Human Brain Tumor Cell Lines Research Articles

Expression and estrogen regulation of G protein-coupled estrogen receptor in human glioblastoma cells.

Glioblastoma (GB) is the most frequent primary brain tumor with a very poor prognosis. Sex hormones are crucial players in the development of GBs. 17 β-estradiol (E2) signaling is involved through its corresponding intracellular receptors [estrogen receptor α (ERα) and β (ERβ)] in GB cell proliferation and progression. E2 activates G-protein coupled estrogen receptor (GPER), leading to rapidly occurring effects, independently of gene transcription. GPER activation is involved in tumor progression in various cancer types. Currently, available data concerning the occurrence and role of GPER in GB are very limited. In the present study, it was observed that GPER was expressed in human brain tumor cell lines [U251 (astrocytoma-derived cell line), U87, LN229 and T98 (glioblastoma-derived cell line)]. Immunofluorescence assays revealed that GPER localizes in the plasma membrane, cytoplasm and nucleus. An in silico analysis identified two potential E2 response elements in the promoter region of the GPER gene. E2 increased GPER expression in the U251, U87 and LN229 cell lines. Molecular modeling data derived from in silico analysis predicted the three-dimensional conformation of GPER, and docking analysis identified potential binding sites of E2 and its specific agonist, G1. Taken together, these results indicate that GPER may be differentially expressed in human GB cell lines with E2 possibly upregulating GPER expression. The present study raises further questions about the implications of GPER-mediated E2 signaling in the biology of GBs.

Strigolactone Analogs: Two New Potential Bioactiphores for Glioblastoma

Strigolactones (SLs), carotenoid-derived phytohormones, control the plant response and signaling pathways for stressful conditions. In addition, they impact numerous cellular processes in mammalians and present new scaffolds for various biomedical applications. Recent studies demonstrated that SLs possess potent antitumor activity against several cancer cells. Herein, we sought to elucidate the inhibitory effects of SL analogs on the growth and survival of human brain tumor cell lines. Among four tested SLs, we showed for the first time that two lead bioactiphores, indanone-derived SL and EGO10, can inhibit cancer cell proliferation, induce apoptosis, and induce G1 cell cycle arrest at low concentrations. SL analogs were marked by increased expression of Bax/Caspase-3 genes and downregulation of Bcl-2. In silico studies were conducted to identify drug-likeness, blood–brain barrier penetrating properties, and molecular docking with Bcl-2 protein. Taken together, this study indicates that SLs may be promising antiglioma agents, presenting novel pharmacophores for further preclinical and clinical assessment.

BRAIN Surgical Tissue for Advanced Tumour models in Precision medicine: Developing the BRAIN-STAT pathway

