Cultured Glioma Cells Research Articles

Erucic acid, a component of Lorenzo's oil and PPAR-δ ligand modifies C6 glioma growth and toxicity of doxorubicin. Experimental data and a comprehensive literature analysis

BackgroundPPAR-δ is a transcription factor which has crucial roles in stimulating oligodendroglial differentiation and myelination and its activation was also shown to differentiate malignant C6 glioma cells into oligodendrocytes. ObjectiveOne of the ligands of PPAR-δ is erucic acid (EA), an edible omega-9 fatty acid consumed more by Asian populations and exists highly in Chinese womens milk. There exist epidemiological evidence that pediatric brain tumor incidence is among the lowest in the Chinese population. EA is also an ingredient of Lorenzo's oil used against adrenoleukodystrophy, a pediatric demyelinating disease. EA was inappropriately assumed as a strong cardiotoxin based on Spanish oil syndrome, caused by toxic-aniline dye refined rapeseed oil. In this study, we studied whether EA is capable to block growth of C6 glioma cells and modify cardiotoxicity of doxorubicin. Materials and methodsWe studied effects of EA on the 3-dimensional appearance of the adherent cells, soft agar colony formation and S-phase in the 3-dimensional spheroids in C6 glioma cell cultures. We also investigated the effects of EA on hepatic and cardiac toxicity of doxorubicin. ResultsEA decreased in vitro growth of C6 glioma cells at therapeutically achievable concentrations. EA effects were more prominent in 3D-assays (soft agar colonies and spheroids) and induced cell fusions in monolayer cultures. EA decreased S-phase inhibitory potency of doxorubicin (DOX), yet augmented its efficacy to induce a senescent morphology (as assessed by scanning electron microscopy) in monolayer and to increase iNOS and eNOS expression in spheroids. In our study, EA reduced DOX-induced necrosis in mice heart and liver and induced healthier morphology of heart mitochondria (as assessed by transmission electron microscopy); yet intercalated disks (ID) were more disturbed with DOX + EA. ConclusionsBoth the antitumor and cardiac effects of EA may associate with the cell-to-cell contact mechanisms. Combining systemic EA with intrathecal DOX-chemotherapy via Ommaya reservoirs may reduce DOX concentrations in systemic circulation, hinder toxic interactions with EA and induce selective kill of glioma cells.

Alternative splicing in transcriptomes of glioma cell cultures

Alternative splicing in transcriptomes of glioma cell cultures

Abstract 3175: Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy

Abstract Pediatric gliomas, particularly high-grade gliomas, are among the most formidable and devastating cancers in children. These tumors have remained incurable, regardless of the many treatment approaches attempted. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance in pediatric gliomas. Signaling by bone morphogenetic protein 4 (BMP4), an essential molecule for central nervous system (CNS) development, increases GSC therapeutic sensitivity, and whose activation is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated as this can lead to the identification of additional targets for treating malignant gliomas in children. Delivery systems for administering BMP4 in a clinical setting also need to be developed. Here, we show that BMP4 increases chemosensitivity by decreasing H3 lysine 4 trimethylation (H3K4me3) at the promoter of multidrug resistant gene 1 (MDR1) that, in turn, results in increased BMP4 expression. This appears to be the result of BMP4 decreasing levels of hSETD1A, a critical methyltransferase for H3K4me3. Our work also demonstrates the first use of a novel sulfated glycopeptide (glycol-PA) nanostructure as a vector for delivery of BMP4. Glycol-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, our results expand understanding of how BMP4 brings about glioma therapeutic sensitization through epigenetic mechanisms, and show that highly bioactive glyco-PA nanostructures have potential as a novel delivery mechanism for treating pediatric malignant glioma, as well as other tumors. Citation Format: Guifa Xi, Benjamin Best, Sonali Nayak, Mark McClendon, Barbara Mania-Farnell, John Kessler, Charles David James, Samuel Stupp, Tadanori Tomita. Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3175.

