Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.     

Proteins of the Ras pathway as novel potential anticancer therapeutic targets

Ras proteins are molecular switches that constitute a pivotal element in the control of cellular responses to many incoming signals, and in particular mitogenic stimulations. They act through multiple effector pathways that carry out the biological functions of Ras in cells. Since mutations that constitutively activate Ras proteins have been found in a high proportion of human malignancies and participate in oncogenesis, a number of therapeutic anticancer strategies aimed against the activity or action of Ras proteins have been developed. This paper reviews the principal aspects of the Ras signaling pathway and describes some of the attempts to develop antitumor drugs based on this concept. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oxidized phospholipids as potential novel drug targets

The interactions of three therapeutic agents, viz. the antipsychotics HPD and CPZ, and the antineoplastic anthracycline DOX, with oxidatively modified phospholipids were studied by monitoring the quenching of fluorescence of an incorporated pyrene-labeled lipid derivative. All three drugs bound avidly to the two oxidized PCs bearing either an aldehyde or carboxylic function at the end of the sn-2 nonanoyl chain, with the highest affinity measured between CPZ and the latter oxidized lipid. Subsequent dissociation of the above drugs from the oxidized lipids by DNA, acidic phospholipids, and NaCl revealed the binding of these drugs with the aldehyde lipid to be driven by hydrophobicity similarly to their binding to lysophosphatidylcholine, whereas a significant contribution of electrostatics was evident for the lipid with the carboxylic moiety. These results connect to previous experimental data, demonstrating the induction by these drugs of oxidative stress and binding to membrane phospholipids. These issues are elaborated with reference to their clinical use and side effects.     

RNA interference: potential therapeutic targets

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.     

Sphingolipids as modulators of cancer cell death: potential therapeutic targets

Through modifications in the fine membrane structure, cell-cell or cell-matrix interactions, and/or modulation of intracellular signaling pathways, sphingolipids can affect the tumorigenic potential of numerous cell types. Whereas ceramide and its metabolites have been described as regulators of cell growth and apoptosis, these lipids as well as other sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer treatments, and their susceptibility to non-apoptotic cell deaths. This review summarizes our current knowledge on the properties of sphingolipids in the regulation of cancer cell death and tumor development. It also provides an update on the potential perspectives of manipulating sphingolipid metabolism and using sphingolipid analogues in anticancer therapy.     

Cytokines as potential therapeutic targets for inflammatory skin diseases

A network of pro-inflammatory cytokines is a central feature in the pathophysiology of cutaneous inflammatory diseases. Thus, the delineation of precise roles for particular cytokines and the development of cytokine-directed therapeutics have become areas of intense investigation. While anti-TNF therapeutics have proven to be effective for the treatment of psoriasis, clinical investigations have now begun with other cytokine-directed therapies, such as those targeting IFN-g, IL-12p40, and IL-18. In addition to therapeutics that target cytokines directly, strategies that target cytokine signaling pathways are in development too. In this short review, we summarize key findings from a recent workshop on cytokines as potential therapeutic targets for inflammatory skin diseases.     

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

14-3-3 proteins as potential therapeutic targets

The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed.     

Comparative proteomic approach identifies PKM2 and cofilin-1 as potential diagnostic, prognostic and therapeutic targets for pulmonary adenocarcinoma

Lung cancer is the leading cause of cancer-related death in the world. Non-small cell lung carcinomas (Non-SCLC) account for almost 80% of lung cancers, of which 40% were adenocarcinomas. For a better understanding of the molecular mechanisms behind the development and progression of lung cancer, particularly lung adenocarcinoma, we have used proteomics technology to search for candidate prognostic and therapeutic targets in pulmonary adenocarcinoma. The protein profile changes between human pulmonary adenocarcinoma tissue and paired surrounding normal tissue were analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE) based approach. Differentially expressed protein-spots were identified with ESI-Q-TOF MS/MS instruments. As a result, thirty two differentially expressed proteins (over 2-fold, p<0.05) were identified in pulmonary adenocarcinoma compared to normal tissues. Among them, two proteins (PKM2 and cofilin-1), significantly up-regulated in adenocarcinoma, were selected for detailed analysis. Immunohistochemical examination indicated that enhanced expression of PKM2 and cofilin-1 were correlated with the severity of epithelial dysplasia, as well as a relatively poor prognosis. Knockdown of PKM2 expression by RNA interference led to a significant suppression of cell growth and induction of apoptosis in pulmonary adenocarcinoma SPC-A1 cells in vitro, and tumor growth inhibition in vivo xenograft model (P<0.05). In addition, the shRNA expressing plasmid targeting cofilin-1 significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. While additional works are needed to elucidate the biological significance and molecular mechanisms of these altered proteins identified in this study, PKM2 and cofilin-1 may serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets for pulmonary adenocarcinoma.     

Pattern recognition receptors as potential therapeutic targets in inflammatory rheumatic disease

The pattern recognition receptors of the innate immune system are part of the first line of defence against pathogens. However, they also have the ability to respond to danger signals that are frequently elevated during tissue damage and at sites of inflammation. Inadvertent activation of pattern recognition receptors has been proposed to contribute to the pathogenesis of many conditions including inflammatory rheumatic diseases. Prolonged inflammation most often results in pain and damage to tissues. In particular, the Toll-like receptors and nucleotide-binding oligomerisation domain-like receptors that form inflammasomes have been postulated as key contributors to the inflammation observed in rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus. As such, there is increasing interest in targeting these receptors for therapeutic treatment in the clinic. Here the role of pattern recognition receptors in the pathogenesis of these diseases is discussed, with an update on the development of interventions to modulate the activity of these potential therapeutic targets.     

