Activation of pluripotent genes in hepatic progenitor cells in the transition of nonalcoholic steatohepatitis to pre-malignant lesions.

Nonalcoholic steatohepatitis is considered as a precancerous condition. However, hepatic carcinogenesis from NASH is poorly understood. This study aims to investigate the activation of pluripotent genes (c-Myc, Oct-4, KLF-4, and Nanog) and morphogenic gene (Gli-1) in hepatic progenitor cells from patient specimens and in an animal model to determine the possibility of normal stem/progenitor cells becoming the origin of NASH-HCC. In this study, expression of pluripotent and morphogenic genes in human NASH-HCC tissues was significantly upregulated compared to adjacent non-tumor liver tissues. After feeding high-fat/calorie diet plus high fructose/glucose in drinking water (HFC diet plus HF/G) for up to 12 months, mice developed obesity, insulin resistance, and steatohepatitis with significant necroptotic inflammation and fibrotic progression, as well as occurrence of hyperplastic nodules with dysplasia; and this model represents pathohistologically as a transition from NASH to NASH-HCC in a pre-carcinomatous stage. High expression of pluripotent and morphogenic genes was immunohistochemically visualized in the dysplasia areas of mouse liver, where there were many OV-6-positive cells, indicating proliferation of HOCs in NASH with fibrotic progression. Moreover, oncogenic transcription factors (c-Myc, KLF-4, and Nanog) were co-localized in these hepatic progenitor cells. In conclusion, pluripotent and morphogenic genes may contribute to the reprogramming of hepatic progenitor cells in driving these cells to be the origin of NASH-HCC in a steatotic and inflamed microenvironment.

The chemopreventive capacity of quercetin to induce programmed cell death in hepatocarcinogenesis.

In this study of chemoprevention in the rat modified resistant hepatocyte model, preneoplastic cells were diminished by >60% with quercetin pretreatment compared with those rats treated with N-Diethylnitrosamine (DEN) to induce liver cancer. This decrease occurred associated with an abolished DEN-induced lipid peroxidation as well as activation of caspase 9 and increased caspase 3, as determined by increased expression of cleaved caspase 3 and 9, but not cleaved caspase 8 and increased fragmentation of Poly (ADP-ribose) polymerase (PARP) inducing apoptosis of presumed genetically injured cells, when quercetin was administered before the initiation agent.

Confirmation of drug delivery after liver chemoembolization: direct tissue doxorubicin measurement by UHPLC-MS-MS.

Because liver cancer is rarely suitable for surgery, transcatheter arterial chemoembolization (TACE) is used for palliative therapy. In this procedure, an emulsion of doxorubicin in iodized oil is injected directly into liver tumors through a catheter positioned within the artery supplying blood flow to the tumor. At present, there is limited understanding of factors affecting the delivery and dispersion of doxorubicin within treated tumors during TACE. This study addresses the development and application of an ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) method for rapid confirmation of drug delivery after TACE in a rabbit VX2 liver cancer model. Doxorubicin levels in liver tumors were measured using UHPLC-MS-MS and compared with computed tomography measured levels of iodized oil, a metric used clinically to indicate drug delivery. We found that tissue drug levels determined using UHPLC-MS-MS did not correlate with the regional iodized oil concentration (vehicle) within tumors following TACE, suggesting that chemotherapeutic drugs like doxorubicin spread throughout tumors, and that lack of iodized oil staining in portions of a tumor does not necessarily indicate inadequate therapy during TACE.

Roles for rat hepatocyte malignant transforming factor (HMTF) in late stage of hepatocarcinogenesis.

In a previous study, to identify genes of importance for hepatocellular carcinogenesis, and especially for processes involved in malignant transformation, the authors investigated differences in gene expression between adenomas and carcinomas by DNA microarray. In the present study, the authors investigated AW434047, one of the sequences that was upregulated in carcinomas. The investigation led to the identification of a novel gene, which the authors named hepatocyte malignant transforming factor (HMTF), of unknown function whose expression was increased in hepatocellular carcinomas. Northern blot and in situ hybridization also demonstrated high levels of HMTF in rat hepatocellular carcinoma (HCC) cell lines, lymphocytes in the spleen, colon mucosal epithelia, spermatocytes, and granule cells of the hippocampus. Reduction of HMTF by RNA interference (RNAi) in N1 cells, an HCC cell line, caused suppression of cell proliferation, invasion, and migration. Suppression of proliferation appeared to be due to cell cycle arrest without increased apoptosis. Decreased HMTF expression resulted in down-regulation of STAT3, PCNA, and cyclin D1 and upregulation of p27. These results suggest that HMTF is a new marker for rat HCC and is involved in HCC cell proliferation and may also be linked to cell proliferation in the spleen, colon, brain, and testis.

