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.     

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.     

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.     

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.     

TRPM channels as potential therapeutic targets against pro-inflammatory diseases

The immune system protects our body against foreign pathogens. However, if it overshoots or turns against itself, pro-inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, or diabetes develop. Ions, the most basic signaling molecules, shape intracellular signaling cascades resulting in immune cell activation and subsequent immune responses. Mutations in ion channels required for calcium signaling result in human immunodeficiencies and highlight those ion channels as valued targets for therapies against pro-inflammatory diseases. Signaling pathways regulated by melastatin-like transient receptor potential (TRPM) cation channels also play crucial roles in calcium signaling and leukocyte physiology, affecting phagocytosis, degranulation, chemokine and cytokine expression, chemotaxis and invasion, as well as lymphocyte development and proliferation. Therefore, this review discusses their regulation, possible interactions and whether they can be exploited as targets for therapeutic approaches to pro-inflammatory diseases.     

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.     

Osteosarcoma and chondrosarcoma: new signaling pathways as targets for novel therapeutic interventions

Osteosarcomas and chondrosarcomas are the most common primary bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. Although the prognosis and quality of life have been improved during the last decades, the pathogenesis of these tumours remains elusive. Recent advances in molecular genetics and cytogenetics have brought a wealth of genes and molecular pathways that govern osteoblast and chondroblast differentiation and maturation, providing a better understanding of the biology of osteogenetic and cartilage tumours. In this review we describe the major tumour suppressor and oncogenic pathways, as well as the most important signal transduction cascades implicated in the development and progression of these malignancies. Furthermore, we discuss novel treatment regimens and future, patient-tailored strategies that will add significantly to the current therapeutic armamentarium.     

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.     

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.     

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.     

Microtubules: From understanding their dynamics to using them as potential therapeutic targets

Microtubules (MT) and actin microfilaments are dynamic cytoskeleton components involved in a range of intracellular processes. MTs play a role in cell division, beating of cilia and flagella, and intracellular transport. Over the past decades, much knowledge has been gained regarding MT function and structure, and its role in underlying disease progression. This makes MT potential therapeutic targets for various disorders. Disturbances in MT and their associated proteins are the underlying cause of diseases such as Alzheimer’s disease, cancer, and several genetic diseases. Some of the advances in the field of MT research, as well as the potenti G beta gamma, is needed al uses of MT-targeting agents in various conditions have been reviewed here.     

Caspase-mediated programmed cell death pathways as potential therapeutic targets in cancer

The caspase family is well characterized as playing a crucial role in modulation of programmed cell death (PCD), which is a genetically regulated, evolutionarily conserved process with numerous links to many human diseases, most notably cancer. In this review, we focus on summarizing the intricate relationships between some members of the caspase family and their key apoptotic mediators, involving tumour necrosis factor receptors, the Bcl-2 family, cytochrome c, Apaf-1 and IAPs in cancer initiation and progression. We elucidate new emerging types of cross-talk between several caspases and autophagy-related genes (Atgs) in cancer. Moreover, we focus on presenting several PCD-modulating agents that may target caspases-3, -8 and -9, and their substrates PARP-1 and Beclin-1, which may help us harness caspase-modulated PCD pathways for future drug discovery.     

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.     

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.