Person: Tachado, Souvenir
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Tachado
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Souvenir
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Tachado, Souvenir
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Publication Cigarette smoke induces nuclear translocation of heme oxygenase 1 (HO-1) in prostate cancer cells: Nuclear HO-1 promotes vascular endothelial growth factor secretion(D.A. Spandidos, 2013) Birrane, Gabriel; LI, HUCHUN; Yang, Suping; Tachado, Souvenir; Seng, SeyhaProstate cancer is the second leading cause of male-cancer related death in the United States. Despite a number of evidence-based studies which strongly suggest an association between cigarette smoking and prostate cancer, the underlying biological mechanism is largely unknown. Heme oxygenase 1 (HO-1) has been implicated in maintaining cellular homeostasis, but also in tumor angiogenesis. Nuclear HO-1 protein expression has been observed in various types of tumors including prostate cancer. These studies, however, were reported as clinical and pathological observations, and failed to investigate nuclear HO-1 at the molecular level in cancer. The present study explores the relationship between cigarette smoke and nuclear HO-1-modulated promotion of vascular endothelial growth factor (VEGF) secretion. We have demonstrated that cigarette smoke medium (SM)-induced HO-1 mRNA expression and upregulated HO-1 protein levels in the prostate cancer cell lines DU145 and PC3. We also observed that SM significantly induced nuclear expression of HO-1, and enhanced secretion of VEGF in cells. Nuclear-directed expression of HO-1 activated the transcriptional activity of VEGF and promoted VEGF secretion in prostate cancer cells. This study provides new insights into the molecular mechanism by which cigarette smoke-induced nuclear translocation of HO-1 promotes VEGF secretion in prostate cancer cells. Nuclear HO-1 may, therefore, constitute an attractive therapeutic target to inhibit angiogenesis and the progression of prostate cancer.Publication HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation(Public Library of Science, 2014) Bernard, Mark A.; Han, Xinbing; Inderbitzin, Sonya; Agbim, Ifunanya; Zhao, Hui; Koziel, Henry; Tachado, SouvenirEven though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients.Publication Cigarette smoke modulates PC3 prostate cancer cell migration by altering adhesion molecules and the extracellular matrix(D.A. Spandidos, 2015) YANG, SUPING; LONG, MINICA; Tachado, Souvenir; SENG, SEYHAProstate cancer (PCa) is the second leading cause of cancer-related mortality among American males. Studies suggest that cigarette smoking is associated with the progression of PCa; however, the molecular mechanisms underlying this process have not been extensively investigated. PCa progression is characterized by increased cell migration and alterations in extracellular matrix (ECM)- and cell adhesion molecule (CAM)-related gene expression. In the present study, the influence of cigarette smoke medium (SM) on cell migration and on the expression of ECM- and CAM-related genes in PC3 prostate adenocarcinoma cells was investigated. According to a wound-healing assay, SM treatment promoted PC3 cell migration. RNA expression levels from SM-treated and control cells were analyzed using a polymerase chain reaction (PCR) array. Of 84 genes analyzed, 27.38% (23/84) exhibited a ≥2-fold change in threshold cycle in PC3 cells following 0.5% SM treatment. Functional gene grouping analysis demonstrated that SM treatment modulated the RNA transcription of approximately 18.4% of CAMs and 33.93% of ECM-related genes. Quantitative PCR analysis showed that SM treatment led to a significant decrease in transcription levels of the following genes: Collagen 5 α-1(V), connective tissue growth factor, integrin β-2, kallmann syndrome 1, laminin α 3, matrix metallopeptidase 7 (MMP7), MMP13, secreted protein acidic cysteine-rich, thrombospondin-2 and versican; and that SM significantly increased the transcription levels of MMP2 and MMP12. Furthermore, MMP2 knockdown significantly reduced the migration of SM-treated PC3 cells. The present study provides novel insights into the association of cigarette smoking with PCa progression, via the alteration of ECM/CAM interactions.Publication DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes(Public Library of Science, 2015) Bernard, Mark A.; Wang, Leyu; Tachado, SouvenirMechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM) with substrate inhibition kinetics (Ki=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.Publication \(Leu128^{3.43}\) (L128) and \(Val247^{6.40}\) (V247) of CXCR1 Are Critical Amino Acid Residues for G Protein Coupling and Receptor Activation(Public Library of Science, 2012) Han, Xinbing; Tachado, Souvenir; Koziel, Henryk; Boisvert, William A.CXCR1, a classic GPCR that binds IL-8, plays a key role in neutrophil activation and migration by activating phospholipase C (PLC)β through \(G\alpha_{15}\) and \(G\alpha_i\) which generates diacylglycerol and inositol phosphates (IPs). In this study, two conserved amino acid residues of CXCR1 on the transmembrane domain (TM) 3 and TM6, \(Leu128^{3.43}\) (L128) and \(Val247^{6.40}\) (V247), respectively, were selectively substituted with other amino acids to investigate the role of these conserved residues in CXCR1 activation. Although two selective mutants on Leu128, Leu128Ala (L128A) and Leu128Arg (L128R), demonstrated high binding affinity to IL-8, they were not capable of coupling to G proteins and consequently lost the functional response of the receptors. By contrast, among the four mutants at residue Val247 (TM6.40), replacing Val247 with Ala (V247A) and Asn (V247N) led to constitutive activation of mutant receptors when cotransfected with \(G\alpha_{15}\). The V247N mutant also constitutively activated the \(G\alpha_i\) protein. These results indicate that L128 on TM3.43 is involved in G protein coupling and receptor activation but is unimportant for ligand binding. On the other hand, V247 on TM6.40 plays a critical role in maintaining the receptor in the inactive state, and the substitution of V247 impaired the receptor constraint and stabilized an active conformation. Functionally, there was an increase in chemotaxis in response to IL-8 in cells expressing V247A and V247N. Our findings indicate that \(Leu128^{3.43}\) and \(Val247^{6.40}\) are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation.