Serum serotonin as unexpected potential marker for staging of experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the primary cancer of the liver. The present study aimed to assess the potential role of the endogenous regulators of angiogenesis like neurotransmitters, as possible HCC biomarkers. Five groups of rats were used in this study (8 rats per each): control healthy group (I), four intoxicated groups (II, III, IV, and V) used for induction of HCC with a single IP dose of diethylnitrosamine (DENA), 200 mg/kg. Groups II, III, IV, and V were sacrificed after 8, 16, 24, and 32 weeks of DENA injection respectively. Serum levels of epinephrine, nor-epinephrine, serotonin, and dopamine of all animals were estimated using high performance liquid chromatography technique coupled with fluorescence detector (HPLC-FLD). Development of HCC was confirmed histopathologically. Our results showed a significant increase in 3 neurotransmitters (epinephrine, nor-epinephrine, and serotonin) in DENA intoxicated HCC rat model. Only serotonin exhibited a significant increase in early histological stage HCC development (16 weeks post DENA injection) in comparison to alpha-fetoprotein (AFP), (24 weeks post DENA injection). These results suggest that neurotransmitters (Epinephrine and Norepinephrine) may have a role as a biomarker for late histological stage HCC. Like AFP, while serotonin may be used for early stage HCC.     

Serum pseudouridine as a biochemical marker in patients with hepatocellular carcinoma

The concentration of serum pseudouridine, a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in patients with hepatocellular carcinoma, liver cirrhosis, other benign hepatobiliary diseases, and healthy controls. The serum pseudouridine concentration in patients with hepatocellular carcinoma was significantly higher than that in patients with cirrhosis or the controls. Twenty-seven (51.9%) of 52 patients with hepatocellular carcinoma had serum pseudouridine concentrations higher than the mean value for healthy controls plus 2 SD. Fourteen of the 36 patients who had serum alpha-fetoprotein levels below 400 ng/ml, had elevated serum pseudouridine concentration. In total, 36 of the 52 patients (69.2%) could be detected by combination of these two markers. Two patients who had developed hepatocellular carcinoma during the course of cirrhosis and were continuously negative for alpha-fetoprotein, had higher levels of the pseudouridine concentration when hepatocellular carcinoma occurred. Furthermore, 4 of the 7 patients who had a very small cancer and were negative for alpha-fetoprotein, had elevated serum pseudouridine concentration. These results indicate that serum pseudouridine is a useful biochemical marker and that serum pseudouridine and alpha-fetoprotein in combination are considered to serve as complementary markers, for the diagnosis of hepatocellular carcinoma.     

Serum exosomal long noncoding RNA CRNDE as a prognostic biomarker for hepatocellular carcinoma

BackgroundAccumulating evidence has shown that long noncoding RNA (lncRNA) CRNDE functions as an oncogene in many cancer types. However, its clinical value has not yet been explored in hepatocellular carcinoma (HCC).MethodsA total of 166 patients with HCC and 100 healthy volunteers were enrolled in this study. The expression levels of serum exosomal lncRNA CRNDE were detected in patients with HCC and controls by quantitative real‐time PCR (qRT‐PCR).ResultsThe serum exosomal lncRNA CRNDE expression levels were significantly increased in patients with HCC compared with normal controls. High serum exosomal lncRNA CRNDE expression was significantly associated with tumor size, tumor differentiation, and TNM stage. Receiver operating characteristic (ROC) analysis revealed that an area under the ROC curve (AUC) of 0.839, with a sensitivity and specificity of 69.3% and 85.0%. In addition, the overall survival (OS) and disease‐free survival (DFS) were significantly longer in patients with lower serum exosomal lncRNA CRNDE expression compared to those with higher CRNDE expression. Moreover, HCC patients with cirrhosis had worse OS and DFS than those without cirrhosis. Univariate and multivariate analyses indicated that high serum exosomal lncRNA CRNDE expression was an independent indicator of poor prognosis.ConclusionTaken together, serum exosomal lncRNA CRNDE might serve as a potential biomarker for HCC diagnosis and prognosis.     

