Gamma-enolase: a well-known tumour marker, with a less-known role in cancer Tjasa Vizin, Janko Kos Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2015; 49(3): 217-226. Received 9 May 2015 Accepted 13 July 2015 Correspondence to: Prof. Janko Kos, Ph.D., Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia. E-mail: Janko.kos@ffa.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. Background. Gamma-enolase, known also as neuron-specific enolase (NSE), is an enzyme of the glycolytic pathway, which is expressed predominantly in neurons and cells of the neuroendocrine system. As a tumour marker it is used in diagnosis and prognosis of cancer; however, the mechanisms enrolling it in malignant progression remain elusive. As a cytoplasmic enzyme gamma-enolase is involved in increased aerobic glycolysis, the main source of energy in cancer cells, supporting cell proliferation. However, different cellular localisation at pathophysiological conditions, proposes other cellular engagements. Conclusions. The C-terminal part of the molecule, which is not related to glycolytic pathway, was shown to promote survival of neuronal cells by regulating neuronal growth factor receptor dependent signalling pathways, resulting also in extensive actin cytoskeleton remodelling. This additional function could be important also in cancer cells either to protect cells from stressful conditions and therapeutic agents or to promote tumour cell migration and invasion. Gamma-enolase might therefore have a multifunctional role in cancer progression: it supports increased tumour cell metabolic demands, protects tumour cells from stressful conditions and promotes their invasion and migration. Key words: gamma-enolase; cancer; glycolysis; cell survival; tumour marker Introduction ic or pathophysiological conditions.5 In addition to their innate glycolytic function, many enzymes of Enolases (EC 4.2.1.11) are intracellular enzymes the glycolytic pathway, including enolase, were that catalyse the dehydration of 2-phospho-D- shown to possess various specific regulatory func- glycerate to phosphoenolpyruvate in the catabolic tions and to play a pleiotropic role in physiological direction of the glycolytic pathway, a process con- and pathological processes, including cancer.1,267 verting glucose into pyruvate, which enables the In this paper we review the properties, distribu- formation of high-energy compounds of ATP and tion and function of gamma-enolase and its role in NADH. In the anabolic direction during gluconeo- enhanced glycolysis and proliferation of tumour genesis, they catalyse the reverse reaction of hy- cells. Additionally, we expose new mechanisms dration of phosphoenolpyruvate to 2-phospho-D- through which gamma-enolase may promote glycerate. The glycolytic pathway and its enzymes cancer progression: aiding adaptation of tumour are one of the most conserved and important meta- cells to stressful conditions by activating survival bolic networks in living organisms and therefore, promoting signalling pathways and promoting enolases are among the most ubiquitously and migration of tumour cells. Finally, we discuss the abundantly expressed proteins.1-4 Despite being role of gamma-enolase as a marker of exposure to expressed in most cells, the gene that encodes for carcinogenic pollutants and review the diagnostic enolase is not a housekeeping gene since its expres- and prognostic utility of gamma-enolase in cancer sion varies during several developmental, metabol- patients. Properties and distribution of enolase Enolases are functionally active as dimers, composed of non-covalently linked subunits alpha- (a), beta- (ß) and gamma- (y), facing each other in an antiparallel fashion, which may form five homodi-meric or heterodimeric isoenzymes, expressed in a development and tissue-specific manner. The isoenzyme aa (alpha-enolase) is localized in all foetal and in the majority of adult mammal tissues. During tissue development, it is replaced by other isoforms: in skeletal and heart muscles by aß and ßß (beta-enolase), and in neuronal cells and cells of the diffuse neuroendocrine system by isoenzymes ay and yy (gamma-enolase). In mammals, each of the three isoenzymes is encoded by an independent loci.89 All enolase isoforms have a molecular range between 82 and 100 kDa and share high sequence identity and kinetic properties.1'610-12 However, each isoform possesses characteristic short variable regions, which are situated predominantly on the surface of the molecule and might be the sites of contact with different cytoskeleton elements or other cell components.13 Besides the peptide molecule, enolase requires a divalent metal ion for its stabilisation and catalytic activity. Six divalent metals have been demonstrated to activate enolase: Mg2+, Zn2+, Cd2+, Co2+, Mn2+ and Ni2+. The most abundant is Mg2+, which provides the highest activation strength.11415 The metal ion is not firmly bound into the protein part of the molecule; therefore enolase is not a typical metalloenzyme, but defined as a "metal-ion-activated enzyme complex".16 Enolase has two binding sites for Mg2+, both contributing to catalysis: binding to the first site, Mg2+ induces conformational changes of the active site enabling the binding of the substrates, whereas the binding of a second Mg2+ is an essential part of the catalytic apparatus.117-19 Enolase localizes predominantly in the cytosol however, variations in cellular localisation were observed for all three enolase isoforms. Alpha-enolase was observed in the nucleus, on the cell surface and in extracellular space. It may interact with different cytoplasmic, nuclear and membrane molecules and exhibits several other functions besides catalysis.1,20 The nuclear form of alpha-enolase was recognized as Myc promoter-binding protein-1 (MBP-1), an alternative splicing form involved in regulation of transcription by repressing the function of Myc and acting as a tumour sup-pressor.621-23 Alpha-enolase localizes also on cell surface of neuronal, endothelial and hematopoi- etic cells as well on pancreatic, breast and lung cancer cells. Its surface expression was shown to depend