Chalcones as Anti-Glioblastoma Stem Cell Agent Alone or as Nanoparticle Formulation Using Carbon Dots as Nanocarrier.

The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GSCs) and the failure of most drugs to reach therapeutic levels within the tumor remain formidable obstacles to successful treatment. Chalcones are aromatic ketones demonstrated to reduce malignant properties in cancers including glioblastoma. Nanomedicines can increase drug accumulation and tumor cell death. Carbon-dots are promising nanocarriers that can be easily functionalized with tumor-targeting ligands and anti-cancer drugs. Therefore, we synthesized a series of 4'-amino chalcones with the rationale that the amino group would serve as a "handle" to facilitate covalent attachment to carbon-dots and tested their cytotoxicity toward GSCs. We generated 31 chalcones (22 4'-amino and 9 4' derivatives) including 5 novel chalcones, and found that 13 had an IC50 below 10 µM in all GSC lines. After confirming that the 4-amino group was not part of the active pharmacophore, chalcones were attached to transferrin-conjugated carbon-dots. These conjugates were significantly more cytotoxic than the free chalcones, with the C-dot-transferrin-2,5, dimethoxy chalcone conjugate inducing up to 100-fold more GSC death. Several of the tested chalcones represent promising lead compounds for the development of novel anti-GSC drugs. Furthermore, designing amino chalcones for carbon-dot mediated drug delivery is a rational and effective methodology.

NAMPT Inhibition Induces Neuroblastoma Cell Death and Blocks Tumor Growth.

High-risk neuroblastoma (NB) portends very poor prognoses in children. Targeting tumor metabolism has emerged as a novel therapeutic strategy. High levels of nicotinamide-adenine-dinucleotide (NAD+) are required for rapid cell proliferation. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate-limiting enzyme for NAD+ salvage and is overexpressed in several cancers. Here, we determine the potential of NAMPT as a therapeutic target for NB treatment. NAMPT inhibition cytotoxicity was determined by trypan blue exclusion and LDH assays. Neuroblastoma stem cell self-renewal was evaluated by neurosphere assay. Protein expression was evaluated via Western blot. The effect of targeting NAMPT in vivo was determined using an NB1691-xenografted mouse model. Robust NAMPT expression was demonstrated in multiple N-MYC amplified, high-risk neuroblastoma cell lines. NAMPT inhibition with STF-118804 (STF) decreased ATP, induced apoptosis, and reduced NB stem cell neurosphere formation. STF treatment down-regulated N-MYC levels and abrogated AKT activation. AKT and glycolytic pathway inhibitors in combination with NAMPT inhibition induced robust, greater-than-additive neuroblastoma cell death. Lastly, STF treatment blocked neuroblastoma tumor growth in mouse xenograft models. NAMPT is a valid therapeutic target as inhibition promoted neuroblastoma cell death in vitro and prevented tumor growth in vivo. Further investigation is warranted to establish this therapy's role as an adjunctive modality.

The contribution of ketone bodies to glycolytic inhibition for the treatment of adult and pediatric glioblastoma.

PURPOSE: Glioblastoma (GBM) remains one of the most lethal primary brain tumors in children and adults. Targeting tumor metabolism has emerged as a promising-targeted therapeutic strategy for GBM and characteristically resistant GBM stem-like cells (GSCs). METHODS: Gene expression data was obtained from the online patient-histology database, GlioVis. GSC mitochondria morphology was examined by TEM. Cell viability and effect on GSC self-renewal was determined via MTS assay and neurosphere assay, respectively. Proteins were evaluated by Western Blot. RESULTS: Enzymes necessary for ketone catabolism (BDH1, OXCT1 and ACAT1) are significantly downregulated in adult and pediatric GBM. GSC mitochondrial ultrastructure suggested defects in oxidative phosphorylation. Treatment of both GBM and GSC cell lines resulted in dose-dependent decreases in viability in response to glycolytic inhibitor 2-deoxy-D-glucose (2-DG), and ketone body Acetoacetate (AA), but not ß-hydroxybutyrate (ßHB). AA induced apoptosis was confirmed by western blot analysis, indicating robust caspase activation and PARP cleavage. AA reduced neurosphere formation at concentrations as low as 1 mM. Combined treatment of low dose 2-DG (50 µM) with AA resulted in more cell death than either treatment alone. The effect was greater than additive at low concentrations of AA, reducing viability approximately 50% at 1 mM AA. AA was found to directly upregulate mitochondrial uncoupling protein 2 (UCP2), which may explain this potential drug synergism via multi-faceted inhibition of the glycolytic pathway. CONCLUSION: Targeting the metabolic pathway of GBM via glycolytic inhibition in conjunction with ketogenic diet or exogenous ketone body supplementation warrants further investigation as a promising adjunctive treatment to conventional therapy.

