U87MG Cell Lines Research Articles

Zika virus has an oncolytic activity against human glioblastoma U87 cells

Glioblastoma is a highly lethal brain cancer. Virotherapy with the use of oncolytic viruses has since recently been regarded as a promising approach for the clinic treatment of human glioblastomas. The purpose of this work was to perform a primary evaluation of the Zika virus as a potential oncolytic agent against glioblastomas.In vitroexperiments showed that the Zika virus strain MR 766 is able to selectively infect and lyse neoplastic cells of the human glioblastoma cell line U87 MG. The selectivity index (SI, the ratio of infectious titer for tumor cells to titer on normal untransformed cells) was 2·102. The selectivity of the replicative activity of Zika virus in relation to U87 MG glioblastoma cells was additionally confrmed by indirect immunofluorescence. Using the model of immunodefcient SCID mice with subcutaneous xenografts of human glioblastoma U87 MG, a strong antitumor activity of the Zika virus under a course (daily for 4 days) of intratumoral administration of 5·105 TCID50 of Zika virus was shown. Treatment with Zika virus resulted in more than a 10­fold reduction in mean volumes of tumors. The tumor growth inhibition index was 92.63 %. Recurrences (metastases) of tumor re­growth were not registered within 64 days of observation. This result demonstrated the prospect of further in­depth studies of the Zika virus as a potential oncolytic agent against human glioblastomas.

Autocrine CXCL-8-Dependent Invasiveness Triggers Modulation of Actin Cytoskeletal Network and Cell Dynamics

Background: Glioblastoma (GB) is the most representative form of primary malignant brain tumour. Several studies indicated a pleiotropic role of CXCL-8 in cancer due to its ability to modulate the tumour microenvironment, growth and aggressiveness of tumour cell. Previous studies indicated that CXCR1 (CXCL-8 receptor) induced activation of the PI3K/p-Akt pathway, a crucial event in the regulation of cytoskeleton rearrangement and cell migration. Methods: We used human GB primary cell culture and U-87MG cell line both exposed to a dual allosteric inhibitor of CXCR1/2 receptors. Cell migration assays associated with the analysis of protein involved in cell migration and cytoskeletal dynamics were performed. Findings: The data obtained indicate a specific effect of autocrine CXCL-8 signalling on GB cell invasiveness by the activation of pathways involved in cell migration and cytoskeletal dynamics,. The use of the inhibitor strongly affects cytoskeletal dynamic opposing to invasion. Interpretation: The data obtained support the concept that autocrine CXCL-8 signalling plays a key role in the activation of an aggressive phenotype in glioblastoma cells and U-87MG cell line. The strong effects of the allosteric inhibitor DF2755A in counteracting the key pathways involved in migration and invasion of GB, although not affect cell viability, suggest additional studies to assess the potential of a pharmacological approach targeting CXCR1/CXCR2 pathways to decrease migration and invasion of GB cells in the brain parenchyma, one of the principal mechanisms of recurrence. Funding Statement: This work was supported by the University of L'Aquila (RIA 2018). Declaration of Interests: Declarations of interests: Laura Brandolini and Marcello Allegretti are employees of Dompe Farmaceutici SpA, Italy. The company has interests in the development of CXCR1/2 allosteric modulator for the treatment of oncologic-related diseases. The other authors declare that they have no conflict of interest. Ethics Approval Statement: This study was ethically approved (Hospital Ethics Committee, n. 3729), and all patients voluntary signed informed consent.

The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.

