Ana M. Metelo

Study of the antidiabetic capacity of the VO(dmpp)2 complex.

In this work we report biochemical ex vivo studies with a vanadium compound containing a pyridinone ligand, the bis(1,2-dimethyl-3-hydroxy-4-pyridinonate)oxovanadium (IV), V(IV)O(dmpp)(2), which has shown to have promising antidiabetic activity. The experiments were carried out on primary adipocytes of 6-8 week old Wistar rats. Insulin-stimulated glucose uptake studies were performed using a radioactive assay by measuring the (U)-(14)C-glucose taken up by the isolated adipocytes for 30 min. Adipocytes were incubated with and without insulin and in the presence and absence of different concentrations of V(IV)O(dmpp)(2) (100-500 microM) for 45 min. We observed that in a nontoxic concentration, as demonstrated by the Alamar Blue test, V(IV)O(dmpp)(2) significantly increases glucose uptake, in the absence of insulin, by 5-folds higher than basal, and it has a significant inhibitory effect of 78% on free fatty acid release in isolated adipocytes from normal rats. We also demonstrated that it promotes the phosphorylation of Akt1, a key protein in the insulin signaling cascade. These results were compared with those obtained with another vanadium compound reported in the literature, with a similar structure, the bis(maltolato)oxovanadium (IV) (BMOV), which is now in clinical trials. Our ex vivo results clearly indicate that V(IV)O(dmpp)(2) is a good candidate to be a promising drug for the treatment of diabetes and other metabolic disorders.

VO(dmpp)2 normalizes pre-diabetic parameters as assessed by in vivo magnetic resonance imaging and spectroscopy.

Type 2 diabetes mellitus has been associated with obesity, metabolic syndrome, cardiovascular diseases and cancer. Attempts have been made for early diagnosis and finding effective drugs to prevent severe consequences and ameliorate the symptoms of this disorder. In this work, the pharmacological properties of VO(dmpp)(2), [bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxovanadium(IV)], were in vivo evaluated. For 4 weeks fatty Zucker rats were subjected to a daily dose of VO(dmpp)(2) (44 μmol/kg) and their metabolic profile was followed by assessing different biological parameters at established time points: body weight, subcutaneous fat width and hepatic triglyceride content determined by magnetic resonance imaging and spectroscopy, respectively. A glucose tolerance test was performed at the end of the experiment. After treatment, treated obese rats presented a weight significantly lower than the non-treated obese animals (359.0±11.1 vs. 433.5±6.2g, P<0.05), a thinner subcutaneous fat width, and a statistically significant decrease in hepatic triglyceride content (5.41±0.59 vs. 21.03±1.40%, P<0.0005). Additionally, the glucose intolerant profile of fatty Zucker rats was completely reversed in treated animals (102.3±2.1 vs. 172.4±1.3 mg/100 mL; P<0.0005). These results reinforce the therapeutic action of VO(dmpp)(2) which shows particular effects on lipid metabolism.

Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma

ABSTRACT Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC. Summary: Zebrafish with an inactivating mutation in the vhl gene can be used as a model of early stage clear cell renal cell carcinoma, with applications for genetic studies and drug screens.

Magnetic Resonance Imaging of Gliomas

Brain cancer is a life threatening neurological disorder in which malignant cells, grow, proliferate and invade the original cerebral structures of the host, hampering seriously adequate brain function. Malignant cells generate eventually a dedifferentiated tumoral mass that interferes with vital brain functions as sensory and motor activations, memory and perception and neuroendocrine regulation, among others. The fully developed tumoral mass consumes a significant part of cerebral volume resulting in cerebral compression and serious neurological impairments, such as vision or hearing disturbances and eventually lethal cerebrovascular complications. Most brain tumors remain asymptomatic during early development, revealing their symptoms and lethal nature only at later stages. Therapy is facilitated many times by an early finding, a circumstance making the neuroimaging approaches particularly useful in the detection and handling of these lesions. In the last decades, Magnetic Resonance Imaging (MRI) approaches have evolved into the most powerful and versatile imaging tool for brain tumor diagnosis, prognosis, therapy evaluation, monitoring of disease progression and planning of neurosurgical strategies. MRI methods enable the non invasive assessment of glioma morphology and functionality providing a point of likeness into histopathological grading of the tumor and helping in this way a more successful patient management. This impressive evolution is based not only for the high resolution and quality of the anatomical images obtained, but on the additional possibilities to achieve quantitative functional information on tumoral physiopathology and its repercussions in the sensorial, motor and integrative functions through the brain. The use of conventional paramagnetic or superparamagnetic contrast media allows for the identification of areas with blood-brain barrier (BBB) disruption and the recent molecular imaging approaches enable researchers to visualize molecular events associated to tumor proliferation and invasion, bringing the potentials of diagnostic imaging to the cellular and molecular aspects of tumor biology. Moreover, functional MRI approaches as performed in the clinic are endowed with the potential to detect and characterize the earliest neoangiogenic, metabolic and hemodynamic alterations induced by the neoplasm. Several advanced magnetic resonance (MR) methodologies have been proposed in the last years to assess the functional competence in healthy and pathologic brain tissue. Diffusion and perfusion MRI are probably the two main approaches that have reached a relevant clinical role