PO-020 Functional characterisation of variant of unknown significate in familial breast cancer

IntroductionGlioblastoma (GBM), the common and aggressive human primary brain tumour (WHO grade IV), is highly resistant to standard radio- and chemotherapy. This is partly due to numerous genetic alterations in oncogenes and DNA damage repair systems. Despite progress in understanding the molecular background of GBM and advances in treatment modalities, survival of GMB patients is only 14 months post-diagnosis. RECQL4 belongs to RecQ family of ATP-dependent DNA helicases and plays an important role in genomic integrity and stability maintenance via involvement in DNA replication, repair, recombination, transcription and telomere maintenance. Mutations in the human RecQ genes are linked with cancer predisposition and/or premature ageing. Of all five human helicases, only RECQL4 is found in mitochondria. We explored if alterations of RECQL4 expression or functions contribute to pathogenesis of human GBM.Material and methodsWe determined the RECQL4 expression in various tumour specimens (tumour samples, human primary and established glioma cell cultures) by qPCR and Western Blotting. We determined the effect of RECQL4 depletion on cell viability, proliferation and GBM sphere formation using MTT metabolism, BrdU incorporation and tumour sphere forming assays, respectively.Results and discussionsWe found the upregulated expression of RECQL4 in GBM at mRNA and protein levels when compared to non-transformed human astrocytes. This finding was corroborated by TCGA data analysis. Fractionation of mitochondrial and cytosolic fractions from human glioma cells revealed the presence of RECQL4 in mitochondria. Downregulation (by siRNA) or genetic depletion of RECQL4 (by CRISPRCas9 knockout) in human glioma LN18 and U87-MG cells impaired cell viability and proliferation. We found upregulation of RECQL4 expression in GBM sphere cultures, enriched in glioma stem cells. Transient knock-down of RECQL4 significantly affected tumour sphere formation as evidenced by decreased numbers and sizes of cultured spheres.ConclusionThese data indicate that deregulation of RECQL4 expression or function may play an important role in GBM pathobiology. Our results provide a rationale for further studies of RECQL4 role in gliomagenesis.Supported by National Science Centre grants 2015/19/N/NZ3/02374 (SKK) and 2016/22/M/NZ3/00679 (BK) and the Foundation for Polish Science TEAM-TECH Core Facility project „NGS platform for comprehensive diagnostics and personalised therapy in neuro-oncology’ (AK).

PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma.

Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O6-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome.We have designed and tested both in vitro and in vivo, a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O6-benzylguanine (O6BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O6BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER).In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time.

Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.

Culture of C6 glioma cells and establishment of model

Objectives: To establish a simple, reliable, stable rat C6 glioma model, and to provide an operating platform for studying the pathogenesis and prevention and treatment of glioma. Methods: C6 rat glioma cells were cultured in vitro and cultured in the right caudate putamen of SD rats. After implantation, the survival status of rats was observed and the pathological sections were made. HE is dyeing under light microscope. Results: After 6 days of inoculation of C6 glioma cells, the survival status was good at about 7 days, and the intracranial hypertension was more obvious at about 14 days. Most of them were in the endangered state. In the 18 specimens of the rats, except for the accidental death, the other tumor formation rate was 75%. The tumor structure was obviously displaced with the prolongation of the age of the tumor. HE staining was used to observe the formation of glioma in rat brain. Conclusion: The animal model of C6 glioma is reliable and stable. Its tumor growth characteristics and pathological features are similar to those of human glioma, which can be used as an ideal model for clinical study of glioma.

The melatonin-MT1 receptor axis modulates tumor growth in PTEN-mutated gliomas

More than 40% of glioma patients have tumors that harbor PTEN (phosphatase and tensin homologue deleted on chromosome ten) mutations; this disease is associated with poor therapeutic resistance and outcome. Such mutations are linked to increased cell survival and growth, decreased apoptosis, and drug resistance; thus, new therapeutic strategies focusing on inhibiting glioma tumorigenesis and progression are urgently needed. Melatonin, an indolamine produced and secreted predominantly by the pineal gland, mediates a variety of physiological functions and possesses antioxidant and antitumor properties. Here, we analyzed the relationship between PTEN and the inhibitory effect of melatonin in primary human glioma cells and cultured glioma cell lines. The results showed that melatonin can inhibit glioma cell growth both in culture and in vivo. This inhibition was associated with PTEN levels, which significantly correlated with the expression level of MT1 in patients. In fact, c-fos-mediated MT1 was shown to be a key modulator of the effect of melatonin on gliomas that harbor wild type PTEN. Taken together, these data suggest that melatonin-MT1 receptor complexes represent a potential target for the treatment of glioma.

Induction and Assessment of Hypoxia in Glioblastoma Cells In Vitro.

To simulate and study the hypoxic microenvironment associated with intracerebral glioma in vivo, simple and reproducible methods are described and discussed for inducing hypoxia or chemical pseudohypoxia in glioma cell cultures and assessing their effects on the expression and nuclear translocation of hypoxia-inducible factor (HIF)-1α, a key transcriptional factor of oxygen homeostasis, by Western blot analysis and immunocytochemistry.