Event Abstract Back to Event The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines. Helena Marcelino1, 2, Cecilia R. Santos2, Vanda S. Nogueira2, 3, 4, João F. Fonseca-Gomes3, 4, Joana Tomás2, Maria José Diógenes3, 4, Ana M. Sebastião3, 4 and José F. Cascalheira1, 2* 1 Department of Chemistry, Faculty of Sciences, University of Beira Interior, Portugal 2 Center for Research in Health Sciences, Faculty of Health Sciences, University of Beira Interior, Portugal 3 Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, Portugal 4 Institute of Molecular Medicine João Lobo Antunes, Faculty of Medicine, University of Lisbon, Portugal Adenosine has been implicated in the proliferation and survival of various tumour cells, namely glioblastoma cells. Adenosine usually produces its cellular effects in the nervous system by activating G protein-coupled adenosine receptors. Increased adenosine concentrations are found in stressful conditions such as hypoxic regions in brain tumours. However it isn’t known if there any difference between the effects of increased adenosine concentration on cell proliferation in normal astrocytes when compared with malign transformed cells. This issue will be addressed in present work. Human astrocytes (HA), and human glioblastoma cell lines U-87MG, U-373 MG, SNB19, were treated for 3 days with 30µM-Adenosine, with or without the selective adenosine kinase inhibitor 5-idotubercidin (25µM). Cell proliferation was evaluated using MTT assay while cell death was assessed by measuring the released LDH activity and αII-Spectrin cleavage. 30µM-Adenosine produced a 40%±4% (P<0.005) reduction in HA cell proliferation but had no effect in any of the 3 glioblastoma cell lines (p<0.01, when compared with HA). Adenosine alone did not induce cell death, assessed by LDH release and αII-Spectrin cleavage. Curiously, incubation with 5-iodotubercidin+adenosine produced a strong and similar decrease (ranging from 86±5% for HA, to 75%±4% for U87, P<0.01) on cell proliferation in both HA and glioblastoma cell lines. The results show a strong attenuation of the antiproliferative effect of adenosine in glioblastoma cells compared to HA, probably resulting from an increased adenosine kinase activity in glioblastoma cells, suggesting an adaptation of the tumour cells to the antiproliferative effect of adenosine. Acknowledgements The present work was funded by a CENTRO 2020 and Lisboa 2020 (POCI-01-0145-FEDER-016822) and FCT – Fundação para a Ciência e Tecnologia, I.P. (PTDC/BIM¬ONC/7121/2014) research Grant. Keywords: Adenosine, Astrocytes, Cell Proliferation, Glioblastoma, Homocysteine Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Glia / Neuroinflammation Citation: Marcelino H, Santos CR, Nogueira VS, Fonseca-Gomes JF, Tomás J, Diógenes M, Sebastião AM and Cascalheira JF (2019). The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. José F Cascalheira, Department of Chemistry, Faculty of Sciences, University of Beira Interior, Covilhã, Portugal, jfcascalheira@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Scholar Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira PubMed Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Combined Applications of Repurposed Drugs and Their Detrimental Effects on Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with high rates of recurrence. This study aimed to investigate the effect of repurposed drug combinations on GBM. Viability of U87 MG and 11ST patient-derived GMB cell lines, after valproic acid, tranylcypromine or riluzole alone, in different combinations, as well as combined with standard temozolomide chemotherapy was examined using the MTT assay. Proliferation, mRNA level of tissue factor pathway inhibitor 2 (TFPI2), and cell invasion were evaluated using anti-Ki-67 antibody staining, reverse transcriptase-polymerase chain reaction and xCELLigence system. The strongest effect on cell viability was achieved by the combination of riluzole with valproic acid (U87MG: 27.2%, 11ST: 25.99%). Tranylcypromine significantly enhanced the effect of temozolomide when used in combination, as did valproic acid. The normally high proliferation of GBM significantly declined under treatment with valproic acid with tranylcypromine (p=0.01). Finally, we observed reduction of invasion comparing single tranylcypromine to its combination with valproic acid or riluzole. These results support the idea that combinations of drugs could increase the treatment efficiency of GBM.

Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma.

