Doxorubicin-Mediated Cardiotoxicity: Role of Mitochondrial Connexin 43

Doxorubicin is the highly effective anthracycline, but its clinical use is limited by cardiotoxicity and consequent dysfunction. It has been proposed that the etiology of this is related to mitochondrial dysfunction. Connexin 43 (Cx43), the principal protein building block of cardiac gap junctions and hemichannels, plays an important role in cardioprotection. Recent reports confirmed the presence of Cx43 in the mitochondria as well. In this study, the role of mitochondrial Cx43 was evaluated 3 or 6 h after Doxorubicin administration to the rat heart cell line H9c2. Pharmacological inhibition of Hsp90 demonstrated that the mitochondrial Cx43 conferred cardioprotection by reducing cytosolic and mitochondrial reactive oxygen species production, mitochondrial calcium overload and mitochondrial membrane depolarization and cytochrome c release. In conclusion, our study demonstrates that Cx43 plays an important role in the protection of cardiac cells from Doxorubicin-induced toxicity

Hazelnut (Corylus avellana l.) shells extract: Phenolic composition, antioxidant effect and cytotoxic activity on human cancer cell lines

Hazelnut shells, a by-product of the kernel industry processing, are reported to contain high amount of polyphenols. However, studies on the chemical composition and potential effects on human health are lacking. A methanol hazelnut shells extract was prepared and dried. Our investigation allowed the isolation and characterization of different classes of phenolic compounds, including neolignans, and a diarylheptanoid, which contribute to a high total polyphenol content (193.8 ± 3.6 mg of gallic acid equivalents (GAE)/g of extract). Neolignans, lawsonicin and cedrusin, a cyclic diarylheptanoid, carpinontriol B, and two phenol derivatives, C-veratroylglycol, and β-hydroxypropiovanillone, were the main components of the extract (0.71%-2.93%, w/w). The biological assays suggested that the extract could be useful as a functional ingredient in food technology and pharmaceutical industry showing an in vitro scavenging activity against the radical 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (EC50 = 31.7 µg/mL with respect to α-tocopherol EC50 = 10.1 µg/mL), and an inhibitory effect on the growth of human cancer cell lines A375, SK-Mel-28 and HeLa (IC50 = 584, 459, and 526 µg/mL, respectively). The expression of cleaved forms of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) suggested that the extract induced apoptosis through caspase-3 activation in both human malignant melanoma (SK-Mel-28) and human cervical cancer (HeLa) cell lines. The cytotoxic activity relies on the presence of the neolignans (balanophonin), and phenol derivatives (gallic acid), showing a pro-apoptotic effect on the tested cell lines, and the neolignan, cedrusin, with a cytotoxic effect on A375 and HeLa cells

Hydroxytyrosol Reduces Foam Cell Formation and Endothelial Inflammation Regulating the PPARγ/LXRα/ABCA1 Pathway.

Cholesterol accumulation in macrophages leads to the formation of foam cells and increases the risk of developing atherosclerosis. We have verified whether hydroxytyrosol (HT), a phenolic compound with anti-inflammatory and antioxidant properties, can reduce the cholesterol build up in THP-1 macrophage-derived foam cells. We have also investigated the potential mechanisms. Oil Red O staining and high-performance liquid chromatography (HPLC) assays were utilized to detect cellular lipid accumulation and cholesterol content, respectively, in THP-1 macrophages foam cells treated with HT. The impact of HT on cholesterol metabolism-related molecules (SR-A1, CD36, LOX-1, ABCA1, ABCG1, PPARγ and LRX-α) in foam cells was assessed using real-time PCR (RT-qPCR) and Western blot analyses. Finally, the effect of HT on the adhesion of THP-1 monocytes to human vascular endothelial cells (HUVEC) was analyzed to study endothelial activation. We found that HT activates the PPARγ/LXRα pathway to upregulate ABCA1 expression, reducing cholesterol accumulation in foam cells. Moreover, HT significantly inhibited monocyte adhesion and reduced the levels of adhesion factors (ICAM-1 and VCAM-1) and pro-inflammatory factors (IL-6 and TNF-α) in LPS-induced endothelial cells. Taken together, our findings suggest that HT, with its ability to interfere with the import and export of cholesterol, could represent a new therapeutic strategy for the treatment of atherosclerotic disease

