Serum and urine metabolite profiling reveals potential biomarkers of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.     

The potential biomarkers of drug addiction: proteomic and metabolomics challenges

Drug addiction places a significant burden on society and individuals. Proteomics and metabolomics approaches pave the road for searching potential biomarkers to assist the diagnosis and treatment. This review summarized putative drug addiction-related biomarkers in proteomics and metabolomics studies and discussed challenges and prospects in future studies. Alterations of several hundred proteins and metabolites were reported when exposure to abused drug, which enriched in energy metabolism, oxidative stress response, protein modification and degradation, synaptic function and neurotrasmission, etc. Hsp70, peroxiredoxin-6 and α- and β-synuclein, as well as n-methylserotonin and purine metabolites, were promising as potential biomarker for drug addiction.     

Urine metabolomics analysis for biomarker discovery and detection of jaundice syndrome in patients with liver disease

Metabolomics is a powerful new technology that allows for the assessment of global metabolic profiles in easily accessible biofluids and biomarker discovery in order to distinguish between diseased and nondiseased status information. Deciphering the molecular networks that distinguish diseases may lead to the identification of critical biomarkers for disease aggressiveness. However, current diagnostic methods cannot predict typical Jaundice syndrome (JS) in patients with liver disease and little is known about the global metabolomic alterations that characterize JS progression. Emerging metabolomics provides a powerful platform for discovering novel biomarkers and biochemical pathways to improve diagnostic, prognostication, and therapy. Therefore, the aim of this study is to find the potential biomarkers from JS disease by using a nontarget metabolomics method, and test their usefulness in human JS diagnosis. Multivariate data analysis methods were utilized to identify the potential biomarkers. Interestingly, 44 marker metabolites contributing to the complete separation of JS from matched healthy controls were identified. Metabolic pathways (Impact-value≥0.10) including alanine, aspartate, and glutamate metabolism and synthesis and degradation of ketone bodies were found to be disturbed in JS patients. This study demonstrates the possibilities of metabolomics as a diagnostic tool in diseases and provides new insight into pathophysiologic mechanisms.     

Serum paraoxonase 1 heteroplasmon, a fucosylated, and sialylated glycoprotein in distinguishing early hepatocellular carcinoma from liver cirrhosis patients

Aberrant glycan structure of serum glycoproteins creates unique patterns in different stages of hepatocellular carcinoma (HCC), which provides potential glycan biomarkers for early diagnosis of HCC. In this study, tandem lectin affinity chromatography using aleuria aurantia lectin (AAL) and wheat germ agglutinin (WGA) was processed to purify both fucosylated and sialylated serum glycoproteins from 27 liver cirrhosis (LC) and 27 early HCC patients, in which 122 glycoproteins were finally screened out by liquid chromatography-tandem mass spectrometry (LC-MSMS). Among the 122 proteins identified by LC-MSMS, 8 of them were only identified in HCC serum and another 6 existed only in LC serum. Serum paraoxonase 1 (PON1) was immunoprecipitated from 47 individual patients and blotted by lectins, showing enhanced fucosylation and sialylation in HCC serum than those in LC serum. The area under the ROC curve (AUROC) for AAL-reactive PON1 was 0.892 with a sensitivity of 71.4% and a specificity of 94.7% in differentiating early HCC from LC. Similarly, WGA-reactive PON1 had an AUROC of 0.902 with a sensitivity of 95.2% and a specificity of 78.9%. The data indicated that the glycan differences of serum PON1 might serve as potential glycan biomarkers for distinguishing early HCC from LC patients.     