Background: Chemotherapeutic resistance of glioblastoma has been attributed to a self-renewing subpopulation, the glioma stem cells (GSCs), which is known to be maintained by the Wnt β−catenin pathway. Our previous findings demonstrated that exogeneous addition of the Wnt antagonist, secreted fizzled-related protein 4 (sFRP4) hampered stem cell properties in GSCs. Methods: To understand the molecular mechanism of sFRP4, we overexpressed sFRP4 (sFRP4 OE) in three human glioblastoma cell lines U87MG, U138MG, and U373MG. We also performed chromatin immunoprecipitation (ChIP) sequencing of sFRP4 OE and RNA sequencing of sFRP4 OE and sFRP4 knocked down U87 cells. Results: We observed nuclear localization of sFRP4, suggesting an unknown nuclear role. ChIP-sequencing of sFRP4 pulldown DNA revealed a homeobox Cphx1, related to the senescence regulator ETS proto-oncogene 2 (ETS2). Furthermore, miRNA885, a p53-mediated apoptosis inducer, was upregulated in sFRP4 OE cells. RNA sequencing analysis suggested that sFRP4-mediated apoptosis is via the Fas-p53 pathway by activating the Wnt calcium and reactive oxygen species pathways. Interestingly, sFRP4 OE cells had decreased stemness, but when knocked down in multipotent mesenchymal stem cells, pluripotentiality was induced and the Wnt β-catenin pathway was upregulated. Conclusions: This study unveils a novel nuclear role for sFRP4 to promote apoptosis by a possible activation of DNA damage machinery in glioblastoma.

Cytotoxic butenolides and diphenyl ethers from the endophytic fungus Pestalotiopsis sp.

Chemical investigation of the plant endophytic fungus Pestalotiopsis sp. afforded two new butenolides, pestalolides B and C (1 and 2), two new diphenyl ethers, pestalotethers E and F (3 and 4), together with seven known compounds (5–11). Their structures were established by extensive spectroscopic analyses and the absolute configuration of 1 was further confirmed by ECD calculation. Structurally, compound 1 represents the first example of a new type of butenolide derivatives, bearing an unprecedented carbon scaffold having 13 carbon atoms. Compounds 1–4 were evaluated for the cytotoxic activities, and compound 1 showed remarkable inhibitory potency against human tumor HL60, U87MG, MDA-MB-231, and HEP-3B cell lines, with IC50 values of 1.42, 5.90, 5.90, and 4.29 μM, respectively.

Lipid nanocapsules decorated and loaded with cannabidiol as targeted prolonged release carriers for glioma therapy: In vitro screening of critical parameters

The therapeutic potential of cannabinoids has been truly constrained heretofore due to their strong psychoactive effects and their high lipophilicity. In this context, precisely due to the lack of psychoactive properties, cannabidiol (CBD), the second major component of Cannabis sativa, arises as the phytocannabinoid with the most auspicious therapeutic potential. Hence, the incorporation of CBD in lipid nanocapsules (LNCs) will contribute to overcome the dosing problems associated with cannabinoids.Herein, we have prepared LNCs decorated and loaded with CBD for glioma therapy and screened in vitro their critical parameters. On the one hand, we have encapsulated CBD into the oily core of LNCs to test their in vitro efficacy as extended-release carriers against the human glioblastoma cell line U373MG. The in vitro antitumor effect was highly dependent on the size of LNCs due to its pivotal role in the extent of CBD release. Effectively, a comparison between two differently-sized LNCs (namely, 20-nm and 50-nm sized carriers) showed that the smaller LNCs reduced by 3.0-fold the IC50 value of their 50-nm sized counterparts. On the other hand, to explore the potential of this phytocannabinoid to target any of the cannabinoid receptors overexpressed in glioma cells, we decorated the LNCs with CBD. This functionalization strategy enhanced the in vitro glioma targeting by 3.4-fold in comparison with their equally-sized undecorated counterparts. Lastly, the combination of CBD-loading with CBD-functionalization further reduced the IC50 values. Hence, the potential of these two strategies of CBD incorporation into LNCs deserves subsequent in vivo evaluation.

