Human Glioblastoma U87MG Cells Research Articles

Green Synthesis of Silver Nanoparticles Using Diospyros malabarica Fruit Extract and Assessments of Their Antimicrobial, Anticancer and Catalytic Reduction of 4-Nitrophenol (4-NP).

The green synthesis of silver nanoparticles (AgNPs) has currently been gaining wide applications in the medical field of nanomedicine. Green synthesis is one of the most effective procedures for the production of AgNPs. The Diospyros malabarica tree grown throughout India has been reported to have antioxidant and various therapeutic applications. In the context of this, we have investigated the fruit of Diospyros malabarica for the potential of forming AgNPs and analyzed its antibacterial and anticancer activity. We have developed a rapid, single-step, cost-effective and eco-friendly method for the synthesis of AgNPs using Diospyros malabarica aqueous fruit extract at room temperature. The AgNPs began to form just after the reaction was initiated. The formation and characterization of AgNPs were confirmed by UV-Vis spectrophotometry, XRD, FTIR, DLS, Zeta potential, FESEM, EDX, TEM and photoluminescence (PL) methods. The average size of AgNPs, in accordance with TEM results, was found to be 17.4 nm. The antibacterial activity of the silver nanoparticles against pathogenic microorganism strains of Staphylococcus aureus and Escherichia coli was confirmed by the well diffusion method and was found to inhibit the growth of the bacteria with an average zone of inhibition size of (8.4 ± 0.3 mm and 12.1 ± 0.5 mm) and (6.1 ± 0.7 mm and 13.1 ± 0.5 mm) at 500 and 1000 µg/mL concentrations of AgNPs, respectively. The anticancer effect of the AgNPs was confirmed by MTT assay using the U87-MG (human primary glioblastoma) cell line. The IC50 value was found to be 58.63 ± 5.74 μg/mL. The results showed that green synthesized AgNPs exhibited significant antimicrobial and anticancer potency. In addition, nitrophenols, which are regarded as priority pollutants by the United States Environmental Protection Agency (USEPA), can also be catalytically reduced to less toxic aminophenols by utilizing synthesized AgNPs. As a model reaction, AgNPs are employed as a catalyst in the reduction of 4-nitrophenol to 4-aminophenol, which is an intermediate for numerous analgesics and antipyretic drugs. Thus, the study is expected to help immensely in the pharmaceutical industries in developing antimicrobial drugs and/or as an anticancer drug, as well as in the cosmetic and food industries.

Valproic Acid (VPA) in Combination with Knockdown of AKT3 and PI3KCA Genes Inhibits Proliferation, Induces Apoptosis and Autophagy in T98G and U87MG Glioblastoma Multiforme Cells

Purpose: Glioblastoma Multiforme (GBM) is a heterogenous and highly vascularized brain tumor that avoid apoptosis due to P-glycoprotein (P-gp) mediated multi-drug resistance. Therefore, development of new therapeutic strategies that induce apoptosis, inhibit proliferation, and overcome multi-drug resistance is urgently warranted. We examined the efficacy of combination of Valproic Acid (VPA) and knockdown of AKT3 and PI3KCA genes in human glioblastoma T98G and U87MG cell lines. Material and Methods: T98G and U87MG cells were transfected with AKT3 or PI3KCA siRNAs. Transfection efficiency was assessed using Flow Cytometry (FC) and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNAs in combination with VPA on T98G and U87MG cell viability, proliferation, apoptosis and autophagy was evaluated as well. Alterations in the mRNA expression of apoptosis-related genes (CASP3 and Bid) were analyzed using QRT-PCR. Results: The transfection of T98G and U87MG cells with AKT3 or PI3KCA siRNAs and exposition on VPA led to a significant reduction in cell viability, the accumulation of subG1-phase cells and a reduced fraction of cells in the S and G2/M phases, apoptosis or necrosis induction and induction of autophagy. Conclusions: The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with VPA may offer a novel therapeutic strategy to more effective control the growth of human GBM cells. Thus, knockdown of these genes in combination with valproic acid inhibits proliferation, induces apoptosis and autophagy in T98G and U87MG cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.