on the pathophysiological conditions of the cells and its C-terminal lysine residue acts as a plasminogen-binding receptor modulating peri-cellular fibrinolytic activity and promoting migration and metastasis of cancer cells. The cell surface alpha-enolase is catalytically active, maintaining its active dimeric form. Alpha-enolase was shown also to be secreted from cells by exosomes, cell derived vesicles, proposed to play an important role in intercellular communication.23-25 However, the mechanisms of surface translocation, membrane attachment, cell surface expression or secretion remain unknown.626-32 The properties and function of alpha-enolase in malignant disease have been extensively studied and reviewed.1,262023 Different subcellular localisation and interactions with other proteins were observed also for beta-enolase during maturation, normal function and regeneration of muscles. Specific interactions with macromolecules may address beta-enolase to the subcellular site where ATP, produced through glycolysis, is most needed for muscular contraction or regeneration.33-35 Increased expression of beta-enolase was detected in rhabdomyosarcoma tissue, which is, to our knowledge, the only evidence that this isoform might be involved in cancer.3637 Gamma-enolase Gamma-enolase, is a 433 amino acid long acidic dimeric protein, which includes two enolase isoen-zymes, yy and ay, and is also referred as neuron-specific enolase (NSE). The subunit molecular mass is approximately 39 kDa, whereas Mr of the native form is 78 kDa which might vary on the subunit combination. Gamma-enolase localizes predominantly in neuronal cells and in neuroendocrine cells, particularly in those of the amine precursor uptake and decarboxylation (APUD) lineage, for example in the intestine, lung, thyroid and pituary gland and pancreas.838 It is found in lower amounts also in non-neuronal and non-neuroendocrine tissues or cells, such as erythrocytes, platelets, breast tissue, prostate and uterus.39-41 The yy isoform is found predominantly in mature neurons and is also used as marker of neuronal maturation and differentiation, while the ay isoenzyme localizes in higher amounts in non-neuronal cells.89 The C-terminal end of gamma-enolase contains a PDZ-binding motif (431S-433L: SVL) (Figure 1), which might enable an interaction with several proteins that contain a PDZ-domain and are involved in intracellular redistribution of molecules and signalling pathway events. Different gamma-enolase cellular localisation, which depends on the pathophysiological conditions of the cells, propose other cellular engagement besides glycolysis. In neuronal, glial and astrocytic cells, gamma-enolase was shown to associate with the plasma membrane, or even appear on the surface of cells42-45, which might occur through its hydrophobic domain in the N-terminal region (32A-43Y: AAVPSGASTGIY). Also, on the cell surface alpha-enolase may bind to plasminogen by C-terminal lysine.46 In contrast to alpha-enolase, gamma-enolase has no C-terminal lysine and does not bind plasminogen; therefore it might exert other functions on cell surface.43 46-48 Gamma-enolase was detected also in the nucleus of malignantly transformed urothelial and epithelial breast cells and in glioblastoma cells; however its role remains unknown.4049-51 Significantly higher increase of gamma-enolase antigen levels than its catalytical activity was observed during exponential growth of small-cell lung cancer cells, proposing that cellular gamma-enolase exists also as an enzymatically inactive compound, that might possess other functions.52 C-terminal end FIGURE 1. Position of gamma-enolase catalytical active site and the PDZ-binding motif containing C-terminal end. Subunits of the YY-dimer are represented by separate colours (wheat and violet). The orange part represents the catalytical active site, yellow balls represent Mg2+ ions and the magenta part represents the C-terminal end of the molecule (the last 6 amino acids). For better representation, active site and C-terminal end are shown only in one subunit. The image was created using PyMOL (DeLano LLC Scientific). Gamma-enolase crystal structure (1TE6) was obtained from Protein Data Bank (PDB). The image was prepared by authors and has not been published elsewhere. The function of gamma-enolase in increased glycolysis in cancer It is generally known that glycolysis is drastically enhanced in tumour cells and is a hallmark of cancer progression.53,54 In tumours that outgrow its feeding circulation, cells are exposed to an environment with poor oxygen and nutrients supply50, which leads to a prevalence of aerobic glycolysis over mitochondrial oxidative phospho-rylation.55-57 This metabolic switch referred also to as the Warburg effect, enables tumour cells to produce energy to survive and eventually proliferate regardless the presence of oxygen. Glycolysis alone, however, is energetically less efficient than oxidative phosphorylation. Therefore, reactions of the glycolytic pathway have to be drastically accelerated to satisfy the higher metabolic needs of proliferating tumour cells, which is evident from a net increase in glucose consumption and higher expression of glycolytic enzymes.5558-60 Gamma-enolase is overly-expressed in tumours39 and its major contribution to tumour progression is, no doubt, the participation to accelerated glycolysis of cancer cells. For instance, malignant transformation of astrocytic61, breast40 and urothelial cells49 led to occurrence of gamma-enolase in originally gamma-enolase-negative cells and to colony formation and proliferation, which strongly suggests that transformed cells might obtain the ability to express gamma-enolase in order to adapt to increased metabolic needs of a neoplastic state.6162 Further, malignantly transformed urothelial cells, which were able to proliferate and form tumours when inoculated into immune compromised mice, were shown to express higher levels of gamma-enolase, compared to less active and differentiated cells. Authors proposed that cells, which express gamma-enolase at higher rates, might have an advantage in tumour initiation