[18F]F-DED PET imaging of reactive astrogliosis in neurodegenerative diseases: preclinical proof of concept and first-in-human data.

OBJECTIVES: Reactive gliosis is a common pathological hallmark of CNS pathology resulting from neurodegeneration and neuroinflammation. In this study we investigate the capability of a novel monoamine oxidase B (MAO-B) PET ligand to monitor reactive astrogliosis in a transgenic mouse model of Alzheimer`s disease (AD). Furthermore, we performed a pilot study in patients with a range of neurodegenerative and neuroinflammatory conditions. METHODS: A cross-sectional cohort of 24 transgenic (PS2APP) and 25 wild-type mice (age range: 4.3-21.0 months) underwent 60 min dynamic [18F]fluorodeprenyl-D2 ([18F]F-DED), static 18 kDa translocator protein (TSPO, [18F]GE-180) and ß-amyloid ([18F]florbetaben) PET imaging. Quantification was performed via image derived input function (IDIF, cardiac input), simplified non-invasive reference tissue modelling (SRTM2, DVR) and late-phase standardized uptake value ratios (SUVr). Immunohistochemical (IHC) analyses of glial fibrillary acidic protein (GFAP) and MAO-B were performed to validate PET imaging by gold standard assessments. Patients belonging to the Alzheimer's disease continuum (AD, n = 2), Parkinson's disease (PD, n = 2), multiple system atrophy (MSA, n = 2), autoimmune encephalitis (n = 1), oligodendroglioma (n = 1) and one healthy control underwent 60 min dynamic [18F]F-DED PET and the data were analyzed using equivalent quantification strategies. RESULTS: We selected the cerebellum as a pseudo-reference region based on the immunohistochemical comparison of age-matched PS2APP and WT mice. Subsequent PET imaging revealed that PS2APP mice showed elevated hippocampal and thalamic [18F]F-DED DVR when compared to age-matched WT mice at 5 months (thalamus: + 4.3%; p = 0.048), 13 months (hippocampus: + 7.6%, p = 0.022) and 19 months (hippocampus: + 12.3%, p < 0.0001; thalamus: + 15.2%, p < 0.0001). Specific [18F]F-DED DVR increases of PS2APP mice occurred earlier when compared to signal alterations in TSPO and ß-amyloid PET and [18F]F-DED DVR correlated with quantitative immunohistochemistry (hippocampus: R = 0.720, p < 0.001; thalamus: R = 0.727, p = 0.002). Preliminary experience in patients showed [18F]F-DED VT and SUVr patterns, matching the expected topology of reactive astrogliosis in neurodegenerative (MSA) and neuroinflammatory conditions, whereas the patient with oligodendroglioma and the healthy control indicated [18F]F-DED binding following the known physiological MAO-B expression in brain. CONCLUSIONS: [18F]F-DED PET imaging is a promising approach to assess reactive astrogliosis in AD mouse models and patients with neurological diseases.

CDK6 Degradation Is Counteracted by p16INK4A and p18INK4C in AML.

Cyclin-dependent kinase 6 (CDK6) represents a novel therapeutic target for the treatment of certain subtypes of acute myeloid leukaemia (AML). CDK4/6 kinase inhibitors have been widely studied in many cancer types and their effects may be limited by primary and secondary resistance mechanisms. CDK4/6 degraders, which eliminate kinase-dependent and kinase-independent effects, have been suggested as an alternative therapeutic option. We show that the efficacy of the CDK6-specific protein degrader BSJ-03-123 varies among AML subtypes and depends on the low expression of the INK4 proteins p16INK4A and p18INK4C. INK4 protein levels are significantly elevated in KMT2A-MLLT3+ cells compared to RUNX1-RUNX1T1+ cells, contributing to the different CDK6 degradation efficacy. We demonstrate that CDK6 complexes containing p16INK4A or p18INK4C are protected from BSJ-mediated degradation and that INK4 levels define the proliferative response to CDK6 degradation. These findings define INK4 proteins as predictive markers for CDK6 degradation-targeted therapies in AML.

