Metabolic Roles of HIF1, c-Myc, and p53 in Glioma Cells.

The metabolic reprogramming that promotes tumorigenesis in glioblastoma is induced by dynamic alterations in the hypoxic tumor microenvironment, as well as in transcriptional and signaling networks, which result in changes in global genetic expression. The signaling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK stimulate cell metabolism, either directly or indirectly, by modulating the transcriptional factors p53, HIF1, and c-Myc. The overexpression of HIF1 and c-Myc, master regulators of cellular metabolism, is a key contributor to the synthesis of bioenergetic molecules that mediate glioma cell transformation, proliferation, survival, migration, and invasion by modifying the transcription levels of key gene groups involved in metabolism. Meanwhile, the tumor-suppressing protein p53, which negatively regulates HIF1 and c-Myc, is often lost in glioblastoma. Alterations in this triad of transcriptional factors induce a metabolic shift in glioma cells that allows them to adapt and survive changes such as mutations, hypoxia, acidosis, the presence of reactive oxygen species, and nutrient deprivation, by modulating the activity and expression of signaling molecules, enzymes, metabolites, transporters, and regulators involved in glycolysis and glutamine metabolism, the pentose phosphate cycle, the tricarboxylic acid cycle, and oxidative phosphorylation, as well as the synthesis and degradation of fatty acids and nucleic acids. This review summarizes our current knowledge on the role of HIF1, c-Myc, and p53 in the genic regulatory network for metabolism in glioma cells, as well as potential therapeutic inhibitors of these factors.

Role of Glycolytic and Glutamine Metabolism Reprogramming on the Proliferation, Invasion, and Apoptosis Resistance through Modulation of Signaling Pathways in Glioblastoma.

Glioma cells exhibit genetic and metabolic alterations that affect the deregulation of several cellular signal transduction pathways, including those related to glucose metabolism. Moreover, oncogenic signaling pathways induce the expression of metabolic genes, increasing the metabolic enzyme activities and thus the critical biosynthetic pathways to generate nucleotides, amino acids, and fatty acids, which provide energy and metabolic intermediates that are essential to accomplish the biosynthetic needs of glioma cells. In this review, we aim to explore how dysregulated metabolic enzymes and their metabolites from primary metabolism pathways in glioblastoma (GBM) such as glycolysis and glutaminolysis modulate anabolic and catabolic metabolic pathways as well as pro-oncogenic signaling and contribute to the formation, survival, growth, and malignancy of glioma cells. Also, we discuss promising therapeutic strategies by targeting the key players in metabolic regulation. Therefore, the knowledge of metabolic reprogramming is necessary to fully understand the biology of malignant gliomas to improve patient survival significantly.

Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

Biochemical and Kinetic Characterization of the Glucose-6-Phosphate Dehydrogenase from Helicobacter pylori Strain 29CaP.

Helicobacter pylori (H. pylori) has been proposed as the foremost risk factor for the development of gastric cancer. We found that H. pylori express the enzyme glucose-6-phosphate dehydrogenase (HpG6PD), which participates in glucose metabolism via the pentose phosphate pathway. Thus, we hypothesized that if the biochemical and physicochemical characteristics of HpG6PD contrast with the host G6PD (human G6PD, HsG6PD), HpG6PD becomes a potential target for novel drugs against H. pylori. In this work, we characterized the biochemical properties of the HpG6PD from the H.pylori strain 29CaP and expressed the active recombinant protein, to analyze its steady-state kinetics, thermostability, and biophysical aspects. In addition, we analyzed the HpG6PD in silico structural properties to compare them with those of the HsG6PD. The optimal pH for enzyme activity was 7.5, with a T1/2 of 46.6 °C, at an optimum stability temperature of 37 °C. The apparent Km values calculated for G6P and NADP+ were 75.0 and 12.8 µM, respectively. G6P does not protect HpG6PD from trypsin digestion, but NADP+ does protect the enzyme from trypsin and guanidine hydrochloride (Gdn-HCl). The biochemical characterization of HpG6PD contributes to knowledge regarding H. pylori metabolism and opens up the possibility of using this enzyme as a potential target for specific and efficient treatment against this pathogen; structural alignment indicates that the three-dimensional (3D) homodimer model of the G6PD protein from H. pylori is different from the 3D G6PD of Homo sapiens.

Crosstalk of the Wnt/ß-Catenin Signaling Pathway in the Induction of Apoptosis on Cancer Cells.

