carbocyanines and Carcinoma--Hepatocellular

Carboxylesterases (CESs) and Histone deacetylases (HDACs) are regarded as important signaling enzymes highly associated with the development and progression of multiple cancers, including hepatocellular carcinoma (HCC). In this work, a near-infrared (NIR) fluorescent probe named Lys-HXPI was designed and synthesized, which linked a hemicyanine dye and 6-acetamidohexanoic acid via an ester bond. Lys-HXPI displayed a remarkable increase with a NIR emission at 720 nm, a low detection limit (<10 nM) for HDAC1, HDAC 6, CES1 and CES2, as well as a high selectivity for the target enzymes over other relevant analytes. Furthermore, Lys-HXPI was used to image endogenous target enzymes in living cells, tumor-bearing nude mice and tissue slices. The ability of Lys-HXPI to simultaneous image CESs and HDACs was demonstrated with RT-qPCR and the confocal imaging in Hep G2 and MDA-MB-231. Taking advantage of NIR emission, the probe was also successfully applied to imaging Hep G2 tumor mice and tissue slices. Lys-HXPI is expected to be useful for the effective detecting of CESs and HDACs in complex biosystems.

Topics: Animals; Carbocyanines; Carboxylic Ester Hydrolases; Carcinoma, Hepatocellular; Fluorescent Dyes; Histone Deacetylases; Humans; Liver Neoplasms; Mice; Mice, Nude

Image-guided chemo-photothermal therapy based on near-infrared (NIR) theranostic agents has found promising applications in treating tumors. In this multimodal treatment, it is of critical importance to image real-time distribution of photothermal agents in vivo and to monitor therapeutic outcomes for implementing personalized treatment. In this study, an optimally synthesized dextran-polylactide (DEX-PLA) copolymer was assembled with doxorubicin (DOX) and DiR, a kind of NIR dye, to construct desirable micelles ((DiR + DOX)/DEX-PLA) for performing image-guided chemo-photothermal therapy. These (DiR + DOX)/DEX-PLA micelles had good physical and photothermal stability in aqueous media and showed high photothermal efficiency in vivo. Based on the H22-tumor-bearing mouse model, (DiR + DOX)/DEX-PLA micelles were found to accumulate inside tumors sustainably and to emit strong fluorescence signals for more than three days. The (DiR + DOX)@DEX-PLA micelles together with NIR laser irradiation were able to highly inhibit tumor growth or even eradicate tumors with one injection and two dose-designated 5-minute laser irradiations at the tumor site during 14 days of treatment. Furthermore, they showed almost no impairment to the body of the treated mice. These (DiR + DOX)@DEX-PLA micelles have confirmative translational potential in clinical tumor therapy on account of their persistent image-guided capacity, high antitumor efficacy and good in vivo safety.

Topics: Animals; Antibiotics, Antineoplastic; Carbocyanines; Carcinoma, Hepatocellular; Dextrans; Doxorubicin; Drug Carriers; Drug Compounding; Fluorescent Dyes; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice, Inbred BALB C; Micelles; Photosensitizing Agents; Photothermal Therapy; Spectroscopy, Near-Infrared; Theranostic Nanomedicine; Time Factors; Tumor Burden

Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for widespread fundamental research. However, inhomogeneous signal attenuation due to tissue absorption and scattering hampers its application for accurate in vivo biosensing. Here, lifetime-based in situ hepatocellular carcinoma (HCC) detection in NIR-II region is presented using a tumor-microenvironment (peroxynitrite, ONOO

Topics: Biosensing Techniques; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Fluorescence Resonance Energy Transfer; Humans; Lanthanoid Series Elements; Liver Neoplasms; Luminescence; Magnetic Resonance Imaging; Peroxynitrous Acid

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.

Topics: Base Sequence; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Fluorescent Dyes; Gene Silencing; Gene Transfer Techniques; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Lipid Metabolism; Lipids; Liposomes; Liver Neoplasms; Polymers; RNA Interference; RNA, Small Interfering; Transfection

Herein, a DNAzyme-powered nanomachine responsive to multiple hepatocellular carcinoma (HCC)-related miRNAs derived from clinical samples was designed. Initially, three types of nanomachines were constructed with dye molecule [(fluorescein (FAM), tetramethylrhodamin (TMR), and Cyanine 5 (Cy5)]-labeled DNA-RNA chimeric substrates and a specific recognized probe for the corresponding miRNAs target. Once the target miRNAs were captured by two recognizing probes, the DNA nanomachine was initiated, leading to the hybridization between the DNAzyme and the substrates. With the help of a cofactor, the automatic operation of the nanomachine was driven by cyclic cleavage of the DNAzyme. Meanwhile, we also explored the recognition behavior between the recognizing probe and the target miRNA. Subsequently, these DNAzyme-powered nanomachines were developed for the homogeneous and simultaneous detection of three target miRNAs at the femtomloar level. Furthermore, the potential in clinical diagnosis was proven by the successful determination of target miRNA in real clinical samples. Thus, this nanomachine-based strategy possesses significant potential to be an innovation in miRNA analysis methodology.

Topics: Biosensing Techniques; Carbocyanines; Carcinoma, Hepatocellular; DNA, Catalytic; Fluorescein; Fluorescent Dyes; Gold; Humans; Liver Neoplasms; Metal Nanoparticles; MicroRNAs; Nucleic Acid Hybridization; Rhodamines

Near infrared fluorescence (NIRF) imaging has strong potential for widespread use in noninvasive tumor imaging. Indocyanine green (ICG) is the only Food and Drug Administration (FDA) -approved NIRF dye for clinical diagnosis; however, it is unstable and poorly targets tumors. DZ-1 is a novel heptamethine cyanine NIRF dye, suitable for imaging and tumor targeting. Here, we compared the fluorescence intensity and metabolism of DZ-1 and ICG. Additionally, we assayed their specificities and abilities to target tumor cells, using cultured hepatocellular carcinoma (HCC) cell lines, a nude mouse subcutaneous xenograft model of liver cancer, and a rabbit orthotopic transplantation model. We found that DZ-1 accumulates in tumor tissue and specifically recognizes HCC in subcutaneous and orthotopic models. The NIRF intensity of DZ-1 was one order of magnitude stronger than that of ICG, and DZ-1 showed excellent intraoperative tumor targeting in the rabbit model. Importantly, ICG accumulated at tumor sites, as well as in the liver and kidney. Furthermore, DZ-1 analog-gemcitabine conjugate (NIRG) exhibited similar tumor-specific targeting and imaging properties, including inhibition of tumor growth, in HCC patient-derived xenograft (PDX) mice. DZ-1 and NIRG demonstrated superior tumor-targeting specificity, compared to ICG. We show that DZ-1 is an effective molecular probe for specific imaging, targeting, and therapy in HCC.

Topics: Animals; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Coloring Agents; Humans; Indocyanine Green; Liver; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Optical Imaging; Rabbits

Topics: Amino Acid Sequence; Animals; Bacteriophages; Carbocyanines; Carcinoma, Hepatocellular; Clone Cells; Fluorescence; Glypicans; Hep G2 Cells; Humans; Imaging, Three-Dimensional; Liver Neoplasms; Mice, Nude; Models, Molecular; Peptides; Staining and Labeling; Tissue Distribution; Xenograft Model Antitumor Assays

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Topics: Aged; alpha-Fetoproteins; Antibodies; Area Under Curve; Biomarkers, Tumor; Carbocyanines; Carcinoma, Hepatocellular; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Fluorescent Dyes; Gene Expression; Humans; Liver Neoplasms; Male; Middle Aged; Protein Array Analysis; ROC Curve; Streptavidin

Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) has become a general platform for analysis of various clinical samples such as biofluids and tissues. In comparison to conventional 2-D polyacrylamide gel electrophoresis (2D PAGE), 2D DIGE offers several advantages, such as accuracy and reproducibility between experiments, which facilitate spot-to-spot comparisons. Although whole plasma can be easily obtained, the complexity of plasma samples makes it challenging to analyze samples with good reproducibility. Here, we describe a method for decreasing protein complexity in plasma samples within a narrow pH range by depleting high-abundance plasma proteins. In combination with analysis of differentially expressed spots, trypsin digestion, identification of protein by mass spectrometry, and standard 2D PAGE and DIGE, this method has been optimized for comparison of plasma samples from healthy donors and patients diagnosed with hepatocellular carcinoma.

Topics: Aged; Biomarkers, Tumor; Carbocyanines; Carcinoma, Hepatocellular; Chromatography, Liquid; Coloring Agents; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Liver Neoplasms; Male; Nanotechnology; Proteolysis; Proteome; Proteomics; Staining and Labeling; Tandem Mass Spectrometry; Trypsin; Two-Dimensional Difference Gel Electrophoresis

Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms.

Topics: Animals; Apolipoprotein A-I; Base Sequence; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cloning, Molecular; Disease Models, Animal; Drug Delivery Systems; Flow Cytometry; Hepatitis B Virus, Woodchuck; Hepatitis B, Chronic; Humans; Liver; Liver Neoplasms; Marmota; Mice; Molecular Sequence Data; Molecular Targeted Therapy; Protein Isoforms; Transfection; Virus Replication

Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.

Topics: Annexin A5; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Ethylenediamines; Humans; Liver Neoplasms; Molecular Weight; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Polyethylene Glycols; Polymers; Propidium; Propylene Glycols; Rhodamine 123; Vinblastine

Diterpenes, present in many medicinal plants, have been the focus of continuous studies for the development of new anticancer agents. ZBB-006 is a new synthetic diterpenoid derivative which exhibited significant anti-proliferation activity against various cancer cell lines in our previous study. Here, we investigated the antitumor effect of ZBB-006 and its potential mechanisms in the human hepatocellular carcinoma cell line HepG2, both in vitro and in vivo. We found that oral administration of ZBB-006 effectively suppressed the growth of HepG2 xenograft tumor in nude mice without body weight decline as compared with the control group. Meanwhile, the growth inhibitory effect of ZBB-006 on HepG2 cells was observed with MTT assay. Apoptosis induced by ZBB-006 in HepG2 cells was evidenced by DAPI staining and Annexin V/PI double staining assay. ZBB-006 also dissipated the mitochondrial membrane potential (ΔΨm) apparently as revealed by JC-1 staining. Furthermore, the cleavage of PARP, activation of caspase-3 and caspase-9 but not caspase-8 was demonstrated by western blot assay both in vitro and in vivo. Additionally, the proapoptotic protein Bax was markedly elevated, while the antiapoptotic protein Bcl-2 was downregulated. Collectively, our data indicated that ZBB-006 exerted a strong antitumor effect on HepG2 cells by initiating the mitochondrial-dependent apoptosis, and it has potential to be explored as a new promising therapeutic agent against human hepatoma.

Topics: Animals; Apoptosis; Benzimidazoles; Carbocyanines; Carcinoma, Hepatocellular; Cell Growth Processes; Diterpenes; Hep G2 Cells; Humans; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Xenograft Model Antitumor Assays

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A.

