Giulia Carpinelli

Pharmacological blockade of group II metabotropic glutamate receptors reduces the growth of glioma cells in vivo

U87MG human glioma cells in cultures expressed metabotropic glutamate (mGlu) receptors mGlu2 and mGlu3. Addition of the mGlu2/3 receptor antagonist LY341495 to the cultures reduced cell growth, expression of cyclin D1/2, and activation of the MAP kinase and phosphatidylinositol-3-kinase pathways. This is in line with the evidence that activation of mGlu2/3 receptors sustains glioma cell proliferation. U87MG cells were either implanted under the skin (1x10(6) cells/0.5 ml) or infused into the caudate nucleus (0.5x10(6) cells/5 microl) of nude mice. Animals were treated for 28 days with mGlu receptor antagonists by means of subcutaneous osmotic minipumps. Treatments with LY341495 or (2S)-alpha-ethylglutamate (both infused at a rate of 1 mg/kg per day) reduced the size of tumors growing under the skin. Infusion of LY341495 (10 mg/kg per day) also reduced the growth of brain tumors, as assessed by magnetic resonance imaging analysis carried out every seven days. The effect of drug treatment was particularly evident during the exponential phase of tumor growth, that is, between the third and the fourth week following cell implantation. Immunohistochemical analysis showed that U87MG cells retained the expression of mGlu2/3 receptors when implanted into the brain of nude mice. These data suggest that mGlu2/3 receptor antagonists are of potential use in the experimental treatment of malignant gliomas.

Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high-fat-diet-fed mice

The mineralocorticoid receptor (MR) controls adipocyte function, but its role in the conversion of white adipose tissue (WAT) into thermogenic fat has not been elucidated. We investigated responses to the MR antagonists spironolactone (spiro; 20 mg/kg/d) and drospirenone (DRSP; 6 mg/kg/d) in C57BL/6 mice fed a high‐fat (HF) diet for 90 d. DRSP and spiro curbed HF diet‐induced impairment in glucose tolerance, and prevented body weight gain and white fat expansion. Notably, either MR antagonist induced up‐regulation of brown adipocyte‐specific transcripts and markedly increased protein levels of uncoupling protein 1 (UCP1) in visceral and inguinal fat depots when compared with the HF diet group. Positron emission tomography and magnetic resonance spectroscopy confirmed acquisition of brown fat features in WAT. Interestingly, MR antagonists markedly reduced the autophagic rate both in murine preadipocytes in vitro (10‐5 M) and in WAT depots in vivo, with a concomitant increase in UCP1 protein expression. Moreover, the autophagy repressor bafilomycin A1 (10‐8 M) mimicked the effect of MR antagonists, increasing UCP1 protein expression in primary preadipocytes. Hence, we showed that adipocyte MR regulates brown remodeling of WAT through a modulation of autophagy. These results provide a rationale for the use of MR antagonists to prevent the adverse metabolic consequences of adipocyte dysfunction.—Armani, A., Cinti, F., Marzolla, V., Morgan, J., Cranston, G. A., Antelmi, A., Carpinelli, G., Canese, R., Pagotto, U., Quarta, C., Malorni, W., Matarrese, P., Marconi, M., Fabbri, A., Rosano, G., Cinti, S., Young, M. J., Caprio, M. Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high‐fat‐diet‐fed mice. FASEB J. 28, 3745–3757 (2014). www.fasebj.org

Abnormal Choline Phospholipid Metabolism in Breast and Ovary Cancer:Molecular Bases for Noninvasive Imaging Approaches

Elevated contents of choline phospholipid metabolites are typically detected by nuclear magnetic resonance spectroscopy (MRS) in human and animal tumors. An increase in the intensity of the 1 H-MRS profile of total cholinecontaining compounds (tCho, 3.2 ppm) is today considered as a common feature in different types of cancer, beyond their otherwise wide phenotypic variability. This finding fostered investigations on the molecular mechanisms underlying the observed spectral changes and on correlations between aberrant phospholipid metabolism and tumor progression. At the clinical level, efforts are addressed to evaluate effectiveness and potential use of in vivo localized MRS and choline-based positron emission tomography (Cho-PET) in cancer diagnosis. Aims of this article are: a) to overview recent advances in the identification of biochemical pathways responsible for the altered 1 H-MRS tCho profile in breast and ovary cancer cells, as a basis for interpreting in vivo MR spectra and enhanced uptake of radiolabeled choline in PET; b) to summarize recent developments of in vivo 1 H-MRS methods in breast cancer diagnosis; c) to discuss the potentialities of complementing current diagnostic modalities with noninvasive MRS and Cho-PET methods to monitor biochemical alterations associated with progression, relapse and therapy response in ovary cancer.