Chemical structure of native and modified sulfated heterorhamnans from the green seaweed Gayralia brasiliensis and their cytotoxic effect on U87MG human glioma cells

A water-soluble sulfated heterorhamnan (Gb1) was isolated from the green seaweed Gayralia brasiliensis and purified by ultrafiltration, yielding a homogeneous polysaccharide (Gb1r). Both fractions contained rhamnose, xylose, galacturonic and glucuronic acids, galactose, and glucose. Chemical and spectroscopic methods allowed the determination of Gb1 and Gb1r chemical structure. Their backbones were constituted by 3-, 2-, and 2,3-linked rhamnosyl units (1:0.49:0.13 and 1:0.58:0.17, respectively), which are unsulfated (13.5 and 14.6%), disulfated (16.6 and 17.8%) or monosulfated at C-2 (8 and 8.6%) and C-4 (24.5 and 23.4%). Gb1 was oversulfated giving rise to Gb1-OS, which presented ~2.5-fold higher content of disulfated rhamnosyl units than Gb1, as determined by methylation analyses and NMR spectroscopy. Gb1 and Gb1-OS potently reduced the viability of U87MG human glioblastoma cells. Gb1 caused cell cycle arrest in the G1 phase, increased annexin V-stained cells, and no DNA fragmentation, while Gb1-OS increased the percentage of cells in the S and G2 phases and the levels of fragmented DNA and cells double-stained with annexin V/propidium iodide, suggesting an apoptosis mechanism. The results suggest that the different effects of Gb1 and Gb1-OS were related to differences in the sulfate content and position of these groups along the polysaccharide chains.

Liposomal IR-780 as a Highly Stable Nanotheranostic Agent for Improved Photothermal/Photodynamic Therapy of Brain Tumors by Convection-Enhanced Delivery.

Simple SummaryTo improve the use of hydrophobic photosensitizer IR-780 in photothermal/photodynamic therapy (PTT/PDT), we entrap IR-780 within the lipid bilayer of liposomes (ILs). Compared to free IR-780, ILs showed well-preserved photothermal response by maintaining the photostability of IR-780 from repeated near infrared (NIR) laser exposure both in vitro and in vivo. Combined with fast endocytosis by human glioblastoma cells, ILs demonstrated enhanced cytotoxicity and induced higher cell apoptosis rate toward human glioblastoma cells over free IR-780, due to PTT with overexpression of heat shock protein and PDT with generation of intracellular reactive oxygen species. To overcome the blood–brain barrier, we used convection enhanced delivery (CED) for specific delivery of ILs to brain tumors in intracranial glioma xenograft. Upon three successive NIR laser irradiations, the liposomal IR-780 could significantly improve the anti-cancer efficacy in glioma treatment, leading to diminished intracranial tumor size and prolonged animal survival time.As a hydrophobic photosensitizer, IR-780 suffers from poor water solubility and low photostability under near infrared (NIR) light, which severely limits its use during successive NIR laser-assisted photothermal/photodynamic therapy (PTT/PDT). To solve this problem, we fabricate cationic IR-780-loaded liposomes (ILs) by entrapping IR-780 within the lipid bilayer of liposomes. We demonstrate enhanced photostability of IR-780 in ILs with well-preserved photothermal response after three repeated NIR laser exposures, in contrast to the rapid decomposition of free IR-780. The cationic nature of ILs promotes fast endocytosis of liposomal IR-780 by U87MG human glioblastoma cells within 30 min. For PTT/PDT in vitro, ILs treatment plus NIR laser irradiation leads to overexpression of heat shock protein 70 and generation of intracellular reactive oxygen species by U87MG cells, resulting in enhanced cytotoxicity and higher cell apoptosis rate. Using intracranial glioma xenograft in nude mice and administration of ILs by convection enhanced delivery (CED) to overcome blood-brain barrier, liposomal IR-780 could be specifically delivered to the brain tumor, as demonstrated from fluorescence imaging. By providing a highly stable liposomal IR-780, ILs significantly improved anti-cancer efficacy in glioma treatment, as revealed from various diagnostic imaging tools and histological examination. Overall, CED of ILs plus successive laser-assisted PTT/PDT may be an alternative approach for treating brain tumor, which can retard glioma growth and prolong animal survival times from orthotopic brain tumor models.