and subsequent growth.49 Gamma-enolase was significantly up-regulated also in glioblastoma cells exposed to hypoxia and serum starvation, and additionally, its knock-down significantly diminished cell growth50, supporting the findings that the dependence of tumour cell growth on glycolysis is even more emphasized in stressful conditions.556063 Finally, in non-small cell lung cancer cells, an alternative splicing form of c-H-ras, p19ras, was shown to specifically bind gamma-enolase and inhibit its enzymatic activity, resulting in diminished cell prolif-eration.58 The glycolytic function of gamma-enolase and its impact on promoting tumour cell growth represents a promising target for cancer therapy.64 The pro-survival function of gamma-enolase in cancer gamma-enolase Gamma-enolase was shown to act as a neurotropic factor in neuronal cells.7,65,66 This function is manifested through an additional active site, which is not a part of the catalytical apparatus involved in glycolysis, but localized at the C-terminal end of the molecule. For instance, a 30 amino acid long pep-tide, mimicking the C-terminal part of gamma-eno-lase, was shown to promote survival, differentiation and regeneration of neurons by activating signal transduction pathways which are normally triggered by the activation of Trk receptor: phosphati-dylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Additionally, the C-terminal peptide of gamma-enolase was demonstrated to impair apoptosis and to interact with p75 neurotrophin receptor (p75NTR) and suppress the activation of its downstream effectors in apop-totic signalling. Despite having similar amino acid sequence in the C-terminal part, other enolase iso-forms do not show a neurotropic function.7,43,46,67-69 Gamma-enolase neurotrophic effect is regulated by cathepsin X, a cysteine carboxymonopeptidase, which is frequently expressed in neuronal and glial cells.70,71 Cathepsin X was shown to sequentially cleave the final two amino acids (433L and 432V) at the C-terminal end of gamma-enolase and to disrupt the PDZ motif, through which gamma-enolase binds to the scaffold protein gamma-1-syntrophin. The latter mediates the translocation of gamma-enolase and its association with plasma membrane, which is a prerequisite for neurotrophic activity.43,46 Therefore, only C-terminally uncleaved gamma-enolase has a pro-survival activity. The protective function of gamma-enolase was observed also in brains of a mouse model of Alzheimer disease (Tg2576): C-terminally truncated gamma-enolase localized in immediate plaque vicinity and strongly colocalized with cathepsin X, while uncleaved gamma-enolase exhibiting neuroprotective activity, localized in microglia cells in close proximity of senile plaques. Additionally, using a mouse microglial cell model, gamma-enolase was shown to protect neuronal cells from amyloid-ß peptide toxicity and cathepsin X reversed its function.66 Gamma-enolase has been proposed to act as a pro-survival factor also in cancer cells. It was shown to support glioblastoma cell adaptation to cellular stress, such as serum starvation, hypoxia, chemotherapy and radiotherapy; however, no specific mechanism has yet been proposed.50 Both, starvation and hypoxia have been linked to progression of cathepsin X gamma-enolase + cathepsin X , ^ - r . «t^—-- FIGURE 2. Co-localization of gamma-enolase and cathepsin X in human glioblastoma cells U87-MG grown in serum-free medium for 72 h. U87-MG cells were grown in Eagle's Minimum Essential Medium (EMEM, Sigma), supplemented with 10% (v/v) foetal bovine serum (HyClone), 1% L-glutamine (Sigma) and 1% penicillin/streptomycin (Sigma) at 37°C and humidified atmosphere with 5% CO2. For protein visualization, cells were seeded on glass coverslips at a concentration of 1 x 104 cells/ml in 24 well plates. After 24 h, complete growth medium was replaced with serum-free medium and cells were left to grow for additional 72 h. After treatment cells were fixed with 10% formalin for 30 min at room temperature and then permeabilized by 0.5% Tween®20 in phosphate buffered saline (PBS), pH 7.4 for 10 min. Non-specific binding was blocked with 3% bovine serum albumin (BSA) in PBS, pH 7.4 for 1.5 h at room temperature. Cells were then incubated with primary antibody against N-terminal end of gamma-enolase (10 pg/ml, goat polyclonal, Santa Cruz Biotechnology) and active cathepsin X (10 pg/ml, mouse monoclonal, 2F12) in 3% BSA in PBS pH 7.4 for 2 h at room temperature. After three washes with PBS, pH 7.4, cells were incubated with Alexa Fluor 555 donkey anti-goat (Molecular Probes™) and Alexa Fluor 488 donkey anti-mouse (Molecular Probes™) secondary antibody in 3% BSA in PBS, pH 7.4. After washing with PBS, ProLong® Gold Antifade Mountant with 4',6-diamidino-2-phenylindole, dilactate (DAPI, Molecular Probes™) was used to mount coverslips on glass slides. Fluorescence microscopy was performed by Carl Zeiss LSM 710 confocal microscope (Carl Zeiss Oberkochen) with ZEN 2012 image software. Gamma-enolase (red) and cathepsin X (green) staining showed co-localisation in the perimembrane region. The blue staining with DAPI represents the nucleus. The image was prepared by authors and has not been published elsewhere. cancer and resistance to treatment by inducing biological changes in tumour cells, one of them being increased glycolysis.55,60,72 However, C-terminally uncleaved gamma-enolase might additionally support tumour cell adaptation to stressful conditions by activating survival promoting signalling pathways as it does in neuronal cells, and cathepsin X, which is present also in tumour cells71, might regulate its function (Figure 2). For instance, in glio-blastoma cell lines, exposed to serum starvation or hypoxia, gamma-enolase expression was significantly increased50; moreover, significant increases in protein and phosphoprotein levels were observed also in PI3K/Akt and MAPK/ERK and anti-apop-totic signalling pathways7374, which are triggered by gamma-enolase in neuronal cells. Separate analysis of expression and role of C-terminally uncleaved and truncated gamma-enolase in cancer cells and tumour tissue might provide