CDO,an Hh-Coreceptor,Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling

Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling.     

Enhanced Device Efficiency of Bilayered Inverted Organic Solar Cells Based on Photocurable P3HTs with a Light‐Harvesting ZnO Nanorod Array

Periodically patterned zinc oxide nanorod (P‐ZnO NR) layers are directly prepared from a pre‐patterned ZnO seed layer using a polydimethylsiloxane (PDMS) elastomeric stamp and then applied in inverted organic photovoltaic devices (IOPVs). The IOPV is assembled with a hydrothermally grown zinc oxide nanorod patterns with a (100) preferential crystal orientation as an electron transport buffer layer (ETBL) and photoactive bilayer consisting of methacylate end‐functionalized poly(3‐hexylthiophene) (P3HT‐MA), phenyl‐C₆₀‐butyric acid methyl ester (PC₆₀BM) and indene‐C₆₀ bis‐adduct (IC₆₀BA). In te IOPVs, the P‐ZnO NR is found to induce efficient light harvesting and the photocrosslinkable P3HTs afford solution‐processed bilayer architecture in IOPVs to show improved device stability and performance (PCE_max= 5.95%), as the bilayered structure allowed direct exciton splitting, thus reducing the charge recombination.     

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade

Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536]     

S-adenosyl methionine prevents endothelial dysfunction by inducing heme oxygenase-1 in vascular endothelial cells

S-adenosyl methionine (SAM) is a key intermediate in the metabolism of sulfur amino acids and is a major methyl donor in the cell. Although the low plasma level of SAM has been associated with atherosclerosis, the effect of SAM administration on atherosclerosis is not known. Endothelial dysfunction is an early prerequisite for atherosclerosis. This study was undertaken to investigate the possible preventive effect of SAM on endothelial dysfunction and the molecular mechanism of its action. SAM treatment prevented endothelial dysfunction in high fat diet (HFD)-fed rats. In cultured human aortic endothelial cells, linoleic acid (LA) increased and SAM decreased cell apoptosis and endoplasmic reticulum stress. Both LA and SAM increased heme oxygenase-1 (HO-1) expression in an NF-E2-related factor 2-dependent manner. However, knockdown of HO-1 reversed only the SAM-induced preventive effect of cell apoptosis. The LA-induced HO-1 expression was dependent on PPARα, whereas SAM induced HO-1 in a PPAR-independent manner. These data demonstrate that SAM treatment prevents endothelial dysfunction in HFDfed animals by inducing HO-1 in vascular endothelial cells. In cultured endothelial cells, SAM-induced HO-1 was responsible for the observed prevention of cell apoptosis. We propose that SAM treatment may represent a new therapeutic strategy for atherosclerosis.     

Analysis of the novel VNTR polymorphisms ofMUC8 gene

Analysis of mucin genes has identified the presence of several features that may represent important functional domains in mucin glycoproteins. In the central region of each mucin, there are a variable number of tandem repeats (VNTR; minisatellites). However, their genomic levels are unclear because of complex genomic properties. We report here the distribution of VNTR and polymorphic analysis ofMUC8. We searched for VNTR ofMUC8 using the Tandem Repeat Finder program and found nine VNTR motif. Six (MUC8 MS1～MS6) among the nine VNTRs were evaluated in this study. Each VNTR inMUC8 region was analyzed in genomic DNA obtained from 200 unrelated individuals and multi-generational families. All VNTRs (MUC8 MS1, -MS2, -MS3, -MS4,-MS5 and -MS6) were genotyped as polymorphic. The degree of polymorphism within theMUC8-MS5 showed the highest heterozygosity (h = 0.786) in theMUC8 region. In order to perform a segregation analysis of the VNTRs inMUC8, we analyzed genomic DNA obtained from two generations of five families and from three generations of two families. Six of the polymorphic VNTRs were transmitted through meiosis following a Mendelian inheritance, which suggests that polymorphic VNTRs could be useful markers for paternity mapping and DNA fingerprinting.     

