to the sustained growth of glioblastoma. Our study suggests that downstream signaling from IGFI to HIF1α, which has been the target of many insulin signaling drugs in clinical trials, plays a smaller role in overall tumor growth. These predictions strongly suggest redirecting the focus of glioma drug candidates on controlling the feedback between IGFBP2 and HIF1α. PMID:25884993

  12. P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines.

    PubMed

    Gallo-Oller, Gabriel; Vollmann-Zwerenz, Arabel; Meléndez, Bárbara; Rey, Juan A; Hau, Peter; Dotor, Javier; Castresana, Javier S

    2016-10-10

    Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-β pathway, suggesting a therapeutic potential of P144 for GBM treatment.

  13. P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines.

    PubMed

    Gallo-Oller, Gabriel; Vollmann-Zwerenz, Arabel; Meléndez, Bárbara; Rey, Juan A; Hau, Peter; Dotor, Javier; Castresana, Javier S

    2016-10-10

    Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-β pathway, suggesting a therapeutic potential of P144 for GBM treatment. PMID:27473823

  14. Glioblastoma care in the elderly.

    PubMed

    Jordan, Justin T; Gerstner, Elizabeth R; Batchelor, Tracy T; Cahill, Daniel P; Plotkin, Scott R

    2016-01-15

    Glioblastoma is common among elderly patients, a group in which comorbidities and a poor prognosis raise important considerations when designing neuro-oncologic care. Although the standard of care for nonelderly patients with glioblastoma includes maximal safe surgical resection followed by radiotherapy with concurrent and adjuvant temozolomide, the safety and efficacy of these modalities in elderly patients are less certain given the population's underrepresentation in many clinical trials. The authors reviewed the clinical trial literature for reports on the treatment of elderly patients with glioblastoma to provide evidence-based guidance for practitioners. In elderly patients with glioblastoma, there is a survival advantage for those who undergo maximal safe resection, which likely includes an incremental benefit with increasing completeness of resection. Radiotherapy extends survival in selected patients, and hypofractionation appears to be more tolerable than standard fractionation. In addition, temozolomide chemotherapy is safe and extends the survival of patients with tumors that harbor O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation. The combination of standard radiation with concurrent and adjuvant temozolomide has not been studied in this population. Although many questions remain unanswered regarding the treatment of glioblastoma in elderly patients, the available evidence provides a framework on which providers may base individual treatment decisions. The importance of tumor biomarkers is increasingly apparent in elderly patients, for whom the therapeutic efficacy of any treatment must be weighed against its potential toxicity. MGMT promoter methylation status has specifically demonstrated utility in predicting the efficacy of temozolomide and should be considered in treatment decisions when possible. Cancer 2016;122:189-197. © 2015 American Cancer Society. PMID:26618888

  15. Modulation of human leukocyte antigen and intracellular adhesion molecule-1 surface expression in malignant and nonmalignant human thyroid cells by cytokines in the context of extracellular matrix.

    PubMed

    Miller, A; Kraiem, Z; Sobel, E; Lider, O; Lahat, N

    2000-11-01

    Interactions between malignant cells and their environment are achieved via cell-surface receptors and adhesion molecules. The extracellular matrix (ECM) and ECM-bound cytokines modulate the expression of cell-surface molecules on target malignant cells, which may lead to changes in their susceptibility to cytolysis, in their ability to present antigens, and in the induction of local immune-cell activation and patrol. Eventually, these alterations may culminate in either the destruction, or escape and proliferation, of the tumor. We studied the effects of the ECM and its components in a "naive" form or following binding of the inflammatory cytokines interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) on the surface expression of human leukocyte antigen (HLA) class-I, HLA class-II (HLA-DR), and intracellular adhesion molecule-1 (ICAM-1), on nonmalignant and malignant thyroid cells. The basal expression of HLA class-I molecules was not significantly changed either by naive ECM and its components or by ECM-bound cytokines. ECM synergized with IFNgamma and TNFalpha in inducing HLA-DR molecules on nonmalignant and malignant thyrocytes, with higher HLA-DR levels on the malignant cells. The laminin component, in particular, synergized with IFNgamma. Basal ICAM-1 expression on nonneoplastic cells was not significantly affected by the cytokines when grown in the absence of ECM, but was significantly upregulated when cells were cultured on ECM. In contrast, in malignant thyrocyte cultures, ECM significantly attenuated IFNgamma- and TNFalpha-mediated enhancement of ICAM-1 expression. We concluded that signals derived from ECM-embedded cytokines participate in the regulation of key thyroid cell surface molecules and, thus, may affect the final outcome of human thyroid malignancies. PMID:11128721

  16. 1'-Acetoxychavicol acetate promotes caspase 3-activated glioblastoma cell death by overcoming enhanced cytokine expression.

    PubMed

    Williams, Musa; Tietzel, Illya; Quick, Quincy A

    2013-06-01

    The brain consumes ∼20% of the oxygen utilized in the human body, meaning that brain tumors are vulnerable to paradoxical physiological effects from free radical generation. In the present study, 1'-acetoxychavicol acetate (ACA), a naturally derived antioxidant that inhibits xanthine oxidase, was evaluated for its role as an anti-tumorigenic agent in glioblastomas. The study revealed that ACA inhibited glioblastoma cell proliferation as a consequence of promoting apoptotic cell death by enhancing caspase 3 activity. It was also shown that ACA impaired the migratory ability of glioblastoma cells by decreasing their adhesive properties. Additionally, ACA increased the protein expression levels of the pro-survival signaling cytokines, IL-6 and IL-1α, established cell protectors and survival molecules in brain tumors. Together, these results demonstrate that, despite enhanced expression of compensatory signaling molecules that contribute to tumor cell survival, ACA is an effective pro-apoptotic inducing agent in glioblastomas.