new information on its involvement in tumour progression. The role of samma-enolase in migration oftumour cells Recently, a study on glioma cells showed that gamma-enolase knockdown significantly reduced migration of cells; however, no specific mechanism has been proposed.75 An important prerequisite for cell migration is a dynamic remodelling of actin cytoskeleton. Remodelling is stimulated by several molecules that link migratory signals to the actin filaments and are upregulated in invasive and metastatic cancer cells.76 In neuroblastoma cells, gamma-enolase was shown to co-localize with ac-tin filaments, an interaction that depends on the presence of gamma-1-syntrophin.43 Additionally, gamma-enolase C-terminal peptide was shown to regulate RhoA kinase, a regulator of actin cytoskel-eton organization. Consequently, gamma-enolase induced actin polymerisation and its redistribution to growth cones of neurites.68 Similarly, alpha-eno-lase was shown to bind to actin and tubulin77 and to mediate invasiveness of tumour cells78 and sensitivity to microtubule targeted drugs.79 These results provide evidence, that gamma-enolase might be involved in migration of tumour cells through interactions with actin filaments and regulation of RhoA kinase function. Gamma-enolase as a marker of exposure to environmental carcinogenic pollutants arsenic and cadmium Arsenic and cadmium exposure is linked to breast and bladder cancer occurrence. Exposure of breast epithelial and urothelial cells to As3+ or Cd2+ was shown to induce malignant transformation of cells and an increase of mRNA and protein levels of gamma-enolase in the cytoplasm and nucleus of cells, while expression of alpha-enolase did not change. Authors proposed that gamma-enolase might be translated as a possible biomarker for chronic environmental exposure to As3+ or Cd2+. Its expression in non-malignant cells was influenced also by methylation and histone modifications, induced by a histone deacetylase inhibitor (MS-275) and a methylation inhibitor (5-AZC), which proposed that gamma-enolase gene expression is controlled by methylation and histone modifications. The later provides evidence that environmental carcinogenic pollutants, such as cadmium and arsenic, might cause changes in epigenetic regulation of genes, which specifically affect the expression and function of gamma-enolase in breast epithelial cells and urothelial cells.40 49 Gamma-enolase in tumour tissues Gamma-enolase is typically overexpressed in tumours of neurogenic and neuroendocrine origin and has been used as a marker for detection of neuroendocrine differentiation of tumour cells. It is considered the most important tumour marker for poorly differentiated neuroendocrine tumours, since a tumour is classified as a neuroendocrine tumour only when it expresses at least two neu-roendocrine markers of which one is gamma-enolase.8081 Immunohistochemistry of gamma-enolase is regularly used for differential diagnosis of small-cell lung cancer (SCLC) from other lung cancer histological subtypes (Table 1).82,83 Gamma-enolase increased expression was observed also in other tumours, including breast cancer, with increased staining in lymph node metastases compared to primary breast tumours84 or in glioblasto-mas, with higher levels in advanced stage tumours, which were related to shorter patient survival.50 Nevertheless, immunostaining of gamma-enolase in tumour tissue has limited diagnostic or prognostic utility, since many clinical studies provided contradictory results.8085-87 Gamma-enolase in extracellular fluids of cancer patients In general, gamma-enolase serum levels are better indicators than its tissue expression (Table 1).80 TABLE 1. Use of gamma-enolase as a tumour marker Neuroendocrine cancer Proposed use Use in clinical practice Recommendations Reference Tumour tissues Serum SCLC Other neuroendocrine tumours (neuroblastoma, endocrine pancreatic tumours, seminoma, medullary thyroid carcinoma, phaeochromocytoma, ect.) Differential diagnosis from other lung cancer subtypes Diagnosis or detection of neuroendocrine differentiation of tumour Yes Yes EGTM, NACB SCLC Differential diagnosis from other lung cancer subtypes when biopsy is not possible Yes EGTM, NACB [82, 83] Prognosis Unknown [82, 83, 97] Post-operative surveillance Yes EGTM, NACB [82, 83] Monitoring efficacy of therapy Yes EGTM, NACB [82, 83] Detection of recurrent disease after primary surgery Yes NACB [82, 83] NSCLC Monitoring therapy in advanced disease No [83] Prognosis Unknown [83] Testicular cancer (seminoma) Diagnosis Experimental EGTM [98] Carcinoids Diagnosis Unknown [96, 99] Monitoring efficacy of therapy Yes EGTM [8, 39] Detection of early relapse Yes [8, 39, 96] Medullary thyroid carcinomas Monitoring efficacy of therapy Yes EGTM [8, 39] Detection of early relapse Yes [8, 39] Phaeochromocytoma Monitoring efficacy of therapy Yes EGTM [8, 39] Detection of early relapse Yes [8, 39] Endocrine pancreatic tumours Diagnosis Yes [95, 96] Monitoring efficacy of therapy Yes EGTM [8, 39] Detection of early relapse Unknown [8, 39, 99] Paraganglioma Diagnosis Unknown [99] Neuroblastoma Differential diagnosis Unknown [8] Prognosis Yes ACS [100] Monitoring efficacy of therapy Yes EGTM [8, 100] Detection of recurrent disease Yes [97] ACS = American Cancer Society; EGTM = European Group for Tumour Markers; NACB = National Academy of Clinical Biochemistry; NSCLC = non-small-cell lung cancer; SCLC = small-cell lung cancer Levels of gamma-enolase are elevated in sera from injury or cardiac arrest, gamma-enolase is released patients with various cancers, however, its appear- into the cerebrospinal fluid and eventually into the ance in extracellular fluids without any apparent bloodstream due to damage or death of neuronal cellular damage is not clear.188 After stroke, brain cells or impairment of the blood-brain barrier in- [82, 83] [80, 81, 95, 96] tegrity. For instance, levels of gamma-enolase in cerebrospinal fluid and serum have been used as a biomarker of cerebral injury and for the assessment of neurological disorders.388990 Gamma-enolase is the only neuroendocrine tumour marker, which is