Hemangioblastomas of the Central Nervous System

Abstract Hemangioblastomas are vascular tumors of the central nervous system (CNS) that develop in patients with Von Hippel–Lindau (VHL) disease or sporadically. Loss-of-VHL tumor suppressor gene is a hallmark of both types of hemangioblastomas. The tumors comprise of stromal cells, proliferating vascular endothelial cells and may contain hematopoietic components. The cell of origin and the lineage relations between the different tumor components are currently unclear. Hemangioblastomas are treated with surgical excision or radiation therapy. Currently, there is no targeted therapy available for treatment of hemangioblastomas. Future studies, based on next-generation sequencing and animal models, will discover and validate oncogenic mutations that can be targeted for medical therapy of these tumors.

Abstract B1: Hypoxia inducible factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation

Hypoxia is a hallmark of tumor microenvironment. We showed before that exposure of cells to hypoxia reprograms cell metabolism and directs an IDH1-mediated reductive carboxylation (RC) of glutamine-derived alpha-ketoglutarate (a-KG) for lipogenesis. In addition Von Hippel-Lindau (VHL)-deficient renal cell carcinoma cells use glutamine to generate citrate and lipids through RC of a-KG. To gain insights into the role of HIF and the molecular mechanisms underlying RC we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduces intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate, or knocking-down PDK-1 and ACLY enzymes restored intracellular citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action, to maintain lipogenesis. Expression of HIF renders hypoxic and VHL-deficient cells addicted to glutamine in vitro. VHL-deficient cells displayed significantly less growth than their isogenic counterparts in media containing low glutamine concentration or in the presence of a glutaminase inhibitor. Systemic administration of a glutaminase inhibitor (BPTES) suppressed the growth of VHL-deficient human RCC cell lines as xenografts in nude mice. Lastly, we investigated whether RC occurs in vivo. We metabolically labeled mice bearing VHL-deficient tumors by infusing them with 13C-1-Glutamine for up to 6 hours and we detected the early formation of labeled citrate in the tumors; thus we showed for the first time RC in vivo. Our data provide mechanistic insights into the signaling events that mediate hypoxia-induced RC, strongly suggest that RC is an in vivo phenomenon in growing tumors and highlight potential novel therapeutic approaches for treatment of hypoxic and VHL-deficient tumors based on their metabolic signature. Citation Format: Paulo Gameiro, Ana Metelo, Rojo Perez-Carro, Arreola Alexandra, Zongwei Wang, Kimryn W. Rathmell, Aria Olumi, Pilar Lopez-Larrubia, Gregory Stephanopoulos, Othon Iliopoulos. Hypoxia inducible factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B1.

Abstract 4253: In vivo activity of small molecule HIF2α inhibitors

Hypoxia-inducible factor (HIF) is a central regulator of the cellular response to hypoxia. Hypoxia promotes HIF activity by increasing its stability, transcriptional activity and translation. HIF target genes involved in several cellular processes including proliferation, angiogenesis, metabolism and promotion of the “stem-cell” phenotype. In normoxic conditions, HIF-α is recognized for degradation by the von Hippel-Lindau tumor suppressor protein (VHL). Loss-of-VHL function leads to constitutive expression of HIF regulatory subunits and consequent over-expression of HIF-target genes. We and others have shown that HIF2a is necessary and sufficient for the development of VHL-related renal cell carcinoma. We have developed small molecules that repress HIF-2α translation, in vitro, by enhancing the binding of Iron-Regulatory Protein 1 (IRP1) to the Iron-Responsive Element (IRE) within the 5′-UTR of HIF-2α mRNA (Zimmer M. et al. Molecular Cell 2008;32: 828-848). We showed that these small molecules down-regulate HIF2a expression in vitro, in all renal cell carcinoma cell lines. To validate these small molecules in vivo, we tested their efficacy on wild-type zebrafish embryos. Treatment of 3 days post fertilization zebrafish embryos with 100microM of the hypoxic mimetic DMOG or hypoxia dramatically upregulated the expression of HIF-target genes, such as Vascular Endothelial Growth Factor (Vegf), erythropoietin (Epo) and prolyl-hydroxylase 3 (Phd3). Pre-treatment of embryos with the small molecule HIF2a inhibitor 76 potently decreased the DMOG-induced upregulation of HIF target genes. The IC50 values of these small molecules were significantly less in vivo than in vitro. We are currently testing the efficacy of HIF2a inhibitors in rescuing the VHL zebrafish phenotype and prolonging survival of VHL mutant zebrafish embryos. In addition, data on the efficacy of these inhibitors on the growth of human renal cell carcinoma cells as tumors in the nude mouse xenograft assay will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4253. doi:1538-7445.AM2012-4253