Mariacristina Valerio

Metformin elicits anticancer effects through the sequential modulation of DICER and c-MYC

Diabetic patients treated with metformin have a reduced incidence of cancer and cancer-related mortality. Here we show that metformin affects engraftment and growth of breast cancer tumours in mice. This correlates with the induction of metabolic changes compatible with clear anticancer effects. We demonstrate that microRNA modulation underlies the anticancer metabolic actions of metformin. In fact, metformin induces DICER expression and its effects are severely impaired in DICER knocked down cells. Conversely, ectopic expression of DICER recapitulates the effects of metformin in vivo and in vitro. The microRNAs upregulated by metformin belong mainly to energy metabolism pathways. Among the messenger RNAs downregulated by metformin, we found c-MYC, IRS-2 and HIF1alpha. Downregulation of c-MYC requires AMP-activated protein kinase-signalling and mir33a upregulation by metformin. Ectopic expression of c-MYC attenuates the anticancer metabolic effects of metformin. We suggest that DICER modulation, mir33a upregulation and c-MYC targeting have an important role in the anticancer metabolic effects of metformin.

Protein Aggregation/Folding: The Role of Deterministic Singularities of Sequence Hydrophobicity as Determined by Nonlinear Signal Analysis of Acylphosphatase and Aβ(1–40)

The problem of protein folding vs. aggregation was investigated in acylphosphatase and the amyloid protein Abeta(1-40) by means of nonlinear signal analysis of their chain hydrophobicity. Numerical descriptors of recurrence patterns provided the basis for statistical evaluation of folding/aggregation distinctive features. Static and dynamic approaches were used to elucidate conditions coincident with folding vs. aggregation using comparisons with known protein secondary structure classifications, site-directed mutagenesis studies of acylphosphatase, and molecular dynamics simulations of amyloid protein, Abeta(1-40). The results suggest that a feature derived from principal component space characterized by the smoothness of singular, deterministic hydrophobicity patches plays a significant role in the conditions governing protein aggregation.

Early events in protein aggregation: molecular flexibility and hydrophobicity/charge interaction in amyloid peptides as studied by molecular dynamics simulations.

In a previous article (Zbilut et al., Biophys J 2003;85:3544–3557), we demonstrated how an aggregation versus folding choice could be approached considering hydrophobicity distribution and charge. In this work, our aim is highlighting the mutual interaction of charge and hydrophobicity distribution in the aggregation process. Use was made of two different peptides, both derived from a transmembrane protein (amyloid precursor protein; APP), namely, Aβ(1‐28) and Aβ(1‐40). Aβ(1‐28) has a much lower aggregation propensity than Aβ(1‐40). The results obtained by means of molecular dynamics simulations show that, when submitted to the most “aggregation‐prone” environment, corresponding to the isoelectric point and consequently to zero net charge, both peptides acquire their maximum flexibility, but Aβ(1‐40) has a definitely higher conformational mobility than Aβ(1‐28). The absence of a hydrophobic “tail,” which is the most mobile part of the molecule in Aβ(1‐40), is the element lacking in Aβ(1‐28) for obtaining a “fully aggregating” phenotype. Our results suggest that conformational flexibility, determined by both hydrophobicity and charge effect, is the main mechanistic determinant of aggregation propensity. Proteins 2005.

Metabolomics approach in allergic and rheumatic diseases.

Metabolomics is the analysis of the concentration profiles of low molecular weight compounds present in biological fluids. Metabolites are nonpeptide molecules representing the end products of cellular activity. Therefore, changes in metabolite concentrations reveal the range of biochemical effects induced by a disease or its therapeutic intervention. Metabolomics has recently become feasible with the accessibility of new technologies, including mass spectrometry and high-resolution proton nuclear magnetic resonance, and has already been applied to several disorders. Indeed, it has the advantage of being a nontargeted approach for identifying potential biomarkers, which means that it does not require a preliminary knowledge of the substances to be studied. In this review, we summarize the main studies in which metabolomic approach was used in some allergic (asthma, atopic dermatitis) and rheumatic diseases (rheumatoid arthritis, systemic lupus erythematosus) to explore the feasibility of this technique as a novel diagnostic tool in these complex disorders.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

1H-NMR-Based Metabolomic Study for Identifying Serum Profiles Associated with the Response to Etanercept in Patients with Rheumatoid Arthritis

Objective A considerable proportion of patients with rheumatoid arthritis (RA) do not have a satisfactory response to biological therapies. We investigated the use of metabolomics approach to identify biomarkers able to anticipate the response to biologics in RA patients. Methods Due to gender differences in metabolomic profiling, the analysis was restricted to female patients starting etanercept as the first biological treatment and having a minimum of six months’ follow-up. Each patient was evaluated by the same rheumatologist before and after six months of treatment. At this time, the clinical response (good, moderate, none) was determined according to the EUropean League Against Rheumatism (EULAR) criteria, based on both erythrocyte sedimentation rate (EULAR-ESR) and C-reactive protein (EULAR-CRP). Sera collected prior and after six months of etanercept were analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate data analysis. Results Twenty-seven patients were enrolled: 18 had a good/moderate response and 9 were non responders according to both EULAR-ESR and EULAR-CRP after six months of etanercept. Metabolomic analysis at baseline was able to discriminate good, moderate, and non-responders with a very good predictivity (Q2 = 0.68) and an excellent sensitivity, specificity, and accuracy (100%). In good responders, we found an increase in isoleucine, leucine, valine, alanine, glutamine, tyrosine, and glucose levels and a decrease in 3-hydroxybutyrate levels after six months of treatment with etanercept with respect to baseline. Conclusion Our study confirms the potential of metabolomic analysis to predict the response to biological agents. Changes in metabolic profiles during treatment may help elucidate their mechanism of action.