Alu cell-free DNA concentration,Alu index,and LINE-1 hypomethylation as a cancer predictor

IntroductionThe liquid biopsy approach, a less-invasive diagnostic tool, enables the detection of disease-specific genetic and epigenetic aberrations. Approximately 66–69% of the human genome may be composed of transposable repetitive elements, including Alu and LINE-1. This study aimed to investigate whether Alu-derived cell-free DNA (cfDNA) concentrations, Alu index, and LINE-1 methylation could be used to distinguish patients with cancers from healthy individuals.MethodsTwo sets of primers, shorter and longer Alu fragments, were used to amplify Alu elements, followed by the quantitation of Alu DNA concentration and its integrity index. LINE-1 methylation status was then analyzed with quantitative PCR using methylation- and unmethylation-specific TaqMan probes.ResultsBoth Alu index and LINE-1 methylation level were significantly different in comparison between patients with lung or breast cancer and the healthy controls. The area under the ROC curve of the Alu index and LINE-1 hypomethylation was 0.742 and 0.848 for lung cancer, respectively, and 0.724 and 0.890 for breast cancer, respectively. However, Alu longer fragment DNA concentration was significantly correlated with Alu index in comparison to LINE-1 hypomethylation. Regression analysis suggested that the LINE-1 methylation level, rather than the Alu index, was a good discriminator for lung and breast cancers.ConclusionsThis study investigated the genome-wide Alu index and LINE-1 methylation status; their associations with cancers suggested that these combinatory panels could be implemented as a triage test to discriminate cancer patients from healthy individuals.     

Plasma nitrite/nitrate concentrations as a tumor marker for hepatocellular carcinoma

Since plasma concentrations of nitrite/nitrate, the stable end-products of nitric oxide, increase in patients with hepatocellular carcinoma (HCC) correlatively to tumor volume, we examined the ability of plasma nitrite/nitrate to discriminate between those patients with HCC and those without and compared the diagnostic performance of the parameter with that of serum alpha-fetoprotein (AFP) concentrations. Plasma nitrite/nitrate and serum AFP concentrations were measured using a Griess reaction and a solid phase enzyme immunoassay, respectively. Eighty-nine patients with chronic liver diseases (CLD) with (n=39) or without HCC (n=50) and 50 healthy control subjects participated in the study. A receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value and accuracy. The areas under ROC curves for nitrite/nitrate and AFP were calculated to be 0.758 and 0.812, respectively, which were not significantly different. There was no correlation between the concentrations of plasma nitrite/nitrate and serum AFP. The sensitivity, the specificity, and diagnostic efficiency were 79.5, 72.0, and 75.3%, respectively, for nitrite/nitrate, and 74.4, 76.0, and 75.3%, respectively, for AFP. Based on a partial ROC curve, the clinical utility of plasma nitrite/nitrate as a tumor marker approximated that of serum AFP, but exceeded in AFP-negative patients. Indeed, nitrite/nitrate was positive in 70% of AFP-negative HCC patients. The simultaneous determinations of serum AFP and plasma nitrite/nitrate concentrations gave significant improvement in detection of HCC in CLD patients compared with that of serum AFP alone.     

Metastasis-related miR-185 is a potential prognostic biomarker for hepatocellular carcinoma in early stage

We previously reported that miR-185 is associated with hepatocellular carcinoma (HCC) venous metastasis analysed by miRNA-array profile. The aim of this study is to further investigate the clinicopathological significance and prognostic value of miR-185 in early stage HCC. We classified 95 patients with early stage HCC into treated recurrence group (TR) and none treated recurrence group (NTR), and detected the miR-185 expression levels in TR and NTR groups. We found that low miR-185 expression correlated with more tumor recurrence (37/46), while high miR-185 level led to lower recurrence rate (17/49) (P < 0.05). There was no direct relationship between miR-185 and clinicopathological features, including age, gender, ALT, AFP, liver cirrhosis, tumor size, tumor encapsulation, tumor differentiation (P > 0.05). Kaplan–Meier analysis showed that low miR-185 group had a remarkable lower survival rate and shorter time to recurrence than high miR-185 group (P < 0.05). Univariate and multivariate analysis, using Cox's proportional hazards model, also indicated that low miR-185 expression was a sensitive prognostic factor for survival and recurrence in early stage HCC (P < 0.05). We upregulated or downregulated miR-185 expression by transfected miR-185 mimics or inhibitor into HCC cell lines, and observed the influence of miR-185 on HCC cells in vitro. Our results manifested that miR-185 could suppress the tumor cell growth and invasive ability (P < 0.05). Therefore, miR-185 might be an effective and sensitive biomarker of HCC in early stage, and the upregulation of miR-185 might be considered to be a potentially important molecular treatment strategy for patients with HCC.     