Tristetraprolin Promotes Hepatic Inflammation and Tumor Initiation but Restrains Cancer Progression to Malignancy.

BACKGROUND & AIMS: Tristetraprolin (TTP) is a key post-transcriptional regulator of inflammatory and oncogenic transcripts. Accordingly, TTP was reported to act as a tumor suppressor in specific cancers. Herein, we investigated how TTP contributes to the development of liver inflammation and fibrosis, which are key drivers of hepatocarcinogenesis, as well as to the onset and progression of hepatocellular carcinoma (HCC). METHODS: TTP expression was investigated in mouse/human models of hepatic metabolic diseases and cancer. The role of TTP in nonalcoholic steatohepatitis and HCC development was further examined through in vivo/vitro approaches using liver-specific TTP knockout mice and a panel of hepatic cancer cells. RESULTS: Our data demonstrate that TTP loss in vivo strongly restrains development of hepatic steatosis and inflammation/fibrosis in mice fed a methionine/choline-deficient diet, as well as HCC development induced by the carcinogen diethylnitrosamine. In contrast, low TTP expression fostered migration and invasion capacities of in vitro transformed hepatic cancer cells likely by unleashing expression of key oncogenes previously associated with these cancerous features. Consistent with these data, TTP was significantly down-regulated in high-grade human HCC, a feature further correlating with poor clinical prognosis. Finally, we uncover hepatocyte nuclear factor 4 alpha and early growth response 1, two key transcription factors lost with hepatocyte dedifferentiation, as key regulators of TTP expression. CONCLUSIONS: Although TTP importantly contributes to hepatic inflammation and cancer initiation, its loss with hepatocyte dedifferentiation fosters cancer cells migration and invasion. Loss of TTP may represent a clinically relevant biomarker of high-grade HCC associated with poor prognosis.

Effect of targeted magnetic nanoparticles containing 5-FU on expression of bcl-2, bax and caspase 3 in nude mice with transplanted human liver cancer.

AIM: To investigate the anti-tumor effect and mechanisms of magnetic nanoparticles targeting hepatocellular carcinoma. METHODS: Human hepatocellular carcinoma was induced in nude mice, and the mice were randomly divided into group A receiving normal saline, group B receiving magnetic nanoparticles containing 5-fluorouracil (5-FU), group C receiving 5-FU, and group D receiving magnetic nanoparticles containing 5-FU with a magnetic field built in tumor tissues. The tumor volume was measured on the day before treatment and 1, 4, 7, 10 and 13 d after treatment. Tumor tissues were isolated for examination of the expression of bcl-2, bax and caspase 3 by immunohistochemical method, reverse transcription polymerase chain reaction and Western blotting. RESULTS: The tumor volume was markedly lower in groups C and D than in groups A and B (group C or D vs group A or B, P<0.01). The volume was markedly lower in group D than in group C (P<0.05). The expression of protein and mRNA of bcl-2 was markedly lower in groups C and D than in groups A and B (group C or D vs group A or B, P<0.01), and was markedly lower in group D than in group C (P<0.01). The expression of bax and caspase 3 in groups C and D was significantly increased, compared with that in groups A and B (P<0.01). CONCLUSION: The targeted magnetic nanoparticles containing 5-FU can improve the chemotherapeutic effect of 5-FU against hepatocellular carcinoma by decreasing the expression of bcl-2 gene, and increasing the expression of bax and caspase 3 genes.

Use of short monolithic columns for isolation of low abundance membrane proteins.