Integrating in vitro experiments with in silico approaches for Glioblastoma invasion: the role of cell-to-cell adhesion heterogeneity

Glioblastoma cells adopt migration strategies to invade into the brain parenchyma ranging from individual to collective mechanisms, whose role and dynamics are not yet fully understood. In this work, we explore Glioblastoma heterogeneity and recapitulate its invasive patterns both in vitro, by utilizing primary cells along with the U87MG cell line, and in silico, by adopting discrete, individual cell-based mathematics. Glioblastoma cells are cultured three-dimensionally in an ECM-like substrate. The primary Glioblastoma spheroids adopt a novel cohesive pattern, mimicking perivascular invasion in the brain, while the U87MG adopt a typical, starburst invasive pattern under the same experimental setup. Mathematically, we focus on the role of the intrinsic heterogeneity with respect to cell-to-cell adhesion. Our proposed mathematical approach mimics the invasive morphologies observed in vitro and predicts the dynamics of tumour expansion. The role of the proliferation and migration is also explored showing that their effect on tumour morphology is different per cell type. The proposed model suggests that allowing cell-to-cell adhesive heterogeneity within the tumour population is sufficient for variable invasive morphologies to emerge which remain originally undetectable by conventional imaging, indicating that exploration in pathological samples is needed to improve our understanding and reveal potential patient-specific therapeutic targets.

Time Sequential Changes of Epithelial-Mesenchymal Transition Signaling Induced by Ionizing Radiation using Next-Generation Sequencing in Glioblastoma

Epithelial-mesenchymal transition (EMT) is considered as one of the mechanisms of treatment resistance in glioblastoma. We evaluated a time sequential changes of EMT signaling in glioblastoma induced by ionizing radiation. The cell line U-373 MG (human glioblastoma) was irradiated with 6Gy of high energy X-ray (6MV) with dose rate of 6 Gy/min. Next generation sequencing (NGS) was performed on the cell line before irradiation (baseline), 6 hours, 24 hours, and 48 hours after irradiation and database of essential genes (DEG) was evaluated in each period. The DEGs were compared with those included in the 128 of the GeneSets. P value from the Fisher exact test and Q value from the Benjamini Hochberg methods were calculated. All KEGG map pathways were evaluated and the EMT related KEGG pathways were assessed using the EMT gene database (dbEMT). GeneSet analysis showed that ionizing radiation up-regulated the expression of the GeneSet of the wholemark of EMT at 6 hours and 24 hours after irradiation. On KEGG pathway analysis, extracellular matrix (ECM) receptor interactions increased over time and the expression of miRNA-21 increased at 6, 24, and 48 hours after irradiation resulting in cell growth and survival pathway. Increased RTK expression suggested RTK-induced EMT process. HIF-α induced hypoxia and activated TGF-β signaling pathway, which is related with EMT. TGF- β activated Smad2/3 followed by activation of Smad4. Ionizing radiation may promote EMT in glioblastoma, resulting in treatment related resistance. miRNA-21 may play a critical role in EMT after irradiation. EMT blocking to prevent treatment resistance may be a promising strategy to increase survival in glioblastoma.

Preparation of curcumin-loaded PLGA nanoparticles and investigation of its cytotoxicity effects on human glioblastoma U87MG cells

Curcumin, an herbal derivative, could be used as anticancer agent in drug delivery. Due to hydrophobic properties of curcumin, its bioavailability decreases to cancer cells. The aim of this study was to prepare curcumin loaded poly (lactic-co-glycolic acid) nanoparticles (CUR-PLGA-NPs) as a drug delivery system to overcome the limitation of hydrophobicity of CUR as well as to investigate its toxicity effect on human glioblastoma U87MG cells compared to free CUR. Single emulsion method was used to prepare CUR-PLGA-NPs. Under optimum conditions, the size of nanoparticles was characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Encapsulation efficiency and loading content were 89.77% and 9.06 %, respectively; with a biphasic release behavior. Differential scanning calorimetric (DSC) thermograph of CUR-PLGA-NPs exhibited that CUR was dispersed in amorphous phase. The cytotoxicity studies on U87MG cell line indicated that CUR-PLGA-NPs had high cytotoxicity effects compared to free CUR. The half maximal inhibitory concentration (IC50) after cell incubation for 72 h was 57.99 μg/mL and 32.90 μg/mL for free CUR and CUR-PLGA-NPs, respectively. In conclusion, it is suggested that CUR-PLGA-NPs can be used for drug delivert to cancer cells.

