Histone deacetylase-3 regulates the expression of the amyloid precursor protein and its inhibition promotes neuroregenerative pathways in Alzheimer's disease models.

HDAC3 inhibition has been shown to improve memory and reduce amyloid-ß (Aß) in Alzheimer's disease (AD) models, but the underlying mechanisms are unclear. We investigated the molecular effects of HDAC3 inhibition on AD pathology, using in vitro and ex vivo models of AD, based on our finding that HDAC3 expression is increased in AD brains. For this purpose, N2a mouse neuroblastoma cells as well as organotypic brain cultures (OBCSs) of 5XFAD and wild-type mice were incubated with various concentrations of the HDAC3 selective inhibitor RGFP966 (0.1-10 µM) for 24 h. Treatment with RGFP966 or HDAC3 knockdown in N2a cells was associated with an increase on amyloid precursor protein (APP) and mRNA expressions, without alterations in Aß42 secretion. In vitro chromatin immunoprecipitation analysis revealed enriched HDAC3 binding at APP promoter regions. The increase in APP expression was also detected in OBCSs from 5XFAD mice incubated with 1 µM RGFP966, without changes in Aß. In addition, HDAC3 inhibition resulted in a reduction of activated Iba-1-positive microglia and astrocytes in 5XFAD slices, which was not observed in OBCSs from wild-type mice. mRNA sequencing analysis revealed that HDAC3 inhibition modulated neuronal regenerative pathways related to neurogenesis, differentiation, axonogenesis, and dendritic spine density in OBCSs. Our findings highlight the complexity and diversity of the effects of HDAC3 inhibition on AD models and suggest that HDAC3 may have multiple roles in the regulation of APP expression and processing, as well as in the modulation of neuroinflammatory and neuroprotective genes.

Profiling of circulating glial cells for accurate blood-based diagnosis of glial malignancies.

Here, we describe a blood test for the detection of glial malignancies (GLI-M) based on the identification of circulating glial cells (CGCs). The test is highly specific for GLI-M and can detect multiple grades (II-IV) and subtypes including gliomas, astrocytomas, oligodendrogliomas, oligoastrocytomas and glioblastomas, irrespective of gender and age. Analytical validation of the test was performed as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Real-world performance characteristics of the test were evaluated in four clinical (observational) studies. The test has high analytical sensitivity (95%), specificity (100%) and precision (coefficient of variation [CV] = 13.7% for repeatability and CV = 23.5% for within laboratory precision, both at the detection threshold) and is not prone to interference from common drugs and serum factors. The ability of the test to detect and differentiate GLI-M from non-malignant brain tumours (NBT), brain metastases from primary epithelial malignancies (EPI-M) and healthy individual donors (HD) was evaluated in four clinical cohorts. Across these clinical studies, the test showed 99.35% sensitivity (95% confidence interval [CI]: 96.44%-99.98%) and 100% specificity (95% CI: 99.37%-100%). The performance characteristics of this test support its clinical utility for diagnostic triaging of individuals presenting with intracranial space-occupying lesions (ICSOL).

Enhanced Sestrin expression through Tanshinone 2A treatment improves PI3K-dependent inhibition of glioma growth.

Glioblastomas are a highly aggressive cancer type which respond poorly to current pharmaceutical treatments, thus novel therapeutic approaches need to be investigated. One such approach involves the use of the bioactive natural product Tanshinone IIA (T2A) derived from the Chinese herb Danshen, where mechanistic insight for this anti-cancer agent is needed to validate its use. Here, we employ a tractable model system, Dictyostelium discoideum, to provide this insight. T2A potently inhibits cellular proliferation of Dictyostelium, suggesting molecular targets in this model. We show that T2A rapidly reduces phosphoinositide 3 kinase (PI3K) and protein kinase B (PKB) activity, but surprisingly, the downstream complex mechanistic target of rapamycin complex 1 (mTORC1) is only inhibited following chronic treatment. Investigating regulators of mTORC1, including PKB, tuberous sclerosis complex (TSC), and AMP-activated protein kinase (AMPK), suggests these enzymes were not responsible for this effect, implicating an additional molecular mechanism of T2A. We identify this mechanism as the increased expression of sestrin, a negative regulator of mTORC1. We further show that combinatory treatment using a PI3K inhibitor and T2A gives rise to a synergistic inhibition of cell proliferation. We then translate our findings to human and mouse-derived glioblastoma cell lines, where both a PI3K inhibitor (Paxalisib) and T2A reduces glioblastoma proliferation in monolayer cultures and in spheroid expansion, with combinatory treatment significantly enhancing this effect. Thus, we propose a new approach for cancer treatment, including glioblastomas, through combinatory treatment with PI3K inhibitors and T2A.