  17. miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2

    SciTech Connect

    Li, Xuesong; Gong, Xuhai; Chen, Jing; Zhang, Jinghui; Sun, Jiahang; Guo, Mian

    2015-05-08

    Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3′UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma. - Highlights: • miR-340 is downregulated in glioblastoma samples and cell lines. • miR-340 inhibits glioblastoma cell proliferation. • miR-340 directly targets CDK6, cyclin-D1, and cyclin-D2. • miR-340 regulates glioblastoma cell proliferation via CDK6, cyclin-D1 and cyclin-D2.

  18. Dipterinyl Calcium Pentahydrate Inhibits Intracellular Mycobacterial Growth in Human Monocytes via the C-C Chemokine MIP-1β and Nitric Oxide

    PubMed Central

    Sakala, Isaac G.; Eickhoff, Christopher S.; Blazevic, Azra; Moheno, Phillip; Silver, Richard F.

    2013-01-01

    Tuberculosis remains one of the top three leading causes of morbidity and mortality worldwide, complicated by the emergence of drug-resistant Mycobacterium tuberculosis strains and high rates of HIV coinfection. It is important to develop new antimycobacterial drugs and immunomodulatory therapeutics and compounds that enhance antituberculous immunity. Dipterinyl calcium pentahydrate (DCP), a calcium-complexed pterin compound, has previously been shown to inhibit human breast cancer cells and hepatitis B virus (HBV). DCP inhibitory effects were attributed to induction of apoptosis and/or increased production of interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we tested the ability of DCP to mediate inhibition of intracellular mycobacteria within human monocytes. DCP treatment of infected monocytes resulted in a significant reduction in viability of intracellular but not extracellular Mycobacterium bovis BCG. The antimicrobial activity of DCP was comparable to that of pyrazinamide (PZA), one of the first-line antituberculosis drugs currently used. DCP potentiated monocyte antimycobacterial activity by induction of the cysteine-cysteine (C-C) chemokine macrophage inflammatory protein 1β (MIP-1β) and inducible nitric oxide synthase 2. Addition of human anti-MIP-1β neutralizing antibody or a specific inhibitor of the l-arginase-nitric oxide pathway (NG-monomethyl l-arginine [l-NMMA] monoacetate) reversed the inhibitory effects of DCP on intracellular mycobacterial growth. These findings indicate that DCP induced mycobacterial killing via MIP-1β- and nitric oxide-dependent effects. Hence, DCP acts as an immunoregulatory compound enhancing the antimycobacterial activity of human monocytes. PMID:23509148

  19. Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth.

    PubMed

    Powers, Ciaran; Aigner, Achim; Stoica, Gerald E; McDonnell, Kevin; Wellstein, Anton

    2002-04-19

    Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK.

  20. Using evolutional properties of gene networks in understanding survival prognosis of glioblastoma.

    PubMed

    Upton, Alex; Arvanitis, Theodoros N

    2014-05-01

    Previously, we investigated survival prognosis of glioblastoma by applying a gene regulatory approach to a human glioblastoma dataset. Here, we further extend our understanding of survival prognosis of glioblastoma by refining the network inference technique we apply to the glioblastoma dataset with the intent of uncovering further topological properties of the networks. For this study, we modify the approach by specifically looking at both positive and negative correlations separately, as opposed to absolute correlations. There is great interest in applying mathematical modeling approaches to cancer cell line datasets to generate network models of gene regulatory interactions. Analysis of these networks using graph theory metrics can identify genes of interest. The principal approach for modeling microarray datasets has been to group all the cell lines together into one overall network, and then, analyze this network as a whole. As per the previous study, we categorize a human glioblastoma cell line dataset into five categories based on survival data, and analyze each category separately using both negative and positive correlation networks constructed using a modified version of the WGCNA algorithm. Using this approach, we identified a number of genes as being important across different survival stages of the glioblastoma cell lines.

  1. Arbutin, an intracellular hydroxyl radical scavenger, protects radiation-induced apoptosis in human lymphoma U937 cells.

    PubMed

    Wu, Li-Hua; Li, Peng; Zhao, Qing-Li; Piao, Jin-Lan; Jiao, Yu-Fei; Kadowaki, Makoto; Kondo, Takashi

    2014-11-01

    Ionizing radiation (IR) can generate reactive oxygen species (ROS). Excessive ROS have the potential to damage cellular macromolecules including DNA, proteins, and lipids and eventually lead to cell death. In this study, we evaluated the potential of arbutin, a drug chosen from a series of traditional herbal medicine by measuring intracellular hydroxyl radical scavenging ability in X-irradiated U937 cells. Arbutin (hydroquinone-β-D-glucopyranoside), a naturally occurring glucoside of hydroquinone, has been traditionally used to treat pigmentary disorders. However, there are no reports describing the effect of arbutin on IR-induced apoptosis. We confirmed that arbutin can protect cells from apoptosis induced by X-irradiation. The combination of arbutin and X-irradiation could reduce intracellular hydroxyl radical production and prevent mitochondrial membrane potential loss. It also could down-regulate the expression of phospho-JNK, phospho-p38 in whole cell lysate and activate Bax in mitochondria. Arbutin also inhibits cytochrome C release from mitochondria to cytosol. To verify the role of JNK in X-irradiation-induced apoptosis, the cells were pretreated with a JNK inhibitor, and found that JNK inhibitor could reduce apoptosis induced by X-irradiation. Taken together, our data indicate that arbutin plays an anti-apoptotic role via decreasing intracellular hydroxyl radical production, inhibition of Bax-mitochondria pathway and activation of the JNK/p38 MAPK pathway.