Application of Neurokinin-1 Receptor in Targeted Strategies for Glioma Treatment. Part I: Synthesis and Evaluation of Substance P Fragments Labeled with 99mTc and 177Lu as Potential Receptor Radiopharmaceuticals.

Gliomas, particularly WHO grade IV glioblastoma multiforme, are one of the most common and aggressive primary tumors of the central nervous system. The neuropeptide, substance P (SP), is the physiological ligand of the neurokinin-1 (NK-1) receptor that is consistently overexpressed in glioblastoma cells. The aim of this work was to study physico-chemical and biological properties of different SP analogues labeled with technetium-99m and lutetium-177 radionuclides. The synthesized compounds were characterized in vitro by partition coefficients (logP) and their stability was investigated in various physiological solutions. Biological properties (Kd, Bmax) were characterized using the U373 MG cell line. The obtained lipophilicity values of the [99mTc]NS3/CN-SP and [177Lu]DOTA-SP radiobioconjugates were in the range of −0.3 to +0.6 and −2.5 to −5.0, respectively. The studied radiobioconjugates were stable in PBS buffer and CSF, as well as in 10 mM histidine and/or cysteine solutions whereas in human serum showed enzymatic biodegradation. [177Lu]DOTA-[Thi8,Met(O2)11]SP(1–11), [177Lu]DOTA-SP(4–11) and [177Lu]DOTA-[Thi8,Met(O2)11]SP(5–11) radiobioconjugates bound specifically to NK-1 receptors expressed on glioblastoma cells with affinity in the nanomolar range. To conclude, the shorter analogues of SP can be used as vectors, nevertheless they still do not fulfil all requirements for preparations in nuclear medicine.

Correction: Opposing Effects of PI3K/Akt and Smad-Dependent Signaling Pathways in NAG-1-Induced Glioblastoma Cell Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0096283.].

Investigation of Vipera Anatolica Venom Disintegrin via Intracellular Uptake with Radiolabeling Study and Cell-Based Electrochemical Biosensing Assay

Snake venoms are a natural biological source that has potential therapeutic value with various protein compounds. Disintegrins originally were discovered as a family of proteins from snake venoms composed of cysteine rich low molecular weight polypeptides. Disintegrins exhibit specific binding and higher affinity toward integrin with potential inhibition of function. Trans-membrane receptors of the integrin family may involve in many pathological conditions such as inflammation and tumor progression with important processes related to invasion and migration. Since disintegrins have the ability to bind to integrins, they could be used for cancer detection and treatment, and in monitoring of therapy in select cancer types. The main purpose of the study is to investigate disintegrin containing Vipera anatolica (VAT) crude venom potential for radiolabeling and intracellular uptake as well as electrochemical biosensing assay against U87MG human brain glioblastoma cells. For this purpose, VAT crude venom containing U87MG cell-specific disintegrin was investigated in terms of radiolabeling and intracellular uptake as well as electrochemical biosensing assay in comparison with echistatin (ECT) disintegrin in cells. The interaction between VAT crude venom and ECT with HEK293 human non-tumorigenic embryonic kidney cells and glioblastoma U87MG cells was electrochemically investigated using pencil graphite electrodes (PGEs). The interaction of the VAT crude venom and ECT with HEK293 and U87MG cells was detected according to the changes in oxidation signals. Then, VAT crude venom and echistatin were labeled with 131I via iodogen method. Intracellular uptakes of radiolabeled molecules were investigated in U87MG cell line. 131I-VAT can be an agent for imaging of glioblastoma cancer. Further work will focus on the production of large quantities of pure VAT disintegrin with a biotechnological approach to improving imaging agent.