Radiotracer stereochemistry affects substrate affinity and kinetics for improved imaging of system xC- in tumors.

Amino acid utilization is perturbed in cancer cells, which rewire their metabolism to support cell survival and proliferation. This metabolic reprogramming can be exploited for diagnostic purposes through positron emission tomography imaging of fluorine-18 labeled amino acids. Despite its promise, little is known regarding transporter-recognition of non-natural amino acid stereoisomers or their utility for cancer imaging. We report here the synthesis and in vivo characterization of a radiolabeled amino acid (R)-4-(3-18F-fluoropropyl)-ʟ-glutamate ([18F]FRPG) and compared its tumor imaging properties to the 4S-isomer, [18F]FSPG. Methods: [18F]FRPG and [18F]FSPG uptake was assessed in H460 lung cancer cells, with efflux measured 30 min after removal of exogenous activity. Specificity of [18F]FRPG for system xC- was further examined following transporter inhibition and blocking studies with system xC- substrates. [18F]FRPG and [18F]FSPG pharmacokinetics was next quantified in mice bearing subcutaneous A549, H460, VCAP and PC3 tumors, with mice bearing A549 tumors imaged by PET/CT. To better-understand differential tumor retention, radiometabolite analysis was performed on tissue and blood samples after imaging. Next, [18F]FRPG and [18F]FSPG retention in lipopolysaccharide-treated lungs were compared to an orthotopic H460 lung cancer model. Finally, the sensitivity of [18F]FRPG to manipulation of the redox environment was examined in cell and in vivo models. Results: [18F]FRPG was specifically transported across the plasma membrane by the cystine/glutamate antiporter system xC- and retained at high levels in multiple tumor models. Conversely, [18F]FRPG was rapidly extracted from the blood and cleared from tissues with low system xC- expression. Due to its favorable imaging properties, tumor-to-blood ratios ≥10 were achieved with [18F]FRPG, which were either equal to or greater than [18F]FSPG. In addition, [18F]FRPG retention in orthotopic lung tumors with high system xC- expression was 2.5-fold higher than inflamed tissue, allowing for clear tumor visualization. In vivo, [18F]FRPG and [18F]FSPG were metabolized to a single species, with [18F]FRPG showing a higher percentage of parent radiotracer in tumors compared to [18F]FSPG. [18F]FRPG was sensitive to redox manipulations and tumor retention was reduced following treatment with liposomal doxorubicin in mice bearing ovarian tumors. Conclusions: Given the fast clearance and low background retention of [18F]FRPG throughout the body, this radiotracer holds promise for the imaging of system xC- activity and treatment response monitoring in tumors of the thorax, abdomen, and head and neck. [18F]FRPG PET imaging provides a sensitive noninvasive measure of system xC- and excellent properties for cancer imaging.

Rosuvastatin exposure in female Wistar rats alters uterine contractility and do not show evident (anti)estrogenic effects.

Statins are 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitor drugs that lead to serum-cholesterol-lowering effects. Rosuvastatin, a third-generation statin, has shown better results in reducing cholesterol concentrations when compared to other widely prescribed statins. Recent studies by our group reported that rosuvastatin impairs reproductive function in rats possibly by disrupting the reproductive-endocrine axis. In this study, we evaluated whether rosuvastatin presents estrogenic or antiestrogenic effects, by an in vivo uterotrophic assay in rats, and investigated the direct effect of this drug upon rat uterine tissue contractility both in non-gravid and gravid periods. Rosuvastatin exposure in vivo at doses of 0 (control), 3, and 10 mg/kg/d was not associated with estrogenic or antiestrogenic effects on uterine tissue. However, in vivo (doses of 0, 3, and 10 mg/kg/d) and ex vivo (concentrations of 0, 1, 10, and 100 µg/mL) exposures to this drug were related to alterations in uterine basal contraction pattern. Furthermore, in vivo and ex vivo rosuvastatin exposures potentially modulate the action of uterine contraction inducers carbachol, norepinephrine, and prostaglandin E2. Thus, rosuvastatin can affect uterine physiology not necessarily by an endocrine mechanism related to the estrogen signaling, but possibly by its pleiotropic effects, with indirect tissue and cellular interactions, since in vivo and ex vivo exposures of uterine fragments to rosuvastatin presented different responses in uterine contractile parameters, which require further studies upon the precise mechanism of action of this drug in female reproductive function.

APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.

Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide1. The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2. A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3, but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.

Synthetic Fibrin-Derived Bß15-42 Peptide Delays Thrombus Resolution in a Mouse Model.

[Figure: see text].

Initial evaluation of (4S)-4-(3-[18F]fluoropropyl)-L-glutamate (FSPG) PET/CT imaging in patients with head and neck cancer, colorectal cancer, or non-Hodgkin lymphoma.

PURPOSE: (4S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) measures system xC- transporter activity and shows promise for oncologic imaging. We present data on tumor uptake of this radiopharmaceutical in human subjects with head and neck cancer (HNC), colorectal cancer (CRC), and non-Hodgkin lymphoma (NHL). METHODS: A total of 15 subjects with HNC (n = 5), CRC (n = 5), or NHL (n = 5) were recruited (mean age 66.2 years, range 44-87 years). 301.4 ± 28.1 MBq (8.1 ± 0.8 mCi) of [18F]FSPG was given intravenously to each subject, and 3 PET/CT scans were obtained 0-2 h post-injection. All subjects also had a positive [18F]FDG PET/CT scan within 1 month prior to the [18F]FSPG PET scan. Semi-quantitative and visual comparisons of the [18F]FSPG and [18F]FDG scans were performed. RESULTS: [18F]FSPG showed strong uptake in all but one HNC subject. The lack of surrounding brain uptake facilitated tumor delineation in the HNC patients. [18F]FSPG also showed tumor uptake in all CRC subjects, but variable uptake in the NHL subjects. While the absolute [18F]FDG SUV values were comparable or higher than [18F]FSPG, the tumor-to-background SUV ratios were greater with [18F]FSPG than [18F]FDG. CONCLUSIONS: [18F]FSPG PET/CT showed promising results across 15 subjects with 3 different cancer types. Concordant visualization was mostly observed between [18F]FSPG and [18F]FDG PET/CT images, with some inter- and intra-individual uptake variability potentially reflecting differences in tumor biology. The tumor-to-background ratios were greater with [18F]FSPG than [18F]FDG in the cancer types evaluated. Future studies based on larger numbers of subjects and those with a wider array of primary and recurrent or metastatic tumors are planned to further evaluate the utility of this novel tracer.

Clinical Evaluation of (4S)-4-(3-[18F]Fluoropropyl)-L-glutamate (18F-FSPG) for PET/CT Imaging in Patients with Newly Diagnosed and Recurrent Prostate Cancer.

PURPOSE: (4S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid (18F-FSPG) is a radiopharmaceutical for PET imaging of system xC - activity, which can be upregulated in prostate cancer. We present data on the first evaluation of patients with newly diagnosed or recurrent prostate cancer with this radiopharmaceutical. EXPERIMENTAL DESIGN: Ten patients with primary and 10 patients with recurrent prostate cancer were enrolled in this prospective multicenter study. After injection of 300 MBq of 18F-FSPG, three whole-body PET/CT scans were obtained. Visual analysis was compared with step-section histopathology when available as well as other imaging studies and clinical outcomes. Metabolic parameters were measured semiquantitatively. Expression levels of xCT and CD44 were evaluated by IHC for patients with available tissue samples. RESULTS: 18F-FSPG PET showed high tumor-to-background ratios with a relatively high tumor detection rate on a per-patient (89%) and per-lobe (87%) basis. The sensitivity was slightly higher with imaging at 105 minutes in comparison with 60 minutes. The maximum standardized uptake values (SUVmax) for cancer was significantly higher than both normal (P < 0.005) and benign pathology (P = 0.011), while there was no significant difference between normal and benign pathology (P = 0.120). In the setting of recurrence, agreement with standard imaging was demonstrated in 7 of 9 patients (78%) and 13 of 18 lesions (72%), and revealed true local recurrence in a discordant case. 18F-FSPG accumulation showed moderate correlation with CD44 expression. CONCLUSIONS: 18F-FSPG is a promising tumor imaging agent for PET that seems to have favorable biodistribution and high cancer detection rate in patients with prostate cancer. Further studies are warranted to determine the diagnostic value for both initial staging and recurrence, and how it compares with other investigational radiotracers and conventional imaging modalities.