  2. A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

    PubMed

    Hack, N; Wilkinson, J M; Crawford, N

    1988-03-01

    To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

  3. PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells.

    PubMed

    Mukherjee, Joydeep; Ohba, Shigeo; See, Wendy L; Phillips, Joanna J; Molinaro, Annette M; Pieper, Russell O

    2016-07-01

    The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway. PMID:26874904

  4. Regulation of Cell Proliferation and Migration by miR-203 via GAS41/miR-10b Axis in Human Glioblastoma Cells

    PubMed Central

    Pal, Dhananjaya; Mukhopadhyay, Debasmita; Ramaiah, M. Janaki; Sarma, Pranjal; Bhadra, Utpal; Bhadra, Manika Pal

    2016-01-01

    Glioma amplified sequence 41(GAS41) is a potent transcription factor that play a crucial role in cell proliferation and survival. In glioblastoma, the expression of GAS41 at both transcriptional and post transcriptional level needs to be tightly maintained in response to cellular signals. Micro RNAs (miRNA) are small non coding RNA that act as important regulators for modulating the expression of various target genes. Studies have shown that several miRNAs play role in the post-transcriptional regulation of GAS41. Here we identified GAS41 as a novel target for endogenous miR-203 and demonstrate an inverse correlation of miR-203 expression with GAS41 in glioma cell lines (HNGC2 and U87). Over expression of miR-203 negatively regulates GAS41 expression in U87 and HNGC2 cell lines. Moreover, miR-203 restrained miR-10b action by suppressing GAS41. GAS41 is essential for repressing p53 in tumor suppressor pathway during cell proliferation. Enforced expression of GAS41 produced contradictory effect on miR-203 but was able to enhance p53 tumor suppressor pathway associated protein. It was also found that miR-203 maintains the stability of p53 as knock down of p53 expression using siRNA resulted in down regulation of pri-miR and mature miR-203 expression. Conversely reconstitution of miR-203 expression induced apoptosis and inhibited migratory property of glioma cells. Taken together, we show that miR-203 is a key negative regulator of GAS41 and acts as tumor suppressor microRNA in glioma. PMID:27467502

  5. Regulation of Cell Proliferation and Migration by miR-203 via GAS41/miR-10b Axis in Human Glioblastoma Cells.

    PubMed

    Pal, Dhananjaya; Mukhopadhyay, Debasmita; Ramaiah, M Janaki; Sarma, Pranjal; Bhadra, Utpal; Bhadra, Manika Pal

    2016-01-01

    Glioma amplified sequence 41(GAS41) is a potent transcription factor that play a crucial role in cell proliferation and survival. In glioblastoma, the expression of GAS41 at both transcriptional and post transcriptional level needs to be tightly maintained in response to cellular signals. Micro RNAs (miRNA) are small non coding RNA that act as important regulators for modulating the expression of various target genes. Studies have shown that several miRNAs play role in the post-transcriptional regulation of GAS41. Here we identified GAS41 as a novel target for endogenous miR-203 and demonstrate an inverse correlation of miR-203 expression with GAS41 in glioma cell lines (HNGC2 and U87). Over expression of miR-203 negatively regulates GAS41 expression in U87 and HNGC2 cell lines. Moreover, miR-203 restrained miR-10b action by suppressing GAS41. GAS41 is essential for repressing p53 in tumor suppressor pathway during cell proliferation. Enforced expression of GAS41 produced contradictory effect on miR-203 but was able to enhance p53 tumor suppressor pathway associated protein. It was also found that miR-203 maintains the stability of p53 as knock down of p53 expression using siRNA resulted in down regulation of pri-miR and mature miR-203 expression. Conversely reconstitution of miR-203 expression induced apoptosis and inhibited migratory property of glioma cells. Taken together, we show that miR-203 is a key negative regulator of GAS41 and acts as tumor suppressor microRNA in glioma.

  6. MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2.

    PubMed

    Liao, Hongzhan; Bai, Yifeng; Qiu, Shengcong; Zheng, Lei; Huang, Lianyan; Liu, Tianzhu; Wang, Xin; Liu, Yanting; Xu, Ningbo; Yan, Xiaohui; Guo, Hongbo

    2015-04-20

    Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM.

  7. PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells.

    PubMed

    Mukherjee, Joydeep; Ohba, Shigeo; See, Wendy L; Phillips, Joanna J; Molinaro, Annette M; Pieper, Russell O

    2016-07-01

    The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway.

  8. Polish Natural Bee Honeys Are Anti-Proliferative and Anti-Metastatic Agents in Human Glioblastoma multiforme U87MG Cell Line

    PubMed Central

    Moskwa, Justyna; Borawska, Maria H.; Markiewicz-Zukowska, Renata; Puscion-Jakubik, Anna; Naliwajko, Sylwia K.; Socha, Katarzyna; Soroczynska, Jolanta

    2014-01-01

    Honey has been used as food and a traditional medicament since ancient times. However, recently many scientists have been concentrating on the anti-oxidant, anti-proliferative, anti-inflammatory and other properties of honey. In this study, we investigated for the first time an anticancer effect of different honeys from Poland on tumor cell line - glioblastoma multiforme U87MG. Anti-proliferative activity of honeys and its interferences with temozolomide were determined by a cytotoxicity test and DNA binding by [H3]-thymidine incorporation. A gelatin zymography was used to conduct an evaluation of metalloproteinases (MMP-2 and MMP-9) expression in U87MG treatment with honey samples. The honeys were previously tested qualitatively (diastase activity, total phenolic content, lead and cadmium content). The data demonstrated that the examined honeys have a potent anti-proliferative effect on U87MG cell line in a time- and dose-dependent manner, being effective at concentrations as low as 0.5% (multifloral light honey - viability 53% after 72 h of incubation). We observed that after 48 h, combining honey with temozolomide showed a significantly higher inhibitory effect than the samples of honey alone. We observed a strong inhibition of MMP-2 and MMP-9 for the tested honeys (from 20 to 56% and from 5 to 58% compared to control, respectively). Our results suggest that Polish honeys have an anti-proliferative and anti-metastatic effect on U87MG cell line. Therefore, natural bee honey can be considered as a promising adjuvant treatment for brain tumors. PMID:24594866

  9. Polish natural bee honeys are anti-proliferative and anti-metastatic agents in human glioblastoma multiforme U87MG cell line.