CHANGES OF PHOSPHATIDYLCHOLINE-SPECIFIC PHOSPHOLIPASE C IN HEPATOCARCINOGENESIS AND IN THE PROLIFERATION AND DIFFERENTIATION OF RAT LIVER CANCER CELLS

The biological significance of phosphatidylcholine-specific phospholipase C (PC-PLC) in hepatocarcinogenesis and the proliferation and differentiation of rat liver cancer cells was investigated. The Ca²⁺-dependent activities of PC-PLC gradually increased during N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis and peaked at weeks 18–20 when the tumour formed. There was a close relationship between Ca²⁺-dependent PC-PLC activities and cellular DNA content, membranous γ-glutamyltranspeptidase (γ-GT), and tyrosine protein kinase. In contrast, Ca²⁺-independent PC-PLC activities decreased during hepatocarcinogenesis. Similarly, when CBRH-7919 rat liver cancer cells were treated with phorbol 12-myristate 13-acetate, a proliferation stimulator of the cells, γ-GT and Ca²⁺-dependent activities of PC-PLC and the expression of α-fetoprotein increased significantly. However, when these cells were induced by retinoic acid to differentiate, Ca²⁺-dependent PC-PLC and γ-GT activities decreased significantly, together with α-fetoprotein expression. There was a close relationship between Ca²⁺-dependent PC-PLC and γ-GT activities during differentiation as there was during proliferation. We suppose that Ca²⁺-dependent PC-PLC is involved in rat hepatocarcinogenesis induced by DEN and that it plays an important role in the phorbol ester-induced proliferation or retinoic acid-induced differentiation of liver cancer cells.     

A Comparative Metabolomic Evaluation of Behcet’s Disease with Arthritis and Seronegative Arthritis Using Synovial Fluid

Behcet’s disease (BD) with arthritis is often confused with seronegative arthritis (SNA) because of shared clinical symptoms and the lack of definitive biomarkers for BD. To investigate possible metabolic patterns and potential biomarkers of BD with arthritis, metabolomic profiling of synovial fluid (SF) from 6 patients with BD with arthritis and 18 patients with SNA was performed using gas chromatography/time-of-flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. A total of 123 metabolites were identified from samples. Orthogonal partial least square-discriminant analysis showed clear discrimination between BD with arthritis and SNA. A set of 11 metabolites were identified as potential biomarkers for BD using variable importance for projection values and the Wilcoxon-Mann-Whitney test. Compared with SNA, BD with arthritis exhibited relatively high levels of glutamate, valine, citramalate, leucine, methionine sulfoxide, glycerate, phosphate, lysine, isoleucine, urea, and citrulline. There were two markers identified, elevated methionine sulfoxide and citrulline, that were associated with increased oxidative stress, providing a potential link to BD-associated neutrophil hyperactivity. Glutamate, citramalate, and valine were selected and validated as putative biomarkers for BD with arthritis (sensitivity, 100%; specificity, 61.1%). This is the first report to present potential biomarkers from SF for discriminating BD with arthritis from SNA. The metabolomics of SF may be helpful in searching for potential biomarkers and elucidating the clinicopathogenesis of BD with arthritis.     

Serum metabonomics study of pregnant women with gestational diabetes mellitus based on LC-MS

ObjectiveThrough metabolomics method, the objective of the paper is to differentially screen serum metabolites of GDM patients and healthy pregnant women, to explore potential biomarkers of GDM and analyze related pathways, and to explain the potential mechanism and biological significance of GDM.MethodsThe serum samples from 30 GDM patients and 30 healthy pregnant women were selected to conduct non-targeted metabolomics study by liquid chromatography-mass spectrometry. The differential metabolites between the two groups were searched and the metabolic pathway was analyzed by KEGG database.ResultsMultivariate statistical analysis found that serum metabolism in GDM patients was different significantly from healthy pregnant women, 36 differential metabolites and corresponding metabolic pathways were identified in serum, which involved several metabolic ways like, fatty acid metabolism, butyric acid metabolism, bile secretion, and amino acid metabolism.ConclusionThe discovery of these biomarkers provided a new theoretical basis and experimental basis for further study of the early diagnosis and pathogenesis of GDM. At the same time, LC-MS-based serum metabolomics methods also showed great application values in disease diagnosis and mechanism research.     