Genistein and Temozolomide-Loaded Polymeric Nanoparticles: A Synergistic Approach For Improved Anti-Tumor Efficacy Against Glioblastoma

Glioblastoma multiforme (GBM) is one of the most malignant type of brain cancer. Highly invasive nature of GBM and poor drug penetration from physiological barriers makes GBM treatment challenging. In this study, temozolomide (TMZ) and genistein (Gen) dual-drug-loaded poly(lactic-co-glycolic-acid) nanoparticle systems (Gen-TMZ-NPs) were developed to achieve enhanced anti-tumor activity in U87MG human glioblastoma cells. For this aim, Gen-TMZ-NPs alongside TMZ and Gen single-drug-loaded PLGA-NPs were produced and physico-chemical properties of fabricated nanoparticles were examined by polydispersity index, mean particle size, zeta potential and FT-IR spectroscopy. In vitro cytotoxic activity of Gen-NP, TMZ-NP and Gen-TMZ-NPs on U87MG cells were assessed and an increased cytotoxicity was observed in Gen-TMZ-NP treated U87MG cells, with IC50 values 35.2 μg/ml for 24 h and 8.7 μg/ml for 48 h. U87MG cell migration activity was significantly inhibited in Gen-TMZ-NP treated group with 6.80% wound closure rate after 48 h treatment and BrdU incorporation assay established a decreased cell proliferation. Moreover, quantitative real-time PCR analysis and western blotting confirmed cytochrome-c mediated activation of apoptosis in Gen-TMZ-NP treated U87MG cells. Our results showed that Gen-TMZ-NPs exhibited improved anti-tumor activity in U87MG cells and that dual nano drug delivery might be a promising strategy against glioblastoma.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

SN38 loaded nanostructured lipid carriers (NLCs); preparation and in vitro evaluations against glioblastoma

SN38 is the active metabolite of irinotecan with 1000-fold greater cytotoxicity compared to the parent drug. Despite the potential, its application as a drug is still seriously limited due to its stability concerns and low solubility in acceptable pharmaceutical solvents. To address these drawbacks here nanostructured lipid carrier (NLC) containing SN38 was prepared and its cytotoxicity against U87MG glioblastoma cell line was investigated. The formulations were prepared using hot ultrasonication and solvent evaporation/emulsification methods. NLCs with a mean size of 140 nm and particle size distribution (PDI) of 0.25 were obtained. The average loading efficiency was 9.5% and its entrapment efficiency was 81%. In order to obtain an accurate determination of released amount of SN38 a novel medium and extraction method was designed, which lead to an appropriate in vitro release profile of the drug from the prepared NLCs. The MTT test results revealed the significant higher cytotoxicity of NLCs on U87MG human glioblastoma cell line compared with the free drug. The confocal microscopy images confirmed the proper penetration of the nanostructures into the cells within the first 4 h. Consequently, the results indicated promising potentials of the prepared NLCs as a novel treatment for glioblastoma.

Enhancement of T2* Weighted MRI Imaging Sensitivity of U87MG Glioblastoma Cells Using γ-Ray Irradiated Low Molecular Weight Hyaluronic Acid-Conjugated Iron Nanoparticles.

IntroductionIt has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection.MethodsLMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs.ResultsFlorescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells.DiscussionOverall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.

WDR81 Gene Silencing Can Reduce Exosome Levels in Human U87-MG GlioblastomaCells.