Effects of aloe emodin on U87MG glioblastoma cell growth: In vitro and in vivo study.

Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence other approaches have been investigated to target more pathways involved in glioblastoma development and progression. Here we investigate the anticancer effect of Aloe-Emodin (AE), an anthraquinone compound presents in the leaves of Aloe arborescens, on human glioblastoma cell line U87MG. U87MG were treated with various concentrations of AE (20 and 40 μM) for different times (24, 48, and 72 hr). Cell growth was monitored by daily cell count after treatments. Growth analysis showed that AE significantly decrease proliferation of U87MG in a time and dose dependent manner. FACS analysis demonstrates a block of cell cycle in S and G2/M phase. AE probably induced also apoptosis by releasing of apoptosis-inducing factor: PARP and Lamin activation leading to nuclear shrinkage. In addition, exposure of U87MG to AE reduced pAKT phosphorylation. AE inhibition of U87MG growth is a result of more mechanism together. Here we report that AE has a specific growth inhibition on U87MG also in in vivo. The growth of U87MG, subcutaneously injected in nude mice with severe combined immunodeficiency, is inhibited without any appreciable toxic effects on the animals after AE treatment. AE might represent a conceptually new lead antitumor adjuvant drug.

CEST MRI of 3-O-methyl-D-glucose uptake and accumulation in brain tumors.

3-O-Methyl-D-glucose (3-OMG) is a nonmetabolizable structural analog of glucose that offers potential to be used as a CEST-contrast agent for tumor detection. Here, we explore it for CEST-detection of malignant brain tumors and compare it with D-glucose. Glioma xenografts of a U87-MG cell line were implanted in five mice. Dynamic 3-OMG weighted images were collected using CEST-MRI at 11.7T at a single offset of 1.2 ppm, showing the effect of accumulation of the contrast agent in the tumor, following an intravenous injection of 3-OMG (3g/kg). Tumor regions showed higher enhancement as compared to contralateral brain. The CEST contrast enhancement in the tumor region ranged from 2.5-5.0%, while it was 1.5-3.5% in contralateral brain. Previous D-glucose studies of the same tumor model showed an enhancement of 1.5-3.0% and 0.5-1.5% in tumor and contralateral brain, respectively. The signal gradually stabilized to a value that persisted for the length of the scan. 3-OMG shows a CEST contrast enhancement that is approximately twice as much as that of D-glucose for a similar tumor line. In view of its suggested low toxicity and transport properties across the BBB, 3-OMG provides an option to be used as a nonmetallic contrast agent for evaluating brain tumors.

An Efficient Bivalent Cyclic RGD-PIK3CB siRNA Conjugate for Specific Targeted Therapy against Glioblastoma In Vitro and In Vivo

The PI3K-AKT-mTOR-signaling pathway is frequently activated in glioblastoma (GBM). Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB)/p110β (a PI3K catalytic isoform) by RNAi substantially suppresses GBM growth with less toxicity to normal astrocytes. However, insufficient and non-specific small interfering RNA (siRNA) delivery may limit the efficacy of RNAi-based therapies against GBM. Here we prepared a novel methoxy-modified PIK3CB siRNA molecule (siPIK3CB) that was covalently conjugated to a [cyclo(Arg-Gly-Asp-D-Phe-Lys)-Ahx]2-Glu-PEG-MAL (biRGD) peptide, which selectively binds to integrin αvβ3 receptors. The αvβ3-positive U87MG cell line was selected as a representative for GBM. An orthotopic GBM xenograft model based on luciferase-expressing U87MG was established and validated in vivo to investigate bio-distribution and anti-tumor efficacy of biRGD-siPIK3CB. In vitro, biRGD-siPIK3CB specifically entered and silenced PIK3CB expression in GBM cells in an αvβ3 receptor-dependent manner, thus inhibiting cell cycle progression and migration and enhancing apoptosis. In vivo, intravenously injected biRGD-siPIK3CB substantially slowed GBM growth and prolonged survival by reducing tumor viability with silencing PIK3CB expression. Furthermore, biRGD-siPIK3CB led to mild tubulointerstitial injury in the treatment of GBM without obvious hepatotoxicity, whereas co-infusion of Gelofusine obviously alleviated this injury without compromising anti-tumor efficacy. These findings revealed a great translational potential of biRGD-siPIK3CB conjugate as a novel molecule for GBM therapy.