The Characterization of 18F-hGTS13 for Molecular Imaging of xC- Transporter Activity with PET.

The aim of this study was development of an improved PET radiotracer for measuring xC- activity with increased tumor uptake and reduced uptake in inflammatory cells compared with (S)-4-(3-18F-fluoropropyl)-l-glutamate (18F-FSPG). Methods: A racemic glutamate derivative, 18F-hGTS13, was evaluated in cell culture and animal tumor models. 18F-hGTS13 was separated into C5 epimers, and the corresponding 18F-hGTS13-isomer1 and 18F-hGTS13-isomer2 were evaluated in H460 tumor-bearing rats. Preliminary studies investigated the cellular uptake of 18F-hGTS13-isomer2 in multiple immune cell populations and states. Results:18F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor-associated radioactivity was significantly higher for 18F-hGTS13 (7.5 ± 0.9 percentage injected dose [%ID]/g, n = 3) than for 18F-FSPG (4.6 ± 0.7 %ID/g, n = 3, P = 0.01). 18F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3 ± 1.1 %ID/g, n = 3) and significantly reduced uptake in multiple immune cell populations relative to 18F-FSPG. 18F-hGTS13-isomer2 exhibited increased liver uptake relative to 18F-FSPG (4.6 ± 0.8 vs. 0.7 ± 0.01 %ID/g), limiting its application in hepatocellular carcinoma. Conclusion:18F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of xC- activity that may provide information on tumor oxidation states. 18F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax because of the low background signal in healthy tissue.

A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif.

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

Mapping the Human Kinome in Response to DNA Damage.

We provide a catalog for the effects of the human kinome on cell survival in response to DNA-damaging agents, covering all major DNA repair pathways. By treating 313 kinase-deficient cell lines with ten diverse DNA-damaging agents, including seven commonly used chemotherapeutics, we identified examples of vulnerability and resistance that are kinase specific. To investigate synthetic lethal interactions, we tested the response to carmustine for 25 cell lines by establishing a phenotypic fluorescence-activated cell sorting (FACS) assay designed to validate gene-drug interactions. We show apoptosis, cell cycle changes, and DNA damage and proliferation after alkylation- or crosslink-induced damage. In addition, we reconstitute the cellular sensitivity of DYRK4, EPHB6, MARK3, and PNCK as a proof of principle for our study. Furthermore, using global phosphoproteomics on cells lacking MARK3, we provide evidence for its role in the DNA damage response. Our data suggest that cancers with inactivating mutations in kinases, including MARK3, are particularly vulnerable to alkylating chemotherapeutic agents.

CDK6 Antagonizes p53-Induced Responses during Tumorigenesis.

Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate TP53 (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists Prmt5, Ppm1d, and Mdm4 The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in TP53 significantly more often than expected.Significance: CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. Cancer Discov; 8(7); 884-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Molecular dissection of effector mechanisms of RAS-mediated resistance to anti-EGFR antibody therapy.

Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), cetuximab and panitumumab, are a mainstay of metastatic colorectal cancer (mCRC) treatment. However, a significant number of patients suffer from primary or acquired resistance. RAS mutations are negative predictors of clinical efficacy of anti-EGFR antibodies in patients with mCRC. Oncogenic RAS activates the MAPK and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways in RAS-mutant CRC is less defined. A better mechanistic understanding of RAS-mediated resistance may guide development of rational intervention strategies. To this end we developed cancer models for functional dissection of resistance to anti-EGFR therapy in vitro and in vivo. To selectively activate MAPK- or AKT-signaling we expressed conditionally activatable RAF-1 and AKT in cancer cells. We found that either pathway independently protected sensitive cancer models against anti-EGFR antibody treatment in vitro and in vivo. RAF-1- and AKT-mediated resistance was associated with increased expression of anti-apoptotic BCL-2 proteins. Biomarkers of MAPK and PI3K/AKT pathway activation correlated with inferior outcome in a cohort of mCRC patients receiving cetuximab-based therapy. Dual pharmacologic inhibition of PI3K and MEK successfully sensitized primary resistant CRC models to anti-EGFR therapy. In conclusion, combined targeting of MAPK and PI3K/AKT signaling, but not single pathways, may be required to enhance the efficacy of anti-EGFR antibody therapy in patients with RAS-mutated CRC as well as in RAS wild type tumors with clinical resistance.

Pilot Preclinical and Clinical Evaluation of (4S)-4-(3-[18F]Fluoropropyl)-L-Glutamate (18F-FSPG) for PET/CT Imaging of Intracranial Malignancies.

PURPOSE: (S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid (18F-FSPG) is a novel radiopharmaceutical for Positron Emission Tomography (PET) imaging. It is a glutamate analogue that can be used to measure xC- transporter activity. This study was performed to assess the feasibility of 18F-FSPG for imaging orthotopic brain tumors in small animals and the translation of this approach in human subjects with intracranial malignancies. EXPERIMENTAL DESIGN: For the small animal study, GS9L glioblastoma cells were implanted into brains of Fischer rats and studied with 18F-FSPG, the 18F-labeled glucose derivative 18F-FDG and with the 18F-labeled amino acid derivative 18F-FET. For the human study, five subjects with either primary or metastatic brain cancer were recruited (mean age 50.4 years). After injection of 300 MBq of 18F-FSPG, 3 whole-body PET/Computed Tomography (CT) scans were obtained and safety parameters were measured. The three subjects with brain metastases also had an 18F-FDG PET/CT scan. Quantitative and qualitative comparison of the scans was performed to assess kinetics, biodistribution, and relative efficacy of the tracers. RESULTS: In the small animals, the orthotopic brain tumors were visualized well with 18F-FSPG. The high tumor uptake of 18F-FSPG in the GS9L model and the absence of background signal led to good tumor visualization with high contrast (tumor/brain ratio: 32.7). 18F-FDG and 18F-FET showed T/B ratios of 1.7 and 2.8, respectively. In the human pilot study, 18F-FSPG was well tolerated and there was similar distribution in all patients. All malignant lesions were positive with 18F-FSPG except for one low-grade primary brain tumor. In the 18F-FSPG-PET-positive tumors a similar T/B ratio was observed as in the animal model. CONCLUSIONS: 18F-FSPG is a novel PET radiopharmaceutical that demonstrates good uptake in both small animal and human studies of intracranial malignancies. Future studies on larger numbers of subjects and a wider array of brain tumors are planned. TRIAL REGISTRATION: ClinicalTrials.gov NCT01186601.

Anxiogenic effects of Tabernaemontana solanifolia A. DC. (Apocynaceae) extracts in rats tested in the elevated plus-maze / Determinación mediante la prueba del laberinto elevado en cruz del efecto ansiogénico en ratas de extractos de Tabernaemontana solanifolia A. DC. (Apocynaceae)