Glioblastoma is a very invasive and prevalent brain tumor that affects 15 in 100,000 persons over the age of 70years. Studies have shown that the expression of the WD repeat domain 81 (WDR81) gene, which is effective in vesicular transport and inhibition of autophagy, is increased in glioblastoma. The decreased autophagy was found to be related to the increased production of exosomes, which is a major factor in the pathogenesis of glioblastoma. The PI-3kinase complex is a pre-autophagic complex that is highly active in the absence of WDR81. The WDR81 gene, as a negative regulator of PI3K activity, prevents autophagy and increases exosome secretion by preventing the formation of the class III PI3K complex. Therefore, targeted reduction of exosomes can be considered an effective strategy for reducing the pathogenesis of glioblastoma. This study aimed to assess the effect of WDR81 gene silencing with siRNA on exosome levels in a U87-MG cell line. Culturing of U87-MG cells was carried out in Dulbecco's modified Eagle medium (DMEM) containing 5% FBS and 1% penicillin/streptomycin. Thereafter, silencing of WDR81 was performed using WDR81 siRNA, whose gene expression level was determined via real-time qRT-PCR. Cell viability was evaluated using the MTT assay. The exosomes were extracted from a cell culture using the Exocib kit. The size accuracy of the exosomes was confirmed by dynamic light scattering (DLS). Finally, the protein content and RNA of the exosomes were assessed. WDR81 gene expression of siRNA-transfected cells was decreased to 82% after 24h compared to the non-transfected control cells. The analysis of the exosomes showed that the concentration of exosomes and their RNA and protein content in the siRNA-transfected cells decreased significantly compared to the non-transfected control cells. No considerable difference was observed in cell viability after transfection with either WDR81-specific siRNAs or scrambled control siRNAs. Our findings showed that silencing the WDR81 gene could reduce the level of exosomes in human U87-MG glioblastoma cells. Therefore, the reduced exosome content may be suggested as a new gene therapy strategy for targeted therapy of glioblastoma by increasing autophagy via activation of PI3KIII. However, more studies are needed in this regard.

Polyphenolic Compounds of Crataegus Berry, Leaf, and Flower Extracts Affect Viability and Invasive Potential of Human Glioblastoma Cells.

Crataegus contains numerous health-promoting compounds that are also proposed to have anti-cancer properties. Herein, we aimed at a contemporaneous evaluation of the effects of polyphenol-rich extracts of berries, leaves, and flowers of six Crataegus species on the viability and invasive potential on the highly aggressive human glioblastoma U87MG cell line. The treatment with the extracts evoked cytotoxic effects, with the strongest in the berry extracts. All extracts not only promoted the apoptosis-related cleavage of poly (ADP-ribose) polymerase 1 (PARP1) but also substantially inhibited the activity of pro-survival kinases, focal adhesion kinase (FAK), and protein kinase B (PKB; also known as Akt), thus indicating the suppression of proliferative and invasive potentials of the examined glioblastoma cells. The qualitative and quantitative characterization of the extracts’ content was also performed and revealed that amongst 37 polyphenolic compounds identified in the examined Crataegus extracts, the majority (29) was detected in berries; the leaf and flower extracts, exerting milder cytotoxic effects, contained only 14 and 13 compounds, respectively. The highest polyphenol content was found in the berries of C. laevigata x rhipidophylla x monogyna, in which flavan-3-ols and phenolic acids predominated. Our results demonstrated that a high content of polyphenolic compounds correlated with the extract cytotoxicity, and especially berries were a valuable source of compounds with anti-cancer potential. This might be a promising option for the development of an effective therapeutic strategy against highly malignant glioblastomas in the future.

PC3-Secreted Microprotein Is Expressed in Glioblastoma Stem-Like Cells and Human Glioma Tissues.

Glioblastoma multiforme (GBM) is the most prevalent malignant primary brain tumor with a high recurrence rate. Despite multimodal therapy including surgical resection, chemotherapy, and radiotherapy, the median survival time after the initial diagnosis of GBM is approximately 14 months. Since cancer stem cells (CSCs) are considered the leading cause of cancer recurrence, glioblastoma stem cell-targeted therapy is a promising strategy for the treatment of GBM. However, because CSC heterogeneity has been implicated in the difficulties of CSC-target therapy, more in-depth knowledge of CSC biology is still required to develop novel therapies. In this study, we established single cell-derived tumorspheres from human glioblastoma U87MG cells. One of these tumorspheres, P4E8 clone, showed CSC-like phenotypes, such as self-renewal capacity, expression of CSC markers, resistance to anti-cancer agents, and in vivo tumorigenicity. Therefore, we used P4E8 cells as a cell-based model of glioblastoma stem cells (GSCs). Gene expression analysis using microarray indicated that the most highly expressed genes in P4E8 cells compared to the parental U87MG were PC3-secreted microprotein (MSMP). Furthermore, MSMP was expressed in patient-derived GSCs and human glioma tissues at the protein level, implying that MSMP might contribute to glioma development and progression.