Accurate prostate cancer detection based on enrichment and characterization of prostate cancer specific circulating tumor cells.

BACKGROUND: The low specificity of serum PSA resulting in the inability to effectively differentiate prostate cancer from benign prostate conditions is a persistent clinical challenge. The low sensitivity of serum PSA results in false negatives and can miss high-grade prostate cancers. We describe a non-invasive test for detection of prostate cancer based on functional enrichment of prostate adenocarcinoma associated circulating tumor cells (PrAD-CTCs) from blood samples followed by their identification by immunostaining for pan-cytokeratins (PanCK), prostate specific membrane antigen (PSMA), alpha methyl-acyl coenzyme-A racemase (AMACR), epithelial cell adhesion molecule (EpCAM), and common leucocyte antigen (CD45). METHODS: Analytical validation studies were performed to establish the performance characteristics of the test using VCaP prostate cancer cells spiked into healthy donor blood (HDB). The clinical performance characteristics of the test were evaluated in a case-control study with 160 known prostate cancer cases and 800 healthy males, followed by a prospective clinical study of 210 suspected cases of prostate cancer. RESULTS: Analytical validation established analyte stability as well as acceptable performance characteristics. The test showed 100% specificity and 100% sensitivity to differentiate prostate cancer cases from healthy individuals in the case control study and 91.2% sensitivity and 100% specificity to differentiate prostate cancers from benign prostate conditions in the prospective clinical study. CONCLUSIONS: The test accurately detects PrAD-CTCs with high sensitivity and specificity irrespective of stage, serum PSA or Gleason score, which translates into low risks of false negatives or overdiagnosis. The high accuracy of the test could offer advantages over PSA based prostate cancer detection.

Traumatic Brain Injury Leads to Alterations in Contusional Cortical miRNAs Involved in Dementia.

There is compelling evidence that head injury is a significant environmental risk factor for Alzheimer's disease (AD) and that a history of traumatic brain injury (TBI) accelerates the onset of AD. Amyloid-ß plaques and tau aggregates have been observed in the post-mortem brains of TBI patients; however, the mechanisms leading to AD neuropathology in TBI are still unknown. In this study, we hypothesized that focal TBI induces changes in miRNA expression in and around affected areas, resulting in the altered expression of genes involved in neurodegeneration and AD pathology. For this purpose, we performed a miRNA array in extracts from rats subjected to experimental TBI, using the controlled cortical impact (CCI) model. In and around the contusion, we observed alterations of miRNAs associated with dementia/AD, compared to the contralateral side. Specifically, the expression of miR-9 was significantly upregulated, while miR-29b, miR-34a, miR-106b, miR-181a and miR-107 were downregulated. Via qPCR, we confirmed these results in an additional group of injured rats when compared to naïve animals. Interestingly, the changes in those miRNAs were concomitant with alterations in the gene expression of mRNAs involved in amyloid generation and tau pathology, such as ß-APP cleaving enzyme (BACE1) and Glycogen synthase-3-ß (GSK3ß). In addition increased levels of neuroinflammatory markers (TNF-α), glial activation, neuronal loss, and tau phosphorylation were observed in pericontusional areas. Therefore, our results suggest that the secondary injury cascade in TBI affects miRNAs regulating the expression of genes involved in AD dementia.

Inhibition of the angiotensin II type 2 receptor AT2R is a novel therapeutic strategy for glioblastoma.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT2R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT2R. We repurposed EMA401, an AT2R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT2R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT2R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or ß-arrestin recruitment. The architecture and interactions of EMA401 in AT2R differ drastically from complexes of AT2R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2-tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

Design Principles Governing the Development of Theranostic Anticancer Agents and Their Nanoformulations with Photoacoustic Properties.