Reprogramming glioma cell cultures with retinoic acid: Additional arguments for reappraising the potential of retinoic acid in the context of personalized glioma therapy

Background: Glioma, notably glioblastoma multiforme, is characterized by extensive inter-and intra-tumoral heterogeneity. Surprisingly, the potential for differentiation of glioma cells has not been systematically analyzed and included in patient stratification methods. In the current study, retinoic acid (RA), a neuronal differentiation agent, was assessed for the pro-differentiative and anti-proliferative effects on glioma cells. Methods: Using RA-responsive glioma culture as an experimental paradigm, we analyzed the differentiation process both by videomicroscopy and at the mRNA (RNA-seq and reverse transcription-quantitative-polymerase chain reaction) and proteomic levels. Results: Glioma cells can differentiate into neurons in response to RA by (i) extending ultra-long cytoplasmic extensions, (ii) using these extensions to move from cell to cell either by perikaryal translocation or in a spider-flight like process, (iii) slowing their cell cycling, (iv) acquiring several neuronal differentiation markers such as MAPT, GAP43, DCX, NRCAM, NeuroD2, NeuroG2, and NeuN, and (v) decreasing the expression of several genes associated with glioma aggressiveness. Conclusion: These results indicate the existence of a subgroup of patients harboring RA-responsive glioma cells amenable to differentiation therapy, and stratifying such patients with a functional test is easily achievable. This provides the first step to reassess the potential of RA in the context of personalized medicine.

Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus’ tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two- and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus’ capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.

IMMU-32. NON-INFLAMMATORY EFFECT OF DIPG ON TUMOR-ASSOCIATED MICROGLIA

Diffuse intrinsic pontine glioma (DIPG) is a diffusely infiltrating high-grade glioma that has a peak incidence from ages 6-8 and only a 9-12 month median survival. Like adult gliomas, DIPG exhibits a significant expansion of myeloid cells, with microglia/macrophages accounting for ~30% of the tumor cell mass (Caretti & Monje, 2014). In adult glioma, these glioma-associated macrophages have been implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). However, genomic and epigenomic characterizations of adult and pediatric high-grade gliomas revealed striking differences in the molecular nature of these tumors (Sturm et al., 2014), and traditional chemotherapeutic approaches to adult glioblastoma are not effective in DIPG, suggesting these are unique diseases with distinct pathophysiology. Our data demonstrates that tumor-associated macrophages from primary DIPG samples exhibit gene expression signatures that are associated with pro-tumorigenic functions, but are also significantly less inflammatory than tumor-associated macrophages isolated from adult glioblastoma samples. Moreover, the cytokine secretion profiles of patient-derived DIPG cell cultures are dramatically less inflammatory when compared to patient-derived adult high-grade glioma cell cultures, and are more similar to the profile of human neural precursor cell cultures. This suggests that DIPG is less immunogenic than adult GBM, which may allow the tumor cells to evade detection and infiltrate healthy tissue. We additionally show that DIPG cells express CD200, a ligand that inhibits myeloid cell activation. Knockdown of DIPG expression of CD200 increases primary microglial cell phagocytosis in direct co-culture experiments, and also inhibits DIPG progression in in vivo xenograft models. Thus, CD200 may represent a mechanism by which DIPG evades normal immune responses to tumor growth. By understanding the DIPG-microglial interaction in molecular detail, we will gain insight into the pathophysiology of DIPG that could direct future targeted therapies.

High LC3/Beclin Expression Correlates with Poor Survival in Glioma: a Definitive Role for Autophagy as Evidenced by In Vitro Autophagic Flux.