Hepatitis C virus-induced cancer stem cell-like signatures in cell culture and murine tumor xenografts

Hepatitis C virus (HCV) infection is a prominent risk factor for the development of hepatocellular carcinoma (HCC). Similar to most solid tumors, HCCs are believed to contain poorly differentiated cancer stem cell-like cells (CSCs) that initiate tumorigenesis and confer resistance to chemotherapy. In these studies, we demonstrate that the expression of an HCV subgenomic replicon in cultured cells results in the acquisition of CSC traits. These traits include enhanced expression of doublecortin and CaM kinase-like-1 (DCAMKL-1), Lgr5, CD133, α-fetoprotein, cytokeratin-19 (CK19), Lin28, and c-Myc. Conversely, curing of the replicon from these cells results in diminished expression of these factors. The putative stem cell marker DCAMKL-1 is also elevated in response to the overexpression of a cassette of pluripotency factors. The DCAMKL-1-positive cells isolated from hepatoma cell lines by fluorescence-activated cell sorting (FACS) form spheroids in Matrigel. The HCV RNA abundance and NS5B levels are significantly reduced by the small interfering RNA (siRNA)-led depletion of DCAMKL-1. We further demonstrate that HCV replicon-expressing cells initiate distinct tumor phenotypes compared to the tumors initiated by parent cells lacking the replicon. This HCV-induced phenotype is characterized by high-level expression/coexpression of DCAMKL-1, CK19, α-fetoprotein, and active c-Src. The results obtained by the analysis of liver tissues from HCV-positive patients and liver tissue microarrays reiterate these observations. In conclusion, chronic HCV infection appears to predispose cells toward the path of acquiring cancer stem cell-like traits by inducing DCAMKL-1 and hepatic progenitor and stem cell-related factors. DCAMKL-1 also represents a novel cellular target for combating HCV-induced hepatocarcinogenesis.     

Serum UPLC-MS/MS metabolic profiling in an experimental model for acute-liver injury reveals potential biomarkers for hepatotoxicity

A key interest in clinical diagnosis and pharmaceutical industry is to have a repertoire of noninvasive biomarkers to??individually or in combination??be able to infer or predict the degree of liver injury caused by pathological conditions or drugs. Metabolomics??a comprehensive study of global metabolites??has become a highly sensitive and powerful tool for biomarker discovery thanks to recent technological advances. An ultra-performance liquid chromatography/time-of-flight tandem mass spectrometry (UPLC/TOF MS/MS)-based metabolomics approach was employed to investigate sera from galactosamine-treated rats to find potential biomarkers for acute liver injury. Hepatic damage was quantified by determining serum transaminase activity and in situ liver histological lesions. Principal component analysis in combination with coefficient of correlation analysis was used for biomarker selection and identification. According to the data, serum levels of several metabolites including glucose, amino acids, and membrane lipids were significantly modified, some of them showing a high correlation with the degree of liver damage determined by histological examination of the livers. In conclusion, this study supports that UPLC-MS/MS based serum metabolomics in experimental animal models could be a powerful approach to search for biomarkers for drug- or disease-induced liver injury.     

The expression of alpha-fetoprotein and albumin genes in rat liver during chemical carcinogenesis

The effect of the hepatocarcinogen 3′-methyl-4-dimethylaminoazobenzene on α-fetoprotein (AFP) and albumin gene expression in rat liver was studied. Serum concentrations of AFP and albumin were measured. Amounts of AFP mRNA and albumin mRNA in rat livers were determined by hybridization of total cytoplasmic RNAs to their cDNAs. Dramatic increases in serum AFP concentrations coincided with increases in AFP biosynthesis and amount of AFP mRNA in livers of carcinogen-treated rats. In contrast, no or little change in albumin mRNA concentration was found in livers of rats treated with 3′-methyl-4-dimethylaminoazobenzene. Concomitantly, there was little change in liver albumin biosynthesis or serum albumin concentrations during hepatocarcinogenesis.     