Topics: Actin Cytoskeleton; Antibodies, Monoclonal; Carbocyanines; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Colchicine; Cytochalasin B; Fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique, Direct; Fluorescent Dyes; Hepacivirus; Humans; Indoles; Kidney; Liver Neoplasms; Microtubules; Replicon; RNA Helicases; RNA, Viral; Serine Endopeptidases; Tubulin; Tubulin Modulators; Viral Nonstructural Proteins; Virus Replication

Scavenger receptor class B type I (SR-BI) is the high-affinity high-density lipoprotein (HDL) receptor, and CLA-1 is the human homologue of the murine SR-BI. CLA-1/SR-BI receptor has been suggested as a new preventative and/or therapeutic target for atherosclerosis due to its pivotal role in overall HDL cholesterol (HDL-C) metabolism and its antiatherogenic activity in vivo. To search for active compounds that can increase CLA-1 transcription, a novel cell-based assay was developed for application in high-throughput screening (HTS). Human hepatoma HepG2 cells were transfected with a CLA-1-promoter-luciferase reporter gene construct, and the stable transfected cell line was selected and named CLAp-LUC HepG2. With rosiglitazone as a positive control, this stable cell line was used to establish a specific CLA-1 gene expression assay in a 96-well microplate format. The evaluating parameter Z' value of 0.64 showed that this cell-based HTS assay was robust and reliable. Screening of 6000 microbial secondary metabolite crude extracts identified 8 positive strains. Between 2 identified CLA-1 up-regulators produced by actinomycete strain 04-4776, 4776B may stimulate not only the expression of CLA-1 on the transcriptional and translational levels but also the activity of CLA-1 to uptake the HDL-C in HepG2 cells. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new antiatherosclerosis agents.

Topics: Actinomycetaceae; Biological Assay; Carbocyanines; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fermentation; Fluorescent Dyes; Gene Expression Regulation; Genes, Reporter; Humans; Hydroxyl Radical; Isoflavones; Lipoproteins, HDL; Liver Neoplasms; Luciferases; PPAR gamma; Receptors, Lipoprotein; Recombinant Fusion Proteins; Rosiglitazone; Scavenger Receptors, Class B; Thiazolidinediones; Transcription, Genetic; Up-Regulation

To evaluate the effects of 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.. DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.. DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC(50) value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC(50) value of 2.09 micromol/L. Furthermore, DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53, Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.. The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.

Topics: Apoptosis; Carbocyanines; Carcinoma, Hepatocellular; Caspase 3; Caspases; Cell Division; Cell Line, Tumor; DNA Damage; DNA Primase; Flow Cytometry; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Microscopy, Electron; Microtubule-Associated Proteins; Neoplasm Proteins; Nucleosomes; Proto-Oncogene Proteins c-bcl-2; Survivin; Tumor Suppressor Protein p53

Hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes the potentially oncogenic HBx protein, which mainly functions as a transcriptional co-activator involving in multiple gene deregulations. However, mechanisms underlying HBx-mediated oncogenicity remain unclear. To determine the role(s) of HBx in the early genesis of HCC, we utilized the NCI Oncochip microarray that contains 2208 human cDNA clones to examine the gene expression profiles in either freshly isolated normal primary adult human hepatocytes (Hhep) or an HCC cell line (SK-Hep-1) ecotopically expressing HBx via an adenoviral system. The gene expression profiles also were determined in liver samples from HBV-infected chronic active hepatitis patients when compared with normal liver samples. The microarray results were validated through Northern blot analysis of the expression of selected genes. Using reciprocally labeling hybridizations, scatterplot analysis of gene expression ratios in human primary hepatocytes expressing HBx demonstrates that microarrays are highly reproducible. The comparison of gene expression profiles between HBx-expressing primary hepatocytes and HBV-infected liver samples shows a consistent alteration of many cellular genes including a subset of oncogenes (such as c-myc and c-myb) and tumor suppressor genes (such as APC, p53, WAF1 and WT1). Furthermore, clustering algorithm analysis showed distinctive gene expression profiles in Hhep and SK-Hep-1 cells. Our findings are consistent with the hypothesis that the deregulation of cellular genes by oncogenic HBx may be an early event that favors hepatocyte proliferation during liver carcinogenesis.