    PubMed

    Moskwa, Justyna; Borawska, Maria H; Markiewicz-Zukowska, Renata; Puscion-Jakubik, Anna; Naliwajko, Sylwia K; Socha, Katarzyna; Soroczynska, Jolanta

    2014-01-01

    Honey has been used as food and a traditional medicament since ancient times. However, recently many scientists have been concentrating on the anti-oxidant, anti-proliferative, anti-inflammatory and other properties of honey. In this study, we investigated for the first time an anticancer effect of different honeys from Poland on tumor cell line - glioblastoma multiforme U87MG. Anti-proliferative activity of honeys and its interferences with temozolomide were determined by a cytotoxicity test and DNA binding by [H3]-thymidine incorporation. A gelatin zymography was used to conduct an evaluation of metalloproteinases (MMP-2 and MMP-9) expression in U87MG treatment with honey samples. The honeys were previously tested qualitatively (diastase activity, total phenolic content, lead and cadmium content). The data demonstrated that the examined honeys have a potent anti-proliferative effect on U87MG cell line in a time- and dose-dependent manner, being effective at concentrations as low as 0.5% (multifloral light honey - viability 53% after 72 h of incubation). We observed that after 48 h, combining honey with temozolomide showed a significantly higher inhibitory effect than the samples of honey alone. We observed a strong inhibition of MMP-2 and MMP-9 for the tested honeys (from 20 to 56% and from 5 to 58% compared to control, respectively). Our results suggest that Polish honeys have an anti-proliferative and anti-metastatic effect on U87MG cell line. Therefore, natural bee honey can be considered as a promising adjuvant treatment for brain tumors.

  10. CXCL12 modulation of CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of the tumor microenvironment.

    PubMed

    Würth, Roberto; Bajetto, Adriana; Harrison, Jeffrey K; Barbieri, Federica; Florio, Tullio

    2014-01-01

    Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells. Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem-like cells (CSCs) is believed to be the main responsible for tumor cell dissemination to the brain. GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g., CXCL12) causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4. This review covers recent developments on the role of CXCL12/CXCR4-CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include chemokine

  11. CXCL12 modulation of CXCR4 and CXCR7 activity in human glioblastoma stem-like cells and regulation of the tumor microenvironment

    PubMed Central

    Würth, Roberto; Bajetto, Adriana; Harrison, Jeffrey K.; Barbieri, Federica; Florio, Tullio

    2014-01-01

    Chemokines are crucial autocrine and paracrine players in tumor development. In particular, CXCL12, through its receptors CXCR4 and CXCR7, affects tumor progression by controlling cancer cell survival, proliferation and migration, and, indirectly, via angiogenesis or recruiting immune cells. Glioblastoma (GBM) is the most prevalent primary malignant brain tumor in adults and despite current multimodal therapies it remains almost incurable. The aggressive and recurrent phenotype of GBM is ascribed to high growth rate, invasiveness to normal brain, marked angiogenesis, ability to escape the immune system and resistance to standard of care therapies. Tumor molecular and cellular heterogeneity severely hinders GBM therapeutic improvement. In particular, a subpopulation of chemo- and radio-therapy resistant tumorigenic cancer stem–like cells (CSCs) is believed to be the main responsible for tumor cell dissemination to the brain. GBM cells display heterogeneous expression levels of CXCR4 and CXCR7 that are overexpressed in CSCs, representing a molecular correlate for the invasive potential of GBM. The microenvironment contribution in GBM development is increasingly emphasized. An interplay exists between CSCs, differentiated GBM cells, and the microenvironment, mainly through secreted chemokines (e.g., CXCL12) causing recruitment of fibroblasts, endothelial, mesenchymal and inflammatory cells to the tumor, via specific receptors such as CXCR4. This review covers recent developments on the role of CXCL12/CXCR4–CXCR7 networks in GBM progression and the potential translational impact of their targeting. The biological and molecular understanding of the heterogeneous GBM cell behavior, phenotype and signaling is still limited. Progress in the identification of chemokine-dependent mechanisms that affect GBM cell survival, trafficking and chemo-attractive functions, opens new perspectives for development of more specific therapeutic approaches that include chemokine

  12. MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    PubMed Central

    Qiu, Shengcong; Zheng, Lei; Huang, Lianyan; Liu, Tianzhu; Wang, Xin; Liu, Yanting; Xu, Ningbo; Yan, Xiaohui; Guo, Hongbo

    2015-01-01

    Epithelial-mesenchymal transition (EMT) has been recognized as a key element of cell migration, invasion, and drug resistance in several types of cancer. In this study, our aim was to clarify microRNAs (miRNAs)-related mechanisms underlying EMT followed by acquired resistance to chemotherapy in glioblastoma (GBM). We used multiple methods to achieve our goal including microarray analysis, qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, wound-healing assay and invasion assay. We found that miR-203 expression was significantly lower in imatinib-resistant GBM cells (U251AR, U87AR) that underwent EMT than in their parental cells (U251, U87). Ectopic expression of miR-203 with miRNA mimics effectively reversed EMT in U251AR and U87AR cells, and sensitized them to chemotherapy, whereas inhibition of miR-203 in the sensitive lines with antisense oligonucleotides induced EMT and conferred chemoresistance. SNAI2 was identified as a direct target gene of miR-203. The knockdown of SNAI2 by short hairpin RNA (shRNA) inhibited EMT and drug resistance. In GBM patients, miR-203 expression was inversely related to SNAI2 expression, and those tumors with low expression of miR-203 experienced poorer clinical outcomes. Our findings indicate that re-expression of miR-203 or targeting SNAI2 might serve as potential therapeutic approaches to overcome chemotherapy resistance in GBM. PMID:25871397

  13. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM.