French Roadmap for complex Systems 2008-2009

This second issue of the French Complex Systems Roadmap is the outcome of the Entretiens de Cargese 2008, an interdisciplinary brainstorming session organized over one week in 2008, jointly by RNSC, ISC-PIF and IXXI. It capitalizes on the first roadmap and gathers contributions of more than 70 scientists from major French institutions. The aim of this roadmap is to foster the coordination of the complex systems community on focused topics and questions, as well as to present contributions and challenges in the complex systems sciences and complexity science to the public, political and industrial spheres

Lumacaftor and Matrine: Possible Therapeutic Combination to Counteract the Inflammatory Process in Cystic Fibrosis

Cystic fibrosis is a monogenic, autosomal, recessive disease characterized by an alteration of chloride transport caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The loss of Phe residue in position 508 (ΔF508-CFTR) causes an incorrect folding of the protein causing its degradation and electrolyte imbalance. CF patients are extremely predisposed to the development of a chronic inflammatory process of the bronchopulmonary system. When the cells of a tissue are damaged, the immune cells are activated and trigger the production of free radicals, provoking an inflammatory process. In addition to routine therapies, today drugs called correctors are available for mutations such as ΔF508-CFTR as well as for others less frequent ones. These active molecules are supposed to facilitate the maturation of the mutant CFTR protein, allowing it to reach the apical membrane of the epithelial cell. Matrine induces ΔF508-CFTR release from the endoplasmic reticulum to cell cytosol and its localization on the cell membrane. We now have evidence that Matrine and Lumacaftor not only restore the transport of mutant CFTR protein, but probably also counteract the inflammatory process by improving the course of the disease

Vx-809, a CFTR Corrector, Acts through a General Mechanism of Protein Folding and on the Inflammatory Process

Correct protein folding is the basis of cellular well-being; thus, accumulation of misfolded proteins within the endoplasmic reticulum (ER) leads to an imbalance of homeostasis that causes stress to the ER. Various studies have shown that protein misfolding is a significant factor in the etiology of many human diseases, including cancer, diabetes, and cystic fibrosis. Misfolded protein accumulation in the ER triggers a sophisticated signal transduction pathway, the unfolded protein response (UPR), which is controlled by three proteins, resident in ER: IRE1α, PERK, and ATF6. Briefly, when ER stress is irreversible, IRE1α induces the activation of pro-inflammatory proteins; PERK phosphorylates eIF2α which induces ATF4 transcription, while ATF6 activates genes encoding ER chaperones. Reticular stress causes an alteration of the calcium homeostasis, which is released from the ER and taken up by the mitochondria, leading to an increase in the oxygen radical species production, and consequently, to oxidative stress. Accumulation of intracellular calcium, in combination with lethal ROS levels, has been associated with an increase of pro-inflammatory protein expression and the initiation of the inflammatory process. Lumacaftor (Vx-809) is a common corrector used in cystic fibrosis treatment which enhances the folding of mutated F508del-CFTR, one of the most prevalent impaired proteins underlying the disease, promoting a higher localization of the mutant protein on the cell membrane. Here, we demonstrate that this drug reduces the ER stress and, consequently, the inflammation that is caused by such events. Thus, this molecule is a promising drug to treat several pathologies that present an etiopathogenesis due to the accumulation of protein aggregates that lead to chronic reticular stress

Overexpression of hsa-miR-663a reduces expression of PLOD3 encoded LH3 protein and secretion of Collagen IV