The unmet need to develop novel approaches for cancer diagnosis and treatment has led to the evolution of theranostic agents, which usually include, in addition to the anticancer drug, an imaging agent based mostly on fluorescent agents. Over the past few years, a non-invasive photoacoustic imaging modality has been effectively integrated into theranostic agents. Herein, we shed light on the design principles governing the development of theranostic agents with photoacoustic properties, which can be formulated into nanocarriers to enhance their potency. Specifically, we provide an extensive analysis of their individual constituents including the imaging dyes, drugs, linkers, targeting moieties, and their formulation into nanocarriers. Along these lines, we present numerous relevant paradigms. Finally, we discuss the clinical relevance of the specific strategy, as also the limitations and future perspectives, and through this review, we envisage paving the way for the development of theranostic agents endowed with photoacoustic properties as effective anticancer medicines.

Arginine deprivation alters microglial polarity and synergizes with radiation to eradicate non-arginine-auxotrophic glioblastoma tumors.

New approaches for the management of glioblastoma (GBM) are an urgent and unmet clinical need. Here, we illustrate that the efficacy of radiotherapy for GBM is strikingly potentiated by concomitant therapy with the arginine-depleting agent ADI-PEG20 in a non-arginine-auxotrophic cellular background (argininosuccinate synthetase 1 positive). Moreover, this combination led to durable and complete radiological and pathological response, with extended disease-free survival in an orthotopic immune-competent model of GBM, with no significant toxicity. ADI-PEG20 not only enhanced the cellular sensitivity of argininosuccinate synthetase 1-positive GBM to ionizing radiation by elevated production of nitric oxide (˙NO) and hence generation of cytotoxic peroxynitrites, but also promoted glioma-associated macrophage/microglial infiltration into tumors and turned their classical antiinflammatory (protumor) phenotype into a proinflammatory (antitumor) phenotype. Our results provide an effective, well-tolerated, and simple strategy to improve GBM treatment that merits consideration for early evaluation in clinical trials.

Tonsillar Carcinoma Spreading Metastases to Central Nervous System: Case Report and Literature Review.

We present a case report of a 51-year-old left-handed male with a background of human papillomovairus 16-positive tonsil squamous cell carcinoma presenting with tonic-clonic seizure and a radiological diagnosis of secondary metastatic deposits. These were initially treated with stereotactic radiosurgery and subsequently with surgery. Surgical resection was performed under general anesthesia with right-sided temporal and parietal approaches. Both the parietal and temporal deposits were removed, while the intraventricular mass was intentionally left to avoid postoperative deficits. Adjuvant radiotherapy and chemotherapy were administered postoperatively. The patient experienced a satisfactory recovery postoperatively and was reoperated for recurrence 4 months later. He maintained a good quality of life and an excellent performance status throughout, but unfortunately he passed away in November 2018 due to septic complications. This case history stresses the difficulty in managing squamous cell carcinomas (SCC) with brain metastatic deposits. There are no current guidelines about the management of patients presenting with such a rare condition. More data are thus desirable to better define treatment guidelines and protocols when SCC brain metastases are present.

The bitter side of epigenetics: variability and resistance to chemotherapy.

One of the major obstacles to the development of effective new cancer treatments and the main factor for the increasing number of clinical trial failures appears to be the paucity of accurate, reproducible and robust drug resistance testing methods. Most research assessing the resistance of cancers to chemotherapy has concentrated on genetic-based molecular mechanisms, while the role of epigenetics in drug resistance has been generally overlooked. This is rather surprising given that an increasing body of evidence pointing to the fact that epigenetic mechanism alterations appear to play a pivotal role in cancer initiation, progression and development of chemoresistance. This resulted in a series of clinical trials involving epi-drug as single treatment or combined with cancer conventional drugs. In this review, we provided the main mechanisms by which the epigenetic regulators control the resistance to cancer drugs.

Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability, and Therapeutic Efficacy Against Glioblastoma Multiforme.