    PubMed

    Jung, Kyung Oh; Youn, Hyewon; Kim, Seung Hoo; Kim, Young-Hwa; Kang, Keon Wook; Chung, June-Key

    2016-08-26

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and (64)Cu. HT29 (hTERT+) and U2OS (hTERT-) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo.

  14. Cystatins--Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells.

    PubMed

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte K; Wassélius, Johan; Ekström, Ulf; Abrahamson, Magnus

    2010-11-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the

  15. Recurrent Glioblastoma: Where we stand

    PubMed Central

    Roy, Sanjoy; Lahiri, Debarshi; Maji, Tapas; Biswas, Jaydip

    2015-01-01

    Current first-line treatment regimens combine surgical resection and chemoradiation for Glioblastoma that provides a slight increase in overall survival. Age on its own should not be used as an exclusion criterion of glioblastoma multiforme (GBM) treatment, but performance should be factored heavily into the decision-making process for treatment planning. Despite aggressive initial treatment, most patients develop recurrent diseases which can be treated with re-resection, systemic treatment with targeted agents or cytotoxic chemotherapy, reirradiation, or radiosurgery. Research into novel therapies is investigating alternative temozolomide regimens, convection-enhanced delivery, immunotherapy, gene therapy, antiangiogenic agents, poly ADP ribose polymerase inhibitors, or cancer stem cell signaling pathways. Given the aggressive and resilient nature of GBM, continued efforts to better understand GBM pathophysiology are required to discover novel targets for future therapy. PMID:26981507

  16. Pharmacological inhibition of lipid droplet formation enhances the effectiveness of curcumin in glioblastoma.

    PubMed

    Zhang, Issan; Cui, Yiming; Amiri, Abdolali; Ding, Yidan; Campbell, Robert E; Maysinger, Dusica

    2016-03-01

    Increased lipid droplet number and fatty acid synthesis allow glioblastoma multiforme, the most common and aggressive type of brain cancer, to withstand accelerated metabolic rates and resist therapeutic treatments. Lipid droplets are postulated to sequester hydrophobic therapeutic agents, thereby reducing drug effectiveness. We hypothesized that the inhibition of lipid droplet accumulation in glioblastoma cells using pyrrolidine-2, a cytoplasmic phospholipase A2 alpha inhibitor, can sensitize cancer cells to the killing effect of curcumin, a promising anticancer agent isolated from the turmeric spice. We observed that curcumin localized in the lipid droplets of human U251N glioblastoma cells. Reduction of lipid droplet number using pyrrolidine-2 drastically enhanced the therapeutic effect of curcumin in both 2D and 3D glioblastoma cell models. The mode of cell death involved was found to be mediated by caspase-3. Comparatively, the current clinical chemotherapeutic standard, temozolomide, was significantly less effective in inducing glioblastoma cell death. Together, our results suggest that the inhibition of lipid droplet accumulation is an effective way to enhance the chemotherapeutic effect of curcumin against glioblastoma multiforme.

  17. Targeting JNK for therapeutic depletion of stem-like glioblastoma cells

    PubMed Central

    Matsuda, Ken-ichiro; Sato, Atsushi; Okada, Masashi; Shibuya, Keita; Seino, Shizuka; Suzuki, Kaori; Watanabe, Eriko; Narita, Yoshitaka; Shibui, Soichiro; Kayama, Takamasa; Kitanaka, Chifumi

    2012-01-01

    Control of the stem-like tumour cell population is considered key to realizing the long-term survival of patients with glioblastoma, one of the most devastating human malignancies. To date, possible therapeutic targets and targeting methods have been described, but none has yet proven to target stem-like glioblastoma cells in the brain to the extent necessary to provide a survival benefit. Here we show that targeting JNK in vivo, the activity of which is required for the maintenance of stem-like glioblastoma cells, via transient, systemic administration of a small-molecule JNK inhibitor depletes the self-renewing and tumour-initiating populations within established tumours, inhibits tumour formation by stem-like glioblastoma cells in the brain, and provide substantial survival benefit without evidence of adverse events. Our findings not only implicate JNK in the maintenance of stem-like glioblastoma cells but also demonstrate that JNK is a viable, clinically relevant therapeutic target in the control of stem-like glioblastoma cells. PMID:22816039

  18. Gene expressions of TRP channels in glioblastoma multiforme and relation with survival.

    PubMed

    Alptekin, M; Eroglu, S; Tutar, E; Sencan, S; Geyik, M A; Ulasli, M; Demiryurek, A T; Camci, C

    2015-12-01

    Glioblastoma multiforme (GBM) is one of the most lethal forms of cancer in humans, with a median survival of 10 to 12 months. Glioblastoma is highly malignant since the cells are supported by a great number of blood vessels. Although new treatments have been developed by increasing knowledge of molecular nature of the disease, surgical operation remains the standard of care. The TRP (transient receptor potential) superfamily consists of cation-selective channels that have roles in sensory physiology such as thermo- and osmosensation and in several complex diseases such as cancer, cardiovascular, and neuronal diseases. The aim of this study was to investigate the expression levels of TRP channel genes in patients with glioblastoma multiforme and to evaluate the relationship between TRP gene expressions and survival of the patients. Thirty-three patients diagnosed with glioblastoma were enrolled to the study. The expression levels of 21 TRP genes were quantified by using qRT-PCR with dynamic array 48 × 48 chip (BioMark HD System, Fluidigm, South San Francisco, CA, USA). TRPC1, TRPC6, TRPM2, TRPM3, TRPM7, TRPM8, TRPV1, and TRPV2 were found significantly higher in glioblastoma patients. Moreover, there was a significant relationship between the overexpression of TRP genes and the survival of the patients. These results demonstrate for the first time that TRP channels contribute to the progression and survival of the glioblastoma patients. PMID:26088448

  19. Computational Trials: Unraveling Motility Phenotypes, Progression Patterns, and Treatment Options for Glioblastoma Multiforme

    PubMed Central

    Raman, Fabio; Scribner, Elizabeth; Saut, Olivier; Wenger, Cornelia; Colin, Thierry; Fathallah-Shaykh, Hassan M.