Genipin induces cell death via intrinsic apoptosis pathways in human glioblastoma cells.

Genipin, a compound derived from Gardenis jasminoides Ellis fruits, was demonstrated to be the specific uncoupling protein 2 (UCP2) inhibitor. UCP2 is a mitochondrial carrier protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) synthesis. Several studies revealed that UCP2 is broadly over-expressed in leukemia, colorectal, lung, ovarian, prostate, testicular, and bladder cancers. However, the effect of genipin still needs to be elucidated in neurological malignancies. In this study, we investigated the anticancer effect of genipin in U87MG and A172 cell lines. The anticancer effect of genipin on these cell lines was measured by microculture tetrazoliumtest (MTT), Trypan blue exclusion, and colony formation assays, in the presence of various concentrations of genipin at different time intervals. We assessed apoptosis and measure intracellular reactive oxygen species (ROS) by flow cytometry. Expression of UCP2 and some of the genes involved in apoptosis was analyzed by real-time quantitative polymerase chain reaction (PCR). Results of the MTT assay showed that genipin moderately reduced metabolic activity of both cell lines in dose- and time-dependent manner. Result of Trypan blue exclusion test indicated that the viable cell count decreased in the treated group in a concentration-dependent manner. Genipin also significantly decreased colony formation ability of these cells in a concentration-dependent manner. Result of morphological changes showed that there were significant differences in cell number and morphology in treated groups as compared with the untreated groups. Flow cytometric analysis of U87MG and A172 cells with annexin V/propidium iodide staining, 48 hours after treatment with genipin, displays 22.4% and 26.1% apoptotic population, respectively, in treated cells, in comparison to 7.42% and 9.31% apoptotic cells of untreated cells. After treatment, UCP2 and B-cell lymphoma 2 (BCL 2 ) genes are downregulated, and BCL 2 associated X protein, BCL 2 antagonist/killer, BCL 2 interacting killer, and Cytochrome c genes are upregulated. Genipin treatment increased mitochondrial ROS levels and also induced apoptosis through caspase-3 upregulation. In conclusion, the antiproliferative effects of genipin on the growth of both glioblastoma cell lines have been shown in all of these assays, and genipin profoundly induced apoptosis in both cell lines via the UCP2-related mitochondrial pathway through the induction of intracellular ROS.

Effects of telomerase inhibitor on epigenetic chromatin modification enzymes in malignancies.

Telomerase has a critical role in cell proliferation, tumor maintaining, and therapy resistance, which act by modifying many signaling pathways. 2-[(E)-3-Naphtalen-2-yl-but-2-enoylamino]-benzoic acid (BIBR1532) is one of the most studied telomerase inhibitors, and it targets telomerase components TERC and TERT. In this novel study, we aimed to investigate the epigenetic effects of BIBR1532 on both hematologic malignancies and solid tumors. K-562 human chronic myeloid leukemia cell line and U87MG glioblastoma cell line were compared with control groups without BIBR1532 treatment. Cytotoxic effects of BIBR1532 were determined by using WST-1 assay. Apoptotic effects of BIBR1532 were detected by using annexin V method. To assess expression changes in the human epigenetic chromatin modification enzyme genes, total RNA was isolated from K-562 and U87MG cells treated with BIBR1532 and untreated control cells. BIBR1532 induced 2.41-fold apoptotic cell death in U87MG cell lines compared with control groups. Apoptosis was slightly induced in K-562 cells with BIBR1532 treatment compared with control cells. We observed that BIBR1532 also regulates similar genes in both cell lines, and it is useful on epigenetic mechanisms. As a result, telomerase inhibitor BIBR1532 has a significant effect on both hematological malignancies and solid tumors.