Cancer occupies a high rank in the global morbidity and mortality scale with glioblastoma multiforme (GBM) accounting for almost 80% of all primary tumors of the brain. Despite the increasing availability of targeted and immunotherapeutic agents, chemotherapy still plays an important role in the treatment of neoplastic diseases. Limitations to the effective use of chemotherapy such as low aqueous solubility and high toxicity have directed the scientific community's interest to the development of new therapeutic agents with enhanced efficacy and limited toxicity. Supramolecular chemistry has offered an alternative way on the design and development of new therapeutic agents as a result of their unique properties. Supramolecules can be used as drug carriers since their cavities can host a wide range of small drugs and surpass in this way the drawbacks of current therapeutic agents. Herein, we present the principles that should be followed for the encapsulation of small drugs in supramolecules with enhanced physicochemical properties and increased efficacy against glioblastoma multiforme.

Development of programmable gemcitabine-GnRH pro-drugs bearing linker controllable "click" oxime bond tethers and preclinical evaluation against prostate cancer.

Peptide-drug conjugates (PDCs) are gaining considerable attention as anti-neoplastic agents. However, their development is often laborious and time-consuming. Herein, we have developed and preclinically evaluated three PDCs with gemcitabine as the anticancer cytotoxic unit and D-Lys6-GnRH (gonadotropin-releasing hormone; GnRH) as the cancer-targeting unit. These units were tethered via acid-labile programmable linkers to guide a differential drug release rate from the PDC through a combination of ester or amide and "click" type oxime ligations. The pro-drugs were designed to enable the selective targeting of malignant tumor cells with linker guided differential drug release rates. We exploited the oxime bond responsiveness against the acidic pH of the tumor microenvironment and the GnRH endocytosis via the GnRH-R GPCR which is overexpressed on cancer cells. The challenging metabolic properties of gemcitabine were addressed during design of the PDCs. We developed a rapid (1 hour) and cost-effective "click" oxime bond ligation platform to assemble in one-pot the 3 desired PDCs that does not require purification, surpassing traditional time-ineffective and low yield methods. The internalization of the tumor-homing peptide unit in cancer cells, overexpressing the GnRH-R, was first validated through confocal laser microscopy and flow cytometry analysis. Subsequently, the three PDCs were evaluated for their in vitro antiproliferative effect in prostate cancer cells. Their stability and the release of gemcitabine over time were monitored in vitro in cell culture and in human plasma using LC-MS/MS. We then assessed the ability of the developed PDCs to internalize in prostate cancer cells and to release gemcitabine. The most potent analog, designated GOXG1, was used for pharmacokinetic studies in mice. The metabolism of GOXG1 was examined in liver microsomes, as well as in buffers mimicking the pH of intracellular organelles, resulting in the identification of two metabolites. The major metabolite at low pH emanated from the cleavage of the pH-labile oxime bond, validating our design approach. NMR spectroscopy and in vitro radioligand binding assays were exploited for GOXG1 to validate that upon conjugating the drug to the peptide, the peptide microenvironment responsible for its GnRH-R binding is not perturbed and to confirm its high binding potency to the GnRH-R. Finally, the binding of GOXG1 to the GnRH-R and the associated elicitation of testosterone release in mice were also determined. The facile platform established herein for the rapid assembly of PDCs with linker controllable characteristics from aldehyde and aminooxy units through rapid "click" oxime ligation, that does not require purification steps, could pave the way for a new generation of potent cancer therapeutics, diagnostics and theranostics.

Comprehending Meningioma Signaling Cascades Using Multipronged Proteomics Approaches & Targeted Validation of Potential Markers.

