Apoptosis and proliferation in thyroid carcinoma: correlation with bcl-2 and p53 protein expression.

The aim of this study was to determine the apoptotic cell death in 92 thyroid carcinomas of different histotypes (42 papillary, PTC; 12 poorly differentiated, PDC: 21 undifferentiated, UC; and 17 medullary, MC) by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL). Apoptotic index (Al, evaluated as a percentage of TUNEL-positive cells of neoplastic cells) was calculated in each tumour. The AI was very low in all subtypes of thyroid carcinoma, ranging from a median value of 0.2 in PTC to 1.4 in UC. The proliferative activity was determined by immunohistochemistry using monoclonal antibody, MIB-1. The percentage of proliferating cells was significantly different among the histotypes, increasing with tumour aggressiveness (from the mean value of 3.1 for PTC to 5.6 for PDC and 51.8 for UC). In addition, the ratio between proliferative activity and apoptosis was significantly higher in UC than in the other histotypes. The expression of bcl-2 and p53 protein (important in the modulation of cell death) was correlated (bcl-2, inverse correlation, r2 = 0.1, P = 0.04; p53, direct correlation, r2 = 0.11, P = 0.02) with apoptotic index in PTC

Activation of diacylglycerol kinase alpha is required for VEGF-induced angiogenic signaling in vitro.

Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.

Diacylglycerol kinase-α phosphorylation by Src on Y335 is required for activation, membrane recruitment and Hgf-induced cell motility

n/

Telerehabilitation for Lee Silverman Voice Treatment (Tele-LSVT)-Loud on voice intensity and voice use in daily living in people with multiple sclerosis: A protocol for a feasibility and pilot randomized controlled study

Objective: Alterations in voice intensity and quality may constitute a social life limitation in people with multiple sclerosis (MS), but only 2% of cases receive speech therapy. Especially the Lee Silverman Voice Treatment (LSVT)-Loud is a highly effective intensive method for voice intensity, requiring subjects’ repeated attendance at the clinic. Telerehabilitation may represent a feasible solution to bypass potential barriers related to speech therapy attendance, scaling up the beneficial effects of the treatment to a broader population. The proposed protocol aims to test the feasibility and the pilot efficacy of the LSVT-Loud delivered in telerehabilitation (Tele-LSVT-Loud), compared to the same treatment delivered in the clinic (LSVT-Loud). Methods: A single-blinded, parallel, two-arm, pilot randomized (1:1 ratio) controlled trial will be performed involving 20 people with MS. Patients will be allocated to 4 weeks of Tele-LSVT-Loud by accessing a telerehabilitation platform at home or LSVT-Loud conventionally delivered in the clinic. Feasibility and pilot effectiveness will be evaluated three times: before (T0), after the treatment (T1), and 3-month follow-up (T2). Feasibility measures will include adherence, adverse events, user experience, motivation, engagement, and acceptability. Vocal intensity during a 1-minute monologue will be the primary outcome measure. Secondary outcome measures will be the vocal quality during a 1-minute monologue, sustained /a/ voice intensity, quality and stability, voice use in daily life, voice subjective perception in daily life, and quality of life. Results: Expected results will be (1) high feasibility of Tele-LSVT-Loud and (2) a non-inferiority effect of Tele-LSVT-Loud compared with face-to-face treatment delivery on voice intensity and quality outcomes. Conclusions: Tele-LSVT-Loud may be a feasible intervention for MS alteration in voice intensity and quality with a non-inferior effect compared to LSVT-Loud

The diacylglycerol kinase α/Atypical PKC/β1 integrin pathway in SDF-1α mammary carcinoma invasiveness

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5β1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of β1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and β1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - β1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells

A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy

Simple Summary Selective inhibitors of mutant BRAFV600E (BRAFi) have revolutionized the treatment of metastatic melanoma patients and represent a powerful example of the efficacy of targeted therapy. However, one of the main limitations of BRAFi is that treated cells put in place several adaptive response mechanisms, which initially confer drug tolerance and later provide a gateway for the insurgence of genetically acquired resistance mechanisms. We previously discovered that pigmentation is one of these adaptive response mechanisms. Upon BRAFi treatment, those cells that increase their pigmentation level are more resistant to BRAFi than those that do not. Here, we demonstrate that pigmentation limits BRAFi activity through an increase in the number of intracellular mature melanosomes. We also show that this increase derives from increased maturation and/or trafficking. In addition, we identify the miRNAs and mRNAs that are involved in these biological processes. Finally, we provide the rationale for testing a new combinatorial therapeutic strategy that aims at increasing BRAFi efficacy by blocking the adaptive responses that they elicit. This strategy is based on the combined use of BRAFi with inhibitors of pigmentation, specifically inhibitors of melanosome maturation and/or trafficking. Background: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. Methods: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). Results: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. Conclusion: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells

ACYLATED AND UNACYLATED GHRELIN IMPAIR SKELETAL MUSCLE ATROPHY IN MICE.

Cachexia is a wasting syndrome associated with cancer, AIDS, and multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1aindependent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3K\u3b2-, mTORC2-, and p38-mediated pathways in myotubes. Up-regulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsrdeficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner

Growth Hormone Secretagogues Protect Mouse Cardiomyocytes from in vitro Ischemia/Reperfusion Injury through Regulation of Intracellular Calcium

Background: Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms. Methodology/Principal Findings: Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca ] ) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca ] transient amplitude and caffeine-releasable SR Ca content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca ] transients. Conclusion/Significance: Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury

Polymorphisms of genes coding for ghrelin and its receptor in relation to colorectal cancer risk: a two-step gene-wide case-control study

<p>Abstract</p> <p>Background</p> <p>Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), has two major functions: the stimulation of the growth hormone production and the stimulation of food intake. Accumulating evidence also indicates a role of ghrelin in cancer development.</p> <p>Methods</p> <p>We conducted a case-control study to examine the association of common genetic variants in the genes coding for ghrelin (GHRL) and its receptor (GHSR) with colorectal cancer risk. Pairwise tagging was used to select the 11 polymorphisms included in the study. The selected polymorphisms were genotyped in 680 cases and 593 controls from the Czech Republic.</p> <p>Results</p> <p>We found two SNPs associated with lower risk of colorectal cancer, namely SNPs rs27647 and rs35683. We replicated the two hits, in additional 569 cases and 726 controls from Germany.</p> <p>Conclusion</p> <p>A joint analysis of the two populations indicated that the T allele of rs27647 SNP exerted a protective borderline effect (P_trend= 0.004).</p