used as a serum marker for follow up and monitoring of therapy effectiveness. Increased gamma-enolase levels in extracellular fluids are related to cancer progression and are typical for cancer in advances stages with distant metastases.8,39,80,84-87 The levels of gamma-enolase in non-treated cancer increase proportionally to the tumour mass, stage and number of metastases and are related to worse prognosis, however, the levels are not related to the location of metastases.39 Gamma-enolase is used in clinical practice in patients with SCLC and neuroblastoma. Its levels are significantly elevated compared to healthy subjects; however, specificity and sensitivity are too low to be used in screening.3991 According to the recommendations of expert groups for the use of markers in lung cancer, gamma-enolase is recommended as an auxiliary marker in SCLC for differential diagnosis when biopsy is not possible and when other neuroendocrine tumours are excluded. Further, it is recommended for SCLC postoperative surveillance, for monitoring of therapy in advanced disease and for detection of recurrent disease.8391 During chemotherapy, a transient rise of gamma-enolase serum levels occurs due to cy-tolysis of tumour cells, which disappears in case of successful treatment. However, persistently elevated levels show unsuccessful therapy. Gamma-enolase is not a recommended tumour marker in neuroblastoma; however, it is frequently used for differential diagnosis of neuroblastoma from ne-froblastoma and for disease monitoring.8,91 Gamma-enolase is used as an auxiliary serum marker for follow-up and monitoring of therapy effectiveness in patients with carcinoids, melanoma, seminoma, feocromocitoma, medullary thyroid carcinoma, and endocrine pancreatic tumours. In patients with brain tumours, the levels of gamma-enolase in sera are not elevated, however, increased levels were reported in cerebrospinal fluid.3991 Increased serum levels of gamma-enolase were reported also in patients with cancers of non-neuroendocrine origin, such as T-cell leukaemia92, B-cell lymphoma93 and malignant melanoma.94 In general, higher serum levels of gamma-enolase are related to worse prognosis and are the highest in patients with advanced metastatic stage.39 Gamma-enolase is usually measured in serum samples and less frequently in cerebrospinal fluid, pleural exudate or ascites. Its half-life in serum is estimated to be approximately 30 h.101 The ay isoform is expressed in large amounts also in eryth-rocytes and in platelets, therefore it is important to separate blood cells from plasma or serum within 60 minutes from sample collection to prevent haemolysis of blood samples, which could lead to falsely elevated levels of gamma-enolase.80,102,103 Falsely elevated serum levels of gamma-enolase might be also due to various noncancerous pathological causes104, such as benign pulmonary dis-eases105, renal failure106, brain injuries, seizures, stroke38,107, severe hypoglycaemia108, benign liver diseases109 or systemic sclerosis.110 Concluding remarks Glycolytic enzymes were shown to exert various specific regulatory functions and to play a pleio-tropic role in physiological and pathological processes. Therefore, their participation to accelerated glycolysis could not be the only contribution to tumour progression.2 Alpha-enolase, the most exhaustively studied enolase isoform, was found to be one of the most frequently altered proteins in human pathologies and suggested as a universal cellular sensor that responds to multiple stimuli and reacts through multiple mechanisms.6,111 Gamma-enolase, sharing high-sequence identity with alpha-enolase, is also emerging as a multifunctional molecule. Different cellular localisation and interactions with other molecules strongly suggest its multiple cellular engagements. Gamma-enolase primary role in cancer is the participation to the accelerated glycolysis, which supports increased tumour cell metabolic demands and enables their proliferation. Its C-terminal end might protect tumour cells from stressful conditions and action of therapeutic agents by activating survival-promoting signalling pathways and regulating apoptosis. An additional role of gamma-enolase in cancer progression is its involvement in actin remodelling and consequently in promotion of migration and invasion of tumour cells. These findings suggest that the role of this well-known tumour marker, whose expression is altered during development and progression of a variety of cancers, is pleiotropic and still has to be defined. Future work should be focused on elucidation of gamma-enolase cellular redistribution, interactions with other molecules and involvement in cell signalling. Understanding these processes, together with the tools enabling effective inhibition of gamma-enolase glycolytic activity, might provide new opportunities for cancer treatment. Acknowledgements We thank Dr. Bojan Doljak for constructing Figure 1. This project was supported by Research Agency of the Republic of Slovenia (grants P4-0127 and J4-4123 to JK). References 1. Pancholi V. Multifunctional alpha-enolase: its role in diseases. Cell Mol Life Sci 2001; 58: 902-20. 2. Kim JW, Dang CV. Multifaceted roles of glycolytic enzymes. Trends Biochem Sci 2005; 30: 142-50. 3. Masoudi-Nejad A, Asgari Y. Metabolic cancer biology: Structural-based analysis of cancer as a metabolic disease, new sights and opportunities for disease treatment. Semin Cancer Biol 2015; 30: 21-9. 4. Dang CV, Semenza GL. Oncogenic alterations of metabolism. Trends Biochem Sci 1999; 24: 68-72. 5. McAlister L, Holland MJ. Targeted deletion of a yeast enolase structural gene. Identification and isolation of yeast enolase isozymes. J Biol Chem 1982; 257: 7181-8. 6. Diaz-Ramos A, Roig-Borrellas A, Garcia-Melero A, Lopez-Alemany R. Alpha-enolase, a multifunctional protein: its role on pathophysiological situations. J Biomed Biotechnol 2012; 2012: 156795. 7. Hattori T, Takei N, Mizuno Y, Kato K, Kohsaka S. Neurotrophic and neuro-protective effects of neuron-specific enolase on cultured neurons from embryonic rat brain. Neurosci Res 1995; 21: 191-8. 