Meningiomas are one of the most prevalent primary brain tumors. Our study aims to obtain mechanistic insights of meningioma pathobiology using mass spectrometry-based label-free quantitative proteome analysis to identifying druggable targets and perturbed pathways for therapeutic intervention. Label-free based proteomics study was done from peptide samples of 21 patients and 8 non-tumor controls which were followed up with Phosphoproteomics to identify the kinases and phosphorylated components of the perturbed pathways. In silico approaches revealed perturbations in extracellular matrix remodeling and associated cascades. To assess the extent of influence of Integrin and PI3K-Akt pathways, we used an Integrin Linked Kinase inhibitor on patient-derived meningioma cell line and performed a transcriptomic analysis of the components. Furthermore, we designed a Targeted proteomics assay which to the best of our knowledge for very first-time enables identification of peptides from 54 meningioma patients via SRM assay to validate the key proteins emerging from our study. This resulted in the identification of peptides from CLIC1, ES8L2, and AHNK many of which are receptors and kinases and are difficult to be characterized using conventional approaches. Furthermore, we were also able to monitor transitions for proteins like NEK9 and CKAP4 which have been reported to be associated with meningioma pathobiology. We believe, this study can aid in designing peptide-based validation assays for meningioma patients as well as IHC studies for clinical applications.

Bioinspired tailoring of fluorogenic thiol responsive antioxidant precursors to protect cells against H2O2-induced DNA damage.

Natural antioxidants, like phenolic acids, possess a unique chemical space that can protect cellular components from oxidative stress. However, their polar carboxylic acid chemotype reduces full intracellular antioxidant potential due to limited diffusion through biological membranes. Here, we have designed and developed a new generation of hydrophobic turn-on fluorescent antioxidant precursors that upon penetration of the cell membrane, reveal a more polar and more potent antioxidant core and simultaneously become fluorescent allowing their intracellular tracking. Their activation is stimulated by polarity alteration by sensing intracellular signals and specifically biothiols. In our design, the carboxylic group of phenolic acids that originally restricts cell entrance is derivatized and conjugated through Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) to a coumarin derivative that its fluorescence properties are quenched with a biothiol activatable element. This more hydrophobic precursor readily penetrates cell membrane and once inside the cell the antioxidant core is revealed upon sensing glutathione, its fluorescence is restored in a turn-on manner and the generation of a more polar character traps the molecule inside the cell. This turn-on fluorescent antioxidant precursor that can be applied to phenolic acids, was developed for rosmarinic acid and the conjugate was named as RCG. The selectivity and responsiveness of RCG towards the most abundant biothiols was monitored through a variety of biophysical techniques including UV-Vis, fluorescence and NMR spectroscopy. The electrochemical behavior and free radical scavenging capacity of the precursor RCG and the active compound (RC) was evaluated and compared with the parent compound (rosmarinic acid) through cyclic voltammetry and EPR spectroscopy, respectively. The stability of the newly synthesized bioactive conjugate RC was found significantly higher than the parent rosmarinic acid when exposed to oxygen. Cell uptake experiments were conducted and revealed the internalization of RCG. The degree of intracellular DNA protection offered by RCG and its active drug (RC) on exposure to H2O2 was also evaluated in Jurkat cells.

ß-Hydroxybutyrate Oxidation Promotes the Accumulation of Immunometabolites in Activated Microglia Cells.

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of ß-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.

Enhancement of glioblastoma multiforme therapy through a novel Quercetin-Losartan hybrid.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. Maximal surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide remains the first-line therapy, prolonging the survival of patients by an average of only 2.5 months. There is therefore an urgent need for novel therapeutic strategies to improve clinical outcomes. Reactive oxygen species (ROS) are an important contributor to GBM development. Here, we describe the rational design and synthesis of a stable hybrid molecule tethering two ROS regulating moieties, with the aim of constructing a chemopreventive and anticancer chemical entity that retains the properties of the parent compounds. We utilized the selective AT1R antagonist losartan, leading to the inhibition of ROS levels, and the antioxidant flavonoid quercetin. In GBM cells, we show that this hybrid retains the binding potential of losartan to the AT1R through competition-binding experiments and simultaneously exhibits ROS inhibition and antioxidant capacity similar to native quercetin. In addition, we demonstrate that the hybrid is able to alter the cell cycle distribution of GBM cells, leading to cell cycle arrest and to the induction of cytotoxic effects. Last, the hybrid significantly and selectively reduces cancer cell proliferation and angiogenesis in primary GBM cultures with respect to the isolated parent components or their simple combination, further emphasizing the potential utility of the current hybridization approach in GBM.

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

Multifaceted transforming growth factor-beta (TGFß) signalling in glioblastoma.