Modulations of glycerophosphorylcholine and phosphorylcholine in Friend erythroleukemia cells upon in vitro-induced erythroid differentiation: a 31P NMR study

A 31P NMR study has been carried out on Friend erythroleukemia cells (FLC) induced to undergo erythroid differentiation in vitro. Significant levels of glycerophosphorylcholine (GroPCho) and phosphorylcholine (P‐Cho) were identified both in the untreated cells and in their PCA extracts. In FLC treated 4 days in vitro with either dimethylsulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), the intracellular concentration of P‐Cho was markedly increased, whereas that of GroPCho appeared to be significantly reduced. HMBA was more effective than DMSO in producing this effect. The concomitant modulations of GroPCho and P‐Cho in differentiated FLC suggest the hypothesis that erythroid differentiation involves modifications of the regulatory mechanisms controlling biosynthesis and catabolism of phospholipids.

31P Nuclear Magnetic Resonance Study of Growth and Dimorphic Transition in Candida albicans

A 31P NMR study of the fungal pathogen Candida albicans was carried out. Yeast-form cells at different phases of growth, as well as germ tubes and hyphae were examined. In all cases, the NMR spectra showed well separated resonance peaks arising from phosphorus-containing metabolites, the most prominent being attributable to inorganic phosphate (Pi) polyphosphates, sugar phosphates and mononucleotides, NAD, ADP and ATP. Relevant signals were also detected in the phosphodiester region. The intensity of most signals, as measured relative to that of Pi, was clearly modulated both at the different phases of growth and during yeast-to-mycelium conversion, suggesting significant changes in the intracellular concentration of the corresponding metabolites. In particular, the intensity of the polyphosphate signal was high in exponentially growing, yeast-form cells, then progressively declined in the stationary phase, was very low in germ tubes and, finally, undetectable in hyphae. NMR spectral analysis of the Pi region showed that from early-stationary phase, Pi was present in two different cellular compartments, probably corresponding to the cytoplasm and the vacuole. From the chemical shift of Pi, the pH values of these two compartments could be evaluated. The cytoplasmic pH was generally slightly lower than neutrality (6.7-6.8), whereas the vacuolar pH was always markedly more acidic.

Targeting Estrogen Receptor-α Reduces Adrenocortical Cancer (ACC) Cell Growth in Vitro and in Vivo: Potential Therapeutic Role of Selective Estrogen Receptor Modulators (SERMs) for ACC Treatment

CONTEXT Adrenocortical carcinoma (ACC) is a rare tumor with a very poor prognosis and no effective treatment. ACC is characterized by an increased production of IGF-II and by estrogen receptor (ER)-α up-regulation. OBJECTIVE The objective of this study was to define the role played by ERα in 17β-estradiol (E2)- and IGF-II-dependent ACC growth and evaluate whether selective estrogen receptor modulators are effective in controlling ACC growth in vivo. EXPERIMENTAL DESIGN The human adrenocortical cell line H295R was used as an in vitro model and to generate xenograft tumors in athymic nude mice. RESULTS In H295R cells IGF-II controlled expression of steroidogenic factor-1 that, in turn, increased aromatase transcription and, consequently, estrogen production, inducing cell proliferation. ERα silencing significantly blocked E2- and IGF-II-dependent cell proliferation. This effect was dependent on the regulation of cyclin D1 expression by ERα, activated in response to both E2 and IGF-II. In fact, IGF-II induced ERα activation by phosphorylating serine 118 and 167. Furthermore, we demonstrated that ERα mediated E2-induced nongenomic signaling that stimulated IGF-I receptor (IGF1R), ERK1/2, and AKT phosphorylation, resulting in a ligand-independent activation of the IGF1R-induced pathway. In addition, E2 potentiated this pathway by up-regulating IGF1R expression as a consequence of increased cAMP-responsive element binding protein activation and binding to IGF1R promoter. The estrogen antagonist, hydroxytamoxifen, the active metabolite of tamoxifen, reduced IGF1R protein levels and both E2- and IGF-II-induced cell proliferation. Moreover, H295R xenograft growth was strongly reduced by tamoxifen. CONCLUSION These findings establish a critical role for ERα in E2- and IGF-II-dependent ACC proliferation and provide a rationale for targeting ERα to control the proliferation of ACC.