Targeting Glioblastoma Stem Cells with 2-Deoxy-D-Glucose (2-DG) Potentiates Radiation-Induced Unfolded Protein Response (UPR).

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, and despite optimized treatment options, median survival remains dismal. Contemporary evidence suggests disease recurrence results from expansion of a robustly radioresistant subset of GBM progenitor cells, termed GBM stem cells (GSCs). In this study, we utilized transmission electron microscopy to uncover ultrastructural effects on patient-derived GSC lines exposed to supratherapeutic radiotherapy levels. Elevated autophagosome formation and increased endoplasmic reticulum (ER) internal diameter, a surrogate for ER stress and activation of unfolded protein response (UPR), was uncovered. These observations were confirmed via protein expression through Western blot. Upon interrogating genomic data from an open-access GBM patient database, overexpression of UPR-related chaperone protein genes was inversely correlated with patient survival. This indicated controlled UPR may play a role in promoting radioresistance. To determine if potentiating UPR further can induce apoptosis, we exposed GSCs to radiation with an ER stress-inducing drug, 2-deoxy-D-glucose (2-DG), and found dose-dependent decreases in viability and increased apoptotic marker expression. Taken together, our results indicate GSC radioresistance is, in part, achieved by overexpression and overactivation of ER stress-related pathways, and this effect can be overcome via potentiation of UPR, leading to loss of GSC viability.

Curcumin decreases malignant characteristics of glioblastoma stem cells via induction of reactive oxygen species.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and lethal form of primary brain tumor in adults. Following standard treatment of surgery, radiation and chemotherapy, patients are expected to survive 12-14 months. Theorized cause of disease recurrence in these patients is tumor cell repopulation through the proliferation of treatment-resistant cancer stem cells. Current research has revealed curcumin, the principal ingredient in turmeric, can modulate multiple signaling pathways important for cancer stem cell self-renewal and survival. METHODS: Following resection, tumor specimens were dissociated and glioblastoma stem cells (GSCs) were propagated in neurosphere media and characterized via immunocytochemistry. Cell viability was determined with MTS assay. GSC proliferation, sphere forming and colony forming assays were conducted through standard counting methods. Reactive oxygen species (ROS) production was examined using the fluorescent molecular probe CM-H2DCFA. Effects on cell signaling pathways were elucidated by western blot. RESULTS: We evaluate the effects of curcumin on patient-derived GSC lines. We demonstrate a curcumin-induced dose-dependent decrease in GSC viability with an approximate IC50 of 25 µM. Treatment with sub-toxic levels (2.5 µM) of curcumin significantly decreased GSC proliferation, sphere forming ability and colony forming potential. Curcumin induced ROS, promoted MAPK pathway activation, downregulated STAT3 activity and IAP family members. Inhibition of ROS with the antioxidant N-acetylcysteine reversed these effects indicating a ROS dependent mechanism. CONCLUSIONS: Discoveries made in this investigation may lead to a non-toxic intervention designed to prevent recurrence in glioblastoma by targeting glioblastoma stem cells.

NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation.

The transcription factor NF-kappaB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-kappaB-mediated regulation of gene expression, and the signaling molecules participating in the NF-kappaB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKK(beta) binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-kappaB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKK(beta), but not IKK(alpha) or IKK(gamma). NIBP overexpression potentiates tumor necrosis factor-alpha-induced NF-kappaB activation through increased phosphorylation of the IKK complex and its downstream I(kappa)B(alpha) and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-alpha-induced NF-kappaB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKK(beta), is a new enhancer of the cytokine-induced NF-(kappa)B signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-(kappa)B signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.

TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation.

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.