    2016-01-01

    Glioblastoma multiforme is a malignant brain tumor with poor prognosis and high morbidity due to its invasiveness. Hypoxia-driven motility and concentration-driven motility are two mechanisms of glioblastoma multiforme invasion in the brain. The use of anti-angiogenic drugs has uncovered new progression patterns of glioblastoma multiforme associated with significant differences in overall survival. Here, we apply a mathematical model of glioblastoma multiforme growth and invasion in humans and design computational trials using agents that target angiogenesis, tumor replication rates, or motility. The findings link highly-dispersive, moderately-dispersive, and hypoxia-driven tumors to the patterns observed in glioblastoma multiforme treated by anti-angiogenesis, consisting of progression by Expanding FLAIR, Expanding FLAIR + Necrosis, and Expanding Necrosis, respectively. Furthermore, replication rate-reducing strategies (e.g. Tumor Treating Fields) appear to be effective in highly-dispersive and moderately-dispersive tumors but not in hypoxia-driven tumors. The latter may respond to motility-reducing agents. In a population computational trial, with all three phenotypes, a correlation was observed between the efficacy of the rate-reducing agent and the prolongation of overall survival times. This research highlights the potential applications of computational trials and supports new hypotheses on glioblastoma multiforme phenotypes and treatment options. PMID:26756205

  20. Extracellular vesicles modulate the glioblastoma microenvironment via a tumor suppression signaling network directed by miR-1.

    PubMed

    Bronisz, Agnieszka; Wang, Yan; Nowicki, Michal O; Peruzzi, Pierpaolo; Ansari, Khairul I; Ogawa, Daisuke; Balaj, Leonora; De Rienzo, Gianluca; Mineo, Marco; Nakano, Ichiro; Ostrowski, Michael C; Hochberg, Fred; Weissleder, Ralph; Lawler, Sean E; Chiocca, E Antonio; Godlewski, Jakub

    2014-02-01

    Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem-like glioblastoma cells. An extracellular vesicle-dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle-derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management. PMID:24310399

  1. Human gastric signet ring carcinoma (KATO-III) cell apoptosis induced by Vitex agnus-castus fruit extract through intracellular oxidative stress.

    PubMed

    Ohyama, Kunio; Akaike, Takenori; Imai, Masahiko; Toyoda, Hiroo; Hirobe, Chieko; Bessho, Toshio

    2005-07-01

    We have previously reported that an ethanol extract of the dried ripe fruit of Vitex agnus-castus (Vitex) displays cytotoxic activity against certain kinds of human cancer cell line resulting in the induction of apoptosis. In this paper, we investigate the molecular mechanism of apoptosis induced by Vitex using a human gastric signet ring carcinoma cell line, KATO-III. DNA fragmentation was observed in Vitex-treated KATO-III cells in a time- and dose-dependent manner. DNA fragmentation was accompanied by the following phenomena: elevation in the level of hemeoxygenase-1 protein and thioredoxin reductase mRNA; repression of Mn-superoxide dismutase and catalase mRNAs; release of cytochrome c from mitochondria into the cytosol; activation of caspases-8, -9 and -3; decrease in the level of Bcl-2, Bcl-XL and Bid protein; increase in the level of Bad protein. The intracellular oxidized state, measured using 2',7'-dichlorofluorescin diacetate, increased after Vitex treatment. While the amount of intracellular GSH decreased significantly after treatment with Vitex, the level of GSSG was unaffected. Furthermore, no significant perturbation in the amount of proteins/mRNAs related to glutathione metabolism could be detected. These apoptotic alterations induced by exposure to Vitex were blocked by the presence of an anti-oxidative reagent, N-acetyl-l-cysteine, or the addition of exogenous GSH. Our results demonstrate that intracellular oxidative stress and mitochondrial membrane damage is responsible for Vitex-induced apoptosis, which may be mediated by a diminution of reduced type glutathione within the cell. PMID:15833280

  2. Antitumor effects of minodronate, a third-generation nitrogen-containing bisphosphonate, in synergy with γδT cells in human glioblastoma in vitro and in vivo.

    PubMed

    Nakazawa, Tsutomu; Nakamura, Mitsutoshi; Matsuda, Ryosuke; Nishimura, Fumihiko; Park, Young Soo; Motoyama, Yasushi; Hironaka, Yasuo; Nakagawa, Ichiro; Yokota, Hiroshi; Yamada, Shuichi; Tamura, Kentaro; Takeshima, Yasuhiro; Omoto, Kouji; Tanaka, Yoshitaka; Ouji, Yukiteru; Yoshikawa, Masahide; Tsujimura, Takahiro; Nakase, Hiroyuki