Yolk sac: site of developmental microheterogeneity of mouse alpha-fetoprotein.

α-Fetoprotein was observed to be synthesized in mouse fetal liver and yolk sac by incorporation of radioactive leucine into appropriate tissue cultures. Cultured fetal liver during early (Day 13.5) and late (Day 16.5) development secreted predominantly the maximally sialylated Fp5. In contrast, the yolk sac secreted a developmentally changing array of α-fetoprotein: Day 11.5 yolk sac secreted predominantly the unsialylated Fp1, at Day 13.5, the yolk sac secreted all five electrophoretic forms of α-fetoprotein, and by Day 16.5, only Fp5 was predominantly secreted, as in the fetal liver. To ascertain whether the ³H-labeled proteins that appeared in the regions of α-fetoprotein on polyacrylamide gels represented α-fetoprotein, immunoprecipitations with anti-α-fetoprotein were carried out. After the immunoprecipitations, radioactivity in the regions of marker α-fetoprotein on polyacrylamide gels was decreased to background levels. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates was performed, radioactivity peaks comigrated with marker α-fetoprotein. The undersialylated α-fetoprotein forms do not appear to arise by loss of sialic acid following secretion as determined by mixing experiments of yolk sac and fetal liver in culture. The contribution of α-fetoprotein synthesized and secreted by fetal liver and yolk sac at Days 13.5 and 16.5 of development was compared. Day 13.5 yolk sac incorporated 6.7 times as much radioactivity into secreted α-fetoprotein as did fetal liver at this time. These results suggest that during early development, the yolk sac is primarily responsible for the synthesis and secretion of the undersialylated forms of α-fetoprotein. In addition to the microheterogeneity of α-fetoprotein attributed to the number of sialic acid residues attached to the glycoprotein, there appeared to be other changes in α-fetoprotein: Fp5 synthesized from fetal liver migrated slightly faster on polyacrylamide gels than that from yolk sac.     

Urine metabolomic analysis identifies potential biomarkers and pathogenic pathways in kidney cancer

Kidney cancer is the seventh most common cancer in the Western world, its incidence is increasing, and it is frequently metastatic at presentation, at which stage patient survival statistics are grim. In addition, there are no useful biofluid markers for this disease, such that diagnosis is dependent on imaging techniques that are not generally used for screening. In the present study, we use metabolomics techniques to identify metabolites in kidney cancer patients' urine, which appear at different levels (when normalized to account for urine volume and concentration) from the same metabolites in nonkidney cancer patients. We found that quinolinate, 4-hydroxybenzoate, and gentisate are differentially expressed at a false discovery rate of 0.26, and these metabolites are involved in common pathways of specific amino acid and energetic metabolism, consistent with high tumor protein breakdown and utilization, and the Warburg effect. When added to four different (three kidney cancer-derived and one "normal") cell lines, several of the significantly altered metabolites, quinolinate, α-ketoglutarate, and gentisate, showed increased or unchanged cell proliferation that was cell line-dependent. Further evaluation of the global metabolomics analysis, as well as confirmation of the specific potential biomarkers using a larger sample size, will lead to new avenues of kidney cancer diagnosis and therapy.     

Proteomic profiling of N‐linked glycoproteins identifies ConA‐binding procathepsin D as a novel serum biomarker for hepatocellular carcinoma

