技术资料

Deregulation of apoptosis,the physiological form of cell death,is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways,both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described,the actual molecular events triggering the mitochondrial pathway are largely unknown. Here,we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound,designated FK866,which induces apoptosis by highly specific,noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT),a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues,however,have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT,whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept,FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM,did not directly inhibit mitochondrial respiratory activity,but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme,when partially purified from K562 human leukemia cells,was noncompetitively inhibited by FK866,and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'),respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore,it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days. View Publication

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence,this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes,Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution,the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay,which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2{\%} vs. 9.9{\%},p {\textgreater} 0.05). However,when 100 mM Z-VAD-FMK was added to the post-thaw culture media,there was a significant enhancement in the survival rate from 9.9{\%} to 14.4{\%} (p {\textless} 0.05),which was further increased to 18.7{\%} when Z-VAD-FMK was also added to the freezing solution as well (p {\textless} 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy,and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture. View Publication

The recent emergence of the novel,pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here,we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally,we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. View Publication

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density,but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model,enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common ($\Delta$F508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether,targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking,but also introduces a new tool for deconstructing the ubiquitin code in situ. View Publication

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor,oxfenicine (0.5 mM),in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95{\%} flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p {\textless} 0.003,p {\textless} 0.0002,respectively) increases in diastolic tension during ischemia,without significant effects on developed tension,or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC),products of CPT-1 action,were decreased (p {\textless} 0.05) by oxfenicine,unaffected by 2 microM perhexiline,and increased slightly by 0.5 microM perhexiline. Perhexiline,but not the active metabolite of oxfenicine,also inhibited cardiac CPT-2 with similar IC50 and Emax,although lower Hill slope,compared with CPT-1. Oxfenicine,but not perhexiline,reduced concentrations of the endogenous CPT-1 inhibitor,malonyl-CoA. Perhexiline,but not oxfenicine,inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model,independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2. View Publication

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation,injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate,Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence,we hypothesized that CDDO-Me would reduce lung inflammation,fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis,mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation,while fibrosis and lung function were evaluated on day 21. On day 7,CDDO-Me reduced total BALF protein by 50{\%},alveolar macrophage infiltration by 40{\%},neutrophil infiltration by 90{\%} (p≤0.01),inhibited production of the inflammatory cytokines KC and IL-6 by over 90{\%} (p≤0.001),and excess production of the pro-fibrotic cytokine TGF$\beta$ by 50{\%}. CDDO-Me also inhibited $\alpha$-smooth muscle actin and fibronectin mRNA by 50{\%} (p≤0.05). On day 21,CDDO-Me treatment reduced histological fibrosis,collagen deposition and $\alpha$SMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me,as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases. View Publication

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately,tumor cells acquire PARPi resistance,which is usually associated with the restoration of homologous recombination,loss of PARP1 expression,and/or loss of DNA double-strand break (DSB) end resection regulation. Here,we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGF$\beta$R) kinase on malignant cells,which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-$\beta$1). Genetic and/or pharmacological targeting of the TGF-$\beta$1-TGF$\beta$R kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGF$\beta$R inhibitor in patients receiving PARPis. View Publication

The Drosophila TEAD ortholog Scalloped is required for Yki-mediated overgrowth but is largely dispensable for normal tissue growth,suggesting that its mammalian counterpart may be exploited for selective inhibition of oncogenic growth driven by YAP hyperactivation. Here we test this hypothesis genetically and pharmacologically. We show that a dominant-negative TEAD molecule does not perturb normal liver growth but potently suppresses hepatomegaly/tumorigenesis resulting from YAP overexpression or Neurofibromin 2 (NF2)/Merlin inactivation. We further identify verteporfin as a small molecule that inhibits TEAD-YAP association and YAP-induced liver overgrowth. These findings provide proof of principle that inhibiting TEAD-YAP interactions is a pharmacologically viable strategy against the YAP oncoprotein. View Publication

COVID-19 pathogenesis is associated with an exaggerated immune response. However,the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here,we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio,elevated serum IL-6,MCP-1 and IP-10,and most strikingly,modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme,COX-2,as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover,they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints. View Publication

During surgery,ATP from damaged cells induces the release of interleukin-1$\beta$,a potent pro-inflammatory cytokine that contributes to the development of postoperative systemic inflammation,sepsis and multi-organ damage. We recently demonstrated that C-reactive protein (CRP) inhibits the ATP-induced release of monocytic interleukin-1$\beta$,although high CRP levels are deemed to be a poor prognostic marker. Here,we retrospectively investigated if preoperative CRP levels correlate with postoperative CRP,leukocyte counts and fever in the context of anatomical lung resection and systematic lymph node dissection as first line lung cancer therapy. No correlation was found in the overall results. In men,however,preoperative CRP and leukocyte counts positively correlated on postoperative days one to two,and a negative correlation of CRP and fever was seen in women. These correlations were more pronounced in men taking statins and in statin-na{\{i}}ve women. Accordingly the inhibitory effect of CRP on the ATP-induced interleukin-1$\beta$ release was blunted in monocytes from coronary heart disease patients treated with atorvastatin compared to monocytes obtained before medication. Hence the common notion that elevated CRP levels predict more severe postoperative inflammation should be questioned. We rather hypothesize that in women and statin-na{\"{i}}ve patients high CRP levels attenuate trauma-induced increases in inflammatory markers.""" View Publication

The pancreatic adenocarcinoma is an aggressive and devastating disease,which is characterized by invasiveness,rapid progression,and profound resistance to actual treatments,including chemotherapy and radiotherapy. At the moment,surgical resection provides the best possibility for long-term survival,but is feasible only in the minority of patients,when advanced disease chemotherapy is considered,although the effects are modest. Several studies have shown that thyroid hormone,3,3',5-triiodo-l-thyronine (T(3)) is able to promote or inhibit cell proliferation in a cell type-dependent manner. The aim of the present study is to investigate the ability of T(3) to reduce the cell growth of the human pancreatic duct cell lines chosen,and to increase the effect of chemotherapeutic drugs at conventional concentrations. Three human cell lines hPANC-1,Capan1,and HPAC have been used as experimental models to investigate the T(3) effects on pancreatic adenocarcinoma cell proliferation. The hPANC-1 and Capan1 cell proliferation was significantly reduced,while the hormone treatment was ineffective for HPAC cells. The T(3)-dependent cell growth inhibition was also confirmed by fluorescent activated cell sorting analysis and by cell cycle-related molecule analysis. A synergic effect of T(3) and chemotherapy was demonstrated by cell kinetic experiments performed at different times and by the traditional isobologram method. We have showed that thyroid hormone T(3) and its combination with low doses of gemcitabine (dFdCyd) and cisplatin (DDP) is able to potentiate the cytotoxic action of these chemotherapic drugs. Treatment with 5-fluorouracil was,instead,largely ineffective. In conclusion,our data support the hypothesis that T(3) and its combination with dFdCyd and DDP may act in a synergic way on adenopancreatic ductal cells. View Publication

The thyroid hormone,3,5,3'-Triiodo-L-thyronine (T3),is essential for growth,differentiation,and regulation of metabolic functions in multicellular organisms,although the specific mechanisms of this control are still unknown. In this study,treatment of a human pancreatic duct cell line (hPANC-1) with T3 blocks cell growth by an increase of cells in G(0)/G(1) cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a beta-cell-like phenotype,we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion,our results suggest,for the first time,that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC-1) into pancreatic-beta-cell-like cells. These findings have important implications in cell-therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes. View Publication