Terbium-Based AGuIX-Design Nanoparticle to Mediate X-ray-Induced Photodynamic Therapy.

X-ray-induced photodynamic therapy is based on the energy transfer from a nanoscintillator to a photosensitizer molecule, whose activation leads to singlet oxygen and radical species generation, triggering cancer cells to cell death. Herein, we synthesized ultra-small nanoparticle chelated with Terbium (Tb) as a nanoscintillator and 5-(4-carboxyphenyl succinimide ester)-10,15,20-triphenyl porphyrin (P1) as a photosensitizer (AGuIX@Tb-P1). The synthesis was based on the AGuIX@ platform design. AGuIX@Tb-P1 was characterised for its photo-physical and physico-chemical properties. The effect of the nanoparticles was studied using human glioblastoma U-251 MG cells and was compared to treatment with AGuIX@ nanoparticles doped with Gadolinium (Gd) and P1 (AGuIX@Gd-P1). We demonstrated that the AGuIX@Tb-P1 design was consistent with X-ray photon energy transfer from Terbium to P1. Both nanoparticles had similar dark cytotoxicity and they were absorbed in a similar rate within the cells. Pre-treated cells exposure to X-rays was related to reactive species production. Using clonogenic assays, establishment of survival curves allowed discrimination of the impact of radiation treatment from X-ray-induced photodynamic effect. We showed that cell growth arrest was increased (35%-increase) when cells were treated with AGuIX@Tb-P1 compared to the nanoparticle doped with Gd.

Graphene Oxide/Zinc Oxide Nanocomposite Displaying Selective Toxicity to Glioblastoma Cell Lines

Glioblastoma is considered the most aggressive and prevalent type of glioma. Resistance mechanisms, side effects, and the blood–brain barrier are factors that make its treatment difficult, requiring the development of alternative therapeutic strategies. Herein, we propose a nanocomposite composed of carboxylated graphene oxide nanosheets decorated with zinc oxide nanoparticles and post-functionalized with Pluronic (GOC-ZnO-P) for chemotherapy against U87MG and U138MG human glioblastoma cell lines. The GOC-ZnO-P formulation is fairly stable in the physiological medium (DMEM-FBS) and demonstrates selectivity toward tumor cell lines, even though it is less cytotoxic than free ZnO. Whereas the photothermal activity of the nanocomposite has a negligible cytotoxic effect, images obtained by scanning and transmission electron microscopies reveal that it induces changes in adhesion points and roughness of the tumor cell membrane, and it is uptaken through vesicles and capable of accumulating in the nucleus, a process that can induce cell death by apoptosis, as verified by flow cytometry. In summary, the GOC phase serves as an inert platform for transporting ZnO, which maintains its selectivity for tumor cells and does not interfere with the mechanisms for inducing apoptotic pathways. Therefore, the GOC-ZnO-P nanocomposite offers a strategy for glioblastoma treatment.

Enantioselective effect of cysteine functionalized mesoporous silica nanoparticles in U87 MG and GM08680 human cells and Staphylococcus aureus bacteria.

Chirality is a fundamental phenomenon in biological systems, since most of the biomolecules and biological components and species are chiral and therefore recognize and respond differently depending on the enantiomer present. With increasing research into the use of nanomaterials for biomedical purposes, it is essential to understand the role that chirality of nanoparticles plays at the cellular level. Here, the chiral cysteine functionalization of mesoporous silica nanoparticles has been shown to broadly affect its interaction with U87 MG human glioblastoma cell, healthy human fibroblast (GM08680) and methicillin-resistant S. aureus bacteria. We believe that this research is important to further advancement of nano-biotechnology.

