Gene expression of thyroid-specific transcription factors may help diagnose thyroid lesions but are not determinants of tumor progression.

PURPOSE: The role of thyroid-specific transcription factors in thyroid malignancy is still poorly understood, so we investigate thyroid-specific transcription factors gene expression both in benign and in malignant thyroid nodules, aiming to study a possible clinical utility of these molecules. METHODS: We quantified TTF-1, FOXE1 and PAX8 mRNA levels, relating their expression to diagnostic and prognostic features of thyroid tumors. RNA was extracted from 4 normal thyroid tissues, 101 malignant [99 papillary thyroid carcinomas (PTC) and 2 anaplastic thyroid carcinomas] and 99 benign thyroid lesion tissues [49 goiter and 50 follicular adenomas (FA)]. RESULTS: Levels of mRNA of both FOXE1 (P < 0.0001) and PAX8 (P < 0.0001) genes, but not TTF-1 (P = 0.7056), were higher in benign than in malignant thyroid lesions. FOXE1 was able to identify malignant nodules with 75.8 % sensitivity, 76.1 % specificity, 75.8 % positive predictive value, 76.1 % negative predictive value and 75.9 % accuracy. PAX8 was able to identify malignancy with 60.6 % sensitivity, 81.1 % specificity, 76.9 % positive predictive value, 66.4 % negative predictive value and 70.6 % accuracy. Both FOXE1 and PAX8 gene expression patterns were also able to differentiate FA from the follicular variant of PTC-FVPTC. However, the investigated gene expression was neither associated with any clinical feature of tumor aggressiveness nor associated with recurrence or survival. CONCLUSIONS: We suggest that FOXE1 and PAX8 gene expression patterns may help to diagnose thyroid nodules, identifying malignancy and characterizing follicular-patterned thyroid lesions, but are not determinants of thyroid tumor progression.

Rottlerin and cancer: novel evidence and mechanisms.

Because cancers are caused by deregulation of hundreds of genes, an ideal anticancer agent should target multiple gene products or signaling pathways simultaneously. Recently, extensive research has addressed the chemotherapeutic potential of plant-derived compounds. Among the ever-increasing list of naturally occurring anticancer agents, Rottlerin appears to have great potentiality for being used in chemotherapy because it affects several cell machineries involved in survival, apoptosis, autophagy, and invasion. The underlying mechanisms that have been described are diverse, and the final, cell-specific, Rottlerin outcome appears to result from a combination of signaling pathways at multiple levels. This paper seeks to summarize the multifocal signal modulatory properties of Rottlerin, which merit to be further exploited for successful prevention and treatment of cancer.

Antiproliferative and survival properties of PMA in MCF-7 breast cancer cell.

Although PKCs are assumed to be the main targets of phorbol esters (PMA), additional PMA effectors, such as chimaerins (a family of RacGTPase activating proteins) and RasGRP (exchange factor for Ras/Rap1), can counteract or strengthen the PKC pathways. In this study, we evaluated the proliferative behavior of PMA-treated MCF-7 breast cancer cell and found that: PMA induced growth arrest and inhibited cell death; PMA activated ERKs, which, in turn, induced p21; and inhibitors of ERK (PD98059) and PKC (GF109203X) prevented p21 induction and abolished the PMA survival effect. We conclude that PMA inhibits MCF-7 cell growth and simultaneously stimulates cell survival; both responses are linked to ERK-dependent and p53-independent p21 induction.

Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells.

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Molecular assembly of endogenous and synthetic big atrial natriuretic peptide (ANP) and its amyloidogenic implications.

The aggregation process of alpha-hANP has been investigated in vitro at physiological concentrations by gel chromatographic procedures using a radiolabeled tracer incubated in PBS and in plasma. In PBS big forms of ANP are organized as a peak eluting from both Sephacryl S-100 and S-300 HR in the void volume of the columns; in plasma, besides this major peak, a second radioactive peak is evident, eluting from Sephacryl S-100 HR around the HSA region. After gel chromatography on Sephacryl S-300 HR the major peak appears to consist of three components of different molecular size. Some information about the nature of these peak materials comes from the result of parallel incubations of partially aggregated (seed or nucleus) and aggregate depleted tracer. The comparison between the two time courses of big ANP formation indicates that: (a) ANP aggregation is a nucleation-dependent process, with a lag time longer than 8 days, at picogram peptide levels and (b) the aggregated forms of peptide are those eluting in the void volume, the other plasma peaks being probably expression of a binding, neither saturable or reversible, to some plasma components. The principle of seeded polymerization, used to detect ANP aggregates present in the plasma, indicates that: (a) the endogenous big ANP cannot act as a nucleus for polymerization and it likely consists of non-fibrillar ANP aggregates and/or bound ANP, and (b) this experimental approach can be suitable to evidence ANP binding plasma factors for further characterization studies.

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins.

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.

Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin.

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.

Multiple plasma proteins control atrial natriuretic peptide (ANP) aggregation.

We have recently demonstrated that human alpha-atrial natriuretic peptide (alpha-hANP), an amyloidogenic peptide responsible for isolated atrial amyloidosis, binds to a dimeric form of apo A-I belonging to small high-density lipoproteins (HDL). This binding phenomenon is considered a protective mechanism since it inhibits or strongly reduces the ANP aggregation process. The observation that plasma exhibits at least four times greater amyloid inhibitory activity than HDL prompted us to determine whether small HDL are the only ANP plasma-binding factors. After incubation of whole plasma with labelled ANP, the macromolecular complexes were subjected to two-dimensional gel electrophoresis followed by autoradiography. The results presented here provide novel evidence of additional binding proteins, in addition to apo A-I dimer, able to bind ANP in vitro and to prevent its aggregation. The mass spectrometry analysis of the radioactive spots identified them as albumin, alpha-1 antitrypsin, orosomucoid and apo A-IV-TTR complex. The putative impact of these findings in the amyloidogenic/antiamyloidogenic peptides network is discussed.

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells.

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Functional interactions of protein kinase A and C in signalling networks: a recapitulation.

On the basis of evidence collected from the literature, we propose a general model by which protein kinase (PK) A and the different PKC isoforms can inversely affect cell growth. Molecular switches, which are able to direct the signal towards antiproliferative or mitogenic pathways, are the different isoforms of Raf and PKC. Conflicting data are also reported and discussed in an attempt to reconcile them.

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts.

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Galpha s-mediated stimulation of adenylylcyclase (AC), Galpha q-mediated activation of phospholipase Cbeta (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast proliferation. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways--an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulated by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects.