Analysis of urinary nucleosides as helper tumor markers in hepatocellular carcinoma diagnosis

Hepatocellular carcinoma (HCC) is a common neoplasm in Taiwan, for which early diagnosis is difficult and the prognosis is usually poor. HCC is usually diagnosed by abdominal sonography and serum alpha‐fetoprotein (AFP) detection. Modified nucleosides, regarded as indicators for the whole‐body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies and can serve as tumor markers. We analyzed the excretion patterns of urinary nucleosides from 25 HCC patients and 20 healthy volunteers by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) under optimized conditions. The HPLC/ESI‐MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples. The mean levels of the urinary nucleosides adenosine, cytidine, and inosine were significantly higher in HCC patients than healthy volunteers (average of 1.78‐, 2.26‐, and 1.47‐fold, respectively). However, the mean levels of urinary 1‐methyladenosine, 3‐methylcytidine, uridine, and 2′‐deoxyguanosine were not significantly different. Combined with the determination of serum AFP levels, the higher levels of urinary adenosine, cytidine, and inosine may be additional diagnosis markers for HCC in Taiwanese patients. Copyright © 2009 John Wiley & Sons, Ltd.     

8-Quinolineboronic acid as a potential phosphorescent molecular switch for the determination of alpha-fetoprotein variant for the prediction of primary hepatocellular carcinoma

8-Quinolineboronic acid phosphorescent molecular switch (8-QBA-PMS) in the “off” state emitted weak room temperature phosphorescence (RTP) of 8-QBA on the acetylcellulose membrane (ACM) with the perturbation of Pb²⁺. When 8-QBA-PMS was used to label concanavalin agglutinin (Con A) to form 8-QBA-PMS-Con A based on the reaction between -OH of 8-QBA-PMS and -COOH of Con A, 8-QBA-PMS turned “on” automatically due to its structure change, and RTP of the system increased 2.7 times. Besides, -NH₂ of 8-QBA-PMS-Con A could carry out affinity adsorption (AA) reaction with the -COOH of alpha-fetoprotein variant (AFP-V) to form the product Con A-AFP-V-Con A-8-QBA-PMS containing -NH-CO- bond, causing the RTP of the system to further increase. Moreover, the amount of AFP-V was linear to the ΔI_p of the system in the range of 0.012-2.40 (fg spot⁻¹). Thus, a new affinity sensitive adsorption solid substrate room temperature phosphorimetry using 8-QBA-PMS as labelling reagent (8-QBA-PMS-AASSRTP) for the determination of AFP-V was proposed with the detection limit (LD) of 9 × 10⁻¹⁵ g mL⁻¹. It had been used to determine AFP-V in human serum with the results agreeing with enzyme-link immunoassay (ELISA), showing promise for the prediction of PHC due to the intimate association between AFP-V and primary hepatocellular carcinoma (PHC). The mechanism of the promethod was also discussed.     

Identification and cloning of molecular markers for UV-B tolerant gene in wild sugarcane (Saccharum spontaneum L.)

Previously we have selected wild sugarcane (Saccharum spontaneum L.) sterile lines that are tolerant or susceptible to UV-B radiation based on response index (RI) in a field screening test. The RI was established according to plant height, tiller number, leaf index, total biomass and brix under enhanced ultraviolet-B (UV-B, 280-310 nm) radiation. In this experiment, molecular markers linked to the UV-B tolerant and susceptible genes were identified and cloned. RAPD (Randomly amplified polymorphic DNAs) assay using 100 arbitrary primers followed by clustering analysis separated the tolerant and susceptible lines into two groups at the genetic distance of 0.380. The UV-B tolerant and susceptible gene pools were constructed and compared using the Bulked Segregate Analysis (BSA) approach. Of the 100 arbitrary RAPD primers, primer OPR16 produced polymorphic DNA banding patterns from both gene pools. The OPR16-1200 bp DNA fragment was only amplified from the tolerant lines and the OPR16-800 bp from the susceptible ones. These two PCR fragments were cloned onto T-vector. DNA sequence alignment analysis determined that 42% homology existed between the reverse and forward sequences of the OPR16-1200 bp clone, and 36% homology between the forward sequences of the OPR16-800 bp and OPR16-1200 bp clones. The two DNA clones were determined to be linked to the UV-B tolerant and susceptible genes, and they can be used to develop molecular markers for the associated traits.     