Choline and nicotine increase glioblastoma cell proliferation by binding and activating α7- and α9- containing nicotinic receptors

Glioblastomas (GBMs), the most frequent and aggressive human primary brain tumours, have altered cell metabolism, and one of the strongest indicators of malignancy is an increase in choline compounds. Choline is also a selective agonist of some neuronal nicotinic acetylcholine receptor (nAChR) subtypes.As little is known concerning the expression of nAChR in glioblastoma cells, we analysed in U87MG human grade-IV astrocytoma cell line and GBM5 temozolomide-resistant glioblastoma cells selected from a cancer stem cell-enriched culture, molecularly, pharmacologically and functionally which nAChR subtypes are expressed and,whether choline and nicotine can affect GBM cell proliferation.We found that U87MG and GBM5 cells express similar nAChR subtypes, and choline and nicotine increase their proliferation rate and activate the anti-apoptotic AKT and pro-proliferative ERK pathways. These effects are blocked by the presence of non-cell-permeable peptide antagonists selective for α7- and α9-containing nicotinic receptors. siRNA-mediated silencing of α7 or α9 subunit expression also selectively prevents the effects of nicotine and choline on GBM cell proliferation.Our findings indicate that nicotine and choline activate the signalling pathways involved in the proliferation of GBM cells, and that these effects are mediated by α7 and α9-containing nAChRs. This suggests that these nicotinic receptors may contribute to the aggressive behaviour of this tumor and may indicate new therapeutic strategies against high-grade human brain tumours.

‘Trojan-Horse’ stress-granule formation mediated by manganese oxide nanoparticles

Exposure to nanomaterials is considered as one of the risk factors for neurodegenerative pathology. In vitro inorganic nanoparticles (NPs) absorb intrinsically disordered proteins, many of which are the constituents of stress-granules (SGs). SGs normally form in response to cellular stress and, here, we addressed whether selected inorganic NPs could trigger SGs formation in cells. To this end, we have tested a series of inorganic NPs for their ability to induce SGs formation in human glioblastoma and fibroblast cell lines. Among tested NPs, only Mn3O4 NPs triggered SGs formation in cell-type-specific and metabolic-dependent manner. In human glioblastoma U87 MG cell line, Mn3O4 NPs entered cells within minutes and resided inside intracellular vesicles for at least 48 h. Mn3O4 NPs induced a strong reduction in oxidative phosphorylation rate, but not glycolysis. We showed that Mn3O4 NPs slowly dissolve producing a local net of Mn2+ cations, which are known to inhibit oxidative phosphorylation. Indeed, direct incubation of cells with equimolar amounts of Mn2+ cations triggered SGs formation and reduced cellular respiration rate. However, while SGs formed in response to Mn3O4 NPs persisted for hours, SGs formation by Mn2+ peaked and dropped within minutes. Finally, Mn3O4 NPs mediated SGs formation via the phosphorylation of eIF2α. Thus, we conclude that exposure of U87 MG cells to Mn3O4 NPs caused a ‘Trojan-horse’ prolonged SGs response.

Manganese Oxide Nanoparticles Inhibit the Growth of Subcutaneous U-87MG Glioblastoma Xenografts in Immunodeficient Mouse.

Our previous study demonstrated that manganese oxide nanoparticles (MnO NP) selectively destroyed U-87MG and U251 human glioblastoma cells in vitro. MnO NP were synthesized and studied by electron microscopy. Their antitumor properties were studied in vivo on the model of immunodeficient SCID mice with subcutaneous xenografts of U-87MG human glioblastoma. The mice were injected subcutaneously with MnO NP in doses of 0.96 and 1.92 mg/kg (calculated for Mn) 3 days a week over 3 weeks. In was shown that MnO NP in these doses significantly suppressed the growth of U-87MG glioblastoma xenografts: on day 21 from the start of the treatment, the tumor growth inhibition index was 61.1 and 99.22%, respectively. These results indicate the necessity of the further studies of MnO NP as a potential oncolytic agent for the therapy of human glioblastomas.