8. Suresh MR. Cancer Markers. In: Wild D, editor. The immunoassay handbook. Third edition. Oxford, UK: Elsevier; 2005. p. 664-94. 9. Marangos PJ, Parma AM, Goodwin FK. Functional properties of neuronal and glial isoenzymes of brain enolase. J Neurochem 1978; 31: 727-32. 10. Fletcher L, Rider CC, Taylor CB. Enolase isoenzymes: III. Chromatographic and immunological characteristics of rat brain enolase. Biochim Biophys Acta 1976; 452: 245-52. 11. Giallongo A, Feo S, Moore R, Croce CM, Showe LC. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase. Proc Natl Acad Sci U S A 1986; 83: 6741-5. 12. Feo S, Oliva D, Barbieri G, Xu WM, Fried M, Giallongo A. The gene for the muscle-specific enolase is on the short arm of human chromosome 17. Genomics 1990; 6: 192-4. 13. Lebioda L, Stec B. Mapping of isozymic differences in enolase. Int J Biol Macromol 1991; 13: 97-100. 14. Faller LD, Johnson AM. Calorimetric studies of the role of magnesium ions in yeast enolase catalysis. Proc Natl Acad Sci U S A 1974; 71: 1083-7. 15. Brewer JM. Specificity and mechanism of action of metal ions in yeast enolase. FEBS Letters 1985; 182: 8-14. 16. Vallee BL. Zinc and metalloenzymes. Adv Protein Chem 1955; 10: 317-84. 17. Faller LD, Baroudy BM, Johnson AM, Ewall RX. Magnesium ion requirements for yeast enolase activity. Biochemistry 1977; 16: 3864-9. 18. Brewer JM. Yeast enolase: mechanism of activation by metal ions. CRC Crit Rev Biochem 1981; 11: 209-54. 19. Brewer JM, Ellis PD. 31P-nmr studies of the effect of various metals on substrate binding to yeast enolase. J Inorg Biochem 1983; 18: 71-82. 20. Ko-Jiunn L, Neng-Yao S. The role of enolase in tissue invasion and metastasis of pathogens and tumor cells. J Cancer Mol 2007; 3: 45-8. 21. Ghosh AK, Steele R, Ray RB. Functional domains of c-myc promoter binding protein 1 involved in transcriptional repression and cell growth regulation. Mol Cell Biol 1999; 19: 2880-6. 22. Feo S, Arcuri D, Piddini E, Passantino R, Giallongo A. ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1). FEBS Lett 2000; 473: 47-52. 23. Capello M, Ferri-Borgogno S, Cappello P, Novelli F. Alpha-Enolase: a promising therapeutic and diagnostic tumor target. FEBS J 2011; 278: 1064-74. 24. Mears R, Craven RA, Hanrahan S, Totty N, Upton C, Young SL, et al. Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 2004; 4: 4019-31. 25. Yu X, Harris SL, Levine AJ. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res 2006; 66: 4795-801. 26. Cappello P, Tomaino B, Chiarle R, Ceruti P, Novarino A, Castagnoli C, et al. An integrated humoral and cellular response is elicited in pancreatic cancer by alpha-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen. Int J Cancer 2009; 125: 639-48. 27. He P, Naka T, Serada S, Fujimoto M, Tanaka T, Hashimoto S, et al. Proteomics-based identification of alpha-enolase as a tumor antigen in non-small lung cancer. Cancer Sci 2007; 98: 1234-40. 28. Seweryn E, Pietkiewicz J, Bednarz-Misa IS, Ceremuga I, Saczko J, Kulbacka J, et al. Localization of enolase in the subfractions of a breast cancer cell line. Z Naturforsch C 2009; 64: 754-8. 29. Nakajima K, Hamanoue M, Takemoto N, Hattori T, Kato K, Kohsaka S. Plasminogen binds specifically to alpha-enolase on rat neuronal plasma membrane. J Neurochem 1994; 63: 2048-57. 30. Miles Dahlberg CM, Plescia J, Felez J, Kato K, Plow EF. Role of cell-surface lysines in plasminogen binding to cells: identification of .alpha.-eno-lase as a candidate plasminogen receptor. Biochemistry 1991; 30: 1682-91. 31. Dudani AK, Cummings C, Hashemi S, Ganz PR. Isolation of a novel 45 kDa plasminogen receptor from human endothelial cells. Thromb Res 1993; 69: 185-96. 32. Redlitz A, Fowler BJ, Plow EF, Miles LA. The role of an enolase-related molecule in plasminogen binding to cells. Eur J Biochem 1995; 227: 407-15. 33. Merkulova T, Lucas M, Jabet C, Lamande N, Rouzeau JD, Gros F, et al. Biochemical characterization of the mouse muscle-specific enolase: developmental changes in electrophoretic variants and selective binding to other proteins. Biochem J 1997; 323: 791-800. 34. Keller A, Demeurie J, Merkulova T, Geraud G, Cywiner-Golenzer C, Lucas M, et al. Fibre-type distribution and subcellular localisation of alpha and beta enolase in mouse striated muscle. Biol Cell 2000; 92: 527-35. 35. Merkulova T, Dehaupas M, Nevers MC, Creminon C, Alameddine H, Keller A. Differential modulation of alpha, beta and gamma enolase isoforms in regenerating mouse skeletal muscle. Eur J Biochem 2000; 267: 3735-43. 36. Royds JA, Variend S, Timperley WR, Taylor CB. An investigation of beta enolase as a histological marker of rhabdomyosarcoma. J Clin Pathol 1984; 37: 905-10. 37. Royds JA, Variend S, Timperley WR, Taylor CB. Comparison of beta enolase and myoglobin as histological markers of rhabdomyosarcoma. J Clin Pathol 1985; 38: 1258-60. 38. Tiainen M, Roine RO, Pettila V, Takkunen O. Serum neuron-specific enolase and S-100B protein in cardiac arrest patients treated with hypothermia. Stroke 2003; 34: 2881-6. 39. Lamerz R. NSE (neuron-specific enolase) y-enolase. In: Thomas L, editor. Clinical laboratory diagnostics: use and assessment of clinical laboratory results. 1st. edition. Frankfurt/Main, Germany: TH-Books Verlagsgesellschaft; 1998. p. 979-81. 40. Soh MA, Garrett SH, Somji S, Dunlevy JR, Zhou XD, Sens MA, et al. Arsenic, cadmium and neuron specific enolase (ENO2, y-enolase) expression in breast cancer. Cancer Cell Int 2011; 11: 41. 41. Haimoto H, Takahashi Y, Koshikawa T, Nagura H, Kato K. Immunohistochemical localization of gamma-enolase in normal human tissues other than nervous and neuroendocrine tissues. Lab Invest 1985; 52: 257-63. 42. Vinores SA, Herman MM, Rubinstein LJ. Electron-immunocytochemical localization of neuron-specific enolase in cytoplasm and on membranes of primary and metastatic cerebral tumours and on glial filaments of glioma cells. Histopathology 1986; 10: 891-908. 43. Hafner A, Obermajer N, Kos J. gamma-1-syntrophin mediates trafficking of gamma-enolase towards the plasma membrane and enhances its neuro-trophic activity. Neurosignals 2010; 18: 246-58. 44. Burack WR, Shaw AS. Signal transduction: hanging on a scaffold. Curr Opin Cell Biol 2000; 12: 211-6. 45. Ponting CP, Phillips C, Davies KE, Blake DJ. PDZ domains: targeting signalling molecules to sub-membranous sites. Bioessays 1997; 19: 469-79. 46. Obermajer N, Doljak B, Jamnik P, Fonovic UP, Kos J. Cathepsin X cleaves the C-terminal dipeptide of alpha- and gamma-enolase and impairs survival and neuritogenesis of neuronal cells. Int J Biochem Cell Biol 2009; 41: 1685-96. 47. McAleese SM, Dunbar B, Fothergill JE, Hinks LJ, Day IN. Complete amino acid sequence of the neurone-specific gamma isozyme of enolase (NSE) from human brain and comparison with the non-neuronal alpha form (NNE). Eur J Biochem 1988; 178: 413-7. 48. Butterfield DA, Lange ML. Multifunctional roles of enolase in Alzheimer's disease brain: beyond altered glucose metabolism. J Neurochem 2009; 111: 915-33. 49. Soh M, Dunlevy JR, Garrett SH, Allen C, Sens DA, Zhou XD, et al. Increased neuron specific enolase expression by urothelial cells exposed to or malignantly transformed by exposure to Cd^* or As^*. Toxicol Lett 2012; 212: 66-74. 50. Yan T, Skaftnesmo KO, Leiss L, Sleire L, Wang J, Li X, et al. Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblas-toma cells to radiotherapy and temozolomide. BMC Cancer 2011; 11: 524. 51. Loja T, Chlapek P, Kuglik P, Pesakova M, Oltova A, Cejpek P, et al. Characterization of a GM7 glioblastoma cell line showing CD133 positivity and both cytoplasmic and nuclear localization of nestin. Oncol Rep 2009; 21: 119-27. 52. Splinter TA, Verkoelen CF, Vlastuin M, Kok TC, Rijksen G, Haglid KG, et al. Distinction of two different classes of small-cell lung cancer cell lines by en-zymatically inactive neuron-specific enolase. Br J Cancer 1992; 66: 1065-9. 53. Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell 2008; 13: 472-82. 54. Vesselle H, Schmidt RA, Pugsley JM, Li M, Kohlmyer SG, Vallires E, et al. Lung cancer proliferation correlates with [F-18]fluorodeoxyglucose uptake by positron emission tomography. Clin Cancer Res 2000; 6: 3837-44. 55. Porporato PE, Dhup S, Dadhich RK, Copetti T, Sonveaux P. Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review. Front Pharmacol 2011; 2: 49. 56. Golpour M, Akhavan Niaki H, Khorasani HR, Hajian A, Mehrasa R, Mostafazadeh A. Human fibroblast switches to anaerobic metabolic pathway in response to serum starvation: a mimic of warburg effect. Int J Mol Cell Med 2014; 3: 74-80. 57. Wu C-A, Chao Y, Shiah S-G, Lin W-W. Nutrient deprivation induces the Warburg effect through ROS/AMPK-dependent activation of pyruvate dehydrogenase kinase. Biochim Biophys Acta 2013; 1833: 1147-56. 58. Jang SM, Kim JW, Kim CH, Kim D, Rhee S, Choi KH. p19(ras) Represses proliferation of non-small cell lung cancer possibly through interaction with Neuron-Specific Enolase (NSE). Cancer Lett 2010; 289: 91-8.. 59. Amoedo Ni D, Valencia J P, Rodrigues M F, Galina A, Rumjanek F D. How does the metabolism of tumour cells differ from that of normal cells. Biosci Rep. 2013; 33: e00080. 60. Sedoris KC, Thomas SD, Miller DM. Hypoxia induces differential translation of enolase/MBP-1. BMC Cancer 2010; 10: 157. 61. Vinores SA, Bonnin JM, Rubinstein LJ, Marangos PJ. Immunohistochemical demonstration of neuron-specific enolase in neoplasms of the CNS and other tissues. Arch Pathol Lab Med 1984; 108: 536-40. 62. Vinores SA, Marangos PJ, Bonnin JM, Rubinstein Immunoradiometric and immunohistochemical demonstration of neuron-specific enolase in experimental rat gliomas. Cancer Res 1984; 44: 2595-9. 63. Kondoh H, Lleonart ME, Bernard D, Gil J. Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization. Histol Histopathol 2007; 22: 85-90. 64. Pelicano H, Martin DS, Xu RH, Huang P. Glycolysis inhibition for anticancer treatment. Oncogene 2006; 25: 4633-46. 65. Takei N, Kondo J, Nagaike K, Ohsawa K, Kato K, Kohsaka S. Neuronal survival factor from bovine brain is identical to neuron-specific enolase. J Neurochem 1991; 57: 1178-84. 66. Hafner A, Glavan G, Obermajer N, Zivin M, Schliebs R, Kos J. Neuroprotective role of gamma-enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X. Aging Cell 2013; 12: 604-14. 67. Hattori T, Ohsawa K, Mizuno Y, Kato K, Kohsaka S. Synthetic peptide corresponding to 30 amino acids of the C-terminal of neuron-specific enolase promotes survival of neocortical neurons in culture. Biochem Biophys Res Commun 1994; 202: 25-30. 68. Hafner A, Obermajer N, Kos J. gamma-Enolase C-terminal peptide promotes cell survival and neurite outgrowth by activation of the PI3K/Akt and MAPK/ERK signalling pathways. Biochem J 2012; 443: 439-50. 69. Pišlar AH, Kos J. C-terminal peptide of gamma-enolase impairs amyloid-beta-induced apoptosis through p75(NTR) signaling. Neuromolecular Med 2013; 15: 623-35. 70. Wendt W, Zhu X-R, Lübbert H, Stichel CC. Differential expression of cathep-sin X in aging and pathological central nervous system of mice. Expl Neurol 2007; 204: 525-40. 71. Kos J, Vižin T, Fonovic UP, Pišlar A. Intracellular signaling by cathepsin X: Molecular mechanisms and diagnostic and therapeutic opportunities in cancer. Semin Cancer Biol 2015; 31: 76-83. 72. Amberger-Murphy V. Hypoxia helps glioma to fight therapy. Curr Cancer Drug Targets 2009; 9: 381-90. 73. Levin VA, Panchabhai SC, Shen L, Kornblau SM, Qiu Y, Baggerly KA. Different changes in protein and phosphoprotein levels result from serum starvation of high-grade glioma and adenocarcinoma cell lines. J Proteome Res 2010; 9: 179-91. 74. Levin VA, Panchabhai S, Shen L, Baggerly KA. Protein and phosphoprotein levels in glioma and adenocarcinoma cell lines grown in normoxia and hypoxia in monolayer and three-dimensional cultures. Proteome Sci 2012; 10: 5. 75. Yan T, Skaftnesmo KO, Leiss L, Sleire L, Wang J, Li X, et al. Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes. BMC Cancer 2011; 11: 524. 76. Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta 2007; 1773: 642-52. 77. Walsh JL, Keith TJ, Knull HR. Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta 1989; 999: 64-70. 78. Trojanowicz B, Winkler A, Hammje K, Chen Z, Sekulla C, Glanz D, et al. Retinoic acid-mediated down-regulation of ENO1/MBP-1 gene products caused decreased invasiveness of the follicular thyroid carcinoma cell lines. J Mol Endocrinol 2009; 42: 249-60. 