AimsThere are approximately four thousand neuro-oncology procedures in the UK per annum. Many of these result in tissue and biofluid specimens that are surplus to diagnostic requirement and can be collected as standard of clinical care. However, developing technologies and treatments for precision medicine require access to a range of individualised biospecimens paired with deep clinical phenotyping data. Here, we present Brain Surgical Tissue for Advanced Tumour Models (BRAINSTAT) programme, an infrastructure that has been established between Queen Elizabeth Hospital, Birmingham and the University of Birmingham, to collect, structure and store these resources and also maximise their value for research over the long-term. Using this approach our aim is to provide high-quality, annotated resources to help develop novel treatments for patients with brain tumours.MethodBRAINSTAT infrastructure allows:Prospective consentBiospecimens, including tumour tissue (brain and other primary in the case of metastasis), cyst fluid, dura, skin, CSF, blood (matched “germ-line” and for circulating cell free tumour DNA analysis), urine and saliva can be collected. Consent for long term follow-up, is either via clinic or NHS digital. More limited consent for non-oncological neurosurgical cohorts (e.g. epilepsy or vascular) and healthy volunteers allow healthy access-tissue and biofluids to be collected.B. Rapid transfer of fresh surgical tissue samples:Strong collaborative links and close physical proximity between operating theatre and laboratory allows rapid transfer of biospecimens minimising transit time.C. Standardised annotation across disciplinesThe RedCAP database system allows granular control over data-access, and each specialist research team is provided access only to the sub-sections relevant to them. All users must have Good Clinical Practice certification and GDPR training, prior to access of the BRAINSTAT database.ResultsBetween 25/11/2019-16/03/2020 and 27/07/2020-16/11/2020, 65 patients were consented for BRAINSTAT at the weekly neurosurgical oncology clinic. (Recruitment gaps due to the SARS-COVID 19 pandemic). Pathological diagnosis of surplus tissue collected included: 37 high grade glioma, 3 low grade glioma and 16 brain metastasis including: (6 lung, 6 breast, 2 colorectal, 1 oesophageal, 1 endometrial). Meningioma (5 WHO I; 1 WHO III) 1 patient undergoing anterior temporal lobectomy for hippocampal sclerosis contributed access tissue from the lateral neocortex. 1 patient had a non- neoplastic, non-diagnostic sample. All patients had matched “germ-line” blood samples.Median time from resection to arrival in the laboratory was 10 minutes (range 4-31). Standardised operating protocols to optimise this have been developed.Glioblastoma and breast-brain metastasis tumourspheres and cerebral organoids are currently being validated.ConclusionDespite the challenges of the pandemic we have established a viable tissue pipeline from neurosurgical operating theatre to our university laboratories. We are developing clinically annotated human brain tumour cell lines, stem cells and 3D organoid models, principally for commonly encountered brain tumours such as glioma and metastasis.The research sets the foundation for a multitude of downstream applications including:- Building more complex organoid cultures e.g. by including other cell types such as healthy brain cells and endothelial cells allowing future experiments to more accurately model tumour growth.- Developing high-throughput, patient-specific drug screens of novel drugs and drug combinations using these 3D tumour models aiming to more effectively treat tumour proliferation and spread. These patient avatars will help inform and test more “stratified” personal medical treatments and will provide opportunities to allow earlier intervention with the aim of improving survival, coupled with a better quality of life.

Effects of electroporation on anticancer activity of 5-FU and newly synthesized zinc(II) complex in chemotherapy-resistance human brain tumor cells.

Zn(II) complex of Schiff base derived from the condensation of 4-aminopyrimidine-2(1H)-one with salicylaldehyde was prepared and characterized by various physico-chemical and spectral methods for structure determination. The cytotoxic activity of the Zn(II) complex was investigated in comparison with 5-fluorouracil (5-FU) against two different human brain tumor cell lines (T98G and U118), while primer human dermal fibroblast cells (HDF) was used as control for biocompatibility. Then, the effectiveness of electroporation (EP) on cytotoxic activities of these compounds has been examined. The cytotoxicities of the 5-FU and new Zn(II) complex, alone or in combination with electroporation, were determined by MTT assay. The Zn(II) complex showed good cytotoxicity against T98G and U118 brain tumor cell lines with IC50 = 282.47 and 297.91μM respectively, while it was safe on HDF healthy cells with IC50 = 826.72μM. The 5-FU exhibited less cytotoxicity compared to the Zn(II) complex against T98G (IC50 = 382.35μM) and U118 (IC50 = 396.56μM) tumor cell lines. The combined application of Zn (II) + EP decreased the IC50 value by 5.96-fold in T98G cells and 4.76-fold in U118 cells. EP showed a similar effect in its combined application with 5-FU, resulting in a decrease of the IC50 value of 4.22-fold in the T98G cells and 3.84-fold in the U118 cells. In a conclusion, the Zn(II) complex exhibited an anticancer potential against both brain tumor cell lines (T98G and U118) and EP greatly increased the cytotoxicity of Zn(II) complex and 5-FU on these chemotherapy-resistant cells.