Novel solution-phase immunoassays for molecular analysis of tumor markers

at 3 x 10(9) M(-1) and a step-wise binding process with PSA-free MAB. Thus, this solution-phase quantitative ECL immunoassay allowed measurement of the affinity of serum PSAs with their MABs and screening of PSAs based upon their affinity to MABs. Unlike other immunoassays, this immunoassay demonstrated one-step rapid analysis while simultaneously eliminating immobilization, separation and washing steps and detected PSA at a level of 1.7 pg mL(-1), which is 1000-fold more sensitive than current PSA immunoassays. Furthermore, single-molecule (SM) phosphorescence microscopy was developed to detect single serum PSA-free and PSA-complex molecules in solution with no use of antibody showing that PSA-free molecules diffused faster than PSA-complex molecules in solution. This finding is consistent with ECL measurements and implies the possibility of screening individual analytes in a complex mixture using their distinct SM diffusion distance. This is the first report describing the detection of single protein molecules labeled with a metal-complex using phosphorescence microscopy and also the screening of serum tumor markers using ECL and SM phosphorescence solution-phase assays.     

Studies of potential cerebrospinal fluid molecular markers for Alzheimer's disease

There is a need for a reliable, molecular-based ante mortem diagnostic test for Alzheimer's disease (AD). In this study, we examined the use of two-dimensional protein electrophoresis for generating molecular barcodes which may be useful for the clinical differentiation of AD patients from normals. We compared cerebrospinal fluid samples taken from AD patients with confirmed post mortem pathology to comparable specimens from normal volunteers. Using canonical correlation analysis, a panel of nine molecular markers were identified which segregated diseased cases from normal controls. Using the scaled volume image analysis variable, a principal factor analysis was also used to distinguish normal from AD spinal fluid, based on molecular markers identified using a heuristic clustering algorithm. The use of panels of molecular markers derived from proteomic analysis may offer the best prospect for developing molecular diagnostic tests for complex neurodegenerative disorders such as AD.     

Advanced radionuclides in diagnosis and therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver, but early diagnosis and effective treatment are still difficult. With the development of radionuclide applications in medicine, nuclear medicine is playing an increasingly important role in the diagnosis and treatment of HCC. Radionuclide-based positron emission tomography-computed tomography and single-photon emission computed tomography-computed tomography molecular imaging are indispensable for assessing progression, staging, differentiation, preoperative planning, postoperative prediction, and evaluation of HCC in clinical applications. Moreover, radionuclide-based endoradiotherapy provides an objective therapeutic strategy for patients with unresectable advanced HCC. This review highlights the application and development of radionuclides in the diagnosis and treatment of HCC. More efforts are warranted for the development of advanced radionuclides to make significant contributions in the treatment of HCC.     

Identification of gasolines with hydrocarbon markers

A procedure for the identification of gasoline samples containing hidden chemical markers based on the results of fuel analysis by gas-liquid chromatography with sample preevaporation is proposed. Tetracosane (C₂₄H₅₀) in a concentration of 2.0 × 10⁻³ wt % was used as a marker. The procedure allowed us to detect the presence of the marker in gasoline samples subjected to uncontrollable evaporation in storage or on fire.     

Serum exosomal microRNAs as novel biomarkers for hepatocellular carcinoma

Recent studies have shown that circulating microRNAs are a potential biomarker in various types of malignancies. The aim of this study was to investigate the feasibility of using serum exosomal microRNAs as novel serological biomarkers for hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). We measured the serum exosomal microRNAs and serum circulating microRNAs in patients with CHB (n=20), liver cirrhosis (LC) (n=20) and HCC (n=20). Serum exosomal microRNA was extracted from 500 μl of serum using an Exosome RNA Isolation kit. The expression levels of microRNAs were quantified by real-time PCR. The expression levels of selected microRNAs were normalized to Caenorhabditis elegans microRNA (Cel-miR-39). The serum levels of exosomal miR-18a, miR-221, miR-222 and miR-224 were significantly higher in patients with HCC than those with CHB or LC (P<0.05). Further, the serum levels of exosomal miR-101, miR-106b, miR-122 and miR-195 were lower in patients with HCC than in patients with CHB (P=0.014, P<0.001, P<0.001 and P<0.001, respectively). There was no significant difference in the levels of miR-21 and miR-93 among the three groups. Additionally, the serum levels of circulating microRNAs showed a smaller difference between HCC and either CHB or LC. This study suggests that serum exosomal microRNAs may be used as novel serological biomarkers for HCC.     

Facile preparation of 177Lu-microspheres for hepatocellular carcinoma radioisotope therapy

As one of the most common cancers in the world, hepatocellular carcinoma (HCC) has become a major threat to human health. Radioembolization is a first-line option for the treatment of HCC, especially when other conventional treatments fail or there exist some relative contraindications. Herein, we developed a facile and efficient method for preparing ¹⁷⁷Lu-microspheres potentially useful for precise radioembolization therapy of HCC. The radiolabeling efficiency of ¹⁷⁷Lu-microspheres was as high as 96.8% ± 0.5%, and the radiolabeling process did not alter the morphology of the mother microspheres. The SPECT/CT studies enabled by the unique emissions of ¹⁷⁷Lu suggested that almost no ¹⁷⁷Lu ion loaded by the microspheres was released over more than 32 d in vivo, which led to remarkable inhibition effect on the growth of HepG2 tumors subcutaneously transplanted in mice. The current approach may thus offer promising ¹⁷⁷Lu-microspheres for clinical radioembolization of HCC.     

Generation of multicolored,prestained molecular weight markers for gel electrophoresis

Molecular weight marker proteins are routinely used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to estimate the relative molecular mass of specific proteins within a sample. This report describes a simple procedure for the generation of multicolored molecular weight proteins using a variety of Remazol-reactive textile dyes. These multicolored proteins provide a set of unambiguous markers for gel electrophoresis. Furthermore, the colored markers can be used in conjunction with Western blotting techniques to provide a visual display of marker proteins on the transfer membrane.     

Identification of the Schistosoma mansoni molecular target for the antimalarial drug artemether

Plasmodium falciparum and Schistosoma mansonii are the parasites responsible for most of the malaria and schistosomiasis cases in the world. Notwithstanding their many differences, the two agents have striking similarities in that they both are blood feeders and are targets of an overlapping set of drugs, including the well-known artemether molecule. Here we explore the possibility of using the known information about the mode of action of artemether in Plasmodium to identify the molecular target of the drug in Schistosoma and provide evidence that artemether binds to SmSERCA, a putative Ca2?-ATPase of Schistosoma . We also predict the putative binding mode of the molecule for both its Plasmodium and Schistosoma targets. Our analysis of the mode of binding of artemether to Ca2?-ATPases also provides an explanation for the apparent paradox that, although the molecule has no side effect in humans, it has been shown to possess antitumoral activity.     

Investigation on the viscoelasticity of synchronous hepatocellular carcinoma cells

Having made use of the micropipette aspiration technique, we here investigated the viscoelastic properties of hepatocellular carcinoma (HTC) cells from the view of cell cycle. The synchronous G₁ and S phase cells were achieved through thymine-2-desoryriboside and colchicine sequential blockage method and double thymine-2-desoryriboside blockage method, respectively. The synchronization results detected with flow cytometer showed that it could meet the requirements of the experiments nicely. Experimental results were analyzed with a standard linear solid viscoelastic model, in which an elastic element, K₁, is in parallel with a Maxwell element composed of another elastic element, K₂, in series with a viscous element, μ. The results indicated that high K₁, K₂ values and low μ value was the general characteristics of the cells; G₁ phase cells had higher K₁ values and low μ value than S phase cells, which endowed G₁ phase cells with higher elasticity and faster passive deformability than S phase cells. The results maybe also reflected the difference of cytoskeleton between G₁ and S phase cells, and suggested that G₁ phase cells were more suitable for surviving and metastasis in blood circulation than S phase cells.     

Development of lactobionic acid conjugated-copper chelators as anticancer candidates for hepatocellular carcinoma

Hepatic copper deposition leads to metabolic disorders, rapid increase in reactive oxygen species (ROS) levels, and even the occurrence and metastasis of hepatocellular carcinoma. Copper chelation or copper transporter inhibition have already been developed into an effective method to control the canceration of hepatocytes and kill the hepatocarcinoma cells. Here, we designed three novel lactobionic acid conjugated copper chelators (GT1, 9 and 10), which have the potential to be recognized by asialoglycoprotein receptor (ASGPR), a high-capacity C-type lectin receptor selectively expressed in liver. Both GT1, 9 and 10 can selectively and efficiently coordinate with copper in solution and in the high-copper treated hepatocellular carcinomas model (HC HepG2 cells). The thiosemicarbazone-based chelator GT1 should more effectively eliminate copper and promote apoptosis of HC HepG2 cells, which might have application prospects in preventing cancerization and other pathological lesions caused by copper deposition of liver. Moreover, our results also revealed the potential of GT1 to be harnessed as preventive leading structures of the hepatocellular carcinomas.     