Evaluation of combined effect of hyperthermia and ionizing radiation on cytotoxic damages induced by IUdR-loaded PCL-PEG-coated magnetic nanoparticles in spheroid culture of U87MG glioblastoma cell line

Purpose: Glioblastoma multiform (GBM) is the most prevalent and aggressive type of primary brain tumor. None of the current conventional treatment methods has improved treatment considerably. Therefore, in this study the effect of magnetic nanoparticles coated with poly (caprolactone)-poly (ethylene glycol) (PCL-PEG) as a 5-iodo 2′deoxyuridine (IUdR) carrier in the presence of hyperthermia and 6 MV (megavoltage) X-ray radiation, were investigated in a spheroid model of U87MG glioblastoma cell line using colony formation assay.Materials and methods: First, the human glioblastoma cell line U87MG was cultured as a spheroid using the liquid overlay technique. Spheroids on day 10 with 100 mm diameters were treated with 1 µM IUdR or nanoparticles as IUdR carriers for one volume doubling time (VDT) of spheroids (67 h) and hyperthermia at 43 °C for 1 h, and then irradiated with 2 Gy of 6 MV X-ray in different groups. Finally, the effect of treatment on colony-forming ability was obtained by colony formation and alkaline assay.Results: Our results revealed that hyperthermia in combination with radiation could significantly reduce the colony number of glioblastoma spheroid cells treated with IUdR or nanoparticles as IUdR carriers. However, the extent of reduction in colony number following treatment with IUdR-loaded nanoparticles in combination with hyperthermia and then X-ray radiation was significantly more than free IUdR.Conclusion: According to this study, PCL-PEG-coated magnetic nanoparticles are effective delivery vehicles for IUdR into cells. Moreover, they can act as a radiosensitizer and thermosensitizer in the treatment of the glioblastoma cell line.

Evaluation of Antimicrobial, Enzyme Inhibitory, Antioxidant and Cytotoxic Activities of Partially Purified Volatile Metabolites of Marine Streptomyces sp.S2A.

In the present study, marine actinobacteria Streptomyces sp.S2A was isolated from the Gulf of Mannar, India. Identification was carried out by 16S rRNA analysis. Bioactive metabolites were extracted by solvent extraction method. The metabolites were assayed for antagonistic activity against bacterial and fungal pathogens, inhibition of α-glucosidase and α-amylase enzymes, antioxidant activity and cytotoxic activity against various cell lines. The actinobacterial extract showed significant antagonistic activity against four gram-positive and two gram-negative pathogens. Excellent reduction in the growth of fungal pathogens was also observed. The minimum inhibitory concentration of the partially purified extract (PPE) was determined as 31.25 μg/mL against Klebsiella pneumoniae, 15.62 μg/mL against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. The lowest MIC was observed against Micrococcus luteus as 7.8 μg/mL. MIC against fungal pathogens was determined as 62.5 μg/mL against Bipolaris maydis and 15.62 μg/mL against Fusarium moniliforme. The α-glucosidase and α-amylase inhibitory potential of the fractions were carried out by microtiter plate method. IC50 value of active fraction for α-glucosidase and α-amylase inhibition was found to be 21.17 μg/mL and 20.46 μg/mL respectively. The antioxidant activity of partially purified extract (PPE) (DPPH, ABTS, FRAP and Metal chelating activity) were observed and were also found to have significant cytotoxic activity against HT-29, MDA and U-87MG cell lines. The compound analysis was performed using gas chromatography-mass spectrometry (GC-MS) and resulted in three constituents; pyrrolo[1–a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl)-, being the main component (80%). Overall, the strain possesses a wide spectrum of antimicrobial, enzyme inhibitory, antioxidant and cytotoxic activities which affords the production of significant bioactive metabolites as potential pharmacological agents.