Abstract 2806: Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo

Abstract While the use of bioluminescent proteins for molecular imaging is a powerful technology to support our knowledge of complex processes, with the advent of near infrared-labeling capabilities optical imaging can be pursuit with deeper tissue penetration, a better signal to background ratio and a quicker image acquisition without the need to inject substrates in preclinical oncology mouse models. This is specifically important in orthoptic brain tumors, when the substrate concentration in the tumor tissue might be influenced by the blood-brain barrier. In our recent study, we validated the feasibility of stable or transient transfection of human brain tumor cell lines and patient derived xenografts to determine tumor load in an intracranial setting. As a proof-of principle, we stably transfected the human glioblastoma cell line U87MG with an iRFP713 construct and followed tumor growth of intracranially implanted NSG mice. As a follow-up, five PDX of pediatric brain tumors (two Ependymoma, two Medulloblastoma, one high grade Glioblastoma) were transiently transfected with the same construct 24h before intracranial implantation. Tumor load was determined twice a week with the Pearl trilogy system (LiCor, Germany) and animals were examined for neurological symptoms every day. When the overall condition of the mice got deteriorated, animals were taken down and brains fixed in formalin. The tumor load and localization was confirmed by H&E and human-specific IHC for LamininB. The comparison between non-transfected and transfected lines of one specific tumor model, revealed that the tumor growth was very similar in both settings. The tumor take rate was as well not affected. In the subsequent analyses we could show that the tumor load linearly correlated with the imaging signal. Moreover, the iRFP713 signal was stable post-resection of the tumor. Thus, it was possible to correlate the tumor load quantification via iRFP713-Protein expression and LamininB-IHC within one slide. The transient transfection was successful for the five tested models. When termination criteria were reached, over 90% of the tumor tissue were expressing the iRFP713 protein. The more general applicability for larger cohorts of mice (> 20) and drug testing experiments will be evaluated soon. We believe the superior optical properties of iRFP713 will be a valuable asset to overcome some of the complications inherent to imaging live animals and a powerful tool for preclinical drug development in clinical relevant mouse models of brain cancer. Citation Format: Kanstantsin Lashuk, Jacqueline Bersano, Dorothee Lenhard, Eva Oswald, Kerstin Klingner, Julia Schüler. Development of an iRFP713-based orthotopic patient derived brain cancer model to study tumor development in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2806.

Tumor-specific overexpression of histone gene, H3C14 in gastric cancer is mediated through EGFR-FOXC1 axis

Incorporation of different H3 histone isoforms/variants have been reported to differentially regulate gene expression via alteration in chromatin organization during diverse cellular processes. However, the differential expression of highly conserved histone H3.2 genes, H3C14 and H3C13 in human cancer has not been delineated. In this study, we investigated the expression of H3.2 genes in primary human gastric, brain, breast, colon, liver, and head and neck cancer tissues and tumor cell lines. The data showed overexpression of H3.2 transcripts in tumor samples and cell lines with respect to normal counterparts. Furthermore, TCGA data of individual and TCGA PANCAN cohort also showed significant up-regulation of H3.2 genes. Further, overexpressed H3C14 gene coding for H3.2 protein was regulated by FOXC1 transcription factor and G4-cassette in gastric cancer cell lines. Elevated expression of FOXC1 protein and transcripts were also observed in human gastric cancer samples and cell lines. Further, FOXC1 protein was predominantly localized in the nuclei of neoplastic gastric cells compared to normal counterpart. In continuation, studies with EGF induction, FOXC1 knockdown, and ChIP-qPCR for the first time identified a novel axis, EGFR-FOXC1-H3C14 for regulation of H3C14 gene overexpression in gastric cancer. Therefore, the changes the epigenomic landscape due to incorporation of differential expression H3 variant contributes to change in gene expression pattern and thereby contributing to pathogenesis of cancer.

Nucleolin targeted delivery of aptamer tagged Trichoderma derived crude protein coated gold nanoparticles for improved cytotoxicity in cancer cells

The present work demonstrates how the fungal crude protein extract (FE) mediated gold nanoparticles (AuNPs) functionalized with aptamer (APT) improved cytotoxicity in human lung cancer cell line (A549) and brain tumor cell line (LN229). The agarose gel electrophoresis (AGE) confirmed the conjugation of APT in FE-AuNPs. The FTIR results revealed the involvement of FE in the amalgamation of FE-AuNPs. FE-AuNPs (λmax 521 nm) had Z-average size of 19.72 ± 0.26 d. nm and PDI of 0.58 ± 0.01 with a zeta potential of -22.20 mV, whereas APT-FE-AuNPs (λmax521 nm) had the Z-average size of 167.63 ± 1.40 d. nm and PDI of 0.21 ± 0.01 with a zeta potential of -36.36 mV. Both synthesized nanoparticles exhibited less cytotoxicity in normal NIH3T3 cells, while the APT-FE-AuNPs (λmax 521 nm) showed significant cytotoxicity in A549 and LN229 cells. The cells treated with APT-FE-AuNPs (λmax 521 nm) displayed higher percentage of early apoptosis (6.97 %), apoptosis (7.08 %), and necrosis (6.63 %) than those treated with bare or APT-FE-AuNPs (λmax 565 nm) in A549 cells. The results demonstrated that the DNA aptamer (AS1411) tagged with FE-AuNPs (λmax 521 nm) increased their accumulation in cancer cells, which triggered nucleolin targeted cell cytotoxicity through nucleus damage and induction of oxidative stress.