Introduction: Tabernaemontana solanifolia A. DC. (Apocynaceae) grows in the Cerrado (savanna), Caatinga (shrublands), and Atlantic Forest of Brazil. Objective: to investigate the potential anxiolytic activity of the crude extracts and ß-amyrin acetate obtained from the leaves of Tabernaemontana solanifolia in the elevated plus-maze (EPM) test for anxiety in rats. Methods: the crude extracts (aqueous, ethanol, and hexane), as well as ß-amyrin acetate, obtained from Tabernaemontana solanifolia leaves were evaluated for possible anxiolytic effects in rats tested in the elevated plus-maze. Acute toxicity in rats was determined by OECD 423 guidelines. The leaves of T. solanifolia specimens collected in Brasília, Brazil, were air dried at 40 °C and macerated with hexane or ethanol. After filtration, the solvents were removed under reduced pressure, yielding the crude hexane and ethanolic extracts (5.96 and 18.62 % yield, respectively). The crude aqueous extract was obtained by infusion, followed by lyophilization (13.5 % yield). Thirty minutes before the elevated plus-maze experiments, the animals were treated with the crude ethanol extract (1 000 mg/kg of body weight [bw], p.o.), the crude hexane extract (1 000 mg/kg bw, p.o.), the crude extract (1 000 mg/kg bw, p.o.), or ß-amyrin acetate (21 mg/kg bw, p.o.). Results: the extracts cauded no mortality up to 2 000 mg/kg, so half of DL50 doses were selected for the present study. The crude extracts (hexane, ethanolic and aqueous) (1.0 g/kg) as well as the substance ß-amyrin acetate (21 mg/kg) were administered once, increasing the number of entries into and the time spent in the closed arms of the elevated plus-maze. Conclusion: the oral administration of the extracts and ß-amyrin acetate obtained from Tabernaemontana solanifolia had an anxiogenic effect in rats. Further studies are needed in order to identify and characterize the anxiogenic properties of Tabernaemontana solanifolia and to understand the anxiogenic mechanisms of the extracts.

Introducción: Tabernaemontana solanifolia A. DC. (Apocynaceae) crece en el Cerrado (sabana), Caatinga (matorrales) y la Mata Atlántica de Brasil. Objetivo: investigar la posible actividad ansiolítica de los extractos crudos y el acetato de ß-amirina obtenidos de las hojas de Tabernaemontana solanifolia en la prueba del laberinto elevado en cruz (EPM) para la ansiedad en ratas. Métodos: los extractos crudos (etanol acuoso y hexano), así como el acetato de ß-amirina, se evaluaron para posibles efectos ansiolíticos en ratas mediante la prueba del laberinto elevado en cruz. La toxicidad aguda en ratas se determinó por las directrices OCDE 423. Las hojas de Tabernaemontana solanifolia se secaron al aire a 40 °C y fueron maceradas con hexano y etanol. Después de la filtración, los disolventes se eliminaron bajo presión reducida, produciendo los extractos crudos hexano y etanólico (5,96 y 18,62 % de rendimiento, respectivamente). El extracto acuoso bruto se obtuvo por infusión, seguida por liofilización (13,5 % de rendimiento). A 30 min antes de los experimentos de laberinto elevado en cruz, los animales se trataron con el extracto de etanol crudo (1 000 mg/kg de peso corporal [bw], p.o.), el extracto de hexano crudo (1 000 mg/kg de peso corporal, por vía oral), el extracto acuoso (1 000 mg/kg de peso corporal, por vía oral) o ß-amirina acetato (21 mg/kg de peso corporal, por vía oral). Resultados: los extractos no produjeron mortalidad hasta 2 000 mg/kg, por lo que para el presente estudio se seleccionó la mitad de la dosis DL50. Los extractos crudos (hexano, etanol y acuoso) (1 000 mg/kg), así como la sustancia acetato de ß-amirina (21 mg/kg) se administraron una vez, aumentando el número de entradas y el tiempo pasado en los brazos cerrados de la prueba del laberinto elevado en cruz. Conclusión: la administración oral de los extractos y el acetato de ß-amirina obtenidos de Tabernaemontana solanifolia tuvo un efecto ansiogénico en ratas. Se necesitan estudios adicionales para identificar y caracterizar las propiedades ansiogénicas de Tabernaemontana solanifolia y para entender los mecanismos ansiogénicos de los extractos.

Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.

The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi.

Preclinical evaluation of a novel c-Met inhibitor in a gastric cancer xenograft model using small animal PET.