Amplification and overexpression of the EMS 1 oncogene,a possible prognostic marker,in human hepatocellular carcinoma

DNA amplification in cancer cells frequently involves oncogenes whose increased expression confers a selective advantage on tumor cell growth. In an attempt to identify novel oncogenes involved in hepatocarcinogenesis, representational difference analysis (RDA) was performed using DNA from a primary human hepatocellular carcinoma (HCC) that showed high-level DNA amplifications on chromosomes 1p32 and 11q13 by comparative genomic hybridization. Ten amplification fragments were isolated by RDA, and when used to probe Southern blots of tumor DNA, there was a 5- to 50-fold increase in hybridization intensity relative to normal DNA. The sequence of one amplification product matched that of the EMS1 oncogene, which is located on chromosome 11q13 and is amplified in other cancers. We detected EMS1 amplification in 3 of 17 primary HCC. Overexpression of EMS1 mRNA was observed in 12 of 14 HCC cell lines in the absence of gene amplification or an increased copy-number of the gene. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development in HCC. EMS1 amplification and overexpresion is indicative of unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer.     

Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential

BACKGROUND: This article reviews research conducted on the immunogenicity of the nucleosides of DNA, especially guanosine, the most immunologically active nucleoside. Discussed is the relationship between circulating antibodies to guanosine, their potential role in SLE disease activity, the binding properties of monoclonal antiguanosine antibody (4H2) compared to polyclonal antiguanosine antibodies in humans with SLE, cell membrane penetration by these antibodies and their interference with signal transduction possibly related to their binding to mitochondria and their apparent GTPase activity. METHODS: Enzyme-linked immunosorbent assay methodology was used to show clinical relationships between antiguanosine antibody levels and disease activity in SLE. These results are discussed along with methods of detecting cell penetration by this antibody using special staining techniques, laser-scanning microscope detection of mitochondrial localization, and interference of cAMP and pKA production/activation. Additionally, there is some discussion regarding the assay used to detect enzymatic activity of antiguanosine antibodies. RESULTS: Enhanced circulating levels of antiguanosine antibodies in patients with SLE correlate closely with SLE disease activity. Other factors are discussed that support the pathogenic potential of these antibodies, including their ability to penetrate lymphocytes, bind to mitochondria, inactivate mitochondrial function, interfere with signal transduction, and their potential enzymatically activity. CONCLUSIONS: Antiguanosine antibodies correlate with SLE disease activity and may be pathogenically important in SLE by interfering with signal transduction, inactivating mitochondrial and cell function in patients with SLE.     

Procalcitonin as a diagnostic marker and IL-6 as a prognostic marker for sepsis

The diagnosis and prognosis of sepsis after antimicrobial therapy among systemic inflammatory response syndrome (SIRS) patients were evaluated with the biomarkers procalcitonin (PCT), interleukin-6 (IL-6), C-reactive protein (CRP), erythrocyte sedimentation rate, and white blood cell counts.     

Rapid and reliable detection of LINE-1 hypomethylation using high-resolution melting analysis

Objectives

The aim of the present study was to evaluate the precision and reproducibility of the LINE-1 high-resolution melting (HRM) assay to detect LINE-1 hypomethylation.

Design and methods

We first evaluated a methylated DNA dilution matrix and a panel of human cancer cell lines. We then applied this LINE-1 HRM assay to a set of 37 archival prostate cancer tissue samples.

Results

Our LINE-1 HRM assay revealed small and reproducible run-to-run and bisulfite-to-bisulfite variations. As expected, we found a large variation in methylation levels between different cancer cell lines. All results were confirmed with MethyLight and pyrosequencing as indicated by the high correlation coefficient. Finally, we successfully applied the LINE-1 HRM assay to archival prostate cancer tissues.

Conclusions

The present LINE-1 HRM assay represents a novel, accurate, and cost-effective method to measure global hypomethylation, which makes it suitable for high- and low-throughput laboratories.     

Cyclin B1 acts as a tumor microenvironment-related cancer promoter and prognostic biomarker in hepatocellular carcinoma

ObjectivesThe present study aimed to develop a gene signature based on the ESTIMATE algorithm in hepatocellular carcinoma (HCC) and explore possible cancer promoters.MethodsThe ESTIMATE and CIBERSORT algorithms were applied to calculate the immune/stromal scores and the proportion of tumor-infiltrating immune cells (TICs) in a cohort of HCC patients. The differentially expressed genes (DEGs) were screened by Cox proportional hazards regression analysis and protein–protein interaction (PPI) network construction. Cyclin B1 (CCNB1) function was verified using experiments.ResultsThe stromal and immune scores were associated with clinicopathological factors and recurrence-free survival (RFS) in HCC patients. In total, 546 DEGs were up-regulated in low score groups, 127 of which were associated with RFS. CCNB1 was regarded as the most predictive factor closely related to prognosis of HCC and could be a cancer promoter. Gene Set Enrichment Analysis (GSEA) and CIBERSORT analyses indicated that CCNB1 levels influenced HCC tumor microenvironment (TME) immune activity.ConclusionsThe ESTIMATE signature can be used as a prognosis tool in HCC. CCNB1 is a tumor promoter and contributes to TME status conversion.     