79. Georges E, Bonneau AM, Prinos P. RNAi-mediated knockdown of alpha-enolase increases the sensitivity of tumor cells to antitubulin chemothera-peutics. Int J Biochem Mol Biol 2011; 2: 303-8. 80. Kasprzak A, Zabel M, Biczysko W. Selected markers (chromogranin A, neuron-specific enolase, synaptophysin, protein gene product 9.5) in diagnosis and prognosis of neuroendocrine pulmonary tumours. Pol J Pathol 2007; 58: 23-33. 81. Tapia FJ, Polak JM, Barbosa AJ, Bloom SR, Marangos PJ, Dermody C, et al. Neuron-specific enolase is produced by neuroendocrine tumours. Lancet 1981; 1: 808-11. 82. Lopez J. Carl A. Burtis, Edward R. In: Ashwood and David E. Bruns, editors. Tietz textbook of clinical chemistry and molecular diagnosis. 5th edition. St. Louis, USA: Elsevier; 2012. 83. Stieber P, Hatz R, Holdenrieder S, Molina R, Nap M, von Pawel J, et al. National Academy of Clinical Biochemistry Guidelines for the use of tumor markers in lung cancer. Section 3P. AACC press; 2006. [citated 2015 Jan 25]. Available at http://www.nacb.org. 84. Hao X, Sun B, Hu L, Lahdesmaki H, Dunmire V, Feng Y, et al. Differential gene and protein expression in primary breast malignancies and their lymph node metastases as revealed by combined cDNA microarray and tissue microarray analysis. Cancer 2004; 100: 1110-22. 85. Miremadi A, Pinder SE, Lee AH, Bell JA, Paish EC, Wencyk P, et al. Neuroendocrine differentiation and prognosis in breast adenocarcinoma. Histopathology 2002; 40: 215-22. 86. Sawaki M, Yokoi K, Nagasaka T, Watanabe R, Kagawa C, Takada H, et al. Prognostic importance of neuroendocrine differentiation in Japanese breast cancer patients. Surg Today 2010; 40: 831-5. 87. Allen FJ, Van Velden DJ, Heyns CF. Are neuroendocrine cells of practical value as an independent prognostic parameter in prostate cancer? Br J Urol 1995; 75: 751-4. 88. Marangos PJ, Schmechel DE. Neuron specific enolase, a clinically useful marker for neurons and neuroendocrine cells. Annu Rev Neurosci 1987; 10: 269-95. 89. Rundgren M, Cronberg T, Friberg H, Isaksson A. Serum neuron specific enolase - impact of storage and measuring method. BMC Res Notes 2014; 7: 726. 90. Yuan SM. Biomarkers of cerebral injury in cardiac surgery. Anadolu Kardiyol Derg 2014; 14: 638-45. 91. Sturgeon C. Practice guidelines for tumor marker use in the clinic. Clin Chem 2002; 48: 1151-9. 92. Fujiwara H, Arima N, Ohtsubo H, Matsumoto T, Kukita T, Kawada H, et al. Clinical significance of serum neuron-specific enolase in patients with adult T-cell leukemia. Am J Hematol 2002; 71: 80-4. 93. Wang L, Liu P, Chen X, Geng Q, Lu Y. Serum neuron-specific enolase is correlated with clinical outcome of patients with non-germinal center B celllike subtype of diffuse large B-cell lymphoma treated with rituximab-based immunochemotherapy. Med Oncol 2012; 29: 2153-8. 94. Lorenz J, Dippold W. Neuron-specific enolase-a serum marker for malignant melanoma. J Natl Cancer Inst 1989; 81: 1754-5. 95. Ro C, Chai W, Yu VE, Yu R. Pancreatic neuroendocrine tumors: biology, diagnosis, and treatment. Chin J Cancer 2013; 32: 312-24. 96. Massironi S, Sciola V, Peracchi M, Ciafardini C, Spampatti MP, Conte D. Neuroendocrine tumors of the gastro-entero-pancreatic system. World J Gastroenterol 2008; 14: 5377-84. 97. DeYoung C, Edelman M. Prognostic Factors for Small-Cell Lung Cancer. In: Syrigos K, Nutting C, Roussos C, editors. Tumors of the chest. Berlin, Heidelberg: Springer; 2006. p. 189-97. 98. Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brunner N, Chan DW, et al. National Academy of Clinical Biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers. Clin Chem 2008; 54: e11-79. 99. Lamberts SWJ, Hofland L^, Nobels FRE. Neuroendocrine tumor markers. Front Neuroendocrinol 2001; 22: 309-39. 100. Riley RD, Heney D, Jones DR, Sutton AJ, Lambert PC, Abrams KR, et al. A systematic review of molecular and biological tumor markers in neuroblastoma. Clin Cancer Res 2004; 10: 4-12. 101. Johnsson P, Blomquist S, Lührs C, Malmkvist G, Alling C, Solem J-O, et al. Neuron-specific enolase increases in plasma during and immediately after extracorporeal circulation. Ann ThoracSurg 2000; 69: 750-4. 102. Ramont L, Thoannes H, Volondat A, Chastang F, Millet MC, Maquart FX. Effects of hemolysis and storage condition on neuron-specific enolase (NSE) in cerebrospinal fluid and serum: implications in clinical practice. Clin Chem Lab Med 2005; 43: 1215-7. 103. Marangos PJ, Campbell IC, Schmechel DE, Murphy DL, Goodwin FK. Blood platelets contain a neuron-specific enolase subunit. J Neurochem 1980; 34: 1254-8. 104. Trape J, Filella X, Alsina-Donadeu M, Juan-Pereira L, Bosch-Ferrer A, Rigo-Bonnin R. Increased plasma concentrations of tumour markers in the absence of neoplasia. Clin Chem Lab Med 2011; 49: 1605-20. 105. Collazos J, Esteban C, Fernandez A, Genolla J. Measurement of the serum tumor marker neuron-specific enolase in patients with benign pulmonary diseases. Am J Respir Crit Care Med 1994; 150: 143-5. 106. Filella X, Cases A, Molina R, Jo J, Bedini JL, Revert L, et al. Tumor markers in patients with chronic renal failure. Int J Biol Markers 1990; 5: 85-8. 107. DeGiorgio CM, Gott PS, Rabinowicz AL, Heck CN, Smith TD, Correale JD. Neuron-specific enolase, a marker of acute neuronal injury, is increased in complex partial status epilepticus. Epilepsia 1996; 37: 606-9. 108. Strachan MW, Abraha HD, Sherwood RA, Lammie GA, Deary IJ, Ewing FM, et al. Evaluation of serum markers of neuronal damage following severe hypoglycaemia in adults with insulin-treated diabetes mellitus. Diabetes Metab Res Rev 1999; 15: 5-12. 109. Collazos J, Genolla J, Ruibal A. Neuron-specific enolase concentrations in serum in benign liver diseases. Clin Chem 1991; 37: 579-81. 110. Massabki PS, Silva NP, Lourenco DM, Andrade LE. Neuron specific enolase concentration is increased in serum and decreased in platelets of patients with active systemic sclerosis. J Rheumatol 2003; 30: 2606-12. 111. Petrak J, Ivanek R, Toman O, Cmejla R, Cmejlova J, Vyoral D, et al. Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins. Proteomics 2008; 8: 1744-9.