Abstract 4176: Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines

Abstract Introduction: Gallium maltolate (GaM) is a novel small molecule metallocompound that acts as an iron mimetic Trojan Horse to disrupt iron-dependent proteins essential in tumor biology. We recently reported that GaM retards the growth of orthotopically implanted adult human glioblastoma (GBM) xenografts in a rodent brain tumor model and is cytotoxic to glioblastoma stem cells (Mol Cancer Ther 17:1240, 2018). GaM's mechanisms of action include inhibition of both mitochondrial function and iron-dependent ribonucleotide reductase. In the present study, we further explored whether GaM has antitumor activity in pediatric brain tumor cells. Experimental procedures: The cytotoxicity of GaM in pediatric GBM and Atypical Teratoid Rhabdoid Tumor (ATRT) cell lines was examined by MTT and clonogenic assays. Apoptotic cell death was assessed by flow cytometry with annexin V (AV) and propidium iodide (PI) staining. Cellular bioenergetics was measured using a Seahorse XF96 Extracellular Flux Analyzer. GaM interaction with lysosomes was analyzed by immunofluorescence and electron microscopy (EM). Results: In a panel of pediatric brain tumor cell lines, CHLA-266 ATRT and GBM cells displayed the greatest sensitivity to GaM. Complete inhibition of colony growth was achieved with 6 - 7 µM GaM over a 96-hour incubation. With a shorter incubation of 48-h, 25 µM GaM induced apoptosis in > 60% of CHLA-266 ATRT cells. In contrast, a longer incubation of 96-h with 25 µM GaM was required to induce apoptosis in > 50% of CHLA-200 GBM, 69% of SF188 GBM and 60% of SJ-GBM2 cell lines. After a 24-h of incubation with GaM, prior to detectable cell death, CHLA-266 and GBM cells displayed a marked reduction in mitochondrial reserve capacity, glycolysis, and glycolytic capacity. Immunofluorescence staining for the lysosomal marker LAMP1 showed that GaM produced an increase in lysosome size and number after a 24-h or 48-h incubation relative to control cells. EM studies demonstrated a gallium signal within the lysosomal compartment as early as 6-h of incubation. EM findings consistent with GaM-induced apoptosis were noted after a 24-h incubation, thus confirming the results of the AV/PI assay. After 24-h however, gallium was no longer detectable in the lysosomes, suggesting that lysosomal disruption led to the release of gallium into the cytoplasm. Conclusions: Our results identify GaM as a novel compound with significant activity against a panel of cell lines representative of pediatric brain tumors. Most of the cell lines used in our study were derived from tumors taken from heavily pre-treated patients and were resistant to conventional chemotherapeutic drugs. Their sensitivity of GaM suggests that GaM does not share cross-resistance with other antineoplastic drugs. Our study also provides new insight into the lysosomes as a target for gallium. The prognosis of adult and pediatric brain tumors is dismal and there is an urgent need to develop newer drugs for this malignancy. Further investigation of GaM as a therapeutic agent for brain tumors is warranted. Citation Format: Salvatore Molino, Christopher R. Chitambar. Involvement of the lysosome in the cytotoxicity of Gallium Maltolate in human pediatric brain tumor cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4176.

Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

RETRACTED ARTICLE: One-Dimensional Ag(I) and Cu(I) Coordination Polymers Constructed from the Two N-donor Ligands: Structural Insights and Anti-brain Tumor Activity Evaluation

This study reports the synthesis and characterization of two new Ag(I) and Cu(I)-based coordination polymers (CPs) with the chemical formulas of [Ag(bipp)(ClO4)]n (1, bipp = bis(4-pyridyl) propane) and [Cu2(bib)(CN)](NO3)(H2O)2 (2, bib = 4,4′-bisimidazolylbiphenyl). The molecular structures of the as-prepared CPs were determined by the single crystal X-ray diffraction studies and their phase purities have been confirmed via the power X-day diffraction analysis. The crystal structure analysis reveals that the both CPs reveal 1D chain-like structures and their frameworks are stabilized via the π–π interactions and H-bonding interactions. Furthermore, the size of the two CPs could be easily downsized into the nanometer via a simple reflux treatment. In connection to these, the as-prepared two nanostructural CPs were tested for their in vitro anticancer activities against three human brain tumor cell lines (SF17, SF767 and F126) via the MTT assay.