The aim of this study was to identify novel biomarkers for the diagnosis of, and potential therapeutic targets for, hepatocellular carcinoma (HCC). Multilectin affinity chromatography was used to enrich N‐linked glycoproteins from nontumorous liver and HCC tissues followed by 2DE and protein identification by MS. Twenty‐eight differentially expressed proteins were identified. Western blotting validated consistently lower concentrations of human liver carboxylesterase 1 and haptoglobin, and higher concentration of procathepsin D (pCD) in HCC tissues. Knockdown of cathepsin D (CD) expression mediated by siRNA significantly inhibited the in vitro invasion of two HCC cell lines, SNU449 and SNU473, which normally secrete high‐levels of CD. Prefractionation using individual lectins demonstrated an elevation in ConA‐binding glycoforms of proCD and CD in HCC tissues. In the serum of HCC patients, “ConA‐binding proCD” (ConA‐pCD) is significantly increased in concentration and this increase is comprised of several distinct upregulated acidic isoforms (pI 4.5–5.5). Receiver operating characteristic analysis showed that the sensitivity and specificity of serum ConA‐pCD for HCC diagnosis were 85% and 80%, respectively. This is the first report that serum ConA‐pCD is increased significantly in HCC and is potentially useful as a serological biomarker for diagnosis of HCC.     

Rapid determination of serological cytokine biomarkers for hepatitis B virus-related hepatocellular carcinoma using antibody microarrays

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with an increasing incidence. The exploration of biomarkers for HCC is one of the main aims for improving the efficacy of diagnosis and treatment. The microarray technology provides a high-throughput platform for parallel exploration of biomarkers for clinics. In this study, we used antibody microarrays to screen the novel cytokine biomarkers of hepatitis B virus (HBV)-related HCC. Cytokine-secreting patterns in sera were determined from 109 cases including 43 HBV-related HCC patients, 33 chronic hepatitis B patients, and 33 normal controls by RayBio Biotin label-based human antibody array. The correlation analysis was performed with conventional clinical diagnostic biomarkers, including serum alanine aminotransferase, alpha-fetoprotein (AFP) and hepatitis B surface antigen. Our results showed that in HBV-related HCC group, which had the highest percentage of AFP positive (>20 ng/ml) ratio, six cytokines were found differentially expressed in HCC patients (P < 0.05), compared with either normal controls or chronic hepatitis B group. Two macrophage-related cytokines, macrophage-derived chemokine (MDC) and macrophage-stimulating protein α (MSPα), displayed significant difference in the HCC group. Furthermore, an HCC diagnostic model for prediction was constructed, by which the combination of MDC and MSPα together with AFP had improved the diagnostic sensitivity from 60% (AFP alone) to 73.2% with similar specificity. Our results suggested that MDC and MSPα screened by antibody microarrays might serve as novel cytokines biomarkers for potential auxiliary diagnosis of HBV-related HCC.     

岩藻糖链与肝癌细胞发生和转移的相关性研究

研究观察了大鼠诱发肝癌过程中,与UEA、LCA凝集素相结合的含岩藻糖糖蛋白尤其是80 ku蛋白的动态变化．在肝癌病人标本中,也观察到了高转移性肝癌细胞比低转移性肝癌细胞表达更多的UEA、LCA相结合的岩藻糖蛋白．岩藻糖寡糖可以构成一些非常重要的黏附分子的结构,如Lewis抗原．继而进一步观察了不同转移潜能的肝癌细胞中Lewis抗原的表达差异,发现高转移性肝癌细胞 (HMCC97H) 比低转移性肝癌细胞(HMCC97L)表达更高的Lewis x 和 b．在肝癌转移动物模型中,转移灶组织中的Lewis抗原合成关键酶α1,3/1,2以及 α1,6 岩藻糖转移酶活性远比对照组高．当肝癌细胞在维甲酸作用以后,细胞表面的Lewis x 或 b 的水平显著下降,α1,3/1,2岩藻糖转移酶活性也显著下降．同时我们观察到Lewis x可以存在于表皮细胞生长因子受体(EGFR)分子上,在维甲酸作用以后,EGFR上的Lewis x抗原和磷酸化水平都显著性下降．上述结果提示岩藻糖化的糖链如Lewis x在肝癌细胞的发生和转移过程中起重要的作用．     