S-Palmitoylation of ��-Secretase Subunits Nicastrin and APH-1

Proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases generates β-amyloid (Aβ) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Aβ production. Previously, we and others reported that the four integral subunits of the γ-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of γ-secretase subunits. We report that in cultured cells and in brain the γ-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 is S-palmitoylated at Cys¹⁸² and Cys²⁴⁵. S-Palmitoylation-defective nicastrin and APH-1 form stable γ-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for γ-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Aβ40, Aβ42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate γ-secretase processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that cause dementia. This debilitating disorder is pathologically characterized by the cerebral deposition of 39–42 amino acid peptides termed Aβ, which are generated by proteolytic processing of amyloid precursor protein (APP)² by β- and γ-secretases (1, 2). The β-site APP cleavage enzyme 1 cleaves full-length APP within its luminal domain to generate a secreted ectodomain leaving behind a C-terminal fragment (β-CTF). γ-Secretase cleaves β-CTF within the transmembrane domain to release Aβ and APP intracellular C-terminal domain (AICD). γ-Secretase is a multiprotein complex, comprising at least four subunits: presenilins (PS1 and PS2), nicastrin, APH-1, and PEN-2 for its activity (3). PS1 is synthesized as a 42–43-kDa polypeptide and undergoes highly regulated endoproteolytic processing within the large cytoplasmic loop domain connecting putative transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and C-terminal fragments (CTF) by an uncharacterized proteolytic activity (4). This endoproteolytic event has been identified as the activation step in the process of PS1 maturation as it assembles with other γ-secretase subunits (3). Nicastrin is a heavily glycosylated type I membrane protein with a large ectodomain that has been proposed to function in substrate recognition and binding (5), but this putative function has not been confirmed by others (6). APH-1 is a seven-transmembrane protein encoded by two human or three rodent genes that are alternatively spliced (7). Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for γ-secretase processing of APP, a type I membrane protein, termed p23 (also referred toTMP21), was recently identified as a γ-secretase component that modulates γ-secretase activity and regulates secretory trafficking of APP (8, 9).A growing number of type I integral membrane proteins has been identified as γ-secretase substrates within the last few years, including Notch1 homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin receptor DCC, and others (10). Mounting evidence suggests that APP processing occurs within cholesterol- and sphingolipid-enriched lipid rafts, which are biochemically defined as detergentresistant membrane microdomains (DRM) (11, 12). Previously we reported that each of the γ-secretase subunits localizes in lipid rafts in post-Golgi and endosome membranes enriched in syntaxin 6 (13). Moreover, loss of γ-secretase activity by gene deletion or exposure to γ-secretase inhibitors results in the accumulation of APP CTFs in lipid rafts indicating that cleavage of APP CTFs likely occurs in raft microdomains (14). In contrast, CTFs derived from Notch1, Jagged2, N-cadherin, and DCC are processed by γ-secretase in non-raft membranes (14). The mechanisms underlying association of γ-secretase subunits with lipid rafts need further clarification to elucidate spatial segregation of amyloidogenic processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as a potential mechanism for regulating raft association, stability, intracellular trafficking, and function of several cytosolic and transmembrane proteins (15–17). S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety to certain cysteine residues through thioester linkage. Cysteines close to transmembrane domains or membrane-associated domains in non-integral membrane proteins are preferred S-palmitoylation sites, although no conserved motif has been identified (18). Palmitoylation modifies numerous neuronal proteins, including postsynaptic density protein PSD-95 (19), a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors (20), nicotinic α7 receptors (21), neuronal t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin (22, 23), neuronal growth-associated protein GAP-43 (24), protein kinase CLICK-III (CL3)/CaMKIγ (25), β-secretase (26), and Huntingtin (27). Although palmitoylation can occur in vitro without the involvement of an enzyme, a family of palmitoyltransferases that specifically catalyze S-palmitoylation has been identified (28, 29).In this study, we have identified S-palmitoylation of γ-secretase subunits nicastrin and APH-1, and characterized its role on DRM association, protein stability, and γ-secretase enzyme activities. We show that nicastrin is S-palmitoylated at Cys⁶⁸⁹, and APH-1 at Cys¹⁸² and Cys²⁴⁵. Mutagenesis of palmitoylation sites results in increased degradation of nascent nicastrin and APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured cells overexpression of S-palmitoylation-deficient nicastrin and APH-1 does not modulate γ-secretase processing of APP or other substrates.     

Role of stretch-activated channels on the stretch-induced changes of rat atrial myocytes

The role of stretch-activated channels (SACs) on the stretch-induced changes of rat atrial myocytes was studied using a computer model that incorporated various ion channels and transporters including SACs. A relationship between the extent of the stretch and the activation of SACs was formulated in the model based on experimental findings to reproduce changes in electrical activity and Ca²⁺ transients by stretch. Action potentials (APs) were significantly changed by the activation of SACs in the model simulation. The duration of the APs decreased at the initial fast phase and increased at the late slow phase of repolarisation. The resting membrane potential was depolarised from −82 to −70 mV. The Ca²⁺ transients were also affected. A prolonged activation of SACs in the model gradually increased the amplitude of the Ca²⁺ transients. The removal of Ca²⁺ permeability through SACs, however, had little effect on the stretch-induced changes in electrical activity and Ca²⁺ transients in the control condition. In contrast, the removal of the Na⁺ permeability nearly abolished these stretch-induced changes. Plotting the peaks of the Ca²⁺ transients during the activation of the SACs along a time axis revealed that they follow the time course of the Na_i⁺ concentration. The Ca²⁺ transients were not changed when the Na_i⁺ concentration was fixed to a control value (5.4 mM). These results predicted by the model suggest that the influx of Na⁺ rather than Ca²⁺ through SACs is more crucial to the generation of stretch-induced changes in the electrical activity and associated Ca²⁺ transients of rat atrial myocytes.     

Lipid raft cholesterol and genistein inhibit the cell viability of prostate cancer cells via the partial contribution of EGFR-Akt/p70S6k pathway and down-regulation of androgen receptor

Soy isoflavones and cholesterol have been reported as dietary factors related to the incidence of prostate cancer. In this study, we investigated whether cell survival could be suppressed by a combination of the dispersion of lipid raft microdomains and treatment with genistein, a well-known potential isoflavone, in LNCaP prostate cancer cells. Cell viability was assayed by the property of reagent change upon reduction of resazurin to resorufin and apoptosis was evaluated by ethidium bromide/acridine orange (EB/AO) staining and PARP and caspase-3 expression. Signal transduction was investigated by immunoblot analysis. Cell viability decreased significantly more following successive double treatment with genistein and the cholesterol-lowering agent 2-hydroxypropyl-beta-cyclodextrin (HPCD) than in response to either agent alone. Apoptotic cell staining and cleavage of PARP and caspase-3 appeared more clearly in double-treated cells than in those treated with genistein alone. In cell signaling, both HPCD and genistein decreased the protein expressions of pAkt as well as the androgen receptors stimulated by EGF and DHT, respectively, in concentration-dependent manners. This pattern was also present in protein levels of pAkt and the androgen receptor located in the lipid raft fraction. Furthermore, the phosphorylation cascade of Akt, GSK-3β and p70S6k was markedly inhibited by the combination treatment. These data suggest that prostate cancer cells could be effectively inhibited by combination treatment of cholesterol-lowering strategies and genistein. The mechanism is likely to be partially via both the EGFR-mediated Akt or p70S6k pathways and a down-regulation of androgen receptor in the lipid raft microdomain.