Topics: Adult; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Fluorescent Dyes; Freezing; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Humans; Liver; Liver Neoplasms; Oligonucleotide Array Sequence Analysis; Staining and Labeling; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins

Stimulatory effects of a novel isobenzofranone, MD-700, on low density lipoprotein (LDL) receptor activity were investigated in vitro and in vivo. MD-700 at 0.03 microg/ml elevated the expression of LDL receptor in HepG2 cells within 4 h. Corresponding to this, uptake of fluorescent labeled-LDL (3,3'-dioctadecylindocarbocyanine-LDL) by the cells increased linearly in time- and dose-dependent manner by MD-700 for up to 12 h. In the experiment using HepG2 cells transiently transfected with promoter-luciferase gene constructs, MD-700 increased luciferase activity in a dose-dependent manner from 0.03 to 0.1 microg/ml. In contrast, luciferase activity was not stimulated by MD-700 in construct with a deleted sterol regulatory element (SRE)-1, suggesting importance of SRE-1 in stimulation of the LDL receptor gene promoter by MD-700. Binding experiments on liver membranes from MD-700-treated hamsters showed about a 60% increase in 125I-labeled LDL binding. A Scatchard plot revealed that MD-700 increased the maximal binding without affecting binding affinity. In contrast to findings with an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin, MD-700 had no effect on the sterol synthesis in hamster liver homogenates. These results suggest that MD-700 stimulates the expression of LDL receptor, presumably in a manner independent of change in sterol metabolism, and thereby promotes LDL clearance. Hypocholesterolemic actions of MD-700 in hamsters were then examined. MD-700 lowered serum cholesterol levels in hamsters fed normal chow or a high-fat diet. Fractionation of serum lipoproteins demonstrated that MD-700 selectively decreased LDL and very low density lipoprotein cholesterol. Dose-dependent decrease in serum cholesterol was also seen in hypercholesterolemic rats. Thus, the hypocholesterolemic action of MD-700 may be attributed to up-regulation of the LDL receptor, based on stimulation of the transcription of the LDL receptor gene. Although pravastatin stimulates LDL uptake and lowers serum cholesterol in a manner similar to that seen with MD-700, the mechanism responsible for hypocholesterolemic action appears to differ.

Topics: Animals; Benzofurans; Blotting, Northern; Carbocyanines; Carcinoma, Hepatocellular; Cell Membrane; Cholesterol; Cricetinae; Disease Models, Animal; DNA Primers; Fluorescent Dyes; Humans; Hypercholesterolemia; Lipoproteins, LDL; Lipoproteins, VLDL; Liver Neoplasms; Male; Promoter Regions, Genetic; Rats; Rats, Wistar; Receptors, LDL; RNA, Messenger; RNA, Neoplasm; Sterols; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation

DiI-LDL (3,3'-dioctadecylindocarbocyanine-low density lipoprotein) has been extensively used in morphological and microscopic studies of receptor-mediated metabolism of LDL in many cell lines. To date the use of this fluorescent probe in a quantitative assay of LDL receptor activity has not been widely used in studies with multiple samples due to the lack of a practical method for quantitatively recovering cell-associated DiI. Therefore, detection by 125I-labeled LDL has remained the method of choice for assaying LDL receptor activity rapidly and reliably. In this paper, we describe a rapid, simple, and nonradioactive assay of LDL receptor activity using DiI-LDL. The increased sensitivity of this method was achieved by modifications to the labeling procedure of LDL and to the extraction of DiI from cells for subsequent fluorescence determination. These modifications did not affect the affinity of DiI-LDL toward HepG2 cells, and the assay was easily adapted to a rapid screen for LDL receptor modulators in this cell model.

Topics: Binding, Competitive; Carbocyanines; Carcinoma, Hepatocellular; Fluorescent Dyes; Humans; Hydroxycholesterols; Iodine Radioisotopes; Lipoproteins, LDL; Liver Neoplasms; Lovastatin; Receptors, LDL; Tumor Cells, Cultured