In the past decade, microRNAs have been uncovered as key regulators of gene expression at post-transcriptional level by the mean of their ability to interact with complementary regions of transcripts and gain repression of translation or RNA degradation. In the attempt to discover novel miRNA induced by ER stress and identify their target genes we performed a combined analysis by the use of miRNA array and 2D-DIGE/mass-spectrometry approach. Comparative analysis of the microRNA profiles revealed that, in the cells exposed to the ER stress inducer Thapsigargin (TG), twenty-four miRNAs are differentially expressed. Among them, hsa-miR-663a turned out to be the most significant TG induced miR. Interestingly, ontological analysis showed that about 26% of predicted miR-663a target genes are related to secretory pathway functions. To identify, miR-663a target genes we performed proteomic analysis by the combined use of 2D-DIGE and mass spectrometry of cells over-expressing miR-663a revealing that miR-663a could potentially target a number of genes having a function within the secretory pathway. In particular, we show that miR-663a, directly targets PLOD3 mRNA encoding Lysyl hydroxylase 3 (LH3) protein, a multifunctional collagen-modifying enzyme. In miR-663a over expressing cells, miR-663a reduces LH3 expression. Reduction of the galactosylhydroxylysine-glucosyltransferase (GGT) activity of LH3 disrupts the localization of type IV collagen, which showed retention in the Endoplasmic Reticulum. Our results suggest that miR-663a could modulate collagen secretion in pathophysiological conditions

Trastuzumab and Doxorubicin Sequential Administration Increases Oxidative Stress and Phosphorylation of Connexin 43 on Ser368

Human epidermal growth factor receptor-2 (HER2) is overexpressed in up to 30% of breast cancer cases, causing a more aggressive tumour growth and poor prognosis. Trastuzumab, the humanized antibody targeted to HER2, increased the life expectancy of patients, but severe cardiotoxicity emerged as a long-term adverse effect. Clinical evidence highlights that Trastuzumab-induced cardiotoxicity drastically increases in association with Doxorubicin; however, the exact mechanisms involved remain incompletely understood. In order to analyse the molecular mechanisms involved and the possible adaptative responses to Trastuzumab and Doxorubicin treatment, in this study, H9c2 cardiomyoblasts were used. Results showed that Trastuzumab and Doxorubicin sequential administration in cardiomyoblast increased cytosolic and mitochondrial ROS production, intracellular calcium dysregulation, mitochondrial membrane depolarization, and the consequent apoptosis, induced by both Trastuzumab and Doxorubicin alone. Furthermore, in these conditions, we observed increased levels of Connexin43 phosphorylated on Ser368 (pCx43). Since phosphorylation on Ser368 decreases gap junction intracellular communication, thus reducing the spread of death signals to adjacent cells, we hypothesized that the increase in pCx43 could be an adaptative response implemented by cells to defend neighbouring cells by Trastuzumab and Doxorubicin sequential administration. However, the other side of the coin is the resulting conduction abnormalities

Endoplasmic reticulum stress reduces COPII vesicles formation and modifies Sec23a cycling at ERESs G. Amodio1, O. Moltedo1, S. Franceschelli1, P. Remondelli2 1Dipartimento di Farmacia, Univ. of Salerno, Italy 2Dipartimento di Medicina e Chirurgia, Univ. of Salerno, Italy

COPII vesicles bud from the ER at ER Exit Sites (ERESs) to mediate the exit from the Endoplasmic Reticulum (ER) of newly synthesized proteins. Previously, we demonstrated that ER Stress rapidly impairs the anterograde transport to the Golgi complex and the formation of COPII vesicles (Amodio et al., 2009). In a recent work (Amodio et al., 2013) we found that the reduced permanence of Sec23a at the ERES could be the mean through which ER Stress modulates COPII assembling and vesicular trafficking. Sec23a is one of the component of the COPII vesicles coat and its GTPase activating function on Sar1 is one of the key mechanisms of COPII assembly. Interestingly, we found that during ER Stress the association to the ER membrane of Sec23a is reduced. Concomitantly, FRAP and FLIP analysis of Sec23a revealed that ER stress accelerates its recycling kinetics on ER membrane. These results prompted us to analyze the role of post-translational modifications of Sec23a in the regulation of its function during ER Stress. Surprisingly, we found that Sec23a is mono-ubiquitinated in mammalian cells on two different cysteines and that the induction of ER stress reduces the amount of mono-ubiquitinated Sec23a. The biological scope of Sec23a cysteine mono-ubiquitination has yet to be elucidated but recent evidences demonstrating that ubiquitination on cysteines regulates signal transduction and membrane translocation (Grou et al., 2008; Shannon and Weerapana, 2013) open new fields of investigation about Sec23a ubiquitination and modulation of COPII function