PURPOSE: Here, we describe the efficacy of the novel small molecule c-Met inhibitor BAY 853474 in reducing tumor growth in the Hs746T gastric cancer xenograft model and tested the suitability of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) versus 3'-deoxy-3'-18F-fluorothymidine ([(18)F]FLT) for response monitoring in a gastric cancer xenograft mouse model using small animal PET. PROCEDURES: The c-Met inhibitor or vehicle control was administered orally at various doses in tumor-bearing mice. Glucose uptake and proliferation was measured using PET before, 48 and 96 h after the first treatment. The PET data were compared to data from tumor growth curves, autoradiography, Glut-1 and Ki-67 staining of tumor sections, and biochemical analysis of tissue probes, i.e., c-Met and ERK phosphorylation and cyclin D1 levels. RESULTS: BAY 853474 significantly reduces tumor growth. [(18)F]FDG uptake in Hs746T tumors was significantly reduced in the groups receiving the drug, compared with the control group. The [(18)F]FLT uptake in the tumor tissue was completely absent 96 h after treatment. Autoradiographic, immunohistochemical, and biochemical analyses confirmed the PET findings. Treatment with the c-Met inhibitor did not affect body weight or glucose levels, and no adverse effects were observed in the animals. CONCLUSION: These preclinical findings suggest that clinical PET imaging is a useful tool for early response monitoring in clinical studies.

(4S)-4-(3-18F-fluoropropyl)-L-glutamate for imaging of xC transporter activity in hepatocellular carcinoma using PET: preclinical and exploratory clinical studies.

UNLABELLED: (4S)-4-(3-(18)F-fluoropropyl)-l-glutamate ((18)F-FSPG, or BAY 94-9392) is a new tracer to assess system x(C)(¯) transporter activity with PET. The aim of this study was to explore the tumor detection rate of (18)F-FSPG, compared with that of (18)F-FDG, in patients with hepatocellular carcinoma (HCC). METHODS: Preclinically, in vivo HCC models of orthotopically implanted Huh7 and MH3924a cancer cells were studied with (18)F-FSPG in Naval Medical Research Institute nude mice (n = 3) and August-Copenhagen Irish rats (n = 4), respectively. Clinically, 5 patients with HCC who had hyper- or isometabolic lesions on (18)F-FDG PET were enrolled for evaluation of the tracer. Dynamic whole-body PET images with (18)F-FSPG were acquired for up to 120 min after injection of approximately 300 MBq of (18)F-FSPG. Immunohistochemical expression levels of the xCT subunit of the system x(C)(¯) and CD44 of HCC were studied in 4 patients with HCC. RESULTS: Strong tumor uptake and low background from nontarget tissue allowed excellent tumor visualization in animal models with orthotopically implanted liver tumors. (18)F-FSPG PET procedures were well tolerated in all patients. (18)F-FSPG PET and (18)F-FDG detected lesions in 5 of 5 and 3 of 5 patients, respectively. The maximal standardized uptake values (SUV) were comparable ((18)F-FSPG, 4.7 ± 3.2; (18)F-FDG, 6.1 ± 2.9). The ratios of maximal SUV of the tumor to mean SUV of normal liver were also comparable ((18)F-FSPG, 3.6 ± 2.2; (18)F-FDG, 2.7 ± 1.3), but the mean SUV of normal liver of (18)F-FSPG was significantly lower than that of (18)F-FDG (P < 0.05). Two patients with HCC who showed both xCT and CD44 expression had moderate or intense accumulation of (18)F-FSPG, but the remaining 2 patients with negative CD44 expression showed mild uptake. CONCLUSION: (18)F-FSPG was successfully translated from preclinical evaluation into patients with HCC. (18)F-FSPG may be a promising tumor PET agent with a high cancer detection rate in patients with HCC.

The maize cystatin CC9 interacts with apoplastic cysteine proteases.

In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

Zn(II)-bis(cyclen) complexes and the imaging of apoptosis/necrosis.

In vivo cell-death imaging is still a challenging issue. Until now, only (99m)Tc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with (18)F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}(4+) complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the (18)F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the (18)F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.