    2016-09-01

    Nitrogen-containing bisphosphonates (N-BPs), which prevent bone resorption, exert direct and γδT cell (GDT)-mediated antitumor effects against several tumor cell types, including glioblastoma (GBM). However, limited information is available regarding the antitumor effects of N-BPs in GBM. Specifically, the antitumor effects of minodronate (MDA), a third-generation N-BP, in GBM are yet unclear. This study aimed to investigate the antitumor effects of MDA in GBM in vitro and in vivo. We performed growth inhibition and apoptosis detection assays using the GBM cell lines U87MG and U138MG. Apoptosis inhibition assays were also conducted. In vivo xenograft assays were performed in highly immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Sug)/Jic mice subcutaneously implanted with U87MG and U138MG cells. Growth inhibition and apoptosis detection assays demonstrated that MDA inhibited GBM cell growth via apoptosis, which was markedly enhanced by ex vivo expanded GDT. A pan-caspase inhibitor, z-VAD-fmk, inhibited MDA-induced U138MG apoptosis and MDA/GDT-induced U87MG and U138MG apoptosis. But z-VAD-fmk increased MDA-induced U87MG apoptosis. MDA/GDT-mediated apoptosis was blocked by the anti-T cell receptor (TCR) Vγ9, mevalonate pathway inhibitor, granzyme B inhibitor, and antitumor necrosis factor (TNF)-α. In vivo xenograft assays showed that combined intraperitoneal administration of MDA/GDT induced antitumor effects on unestablished U87MG-derived subcutaneous tumors. MDA exerted direct and GDT-mediated anti-GBM apoptotic effects in a caspase-dependent manner. GDT recognized MDA-exposed GBM cells via TCRVγ9 and induced apoptosis via granzyme B and TNF-α release. Because MDA elicited anti-GBM effects in synergy with GDT in vivo, a combination of MDA and ex vivo-generated GDT could be an effective treatment in patients with GBM. PMID:27393349

  3. Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis

    PubMed Central

    Cui, Qi; Yang, Su; Ye, Peng; Tian, E.; Sun, Guoqiang; Zhou, Jiehua; Sun, Guihua; Liu, Xiaoxuan; Chen, Chao; Murai, Kiyohito; Zhao, Chunnian; Azizian, Krist T.; Yang, Lu; Warden, Charles; Wu, Xiwei; D'Apuzzo, Massimo; Brown, Christine; Badie, Behnam; Peng, Ling; Riggs, Arthur D.; Rossi, John J.; Shi, Yanhong

    2016-01-01

    Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma. PMID:26838672

  4. Blockade of Human Immunodeficiency Virus Type 1 Production in CD4^+ T Cells by an Intracellular CD4 Expressed Under Control of the Viral Long Terminal Repeat

    NASA Astrophysics Data System (ADS)

    Buonocore, Linda; Rose, John K.

    1993-04-01

    A retroviral vector was constructed in which a gene encoding a mutated soluble CD4 protein that is retained in the endoplasmic reticulum (sCD4-KDEL) is expressed under control of human immunodeficiency virus type 1 (HIV-1) regulatory elements. HIV-1 infection of a human T-cell line transduced with this vector led to induction of sCD4-KDEL synthesis and a block in transport of the HIV envelope protein to the cell surface. There was a complete block to maturation of infectious HIV-1 in the transduced cells, no viral spread, and little or no syncytium formation. Infected cells gradually disappeared from the culture over a period of 2 months. This intracellular trap for HIV has potential application in gene therapy for AIDS.

  5. Control of glioblastoma tumorigenesis by feed-forward cytokine signaling

    PubMed Central

    Jahani-Asl, Arezu; Yin, Hang; Soleimani, Vahab D; Haque, Takrima; Luchman, H Artee; Chang, Natasha C; Sincennes, Marie-Claude; Puram, Sidharth V; Scott, Andrew M; Lorimer, Ian A J; Perkins, Theodore J; Ligon, Keith L; Weiss, Samuel; Rudnicki, Michael A; Bonni, Azad

    2016-01-01

    EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of those mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis. PMID:27110918

  6. Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

    PubMed Central

    Cater, Michael A; Forbes, John; La Fontaine, Sharon; Cox, Diane; Mercer, Julian F B

    2004-01-01

    The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases. PMID:14998371

  7. New facets of keratin K77: interspecies variations of expression and different intracellular location in embryonic and adult skin of humans and mice.

    PubMed

    Langbein, Lutz; Reichelt, Julia; Eckhart, Leopold; Praetzel-Wunder, Silke; Kittstein, Walter; Gassler, Nikolaus; Schweizer, Juergen

    2013-12-01

    The differential expression of keratins is central to the formation of various epithelia and their appendages. Structurally, the type II keratin K77 is closely related to K1, the prototypical type II keratin of the suprabasal epidermis. Here, we perform a developmental study on K77 expression in human and murine skin. In both species, K77 is expressed in the suprabasal fetal epidermis. While K77 appears after K1 in the human epidermis, the opposite is true for the murine tissue. This species-specific pattern of expression is also found in conventional and organotypic cultures of human and murine keratinocytes. Ultrastructure investigation shows that, in contrast to K77 intermediate filaments of mice, those of the human ortholog are not attached to desmosomes. After birth, K77 disappears without deleterious consequences from human epidermis while it is maintained in the adult mouse epidermis, where its presence has so far gone unnoticed. After targeted Krt1 gene deletion in mice, K77 is normally expressed but fails to functionally replace K1. Besides the epidermis, both human and mouse K77 are present in luminal duct cells of eccrine sweat glands. The demonstration of a K77 ortholog in platypus but not in non-mammalian vertebrates identifies K77 as an evolutionarily ancient component of the mammalian integument that has evolved different patterns of intracellular distribution and adult tissue expression in primates. PMID:24057875

  8. A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.

    PubMed

    Amici, Carla; Visintin, Michela; Verachi, Francesca; Paolini, Francesca; Percario, Zulema; Di Bonito, Paola; Mandarino, Angela; Affabris, Elisabetta; Venuti, Aldo; Accardi, Luisa

    2016-03-29

    Single-chain variable fragments (scFvs) expressed as "intracellular antibodies" (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients.A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells.Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors. PMID:26788990

  9. Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain

    SciTech Connect

    Radzimanowski, Jens; Beyreuther, Konrad; Sinning, Irmgard; Wild, Klemens

    2008-05-01

    Alzheimer’s disease is characterized by proteolytic processing of the amyloid precursor protein (APP), which releases the aggregation-prone amyloid-β (Aβ) peptide and liberates the intracellular domain (AICD) that interacts with various adaptor proteins. The crystallized AICD–Fe65-PTB2 complex is of central importance for APP translocation, nuclear signalling, processing and Aβ generation. Alzheimer’s disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid β peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 Å resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

  10. Rhein triggers apoptosis via induction of endoplasmic reticulum stress, caspase-4 and intracellular calcium in primary human hepatic HL-7702 cells.

