Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.     

Cl-IB-MECA enhances TNF-α release in peritoneal macrophages stimulated with LPS

Adenosine receptor A3 (A3R) belongs to the Gi/Gq-coupled receptor family, that leads to the intracellular cAMP reduction and intracellular calcium increase, respectively. A3R is widely expressed and it can play a crucial role in many patho-physiological conditions, including inflammation. Here we investigate the effect of Cl-IB-MECA, A3R agonist, on the production of TNF-α. We found that Cl-IB-MECA enhances LPS-induced TNF-α release in peritoneal macrophages. This effect is reduced by MRS1191, A3R antagonist and by forskolin, activator of adenylyl cyclase. pIκBα increased in LPS+Cl-IB-MECA-treated macrophages, while total IκB kinase-β (IKKβ) reduced. Indeed, p65NF-κB nuclear translocation increased in cells treated with LPS+Cl-IB-MECA. Moreover, IMD 0354, IKKβ inhibitor, significantly abrogated the effect of Cl-IB-MECA on TNF-α release. Inhibition of protein kinase C (PKC) significantly reduced Cl-IB-MECA-induced TNF-α release in LPS-stimulated macrophages. Furthermore, LY-294002, PI3K inhibitor, reduced the TNF-α production enhanced by Cl-IB-MECA, although the phosphorylation status of Akt did not change in cells treated with LPS+Cl-IB-MECA than LPS alone. In summary, these data show that Cl-IB-MECA is able to enhance TNF-α production in LPS-treated macrophages in an NF-κB- dependent manner.     

Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

Background  

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1) predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics.     

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3′UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.Key words: melanoma, microRNA profiling, biomarker, acral, KRAS-variant, SNP     

Phosphoproteomic screen identifies potential therapeutic targets in melanoma

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.     

Constraints in the identification and interpretation of ozone as a significant predictor of effects on the supersensitive indicator Nicotiana tabacum Bel-W3 in biomonitoring studies

Biomonitoring by means of the supersensitive cultivar Nicotiana tabacum Bel-W3 is frequently used to obtain information on ozone effects on plants and estimates of ozone exposure. However, most of biomonitoring surveys do not account for other environmental variables (predictors in a statistical model) and their inherent multicollinearity with ozone. We tested the relative role of different predictors (fixed: time and site; random: ozone, temperature and humidity) on height growth and on the development of visible foliar symptoms of N. tabacum Bel-W3 plants. To do this, we investigated a relatively small area (256 km²), used a random design at every stage of the survey, controlled watering and protected plants from direct solar radiation and wind. QA/QC procedures were adopted at every stage of the investigation. Linear correlation shows that Leaf Injury Index (LII) and height increment (H.I.) positively related to ozone concentration, elevation and temperature, and negatively to relative humidity. All the predictors correlate to each other. However, relationships between response and ozone vary with the site and the monitoring week. The effect of the random factor “ozone” in combination with fixed factors “site” and “time” on the response variables was therefore formally investigated using the ANCOVA model. Besides ozone, the interactions “ozone × site” and “ozone × time” resulted always significant (0.001 < P < 0.05). While the factor “time” emphasize the inherent development of injury and growth through time, the interaction “ozone × site” pointed out the importance of local conditions. When watering, solar radiation, wind and plant characteristics were controlled, the remaining site-specific covariates of interest were temperature (T) and humidity (RH). When T and RH were accounted for by means of partial correlation analysis, no significant relationship was found between ozone and LII. On the other side, O₃ and RH resulted significant for both absolute and relative height increment. In short, LII seemed to be not solely dependent on ozone, T and RH, but showed to integrate their combined effect. On the other side, H.I. seemed to be favoured by high RH and T, and depressed by high ozone. Based on the above results, we recommend caution when handling bioindicator data: if the purpose is to infer ozone concentrations by leaf injury data, results may be affected by a serious bias, as the frequently reported correlations may be partly an artefact due to co-variation between predictors.     

A comparative MudPIT analysis identifies different expression profiles in heart compartments

Cardiomyopathies indistinctly affect atrial and ventricular cardiac compartments with alterations of their mechanical and/or electrical activity. To understand the main mechanisms involved in these pathological alterations, a detailed knowledge of the physiology of the healthy heart is critical. In the present work, we utilize multidimensional protein identification technology to characterize the murine left ventricle (LV), right ventricle (RV), and atria (A) proteomes, identifying thousands of distinct proteins. Moreover, using multidimensional algorithm protein map tool, relative abundances of proteins among the heart chambers were investigated. In sum, we found 16 and 55 proteins were more abundant in LV compared to RV and A, respectively; 47 and 60 proteins were more abundant in RV than LV and A, respectively; and, 81 and 74 proteins were more abundant in A than LV and RV, respectively. This detailed characterization of myocardial compartment proteome represents an important advancement in the knowledge of heart physiology, and may contribute to the identification of key features underlying the onset of cardiomyopathy.     

HMGB1 attenuates cardiac remodelling in the failing heart via enhanced cardiac regeneration and miR-206-mediated inhibition of TIMP-3

Aims

HMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts.

Methods and Results

Adult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit⁺ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20–25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206.

Conclusions

HMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.