Zinc(II)‐N2O2 ligation complex‐based DNA/protein binder and cleaver having enhanced cytotoxic and phosphatase activity

The alkyne arms containing zinc(II) N2O2 ligation complexes (1) and (2) were prepared from 2,2′‐{cyclohexane‐1,2‐diylbis[nitrilo(E)methylylidene]}bis[5‐(prop‐2‐yn‐1‐yloxy)phenol] (L1) and 2,2′‐{1,2‐phenylenebis[nitrilo(E)methylylidene]}bis[5‐(prop‐2‐yn‐1‐yloxy)phenol] (L2) and characterized by analytical and various spectral techniques. The molecular geometry of 1 and 2 was optimized by Density Functional Theory (DFT) at B3LYP/6‐311G(d,p) level and compared with literature. The complexes are stable in solution, and their solution structure was assessed using NMR and ESI‐MS spectroscopy. Topological analysis of the electron density and nature of the bonding in the complexes has been determined by using Bader's AIM method. The interaction and binding modes of calf thymus DNA (CT‐DNA) with complexes (1 and 2) were investigated by using absorption and emission spectral and viscometric studies. The nuclease activity investigated through gel electrophoresis reveals that 1 and 2 exhibits a significant DNA cleavage activity via a hydrolytic pathway and is further confirmed by an experiment performed in the presence of T4 ligase. The protein binding ability of 1 and 2 with bovine serum albumin (BSA) protein evaluated show good protein binding propensity. In‐vitro cytotoxicity of the complexes has shown significant activity against human brain tumor (U87 MG) and breast carcinoma (BT20) cancer cell lines. The comet assay has been used to determine the extent of DNA fragmentation in cancer cells. The phosphatase activity investigated through kinetic measurements establishes that the zinc complexes possess significant hydrolytic efficiency and follow the order 2 > 1. The DFT calculations have also been carried out to support the proposed mechanistic pathway of catalytic activity.

MiR-1254 inhibits proliferation, migration and invasion of human brain tumour cell lines

Purpose: To investigate the expression of miR-1254 in 5 astrocytoma cell lines, and the mechanism involved.Methods: Total RNA was isolated by RNeasy RNA isolation kit while cDNA was prepared by RevertAid cDNA synthesis kit. The transcripts were analysed by real-time polymerase chain reaction (RT-PCR). Transfection of miR-1254 was carried out using FuGENE HD (Promega). Apoptosis was determined by DAPI, acridine orange (AO)/ethidium bromide (EB) and annexin V/PI double staining. Cell migration and invasion were investigated by wound healing and Martigel invasion assays, respectively. Protein expression was measured by western blotting.Results: Expression of miR-1254 was significantly down-regulated in the astrocytoma cell lines when compared to normal astrocyte cells (p < 0.05). Ectopic expression of miR-1254 in astrocytoma SW 1088 cells inhibited cell proliferation via initiation of apoptosis and cell cycle arrest. Over-expression of miR- 1254 also led to significant decrease in cell migration and invasion of SW 1088 astrocytoma cells (p < 0.05).Conclusion: The results show that the expression of miR-1254 is down-regulated in astrocytoma cell lines, but over-expression of miR-1254 inhibits proliferation of the cell lines via cell cycle arrest and apoptosis. Thus, miR-1254 has promising potential for use in the treatment of brain tumour.Keywords: Brain tumour, Astrocytoma, miR-1254, Apoptosis, Cell migration