    PubMed

    KoraMagazi, Arouna; Wang, Dandan; Yousef, Bashir; Guerram, Mounia; Yu, Feng

    2016-04-22

    Rhein is an active component of rhubarb; a traditional Chinese medicine reported to induce apoptosis and cause liver toxicity. However, rhein's apoptotic-inducing effects, as well as its molecular mechanisms of action on hepatic cells need to be further explored. In the present study, rhein was found to trigger apoptosis in primary human hepatic HL-7702 cells as showed by annexin V/PI double staining assay and nuclear morphological changes demonstrated by Hoechst 33258 staining. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was caspase-dependent, presumably via ER-stress associated pathways, as illustrated by up-regulation of glucose-regulated protein 78 (GRP 78), PKR-like ER kinase (PERK), C-Jun N-terminal kinase (JNK) and CCAAT/enhancer-binding protein homologous protein (CHOP). Meanwhile, caspase-4 as a hallmark of ER-stress, was also showed to be activated following by caspase-3 activation. Furthermore, rhein also promoted intracellular elevation of calcium that contributed in apoptosis induction. Interestingly, pre-treatment with calpain inhibitor I reduced the effects of rhein on apoptosis induction and JNK activation. These data suggested that rhein-induced apoptosis through ER-stress and elevated intracellular calcium level in HL-7702 cells. PMID:27003256

  11. Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction.

    PubMed Central

    Gorn, A H; Rudolph, S M; Flannery, M R; Morton, C C; Weremowicz, S; Wang, T Z; Krane, S M; Goldring, S R

    1995-01-01

    Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system. Images PMID:7769107

  12. Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology.

    PubMed

    Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pelà, Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D'Arca, Domenico

    2016-01-01

    Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding. PMID:27250901

  13. Isolation of Microarray-Quality RNA from Primary Human Cells after Intracellular Immunostaining and Fluorescence-Activated Cell Sorting

    PubMed Central

    Iglesias-Ussel, Maria; Marchionni, Luigi; Romerio, Fabio

    2013-01-01

    Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that – although the RNA isolated from these samples presented some degradation – it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis. PMID:23434645

  14. Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

    PubMed Central

    Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pelà, Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D’Arca, Domenico

    2016-01-01

    Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding. PMID:27250901

  15. Aberrant Notch signaling in glioblastoma stem cells contributes to tumor recurrence and invasion.

    PubMed

    Yu, Jian-Bo; Jiang, Hao; Zhan, Ren-Ya

    2016-08-01

    Upregulation of the Notch signaling pathway in cancer stem cells and side population (SP) cells has a major role in maintenance, self-renewal and chemoresistance. The present study isolated a cancer stem cell-like SP accounting for 4.1% of a glioblastoma cell population using a Hoechst 33342 dye exclusion assay. In this glioblastoma SP, the expression of of Notch1 signaling proteins Notch1 intracellular domain and Hes‑1 was markedly upregulated. Furthermore, knockdown of Notch1 by RNA interference significantly diminished the neurosphere formation ability, self‑renewal and chemoresistance of the SP cells. In addition, the expression of the stem‑cell surface genes Oct‑4, Sox2 and Nanog in SP cells was significantly reduced and the sensitivity to the SP cells to chemotherapeutics was enhanced following Notch1 knockdown. In conclusion, the results of the present study suggested that upregulation of Notch1 is involved in the chemotherapy resistance and tumor recurrence of glioblastoma. Hence, the development of novel anti‑cancer drugs targeting the Notch1 signaling pathway may be a promising strategy for curing glioblastoma. PMID:27315154

  16. PINK1 Is a Negative Regulator of Growth and the Warburg Effect in Glioblastoma.

    PubMed

    Agnihotri, Sameer; Golbourn, Brian; Huang, Xi; Remke, Marc; Younger, Susan; Cairns, Rob A; Chalil, Alan; Smith, Christian A; Krumholtz, Stacey-Lynn; Mackenzie, Danielle; Rakopoulos, Patricia; Ramaswamy, Vijay; Taccone, Michael S; Mischel, Paul S; Fuller, Gregory N; Hawkins, Cynthia; Stanford, William L; Taylor, Michael D; Zadeh, Gelareh; Rutka, James T

    2016-08-15

    Proliferating cancer cells are characterized by high rates of glycolysis, lactate production, and altered mitochondrial metabolism. This metabolic reprogramming provides important metabolites for proliferation of tumor cells, including glioblastoma. These biological processes, however, generate oxidative stress that must be balanced through detoxification of reactive oxygen species (ROS). Using an unbiased retroviral loss-of-function screen in nontransformed human astrocytes, we demonstrate that mitochondrial PTEN-induced kinase 1 (PINK1) is a regulator of the Warburg effect and negative regulator of glioblastoma growth. We report that loss of PINK1 contributes to the Warburg effect through ROS-dependent stabilization of hypoxia-inducible factor-1A and reduced pyruvate kinase muscle isozyme 2 activity, both key regulators of aerobic glycolysis. Mechanistically, PINK1 suppresses ROS and tumor growth through FOXO3a, a master regulator of oxidative stress and superoxide dismutase 2. These findings highlight the importance of PINK1 and ROS balance in normal and tumor cells. PINK1 loss was observed in a significant number of human brain tumors including glioblastoma (n > 900) and correlated with poor patient survival. PINK1 overexpression attenuates in vivo glioblastoma growth in orthotopic mouse xenograft models and a transgenic glioblastoma model in Drosophila Cancer Res; 76(16); 4708-19. ©2016 AACR. PMID:27325644

  17. 5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2