Metformin Increases Exosome Biogenesis and Secretion in U87 MG Human Glioblastoma Cells: A Possible Mechanism of Therapeutic Resistance

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor. Metformin, an anti-diabetic drug, can suppress tumor cells. Exosomes from GBM cells contribute to intercellular communication, tumor aggressiveness, and therapeutic resistance. We studied the effect of metformin on the exosomal secretory pathway in U87MGcells. Cell survival against metformin was investigated using MTT assay. Expression of miRNA-21, miRNA-155, and miRNA-182, as well as the genes involved in exosome biogenesis and secretion such as Rab27a, Rab27b, Rab11, CD63, and Alix were calculated by real time-PCR. The expression of CD63 protein was analyzed by western blotting, while the subcellular distribution of CD63 protein was monitored by flow cytometry. Exosomes were characterized by transmission and scanning electron microscopes, and flow cytometry. Amount of exosomes was assayed using acetylcholinesterase activity assay and ELISA. The expression of autophagic markers LC3 and P62 were assessed using ELISA. Data showed that metformin decreased cell survival and expression of miRNA-21, miRNA-155, and miRNA-182 (p <0.05). Expression of Rab27a, Rab27b, Rab11, CD63, and Alix as well as protein level of CD63 up-regulated in treated cells (p <0.05). Concurrently, flow cytometry analysis showed that surface CD63/total CD63 ratio was increased in treated cells (p <0.05). We found that acetylcholinesterase activity and CD63 protein of exosomes from treated cells increased (p <0.05). The expression of LC3 and P62 was not affected by metformin (p >0.05). Data indicates metformin could promote exosome biogenesis and secretion in U87MGcells, proposing the therapeutic response against metformin.

High-voltage 10 ns delayed paired or bipolar pulses for in vitro bioelectric experiments

Recent studies proved that classical bio-effects induced by nanosecond pulsed electric field (nsPEF) can be reduced by the delivery of a negative polarity pulse generated immediately after a positive polarity pulse. This phenomenon is known as “bipolar cancellation” and it was reported for a wide range of bipolar pulses with pulse duration from 2 ns to 900 ns. On the contrary, paired pulses, i.e., two identical pulses with the same polarity, increased traditional nsPEF outcomes. Herein, we propose a novel robust and flexible generator, based on the frozen-wave concept, able to produce a broad range of pulses with the duration of 10 ns and delay between 17 and 360 ns. Numerical simulations and experimental measurements were performed to fully characterize the proposed generator. YO-PROTM-1 uptake was investigated in the U87-MG human glioblastoma cell line as a marker of membrane permeabilization in response to 10 ns, 11.5MV/m nsPEF. Our results showed that bipolar cancellation occurred for delays of 0–30 ns and decreased as a function of the interphase interval. In addition, we observed that cellular response following the application of paired nsPEF was more than two-fold compared to the unipolar pulse response and was independent from the interphase interval.

CARMUSTINE LOADED NANOSIZE LIPID VESICLES SHOWED PREFERENTIAL CYTOTOXICITY AND INTERNALIZATION IN U87MG CELL LINE ALONG WITH IMPROVED PHARMACOKINETIC PROFILE IN MICE: A STRATEGY FOR TREATMENT OF GLIOMA

Objective: Successful treatment of glioma still remains a tough challenge. The present study aims at the development and evaluation of carmustine loaded nanosize phospholipid vesicles (CNLVs) for the treatment of glioma.&#x0D; Methods: The experimental NLVs were developed by conventional lipid layer hydration technique and were characterized by different in vitro tools such as diffraction light scattering (DLS), zeta potential, field emission scanning electron microscopy (FESEM), cryo-transmission electron microscopy (cryo-TEM), in vitro drug loading capacity, drug release study etc. In vitro cytotoxicity and cellular uptake of the optimized drug-loaded NLVs were carried out in U87MG human glioblastoma cell line. In vivo pharmacokinetic study was conducted in Swiss albino mice.&#x0D; Results: DLS data showed an average vesicle diameter of 92 nm with narrow size distribution. Optimized CNLVs were spherical in shape with a smooth surface as depicted from FESEM data. Cryo-TEM study confirmed formation of unilamellar vesicles with intact outer bilayer. A reasonable drug loading of 7.8 % was reported for the optimized CNLVs along with a sustained release of CS over a 48 h study period. In vitro cytotoxicity assay revealed a considerable higher toxicity of CNLVs than free drugs in the U87MG cells. Confocal microscopy showed a satisfactory internalization of the optimized drug-loaded NLVs in the tested cell line. Pharmacokinetic data demonstrated an enhanced mean residence time of optimized CNLVs in blood than free drug.&#x0D; Conclusion: Results depicted the potential of experimental CNLVs for the treatment of glioma after further in vivo tests.