3T multiparametric MRI of the prostate: Does intravoxel incoherent motion diffusion imaging have a role in the detection and stratification of prostate cancer in the peripheral zone?

PURPOSE To evaluate the potential added value of the intravoxel incoherent motion model to conventional multiparametric magnetic resonance protocol in order to differentiate between healthy and neoplastic prostate tissue in the peripheral zone. MATERIAL AND METHODS Mono-exponential and bi-exponential fits were used to calculate ADC and IVIM parameters in 53 patients with peripheral zone biopsy proved tumor. Inferential statistics analysis was performed on T2, ADC and IVIM parameters (D, D*, f) comparing healthy and neoplastic tissues. Linear discriminant analysis was performed for the conventional parameters (T2 and ADC), the IVIM parameters (molecular diffusion coefficient (D), perfusion-related diffusion coefficient (D*), and perfusion fraction (f) and the combined T2-weighted imaging/DWI and IVIM parameters (T2, ADC, D, D* and f). A correlation with Gleason scores was achieved. RESULTS The values of T2, ADC and D were significantly lower in cancerous tissues (2749.82 ± 1324.67 ms, 0.76 ± 0.27 × 10(-3)mm(2)/s and 0.99 ± 0.38 × 10(-3)mm(2)/s respectively) compared to those found in the healthy tissues (3750.70 ± 1735.37 ms, 1.39 ± 0.48 × 10(-3)mm(2)/s and 1.77 ± 0.36 × 10(-3)mm(2)/s respectively); D* parameter was significantly increased in neoplastic compared to healthy tissue (15.56 ± 12.91 × 10(-3)mm(2)/s and 10.25 ± 10.52 × 10(-3)mm(2)/s respectively). The specificity, sensitivity and accuracy of the T2-weighted imaging/DWI and IVIM parameters were 100, 96 and 98%, respectively, compare to 88, 92 and 90% and 96, 92 and 94 for T2-weighted imaging/ADC and IVIM alone. CONCLUSIONS IVIM parameters increase the specificity and sensitivity in the evaluation of peripheral zone prostate cancer. A statistical difference between low grade tumors and high grade tumors has been demostrated in that ADC, D and D* dataset; in particular, D has been found to have the highest significativity.

pH Effects on the Conformational Preferences of Amyloid β-Peptide (1–40) in HFIP Aqueous Solution by NMR Spectroscopy

The structure and aggregation state of amyloid β‐peptide (Aβ) in membrane‐like environments are important determinants of pathological events in Alzheimer’s disease. In fact, the neurotoxic nature of amyloid‐forming peptides and proteins is associated with specific conformational transitions proximal to the membrane. Under certain conditions, the Aβ peptide undergoes a conformational change that brings the peptide in solution to a “competent state” for aggregation. Conversion can be obtained at medium pH (5.0–6.0), and in vivo this appears to take place in the endocytic pathway. The combined use of 1H NMR spectroscopy and molecular dynamics‐simulated annealing calculations in aqueous hexafluoroisopropanol simulating the membrane environment, at different pH conditions, enabled us to get some insights into the aggregation process of Aβ, confirming our previous hypotheses of a relationship between conformational flexibility and aggregation propensity. The conformational space of the peptide was explored by means of an innovative use of principal component analysis as applied to residue‐by‐residue root‐mean‐square deviations values from a reference structure. This procedure allowed us to identify the aggregation‐prone regions of the peptide.

Classification of prostatic diseases by means of multivariate analysis on in vivo proton MRSI and DCE-MRI data.

Multivariate analysis has been applied on proton magnetic resonance spectroscopic imaging (1H‐MRSI) and dynamic contrast enhanced MRI (DCE‐MRI) data of patients with different prostatic diseases such as chronic inflammation, fibrosis and adenocarcinoma. Multivariate analysis offers a global view of the entire range of information coming from both the imaging and spectroscopic side of NMR technology, leading to an integrated picture of the system relying upon the entire metabolic and dynamic profile of the studied samples. In this study, we show how this approach, applied to 1H‐MRSI/DCE‐MRI results, allows us to differentiate among the various prostatic diseases in a non‐invasive way with a 100% accuracy. These findings suggest that multivariate analysis of 1H‐MRSI/DCE‐MRI can significantly improve the diagnostic accuracy for these pathological entities. From a more theoretical point of view, the complementation of a single biomarker approach with an integrated picture of the entire metabolic and dynamic profile allows for a more realistic appreciation of pathological entities. Copyright

Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

BackgroundSolid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia.MethodsThe human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses.ResultsHere we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism.ConclusionsWe show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.