The Wnt/ß-catenin signaling pathway plays a major role in cell survival and proliferation, as well as in angiogenesis, migration, invasion, metastasis, and stem cell renewal in various cancer types. However, the modulation (either up- or downregulation) of this pathway can inhibit cell proliferation and apoptosis both through ß-catenin-dependent and independent mechanisms, and by crosstalk with other signaling pathways in a wide range of malignant tumors. Existing studies have reported conflicting results, indicating that the Wnt signaling can have both oncogenic and tumor-suppressing roles, depending on the cellular context. This review summarizes the available information on the role of the Wnt/ß-catenin pathway and its crosstalk with other signaling pathways in apoptosis induction in cancer cells and presents a modified dual-signal model for the function of ß-catenin. Understanding the proapoptotic mechanisms induced by the Wnt/ß-catenin pathway could open new therapeutic opportunities.

Anti-proliferative, pro-apoptotic and anti-invasive effect of the copper coordination compound Cas III-La through the induction of reactive oxygen species and regulation of Wnt/ß-catenin pathway in glioma.

Gliomas are the most aggressive neoplasms that affect the central nervous system, being glioblastoma multiforme (GBM) the most malignant. The resistance of GBM to therapies is attributed to its high rate of cell proliferation, angiogenesis, invasion, and resistance to apoptosis; thus, finding alternative therapeutic approaches is vital. In this work, the anti-proliferative, pro-apoptotic, and anti-invasive effect of the copper coordination compound Casiopeina III-La (Cas III-La) on human U373 MG cells was determined in vitro and in vivo. Our results indicate that Cas III-La exerts an anti-proliferative effect, promoting apoptotic cell death and inactivating the invasive process by generating reactive oxygen species (ROS), inactivating GSK3ß, activating JNK and ERK, and promoting the nuclear accumulation of ß-catenin. The inhibition of ROS generation by N-acetyl-l-cysteine not only recovered cell migration and viability, but also reduced ß-catenin accumulation and JNK and ERK activation. Additionally, Cas III-La significantly reduced tumor volume, cell proliferation and mitotic indices, and increased the apoptotic index in mice xenotransplanted with U373 glioma cells. Thus, Cas III-La is a promising agent to treat GBM.

Validation and Selection of New Reference Genes for RT-qPCR Analysis in Pediatric Glioma of Different Grades.

Gliomas are heterogeneous, solid, and intracranial tumors that originate from glial cells. Malignant cells from the tumor undergo metabolic alterations to obtain the energy required for proliferation and the invasion of the cerebral parenchyma. The alterations in the expression of the genes related to the metabolic pathways can be detected in biopsies of gliomas of different CNS WHO grades. In this study, we evaluated the expression of 16 candidate reference genes in the HMC3 microglia cell line. Then, statistical algorithms such as BestKeeper, the comparative ΔCT method, geNorm, NormFinder, and RefFinder were applied to obtain the genes most suitable to be considered as references for measuring the levels of expression in glioma samples. The results show that PKM and TPI1 are two novel genes suitable for genic expression studies on gliomas. Finally, we analyzed the expression of genes involved in metabolic pathways in clinical samples of brain gliomas of different CNS WHO grades. RT-qPCR analysis showed that in CNS WHO grade 3 and 4 gliomas, the expression levels of HK1, PFKM, GAPDH, G6PD, PGD1, IDH1, FASN, ACACA, and ELOVL2 were higher than those of CNS WHO grade 1 and 2 glioma biopsies. Hence, our results suggest that reference genes from metabolic pathways have different expression profiles depending on the stratification of gliomas and constitute a potential model for studying the development of this type of tumor and the search for molecular targets to treat gliomas.

Hypoxia as a modulator of cytochromes P450: Overexpression of the cytochromes CYP2S1 and CYP24A1 in human liver cancer cells in hypoxia.