Inhibitory effect of protonic bis(5-amino-1,10-phenanthroline) on proliferation of hepatocellular carcinoma and its molecular mechanism

The incidence of HCC continues to increase rapidly in recent years. the principle of developing anti-HCC drugs is to specifically kill tumor cells, without affecting the function of normal cells. Here we synthesized a novel protonic compound bis(5-amino-1,10-phenanthroline) (P-BAP), and the antitumor activity was explored in vitro and in vivo. The results showed that P-BAP could selectively inhibit the proliferation of tumor cells, and induce HCC apoptosis. In HCC-bearing mice, P-BAP could effectively retard the tumor growth, even completely eliminate the tumors at the dose of 5 mg/kg, and meanwhile P-BAP had no significant effect on mouse body weight. Mechanism analysis revealed that P-BAP could down-regulate the protein expressions of pleomorphic adenoma gene like-2 (PLAGL2), HIF-1α and β-catenin, and up-regulate the levels of pro-apoptotic protein BAX and BNIP3. In addition, P-BAP could reduce mitochondrial respiratory chain complex activities, leading to insufficient ATP production. The study provides a new approach for designing selective antitumor drugs, and suggests that P-BAP would be a potential candidate for HCC therapy.     

Amperometric low potential aptasensor for the fucosylated Golgi protein 73, a marker for hepatocellular carcinoma

The fucosylated Golgi protein 73 (fuc-GP73) has been used as a criterion to distinguish hepatocellular carcinoma (HCC) from other chronic liver diseases. We describe an amperometric aptasensor for ultrasensitive detection of fuc-GP73 that uses a thiolated aptamer against GP73 as the capture probe, and gold nanoparticles (AuNPs) modified with Avidinlens culinaris agglutinin (A-LCA) as the detection probe. The AuNPs on the surface of a gold electrode provide a large surface for immobilization of A-LCA, so that they can be heavily loaded with biotinylated horse radish peroxidase (B-HRP) via avidin-biotin interactions. This results in enhanced analytical sensitivity. Under optimized conditions and a typical working potential as low as 48 mV (vs. SCE), the dynamic response of the electrode covers the 10 pg·mL^?1 to 25 ng·mL^?1 fuc-GP73 concentation range, with a 7 pg·mL^?1 detection limit (for an S/N ratio of 3). The assay is precise, selective and reproducible. It was applied to the determination of fuc-GP73 in serum.

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Graphical abstract Schematic of an electrochemical aptasensor for the determination of fucosylated golgi protein 73 (fuc-gp73) based on the avidin-Lens culinaris agglutinin (A-LCA) and biotinylated horse radish peroxidase (B-HRP). It was applied to serum analysis with good sensitivity, selectivity and reproducibility.

     

Protein phosphatases regulate the liver microenvironment in the development of hepatocellular carcinoma

The liver is a complicated heterogeneous organ composed of different cells. Parenchymal cells called hepatocytes and various nonparenchymal cells, including immune cells and stromal cells, are distributed in liver lobules with hepatic architecture. They interact with each other to compose the liver microenvironment and determine its characteristics. Although the liver microenvironment maintains liver homeostasis and function under healthy conditions, it also shows proinflammatory and profibrogenic characteristics that can induce the progression of hepatitis and hepatic fibrosis, eventually changing to a protumoral microenvironment that contributes to the development of hepatocellular carcinoma (HCC). According to recent studies, phosphatases are involved in liver diseases and HCC development by regulating protein phosphorylation in intracellular signaling pathways and changing the activities and characteristics of liver cells. Therefore, this review aims to highlight the importance of protein phosphatases in HCC development and in the regulation of the cellular components in the liver microenvironment and to show their significance as therapeutic targets.Subject terms: Cancer microenvironment, Post-translational modifications