TMIC-01. ALERTING THE IMMUNE SYSTEM BY REMOVING EPIGENETIC SILENCING OF TH1 CHEMOKINES

Solid tumors employ multiple mechanisms to evade immunologic detection and destruction. However, the potential to enhance the immune response to cancer has been proven in several malignancies and is under investigation in many others, including primary CNS tumors. Brain tumors, in particular, lack robust T-cell infiltration, suggesting poor immune surveillance. Recent studies have found that tumor cells express T-cell attracting chemokines, CXCL9/10, in response to interferon gamma (IFNg). Chemokine expression can be further amplified by drugs that prevent epigenetic silencing, such as histone methyltransferase inhibitors or histone deacetylase (HDAC) inhibitors. This approach increases T-cell trafficking to tumors in vivo and enhances the anti-tumor efficacy of checkpoint blockade. We hypothesized that T-cell trafficking to brain tumors could be increased by epigenetically enhancing CXCL9/10 expression. Assays were performed on human glioma brain tumor cell lines. CXCL9/10 expression were measured by real-time PCR and Western blot. GSK126, a commercially available methyltransferase inhibitor, was utilized to demethylate histone H3 (K9/K27). LB201 (Lixte Biotechnology, Setatuket, NY) was used to inhibit class 1/2 HDAC. Histone methylation and acetylation status were examined using Western blot analysis. T-cell migration was measured using transwell migration assays. expression of CXCL9/10 was induced by IFNg in brain tumor lines. Chemokine expression was further increased when cells were treated with GSK126 and LB201 compared to IFNg alone. Migration assays confirmed that tumor cells treated with GSK126 and LB201 released T-cell chemotactic factors that increased T-cell migration. These studies demonstrate that brain tumors express T-cell attracting chemokines CXCL9/10 in response to IFNg. Further, GSK126 and the combination of GSK126 and LB201 enhanced expression of CXCL9/10, resulting in increased T-cell migration toward tumor cells. Together, these data provide a potential means to enhance the efficacy of immune therapies for brain tumors by promoting T-cell trafficking toward tumor cells.

Screening of some Juniperus extracts for the phenolic compounds and their antiproliferative activities

In this study, the needle and cone of Juniperus species (Juniperus oxycedrus L. subsp. oxycedrus, J. foetidissima Willd., J. excelsa Bieb. and J. communis L.) that are commonly used in Turkish folk medicine were investigated in terms of their phenolic contents and antiproliferative activities. The phenolic constituents of the extracts were determined by HPLC-TOF/MS. Antiproliferative activities of the species were investigated between 5 and 100μg/mL concentrations against human cervix carcinoma (HeLa) and rat brain tumor (C6) cell lines. Catechin and rutin were determined as the main phenolics of Juniperus species. Needle extracts exhibited better activities than cone extracts against HeLa and C6 cells. Needle of J. foetidissima exhibited high antiproliferative activities with an IC50 value of 10.65 against C6 cell lines. For HeLa cells, the highest antiproliferative activity was obtained with needle of J. communis with an IC50 value of 32.96. The results of the present study give a scientific basis to the traditional utilization of these Juniperus species, also demonstrating their potential as sources of natural anticancer compounds.

Abstract 2147: IDH1 mutation-inspired α-ketoglutaric acid mimics for epigenetic therapy of higher grade gliomas

Abstract Mutations at the active site of isocitrate dehydrogenase 1 gene (IDH1; R132H) occur at a high frequency (>70%) early in the oncogenesis of lower grade malignant gliomas, and result in a dramatic accumulation of the oncometabolite D-2 hydroxyglutarate ( D-2HG), that effectively replaces the normal metabolite α-ketoglutarate (α-KG) in cell physiology. Surprisingly, IDH1 mutations bestow superior therapeutic responses to alkylating agents and better patient survival. D-2HG, effectively competes with α-KG and potently inhibits various dioxygenase reactions, including the TET1, TET2 DNA -demethylases and histone demethylases (5m-cytosine to 5-OH cytosine, H3-K-meX to H3-K-meX-1) thereby, re-shaping and reprogramming the epigenetic landscape, and consequent transcriptional silencing; the DNA repair protein O6-methlguanine DNA methyltransferase (MGMT), which confers drug resistance is one major target for such repression. As a novel and innovative strategy of turning the tide against GBMs by exploiting the mechanistic aspects of the oncometabolite, we hypothesized that D-2HG and α-KG derivatives that can replace the natural metabolite in epigenomic dioxygenase reactions will serve as potent anti-glioma drugs either by themselves or in combination with the alkylating agents. To this end, we first synthesized a D-2HG diethyl ester to enhance its cellular uptake and tested its effects on four human brain tumor cell lines (DAOY, T98G, SF188 and UW18). This compound, at 5-10 mM and 24 h treatments, moderately inhibited the DNA repair activity of MGMT, increased temozolomide cytotoxicity by 1.5 to 3-fold, and induced histone H3-methylations as determined by western blot analyses. To increase the potency and design better compounds mimicking the oncometabolite and validate its impact on epigenesis, we synthesized a 2,4-dimethyleneglutaric acid (DMG), a α-KG mimic with methylene groups inserted at the C2 and C4 positions. The hydrophobic DMG ester, by itself, was cytotoxic with IC50 values up to 500 µM against brain tumor cell lines, however, when combined at 100 µM with TMZ resulted in a great synergistic cell killing (generally-9 fold, but 28-fold with UW-18 GBM cells). 0.25 mM DMG inhibited the cellular MGMT activity by >80%, induced degradation of TET1 protein and highly increased the methylation levels of histones (H1K25me1, me2 and H2BK25me2). Currently, experiments to determine the BBB-penetrance of DMG, its ability to induce genomic and MGMT-specific methylations in glioma cells and GBM regression in intracranial xenograft models developed in nude mice are underway. Collectively, these data reveal that acute treatments of α-KG analogs can alter the cellular epigenetic makeup in a manner ascribed to D-2HG, and open up the much-needed novel and exciting avenues of oncometabolite therapy for brain tumors (supported by CPRIT grants RP130266 & RP170207 to KSS). Citation Format: Hanumantha Rao Madala, Surendra Reddy Punganuru, Kalkunte S. Srivenugopal. IDH1 mutation-inspired α-ketoglutaric acid mimics for epigenetic therapy of higher grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2147. doi:10.1158/1538-7445.AM2017-2147