Low levels of oxygen (hypoxia) have been reported in solid tumours. This hypoxic microenvironment modulates the expression of genes linked to a more aggressive disease. However, it is unclear if the expression of drug-metabolizing enzymes as cytochromes P450 (CYPs) is affected by hypoxia in cancer. We aimed to define which cytochromes are affected by hypoxia using a liver cancer model in vitro. For this purpose, we assessed whole-genome expression microarrays of HepG2 liver cancer cell line from free repository databases, looking for gene expression hypoxia-associated profiles and selected those cytochromes with significant differences. Then, we corroborated their mRNA expression and protein levels by RT-qPCR and western blot, respectively, as well as immunofluorescence. Based on microarray analysis, we found that the expression of CYP2S1 and CYP24A1 were up-regulated with at least twice fold change compared with normoxia. The levels of mRNA and protein of CYP2S1 and CYP24A1 were increased significantly in hypoxic conditions (P < .05), and this tendency was also observed by immunofluorescence assays. Our data show that the expression of cytochromes CYP2S1 and CYP24A1 are induced in hypoxia, being the first time that CYP24A1 expression is associated with tumour hypoxia; which might have consequences in cancer progression and drug resistance. SIGNIFICANCE OF THE STUDY: Hypoxia is among the most important factors for cellular adaptation to stress. Especially in cancer, a major public health issue, hypoxia plays a substantial role in angiogenesis, metastasis and resistance to therapy. Tumoral hypoxia has been described at least in the brain, breast, cervical, liver, renal, lung, pancreatic and renal cancer. However, the understanding of how hypoxia drives cancer progression is still a major challenge. One emerging question is the role of hypoxia over the expression of drug-metabolizing enzymes, with a significant impact on drug treatment. In this context, our paper focus on the effect of hypoxia on CYPs, which is an essential group of drug-metabolizing enzymes. We show that hypoxia induces the expression of two members of the CYPs family: CYP2S1 and CYP24A1. Importantly, CYP2S1 is a major metabolizer of carcinogenic substances being relevant that hypoxia could promote this function. Interestingly, CYP24A1 limits the action of the active form of vitamin D, which is an anti-proliferative factor in cancer. Our evidence shows for the first time that hypoxia can induce CYP24A1 expression, with a potential effect on cancer progression. Our contribution clarifies a particular effect of tumoral hypoxia and the implications will be useful in the understanding of the progression of cancer, the resistance to treatment and the development of alternative therapies.

Protein-conformational diseases in childhood: Naturally-occurring hIAPP amyloid-oligomers and early ß-cell damage in obesity and diabetes.

BACKGROUND AND AIMS: This is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when ß-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early ß-cell damage. MATERIALS AND METHODS: We performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis. RESULTS: We demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early ß-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk. CONCLUSIONS: RIAO level increases as the number of complications rises; RIAOs > 3.35 µg/ml is a predictor of changes in the current indicators of ß-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making.

Data mining of pediatric medulloblastoma microarray expression reveals a novel potential subdivision of the Group 4 molecular subgroup.

Medulloblastoma is the most common type of solid brain tumor in children. This type of embryonic tumor is highly heterogeneous and has been classified into 4 molecular subgroups based on their gene expression profiles: WNT, SHH, Group 3 (G3) and Group 4 (G4). WNT and SHH tumors exhibit the specific dysregulation of genes and pathways, whereas G3 and G4 tumors, two of the more frequent subtypes, are the least characterized. Thus, novel markers to aid in the diagnosis, prognosis and management of medulloblastoma are required. In the present study, microarray gene expression data was downloaded from the Gene Expression Omnibus database, including data from the 4 subgroups of medulloblastoma and healthy cerebellum tissue (CT). The data was utilized in an in silico analysis to characterize each subgroup at a transcriptomic level. Using Partek Genomics Suite software, the data were visualized via hierarchical clustering and principal component analysis. The differentially expressed genes were uploaded to the MetaCore portal to perform enrichment analysis using CT gene expression as baseline, with fold change thresholds of <-5 and >5 for differential expression. The data mining analysis of microarray gene expression data enabled the identification of a range of dysregulated molecules associated with each subgroup of medulloblastoma. G4 is the most heterogeneous subgroup, as no definitive pathway defines its pathogenesis; analysis of the gene expression profiles were associated with the G4α and G4ß subcategories. TOX high mobility group box family member 3, synuclein α interacting protein and, potassium voltage-gated channel interacting protein 4 were identified as three novel potential markers for distinguishing the α and ß subcategories of G4. These genes may be associated with medulloblastoma pathogenesis, and thus may provide a basis for researching novel targeted treatment strategies for G4 medulloblastoma.

Diabetes Drug Discovery: hIAPP1-37 Polymorphic Amyloid Structures as Novel Therapeutic Targets.

Human islet amyloid peptide (hIAPP1-37) aggregation is an early step in Diabetes Mellitus. We aimed to evaluate a family of pharmaco-chaperones to act as modulators that provide dynamic interventions and the multi-target capacity (native state, cytotoxic oligomers, protofilaments and fibrils of hIAPP1-37) required to meet the treatment challenges of diabetes. We used a cross-functional approach that combines in silico and in vitro biochemical and biophysical methods to study the hIAPP1-37 aggregation-oligomerization process as to reveal novel potential anti-diabetic drugs. The family of pharmaco-chaperones are modulators of the oligomerization and fibre formation of hIAPP1-37. When they interact with the amino acid in the amyloid-like steric zipper zone, they inhibit and/or delay the aggregation-oligomerization pathway by binding and stabilizing several amyloid structures of hIAPP1-37. Moreover, they can protect cerebellar granule cells (CGC) from the cytotoxicity produced by the hIAPP1-37 oligomers. The modulation of proteostasis by the family of pharmaco-chaperones A-F is a promising potential approach to limit the onset and progression of diabetes and its comorbidities.