技术资料

Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents,including organelles and long-lived proteins,that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62,a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal,a singlet oxygen producer structurally unrelated to verteporfin,also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly,small amounts of crosslinked p62 oligomers were detected in untreated cells,and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy. View Publication

BACKGROUND AND AIM An oral trypsin inhibitor,camostat (CM),has a beneficial effect on chronic pancreatitis,but its mechanism is not yet fully understood. Recently,pancreatic stellate cells (PSC) have been reported to play an essential role in pancreatic fibrosis. An experimental model of pancreatic fibrosis induced by a superoxide dismutase (SOD) inhibitor (diethyldithiocarbamate [DDC]) was developed in rats. Thus,the effect of an oral trypsin inhibitor on pancreatic fibrosis and PSC was investigated. METHODS Pancreatic fibrosis was induced in rats using DDC (DDC rats). DDC + CM rats were administered DDC,and subsequently were fed a diet containing CM. Immunohistochemistry of the pancreas was performed with monoclonal anti-alpha-smooth muscle actin (alpha-SMA) antibody and anti-desmin antibody. RESULTS The DDC rats showed a significant increase in alpha-SMA-positive cells or desmin-positive cells compared with control rats. These significant increases in the fibrotic area improved after treatment with CM. The level of prolyl hydroxylase in the pancreas,which significantly increased as a result of DDC,decreased after treatment with CM. CONCLUSION Camostat has a beneficial effect on pancreatic fibrosis induced by the administration of a SOD inhibitor,which inhibits the proliferation and activation of PSC. View Publication

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO,Ac-DEVD-CHO,Ac-YVAD-CHO,t-butoxycarbonyl-IETD-CHO,and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes,with dissociation constants ranging from 75 pM to {\textgreater}10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor,with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki {\textless} 20 nM) and selective inhibitor of Group I caspases (caspase-1,-4,and -5) and most Group III caspases (caspase-8,-9,and -10),suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response. View Publication

Camostat mesilate (CM),an oral protease inhibitor,has been used clinically for the treatment of chronic pancreatitis in Japan. However,the mechanism by which it operates has not been fully understood. Our aim was to evaluate the therapeutic efficacy of CM in the experimental pancreatic fibrosis model induced by dibutyltin dichloride (DBTC),and we also determined the effect of CM on isolated monocytes and panceatic stellate cells (PSCs). In vivo,chronic pancreatitis was induced in male Lewis rats by single administration of 7 mg/kg DBTC and a special diet containing 1 mg/g CM was fed to the DBTC+CM-treated group from day 7,while the DBTC-treated group rats were fed a standard diet. At days 0,7,14 and 28,the severity of pancreatitis and fibrosis was examined histologically and enzymologically in both groups. In vitro,monocytes were isolated from the spleen of a Lewis rat,and activated with lipopolysaccharide stimulation. Thereafter,the effect of CM on monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) production from monocytes was examined. Subsequently,cultured rat PSCs were exposed to CM and tested to see whether their proliferation,MCP-1 production and procollagen alpha1 messenger RNA expression was influenced by CM. In vivo,the oral administration of CM inhibited inflammation,cytokines expression and fibrosis in the pancreas. The in vitro study revealed that CM inhibited both MCP-1 and TNF-alpha production from monocytes,and proliferation and MCP-1 production from PSCs. However,procollagen alpha1 expression in PSCs was not influenced by CM. These results suggest that CM attenuated DBTC-induced rat pancreatic fibrosis via inhibition of monocytes and PSCs activity. View Publication

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties,yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME,we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway,possibly by modulating availability of hyaluronic acid (HA),the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships,and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions,respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction,whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo,targeting CD44 decreased PI3K-4EBP1-SOX2 signaling,tumor growth,and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally,a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes,respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44,TAM,and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery. View Publication

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however,most patients across cancer types still fail to respond. Consequently,there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy,which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable,we designed an $\alpha$HER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models,desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice,an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus,antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy. View Publication

View Publication

BACKGROUND Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium,especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-$\beta$1,which plays a role in recruitment and activation of inflammatory cells,extracellular matrix (ECM) production and fibrosis development,and is a potent inducer of EMT. OBJECTIVE As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells,the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. METHODS We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. RESULTS Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-$\beta$1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. CONCLUSIONS AND CLINICAL RELEVANCE As an inducer of EMT and an important source of TGF-$\beta$1,neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways. View Publication

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication

Perhexiline maleate,originally classified as a calcium antagonist,is in use as an antianginal agent. The mechanism of its protective effect is unknown,but there is speculation that it involves a modification of myocardial substrate utilization,in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration,as follows: acetoacetate,glucose,lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization,which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption,which suggested an improvement in myocardial efficiency. Thus,it was confirmed by direct measurement that this drug does modify substrate oxidation,which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial. View Publication

The endoplasmic reticular Ca(2+)-ATPase inhibitor,thapsigargin,was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition,confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator,bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus,we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis. View Publication

The mechanism of the anti-anginal effect of perhexiline is unclear but appears to involve a shift in cardiac metabolism from utilization of fatty acid to that of carbohydrate. We tested the hypothesis that perhexiline inhibits the enzyme carnitine palmitoyltransferase-1 (CPT-1),which controls access of long chain fatty acids to the mitochondrial site of beta-oxidation. Perhexiline produced a concentration-dependent inhibition of CPT-1 in rat cardiac and hepatic mitochondria in vitro,with half-maximal inhibition (IC50) at 77 and 148 mumol/L,respectively. Amiodarone,another drug with anti-anginal properties,also inhibited cardiac CPT-1 (IC50 = 228 mumol/L). The rank order of potency for inhibition was malonyl-CoA {\textgreater} 4-hydroxyphenylglyoxylate (HPG) = perhexiline {\textgreater} amiodarone = monohydroxy-perhexiline. Kinetic analysis revealed competitive inhibition of cardiac and hepatic CPT-1 by perhexiline with respect to palmitoyl-CoA but non-competitive inhibition with respect to carnitine. Curvilinear Dixon plots generated apparent inhibitory constant (Ki)" values for perhexiline which indicated a greater sensitivity of the cardiac than the hepatic enzyme to inhibition by perhexiline. Perhexiline inhibition of CPT-1 unlike that of malonyl-CoA and HPG was unaffected by pretreatment with the protease nagarse. These data establish for the first time that two agents with proven anti-anginal effects inhibit cardiac CPT-1. This action is likely to contribute to the anti-ischaemic effects of both perhexiline and amiodarone." View Publication