Abstract 2438: Analysis of IL-13 signaling through IL-13Rα2 in human brain tumor specimens in situ

Abstract Previously, we have demonstrated that IL-13 receptor alpha2 (IL-13Rα2), a high affinity receptor for Th2 cytokine IL-13, is overexpressed in most glioblastoma multiforme (GBM) cell lines and approximately 78% of the patient-derived samples. We have also demonstrated that IL-13Rα2 can be targeted by a number of immunotherapeutic agents including chimeric antigen receptor modified T (CAR-T) cells, targeted lentivirus and adenovirus vectors, and a chimeric fusion immunotoxin consisting of IL-13 and truncated Pseudomonas exotoxin (IL-13-PE). However, the signal transduction initiated by IL-13 in GBM tumor is not known to occur through the IL13Rα2, which has a high affinity for IL-13. We have recently observed that IL-13 can signal through IL-13Rα2 by activating AP-1 transcription factors in human brain tumor cell lines. In this study, we have examined IL-13Rα2 expression in human brain tumor and normal brain specimens, and the subsequent signaling through the AP-1 pathway in situ. Using six human glioblastoma and three astrocytoma specimens, we evaluated the expression of AP-1 transcription factors by immunohistochemistry (IHC) and compared the extent of immunostaining and percent positive fields with three normal brain specimens. Six GBM specimens examined showed high degree of immunostaining for c-Fos, c-Jun, Jun D and Fra-1 (AP-1 family of transcription factors) and a high percentage of positive fields. These specimens also showed strong immunostaining for IL-13Rα2 (4+) in >70% fields (P<0.001 compared to normal brain). Three astrocytoma specimens showed staining for IL-13Rα2 (>2+ and 32% fields, P<0.01 compared to normal brain), but the extent of staining was lower compared to GBM. Similar to IL-13Rα2 expression, the extent of staining and percentage of positive fields for AP-1 transcription factors were highly statistically significant between tumors and normal brain (P<0.001 for GBM compared to normal brain and P<0.01 for astrocytoma compared to normal brain). The extent of immunostaining in GBM was highest for c-Fos (4+, 78% fields) followed by c-Jun (3+, 57% fields), Fra-1 (2+, 70% fields) and Jun-D (2+, 28% fields). Jun-B expression was the lowest among the AP-1 transcription factors (<1+, 7% fields) in GBM specimens. Astrocytoma specimens showed lesser extent of immunostaining for AP-1 members compared to GBM; c-Fos showed 2+ staining and 42% positive fields followed by c-Jun (2+, 12% fields), Fra-1 (2+, 48% fields) and Jun-D (<1+, 18% fields). Jun-B staining intensity was <1+ in only 6% fields. Normal brain specimens showed no immunostaining for AP-1 family members. Our results generally corroborate with data obtained from GBM cell lines and confirm that IL-13 can signal in IL-13Rα2 positive GBM tumors in-situ through the AP-1 pathway, thus indicating that this pathway may be an important target for therapeutic intervention of GBM in addition to targeting IL-13Rα2. Citation Format: Rukmini Bhardwaj, Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Analysis of IL-13 signaling through IL-13Rα2 in human brain tumor specimens in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2017-2438