Metabonomic profiles discriminate hepatocellular carcinoma from liver cirrhosis by ultraperformance liquid chromatography-mass spectrometry

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and usually develops in patients with liver cirrhosis (LC). Biomarkers that discriminate HCC from LC are important but are limited. In the present study, an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS)-based metabonomics approach was used to characterize serum profiles from HCC (n = 82), LC (n = 48), and healthy subjects (n = 90), and the accuracy of UPLC-MS profiles and alpha-fetoprotein (AFP) levels were compared for their use in HCC diagnosis. By multivariate data and receiver operating characteristic curves analysis, metabolic profiles were capable of discriminating not only patients from the controls but also HCC from LC with 100% sensitivity and specificity. Thirteen potential biomarkers were identified and suggested that there were significant disturbances of key metabolic pathways, such as organic acids, phospholipids, fatty acids, bile acids, and gut flora metabolism, in HCC patients. Canavaninosuccinate was first identified as a metabolite that exhibited a significant decrease in LC and an increase in HCC. In addition, glycochenodeoxycholic acid was suggested to be an important indicator for HCC diagnosis and disease prognosis. UPLC-MS signatures, alone or in combination with AFP levels, could be an efficient and convenient tool for early diagnosis and screening of HCC in high-risk populations.     

Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.     

Role of lipids in pathophysiology,diagnosis and therapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive and widespread cancer. Patients with liver cirrhosis of different aetiologies are at a risk to develop HCC. It is important to know that in approximately 20% of cases primary liver tumors arise in a non-cirrhotic liver. Lipid metabolism is variable in patients with chronic liver diseases, and lipid metabolites involved therein do play a role in the development of HCC. Of note, lipid composition of carcinogenic tissues differs from non-affected liver tissues. High cholesterol and low ceramide levels in the tumors protect the cells from oxidative stress and apoptosis, and do also promote cell proliferation. So far, detailed characterization of the mechanisms by which lipids enable the development of HCC has received little attention. Evaluation of the complex roles of lipids in HCC is needed to better understand the pathophysiology of HCC, the later being of paramount importance for the development of urgently needed therapeutic interventions. Disturbed hepatic lipid homeostasis has systemic consequences and lipid species may emerge as promising biomarkers for early diagnosis of HCC. The challenge is to distinguish lipids specifically related to HCC from changes simply related to the underlying liver disease. This review article discusses aberrant lipid metabolism in patients with HCC.     

Metabolic signatures of lung cancer in biofluids: NMR-based metabonomics of urine

In this study, 1H NMR-based metabonomics has been applied, for the first time to our knowledge, to investigate lung cancer metabolic signatures in urine, aiming at assessing the diagnostic potential of this approach and gaining novel insights into lung cancer metabolism and systemic effects. Urine samples from lung cancer patients (n = 71) and a control healthy group (n = 54) were analyzed by high resolution 1H NMR (500 MHz), and their spectral profiles subjected to multivariate statistics, namely, Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Projections to Latent Structures (OPLS)-DA. Very good discrimination between cancer and control groups was achieved by multivariate modeling of urinary profiles. By Monte Carlo Cross Validation, the classification model showed 93% sensitivity, 94% specificity and an overall classification rate of 93.5%. The possible confounding influence of other factors, namely, gender and age, have also been modeled and found to have much lower predictive power than the presence of the disease. Moreover, smoking habits were found not to have a dominating influence over class discrimination. The main metabolites contributing to this discrimination, as highlighted by multivariate analysis and confirmed by spectral integration, were hippurate and trigonelline (reduced in patients), and β-hydroxyisovalerate, α-hydroxyisobutyrate, N-acetylglutamine, and creatinine (elevated in patients relatively to controls). These results show the valuable potential of NMR-based metabonomics for finding putative biomarkers of lung cancer in urine, collected in a minimally invasive way, which may have important diagnostic impact, provided that these metabolites are found to be specifically disease-related.     

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.