Glioblastoma (GBM) is an aggressive and devastating primary brain cancer which responds very poorly to treatment. The average survival time of patients is only 14-15 months from diagnosis so there is a clear and unmet need for the development of novel targeted therapies to improve patient outcomes. The multifunctional cytokine TGFß plays fundamental roles in development, adult tissue homeostasis, tissue wound repair and immune responses. Dysfunction of TGFß signalling has been implicated in both the development and progression of many tumour types including GBM, thereby potentially providing an actionable target for its treatment. This review will examine TGFß signalling mechanisms and their role in the development and progression of GBM. The targeting of TGFß signalling using a variety of approaches including the TGFß binding protein Decorin will be highlighted as attractive therapeutic strategies.

Integrated systems-genetic analyses reveal a network target for delaying glioma progression.

OBJECTIVE: To identify a convergent, multitarget proliferation characteristic for astrocytoma transformation that could be targeted for therapy discovery. METHODS: Using an integrated functional genomics approach, we prioritized networks associated with astrocytoma progression using the following criteria: differential co-expression between grade II and grade III IDH1-mutated and 1p/19q euploid astrocytomas, preferential enrichment for genetic risk to cancer, association with patient survival and sample-level genomic features. Drugs targeting the identified multitarget network characteristic for astrocytoma transformation were computationally predicted using drug transcriptional perturbation data and validated using primary human astrocytoma cells. RESULTS: A single network, M2, consisting of 177 genes, was associated with glioma progression on the basis of the above criteria. Functionally, M2 encoded physically interacting proteins regulating cell cycle processes and analysis of genome-wide gene-regulatory interactions using mutual information and DNA-protein interactions revealed the known regulators of cell cycle processes FoxM1, B-Myb, and E2F2 as key regulators of M2. These results suggest functional disruption of M2 via gene mutation or altered expression as a convergent pathway regulating astrocytoma transformation. By considering M2 as a multitarget drug target regulating astrocytoma transformation, we identified several drugs that are predicted to restore M2 expression in anaplastic astrocytoma toward its low-grade profile and of these, we validated the known antiproliferative drug resveratrol as down-regulating multiple nodes of M2 including at nanomolar concentrations achievable in human cerebrospinal fluid by oral dosing. INTERPRETATION: Our results identify M2 as a multitarget network characteristic for astrocytoma progression and encourage M2-based drug screening to identify new compounds for preventing glioma transformation.

Encapsulation of Temozolomide in a Calixarene Nanocapsule Improves Its Stability and Enhances Its Therapeutic Efficacy against Glioblastoma.

The alkylating agent temozolomide (TMZ) is the first-line chemotherapeutic for glioblastoma (GBM), a common and aggressive primary brain tumor in adults. However, its poor stability and unfavorable pharmacokinetic profile limit its clinical efficacy. There is an unmet need to tailor the therapeutic window of TMZ, either through complex derivatization or by utilizing pharmaceutical excipients. To enhance stability and aqueous solubility, we encapsulated TMZ in a p-sulphonatocalix[4]arene (Calix) nanocapsule and used 1H-NMR, LC-MS, and UV-Vis spectroscopy to chart the stability of this novel TMZ@Calix complex according to FDA and European Medicines Agency guidelines. LC-MS/MS plasma stability assays were conducted in mice to further explore the stability profile of TMZ@Calix in vivo The therapeutic efficacy of TMZ@Calix was compared with that of unbound TMZ in GBM cell lines and patient-derived primary cells with known O6-methylguanine-DNA methyltransferase (MGMT) expression status and in vivo in an intracranial U87 xenograft mouse model. Encapsulation significantly enhanced the stability of TMZ in all conditions tested. TMZ@Calix was more potent than native TMZ at inhibiting the growth of established GBM cell lines and patient-derived primary lines expressing MGMT and highly resistant to TMZ. In vivo, native TMZ was rapidly degraded in mouse plasma, whereas the stability of TMZ@Calix was enhanced threefold with increased therapeutic efficacy in an orthotopic model. In the absence of new effective therapies, this novel formulation is of clinical importance, serving as an inexpensive and highly efficient treatment that could be made readily available to patients with GBM and warrants further preclinical and clinical evaluation.