Abstract 3998: Alerting the immune system by removing epigenetic silencing of Th1 chemokines

Abstract BACKGROUND: Solid tumors employ multiple mechanisms to evade an immune response. However, the potential to enhance the immune response to cancer has been proven in several malignancie and is under investigation in many others, including primary CNS tumors. Brain tumors in particular lack robust T-cell infiltration. Recent studies have found that certain tumors can be induced to express T-cell attracting chemokines, CXCL9 and CXCL10, by interferon gamma (IFNg). This response is further amplified using methyltransferase inhibitors (Peng, et al. Nature 2015. Vol 527: 249-253.), resulting in increased Tcell trafficking to tumors both in vitro and in vivo. We hypothesized that T-cell trafficking to brain tumors could likewise be enhanced with DNA and histone methyltransferase inhibitors to induce CXCL9 and CXCL10 transcription. METHODS: Assays were performed on 7 human glioma brain tumor cell lines. CXCL9 and CXCL10 expression were measured by real-time PCR. Two commercially available methyltransferase inhibitors, 5-AZA-dC and GSK126, were utilized to demethylate DNA and histone H3 (K9 and K27), respectively. Histone methylation status was examined using Western blot. T-cell migration was measured using transwell migration assays. RESULTS: IFNg increased CXCL9 and CXCL10 transcription in brain tumor lines. GSK126 and 5-AZA-dC enhanced expression of CXCL9 and CXCL10 compared to IFNg alone. Migration assays confirmed T-cell trafficking towards chemokines produced by tumor cells in response to methyltransferase inhibitors. CONCLUSIONS: These studies demonstrate that brain tumors express T-cell attracting chemokines CXCL9 and CXCL10 in response to IFNg. Further, GSK126 and the combination of GSK126 and 5-AZA-dC enhanced expression of CXCL9 and CXCL10 transcription by real-time PCR and T-cell trafficking by migration assay. Together, these data provide a potential means to increase T-cell trafficking into tumors and potentially enhances the efficacy of immune therapies for brain tumors. Citation Format: Heather M. Sonnemann, Amber J. Giles, Caitlin M. Reid, Marsha-Kay N. Hutchinson, Deric M. Park, Mark R. Gilbert. Alerting the immune system by removing epigenetic silencing of Th1 chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3998. doi:10.1158/1538-7445.AM2017-3998

Cannabinoid receptor CB1 regulates STAT3 activity and its expression dictates the responsiveness to SR141716 treatment in human glioma patients' cells.

Herein we show that a majority of human brain tumor samples and cell lines over-expressed cannabinoid receptor CB1 as compared to normal human astrocytes (NHA), while uniformly expressed low levels of CB2. This finding prompted us to investigate the therapeutic exploitation of CB1 inactivation by SR141716 treatment, with regard to its direct and indirect cell-mediated effects against gliomas. Functional studies, using U251MG glioma cells and primary tumor cell lines derived from glioma patients expressing different levels of CB1, highlighted SR141716 efficacy in inducing apoptosis via G1 phase stasis and block of TGF-β1 secretion through a mechanism that involves STAT3 inhibition. According to the multivariate role of STAT3 in the immune escape too, interestingly SR141716 lead also to the functional and selective expression of MICA/B on the surface of responsive malignant glioma cells, but not on NHA. This makes SR141716 treated-glioma cells potent targets for allogeneic NK cell-mediated recognition through a NKG2D restricted mechanism, thus priming them for NK cell antitumor reactivity. These results indicate that CB1 and STAT3 participate in a new oncogenic network in the complex biology of glioma and their expression levels in patients dictate the efficacy of the CB1 antagonist SR141716 in multimodal glioma destruction.

Distinctive responses of brain tumor cells to TLR2 ligands:

Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll-like receptors (TLR)-2-mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2-deficient, interferon (IFN)-γ-deficient, and IFNγ-receptor-1-deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2-dependent pathway, not IFNγ-receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2-dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2-associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors.

Cancer Prognosis

Cancer Prognosis