Recent studies suggest the role of autophagy, an evolutionarily conserved catabolic process, in determining the response of gliomas to treatment either positively or negatively. The study attempts to characterize autophagy in low and high-grade glioma by investigating the autophagic flux and clinical significance of autophagy proteins (LC3 and beclin 1) in a group of glioma patients. We evaluated the expression of autophagic markers in resected specimens of low-grade glioma (LGG) and high-grade glioma (HGG) tissues, by immunohistochemistry and Western blotting. Our results show that expression of autophagy proteins were more prominent in HGG than in LGG. Increased level of autophagic proteins in HGG can be due to an increased rate of autophagy or can be because of blockage in the final degradation step of autophagy (defective autophagy). To distinguish these possibilities, the autophagic flux assay which helps to determine the rate of degradation/synthesis of autophagic proteins (LC3-II and p62) over a period of time by blocking the final degradation step of autophagy using bafilomycin A1 was used . The assessment of autophagic flux in ex vivo culture of primary glioma cells revealed for the first time increased turnover of autophagy in high grade compared to low grade-glioma. Though autophagic markers were reduced in LGG, functionally autophagy was non defective in both grades of glioma. We then investigated whether autophagy in gliomas is regulated by nutrient sensing pathways including mTOR and promote cell survival by providing an alternate energy source in response to metabolic stress. The results depicted that the role of autophagy during stress varies with tissue and has a negative correlation with mTOR substrate phosphorylation. We also evaluated the expression of LC3 and beclin 1 with progression free survival (PFS) using Kaplan-Meier survival analysis and have found that patients with low LC3/beclin 1 expression had better PFS than those with high expression of LC3/beclin 1 in their tumors. Together, we provide evidence that autophagy is non-defective in glioma and also show that high LC3/beclin 1 expression correlates with poor PFS in both LGG and HGG.

Abstract A39: Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer

Abstract Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 <30nM, whilst non-deleted cells could readily tolerate μM levels of inhibitor. As such, POMHEX was selected for in vivo experiments. Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in personalized cancer therapy. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

The Protective Effect of Lonicerae flos Extract on Cultured C6 Glioma Cells Damaged by Aluminum of Dementia Inducer

치매유발제인 염화알루미늄(aluminum chloride, $AlCl_3$ )의 세포독성을 대뇌교종세포인 C6 glioma 세포를 대상으로 산화적 손상측면에서 조사하였으며, 또한 $AlCl_3$ 의 세포독성에 대한 금은화(Lonicerae flos, LF) 추출물의 영향을 세포생존율을 비롯한 DPPH-라디칼 소거능, superoxide anion-radical (SAR) 제거...

Soft Substrates Containing Hyaluronan Mimic the Effects of Increased Stiffness on Morphology, Motility, and Proliferation of Glioma Cells.

Unlike many other cancer cells that grow in tumors characterized by an abnormally stiff collagen-enriched stroma, glioma cells proliferate and migrate in the much softer environment of the brain, which generally lacks the filamentous protein matrix characteristic of breast, liver, colorectal, and other types of cancer. Glial cell-derived tumors and the cells derived from them are highly heterogeneous and variable in their mechanical properties, their response to treatments, and their properties in vitro. Some glioma samples are stiffer than normal brain when measured ex vivo, but even those that are soft in vitro stiffen after deformation by pressure gradients that arise in the tumor environment in vivo. Such mechanical differences can strongly alter the phenotype of cultured glioma cells. Alternatively, chemical signaling might elicit the same phenotype as increased stiffness by activating intracellular messengers common to both initial stimuli. In this study the responses of three different human glioma cell lines to changes in substrate stiffness are compared with their responses on very soft substrates composed of a combination of hyaluronic acid and a specific integrin ligand, either laminin or collagen I. By quantifying cell morphology, stiffness, motility, proliferation, and secretion of the cytokine IL-8, glioma cell responses to increased stiffness are shown to be nearly identically elicited by substrates containing hyaluronic acid, even in the absence of increased stiffness. PI3-kinase activity was required for the response to hyaluronan but not to stiffness. This outcome suggests that hyaluronic acid can trigger the same cellular response, as can be obtained by mechanical force transduced from a stiff environment, and demonstrates that chemical and mechanical features of the tumor microenvironment can achieve equivalent reactions in cancer cells.

Anti-glioma Activity of Dapsone and Its Enhancement by Synthetic Chemical Modification.

The sulfone dapsone is an old antibiotic used for the treatment of mycobacterial and protozoal infections. We postulated before that dapsone might possess biological activity exceeding its anti-infectious properties and that it could potentially be repurposed for the treatment of glioma. To test this hypothesis, we treated established and primary cultured glioma cells with dapsone or several dapsone analogues which we previously synthesized (D2-D5) and determined effects on proliferation, anchorage-independent growth and migration. While dapsone and its synthetic analogues D2-D5 displayed only modest anti-proliferative activity, important neoplastic features such as anchorage-independent growth, clonogenic survival and directed migration were significantly inhibited by dapsone treatment. Moreover, dapsone analogues D3, D4 and D5 yielded even enhanced anti-glioma activity against different pro-neoplastic features. Overall these data suggest that dapsone provides activity against glioma which can be further enhanced by molecular modifications. These compounds could potentially serve as a therapeutic adjunct to the treatment of gliomas in a repurposing approach.