Potential Role of in vitro 1H Magnetic Resonance Spectroscopy in the Definition of Malignancy Grading of Human Neuroepithelial Brain Tumours

The increasing sensitivity of neuro-imaging in the diagnosis of brain expanding lesions is not directly related to biopathological specificity and new technological approaches are under study. In particular Magnetic Resonance Spectroscopy (MRS) allows evaluation of some biochemical pathways whose metabolic alterations may be correlated with the nature and malignancy grading of primary brain tumours. In the present study the author performed an in vitro high field 1H MRS (9.4 and 14.1 T) analysis of specimens obtained from stereotactic biopsy or microsurgical removal of primary brain tumours. Different samples derived from heterogeneous areas and/or infiltrated perilesional regions were examined. This study was principally focused on malignancy grading of gliomas and its correlation with the ratio (R) between the resonance band arising from choline containing compounds (between 3.14 and 3.35 ppm) and the total creatine signal (3.0 ppm). Analyses allowed significant discrimination between astrocytomas (R = 2.4 +/- 0.6) and glioblastoma (GBM) (R = 4.4 +/- 1.3) [p < 0.002]; however the results did not allow discrimination between differentiated and anaplastic astrocytomas. The GBM showed the largest spread of values corresponding to their higher level of tissue heterogeneity and de-differentiation. Studies on non astrocytic brain tumours indicated that even higher R values were exhibited by oligodendrogliomas, even in well differentiated forms (p < 0.02 with respect to GBM). Moreover, preliminary observations indicated that signals arising from other metabolites may also contribute to a differential diagnosis of different oncotypes. Among these glycine appears particularly relevant, since higher levels were measured for this amino acid in GBM with respect to both astrocytomas and oligodendrogliomas.

Alterations of lipid composition in Friend leukemia cell tumors in mice treated with tumor necrosis factor-α

Lipid analyses were carried out on transplantable murine Friend leukemia cell tumors, 6 h after intratumoral administration of tumor necrosis factor‐α (TNF). The levels of the major phospholipid classes were uniformly decreased to about 70% of control values; free fatty acids were increased to about 170%; diacylglycerol was decreased to about 50% and triacylglycerol, the main lipid component, was not significantly altered. These results analysed in the light of concomitant alterations in the levels of phospholipid precursors and catabolites (determined in previous 31P NMR studies) and histological modifications demonstrated that at early stages of TNF‐induced inhibition of tumor growth (a) phospholipid catabolism was significantly enhanced; (b) morphological changes were apparently correlated with alterations in the levels of phosphatidylcholine and its catabolic products.

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

BackgroundThe ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.ResultsAn enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.ConclusionThe construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

A 19F nuclear magnetic resonance study of uptake and metabolism of 5-fluorocytosine in susceptible and resistant strains of Candida albicans.

The metabolism of the antifungal drug 5-fluorocytosine (5-FC) was studied in intact viable cells of Candida albicans by 19F nuclear magnetic resonance (NMR). The uptake of the drug and its conversion to the deaminated product 5-fluorouracil (5-FU) were easily observed by NMR analysis of both the cells and the supernatants of the incubation mixture. In the 5-FC-resistant mutant D14 of C. albicans, which lacked cytosine deaminase activity, the resonance peak of 5-FU was not observed. In intact cells of all 5-FC-susceptible strains the metabolism of 5-FU progressed to the formation of other fluorinated derivatives which were visualized as a single, broad resonance band at a lower field with respect to 5-FC and 5-FU. This band was resolved into three distinct peaks in the acid extract of treated cells, one of these peaks being attributable to 5-fluoro-dUMP (5-FdUMP). In strain 72R of C. albicans, which is 5-FC resistant because of a low level of UMP-pyrophosphorylase activity, the broad, low-field resonance band was detected later and with much less intensity than in the 5-FC-sensitive strains. This suggests that, besides 5-FdUMP, this band is also contributed to by 5-FUMP and possibly other phosphorylated derivatives. 19F NMR analysis also revealed that a significant amount of 5-FU is secreted into the external medium, the rate of secretion being higher in 5-FC-resistant strain 72R than in 5-FC-sensitive strain 72S. Although not all resonances were definitely identified, this study shows that 19F NMR spectroscopy may be an important tool for noninvasive analysis of the metabolism of fluorinated drugs in yeasts.