DLK1 as a potential target against cancer stem/progenitor cells of hepatocellular carcinoma

Delta-like 1 homolog (DLK1; Drosophila) is a hepatic stem/progenitor cell marker in fetal livers that plays a vital role in oncogenesis of hepatocellular carcinoma (HCC). The aim of this study is to investigate whether DLK1 could serve as a potential therapeutic target against cancer stem/progenitor cells of HCC. DLK1(+) and DLK1(-) cells were sorted by fluorescence-activated cell sorting and magnetic-activated cell sorting, respectively, and then were evaluated by flow cytometry. The biological behaviors of these isolated cells and those with DLK1 knockdown were assessed by growth curve, colony formation assay, spheroid colony formation, chemoresistance, and in vivo tumorigenicity. Adenovirus-mediated RNA interference was used to knockdown the endogenous DLK1. We found that DLK1(+) population was less than 10% in almost all 17 HCC cell lines examined. DLK1(+) HCC cells showed stronger ability of chemoresistance, colony formation, spheroid colony formation, and in vivo tumorigenicity compared with DLK1(-) cells. The DLK1(+) HCC cells could generate the progeny without DLK1 expression. Furthermore, DLK1 knockdown could suppress the ability of proliferation, colony formation, spheroid colony formation, and in vivo tumorigenicity of Hep3B and Huh-7 HCC cells. Our data suggested that DLK1(+) HCC cells have characteristics similar to those of cancer stem/progenitor cells. RNA interference against DLK1 can suppress the malignant behaviors of HCC cells, possibly through directly disrupting cancer stem/progenitor cells, which suggested that DLK1 could be a potential therapeutic target against the HCC stem/progenitor cells.     

Serum CK-BB as a tumor marker in patients with carcinoma confirmed histologically

We determined serum CK-BB mass concentration using a specific RIA method, in 267 patients with carcinoma confirmed histologically distributed as follows: 46 prostatic adenocarcinoma, 52 lung neoplasies, 70 colon carcinoma, 52 breast carcinoma and 41 gastric carcinoma; and also in 135 patients with histologically proved non-neoplastic diseases distributed as follows: 28 prostatic hyperplasy, 31 lung tuberculosis, 29 inflammatory bowel disease, 27 fibrocystic mastopathy and 20 gastroduodenal ulcer. Reference values in healthy subjects (n = 360) were 5.46 +/- 2.68 (SD) ng/ml. We found that serum CK-BB mass concentration is not a specific tumor marker but it is a valuable indicator of responsing to therapy and metastatic widespread. However, in prostatic carcinoma--prevalence 0.25, predictive positive value (PPV) 0.51 and predictive negative value (PNV) 0.88--and breast carcinoma--prevalence 0.32, PPV 0.60 and PNV 0.87--serum CK-BB can be used as a tumor marker. Only 12 over 268 patients with different neoplastic disease (4.47%) showed detectable serum CK-BB catalytic concentrations.     

The clinical utility of miR-21 as a diagnostic and prognostic marker for renal cell carcinoma

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney. Increasing evidence suggests that microRNAs are dysregulated in RCC and are important factors in RCC pathogenesis. miR-21 is a known oncogene with tumor-promoting effects in many types of cancer. In this study, we analyzed miR-21 in 121 cases of healthy kidney and different RCC subtypes, including clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), and oncocytoma. Total RNA was extracted, and the expression of miR-21 was measured with real-time quantitative RT-PCR using miR-21-specific probes. The expression of miR-21 was significantly up-regulated in RCC compared with healthy kidney. There was a significant difference in the expression levels between RCC subtypes, with the highest levels of expression in ccRCC and pRCC subtypes. miR-21 expression distinguished ccRCC and pRCC from chRCC and oncocytoma with 90% specificity (95% CI, 63.9% to 98.1%) and 83% sensitivity (95% CI, 53.5% to 97.6%). Significantly higher miR-21 levels were associated with higher stage and grade. Patients who were miR-21 positive had statistically significant shorter disease-free and overall survival rates. Thus, miR-21 is up-regulated in RCC, and its expression levels can be used as a diagnostic marker to distinguish ccRCC and pRCC from chRCC and oncocytoma. Moreover, it has potential as a prognostic marker in RCC, although it is not independent of tumor stage and grade.