Synergistic and targeted therapy with a procaspase-3 activator and temozolomide extends survival in glioma rodent models and is feasible for the treatment of canine malignant glioma patients.

PurposeGlioblastoma is a deadly brain cancer with a median survival time of ∼15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas.Experimental DesignUsing cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1.ResultsProcaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed.ConclusionsProcaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.

Abstract 2044: Development of stable and brain-penetrating disulfiram nanoparticles, characterization and efficacy in glioma cell culture and xenograft models

Abstract Blood brain-barrier entry, tumor heterogeneity, need to eliminate the tumorigenic stem cells and inhibit the DNA repair protein MGMT are all essential considerations in glioma treatment. Previously, we showed that alcohol aversion drug disulfiram (DSF) shares all these properties [Carcinogenesis 35, 692, 2014; Oncotarget 4,502, 2013]. DSF was shown to inhibit the MGMT activity in the same way as ALDH by conjugating with the active-site cysteine 145 and sensitize brain tumors to alkylating agents. Although DSF exerts significant anticancer effects in vitro, the drug undergoes rapid decomposition and metabolism. To overcome this problem, we engineered DSF nanoparticles by encapsulating the drug in PEG: PLGA polymer by solvent evaporation method. The formulation was optimized for various process formulation variables. The optimized nano-formulation (NP) ranged from 70-80 nm in particle size as confirmed using dynamic light scattering and transition electron microscopy. Drug loading, encapsulation efficiency, in vitro release were also optimal and the particles showed excellent BBB permeability in vitro. The time course of internalization of particles using a cyanine dye (HITC-1) showed a lysosomal accumulation followed by mitochondria. Live animal imaging after IV injections of HITC-labeled DSF-NPs revealed a selective accumulation in the brain and subcutaneous tumors. Further, the cytotoxicity elicited by DSFNPs against SF188, DAOY, UW18, UW28 and T98G brain tumor cells was extended and greater than obtained with the free drug. When combined with DSF-NPs, temozolomide showed an 8-10-fold increased cytotoxicity against glioma cell lines. The DSFNPs also induced significant degradation of MGMT and other redox-sensitive proteins such as the NF-κB, MDR1 and GSTP1 in a concentration-dependent manner in glioma cells. ROS induction by the encapsulated drug was confirmed through DCF-DA and DHE staining of live cells; co-staining these cells with the mitochondrial membrane potential markers DiOC6 and DHE confirmed the altered integrity. GSTP1 inhibition and ROS induction by DSFNPs led to an activation of JNK/MAPK pathway, culminating in autophagy and apoptosis as verified by western blotting, FACS and immunofluorescence of appropriate markers. Finally, the antitumor efficacy of DSFNPs was tested in intracranial glioblastoma developed in nude mice by injecting luciferase-expressing SF-188 GBM cells. A superior tumor regression was evident in DSFNP-administered animals compared with the free drug after IVIS bioluminescence and histochemical staining of brain tissue. Collectively, our efforts provide strong evidence that DSF nanoparticles can cross the BBB and offer a sustained drug supply to eliminate CNS cancers. Further refinement of DSFNPs by incorporating brain targeting strategies is underway (supported by CPRIT grants RP130266 & RP170207 to KSS). Citation Format: Hanumantha Rao Madala, Surendra R. Punganuru, Francis Ali-Osman, Kalkunte S. Srivenugopal. Development of stable and brain-penetrating disulfiram nanoparticles, characterization and efficacy in glioma cell culture and xenograft models [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 2044. doi:10.1158/1538-7445.AM2017-2044

HP1α is highly expressed in glioma cells and facilitates cell proliferation and survival

Epigenetic alteration plays critical roles in gliomagenesis by regulating gene expression through modifications of Histones and DNA. Trimethylation of H3K9, an essential repressed transcription mark, and one of its methyltransferase, SUV39H1, are implicated in glioma pathogenesis and progression. We find that the protein level of HP1α, a reader of H3K9me3 is elevated in cultured glioma cell lines and glioma tissues. H3K9me3 is also upregulated. Depletion of HP1α and SUV39H1 weakens glioma cell proliferation capacity and results in apoptosis of cells. Furthermore, we find that HP1α and H3K9me3 are enriched in the FAS and PUMA promoters, which suggests that upregulated HP1α and H3K9me3 contribute to cell survival by suppressing apoptotic activators. These data suggests that up-regulated HP1α and H3K9me3 in glioma cells are functionally associated with glioma pathogenesis and progression and may serve as novel biomarkers for diagnostic and therapeutic targeting of brain tumors.