Ceramide synthases as potential targets for therapeutic intervention in human diseases

Ceramide is located at a key hub in the sphingolipid metabolic pathway and also acts as an important cellular signaling molecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amide bond, with the acyl chain varying in length and degree of saturation. The identification of a family of six mammalian ceramide synthases (CerS) that synthesize ceramide with distinct acyl chains, has led to significant advances in our understanding of ceramide biology, including further delineation of the role of ceramide in various pathophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from ceramide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in the diseases in which the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Roles of PPARs in NAFLD: potential therapeutic targets

Non-alcoholic fatty liver disease (NAFLD) is a liver pathology with increasing prevalence due to the obesity epidemic. Hence, NAFLD represents a rising threat to public health. Currently, no effective treatments are available to treat NAFLD and its complications such as cirrhosis and liver cancer. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors which regulate lipid and glucose metabolism as well as inflammation. Here we review recent findings on the pathophysiological role of PPARs in the different stages of NAFLD, from steatosis development to steatohepatitis and fibrosis, as well as the preclinical and clinical evidence for potential therapeutical use of PPAR agonists in the treatment of NAFLD. PPARs play a role in modulating hepatic triglyceride accumulation, a hallmark of the development of NAFLD. Moreover, PPARs may also influence the evolution of reversible steatosis toward irreversible, more advanced lesions. Presently, large controlled trials of long duration are needed to assess the long-term clinical benefits of PPAR agonists in humans. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Periostin: novel diagnostic and therapeutic target for cancer

Periostin is a secreted protein that shares a structural homology to the axon guidance protein fasciclin I (FAS1) in insects and was originally named as osteoblast-specific factor-2 (Osf2). Periostin is particularly highly homologus to Betaig-h3, which promotes cell adhesion and spreading of fibroblasts. It has recently been reported that Periostin was frequently overexpressed in various types of human cancers. Although the detailed function of Periostin is still unclear, Periostin-integrin interaction through FAS1 domain is thought to be involved in tumor development. In addition, Periostin stimulates metastatic growth by promoting cancer cell survival, invasion and angiogenesis. Therefore, Periostin can be a useful marker to predict the behavior of cancer. This review summarizes the recent understanding of Periostin roles in tumor development and speculates on the usefulness of Periostin as a therapeutic and diagnostic target for cancer.     

HDL and triglyceride as therapeutic targets

PURPOSE OF REVIEW: Epidemiological studies have shown that plasma HDL-cholesterol is inversely related to coronary artery disease and that there is an inverse relationship between HDL-cholesterol and triglyceride levels, but it is now demonstrated that hypertriglyceridemia is an independent risk factor for coronary heart disease (CHD). The goal of this review is to discuss if triglycerides and HDL-cholesterol could be therapeutic targets to reduce cardiovascular risk. RECENT FINDINGS: Triglyceride measurement is not informative on the specificity of the triglyceride-rich lipoproteins present in the plasma because some of these are not atherogenic (chylomicrons, large VLDLs) while others are highly atherogenic (small VLDLs, remnants, IDL...). Statins, in addition to reducing LDL-cholesterol, significantly reduced atherogenic remnant lipoprotein cholesterol levels. 4S, CARE+LIPID, and AFCAPS/TexCAPS studies, suggested enhanced therapeutic potential of statins for improving triglyceride and HDL-cholesterol levels in patients with CHD. A fibrate (gemfibrozil) was shown to reduce death from CHD and non-fatal myocardial infarction in secondary prevention of CHD in men with low levels of HDL-cholesterol (VA-HIT); during the treatment these levels predicted the magnitude of reduction in risk for CHD events. SUMMARY: ATP III recommendations state, on triglycerides and HDL-cholesterol as targets to reduce cardiovascular risk: (1) that lowering LDL-cholesterol levels is the primary target of therapy, (2) a secondary target is to achieve a triglyceride level < 150 mg/dL and (3) clinical trial data are considered to be insufficient to support recommended a specific HDL-cholesterol goal even if HDL-cholesterol < 40 mg/dL is considered to be a major risk factor of CHD.     

ENaCs and ASICs as therapeutic targets

The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity. As a physiologist intrigued by the fundamental mechanics of salt and water transport, it was natural that Dale Benos, to whom this series of reviews is dedicated, should have been at the forefront of research into the amiloride-sensitive sodium channel. The cloning of ENaC and subsequently the ASIC channels has revealed a far wider role for this channel family than was previously imagined. In this review, we will discuss the known and potential roles of ENaC and ASIC subunits in the wide variety of pathologies in which these channels have been implicated. Some of these, such as the role of ENaC in Liddle's syndrome are well established, others less so; however, all are related in that the fundamental defect is due to inappropriate channel activity.     

Adenylyl cyclase isoforms as novel therapeutic targets: an exciting example of excitotoxicity neuroprotection

The use of transgenic animals lacking one or multiple adenylyl cyclase (AC) isoforms has provided significant information on the roles of AC-dependent signaling in the central nervous system. A recent study provides evidence that AC type 1 (AC1) might be important in glutamate-induced neuronal toxicity. However, the absolute AC specificity revealed in this study contrasts earlier work examining other forms of neurodegeneration. Nonetheless, these observations suggest that specific AC isoforms may represent novel targets for the treatment of central nervous system disorders. It is anticipated that such findings will help catalyze new drug discovery efforts to identify small-molecule modulators of individual AC isoforms.