Convective interaction media (CIM) monoliths provide a stationary phase with a high binding capacity for large molecules and are capable of high flow rates at a very low pressure drop. Used as anion- and cation-exchangers or with affinity ligands such as antibodies, these columns have the potential for processing large volumes of complex biological mixtures within a short time. In the present report, monoclonal antibodies against several rat liver plasma membrane proteins were bound and cross-linked to protein A or protein G CIM affinity columns with a bed volume of only 60 microL. Antigens recognized by bound antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step from either total plasma membrane extracts or subfractions isolated using anion-exchange CIM disk-shaped columns. The isolated antigens and co-eluting proteins were subsequently identified by immunoblot or by LC-MS/MS.

Identification of members of the annexin family in the detergent-insoluble fraction of rat Morris hepatoma plasma membranes.

For proteomic analysis, plasma membranes of rat hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized according to the previously developed method [D. Josic, K. Zeilinger, Methods Enzymol. 271 (1996) 113-134]. If the Triton X100 insoluble pellet is subsequently extracted, several proteins can be solubilized. These proteins can be classified in two groups according to their molecular size. The proteins with apparent molecular weights in SDS-PAGE between 70 and 75 kDa belong to the first group. Smaller proteins, with apparent molecular weights between 30 and 45 kDa, are members of the second group. The main protein of higher molecular weight was also found in the Triton X100 insoluble extract from normal rat liver plasma membranes. This protein was identified as Annexin A6. The proteins from the second group are practically absent in the Triton X100 insoluble extract from rat liver. These proteins are present in relatively high concentrations in plasma membranes of Morris hepatoma 7777. Both groups of detergent-insoluble proteins from Morris hepatoma 7777 were further analyzed with SELDI-TOF and LC electrospray ionization mass spectrometry. From the first group, Annexin A6, together with two other integral plasma membrane proteins, was identified. In the second group of proteins with apparent molecular weights between 30 and 45kDa, further members of the annexin family, Annexins A1, A2, A4, A5 and A7 were identified. The possible role of these low molecular size annexins as potential cancer biomarkers is discussed.

Arachidonic acid inhibits the store-operated Ca2+ current in rat liver cells.

Vasopressin and other phospholipase-C-coupled hormones induce oscillations (waves) of [Ca2+]cyt (cytoplasmic Ca2+ concentration) in liver cells. Maintenance of these oscillations requires replenishment of Ca2+ in intracellular stores through Ca2+ inflow across the plasma membrane. While this may be achieved by SOCs (store-operated Ca2+ channels), some studies in other cell types indicate that it is dependent on AA (arachidonic acid)-activated Ca2+ channels. We studied the effects of AA on membrane conductance of rat liver cells using whole-cell patch clamping. We found no evidence that concentrations of AA in the physiological range could activate Ca2+-permeable channels in either H4IIE liver cells or rat hepatocytes. However, AA (1-10 microM) did inhibit (IC50=2.4+/-0.1 microM) Ca2+ inflow through SOCs (ISOC) initiated by intracellular application of Ins(1,4,5)P3 in H4IIE cells. Pre-incubation with AA did not inhibit ISOC development, but decreased maximal amplitude of the current. Iso-tetrandrine, widely used to inhibit receptor-activation of phospholipase A2, and therefore AA release, inhibited ISOC directly in H4IIE cells. It is concluded that (i) in rat liver cells, AA does not activate an AA-regulated Ca2+-permeable channel, but does inhibit SOCs, and (ii) iso-tetrandrine and tetrandrine are effective blockers of CRAC (Ca2+-release-activated Ca2+) channel-like SOCs. These results indicate that AA-activated Ca2+-permeable channels do not contribute to hormone-induced increases or oscillations in [Ca2+]cyt in liver cells. However, AA may be a physiological modulator of Ca2+ inflow in these cells.

Lipid compositions of intracellular membranes isolated from rat liver nodules in Wistar rats.

The mevalonate pathway gives rise to important end products for the regulation of growth and resistance to oxidative stress and is, consequently, of importance in carcinogenesis. In this study liver nodules were produced in Wistar rats by intermittent feeding with dietary 2-acetylaminofluorene, and the lipid compositions of isolated microsomes, mitochondria, and lysosomes were examined. The phospholipid compositions of these subfractions were unchanged compared to normal hepatic tissue, but the fatty acid patterns were altered, particularly in microsomes. An increase in the content of palmitic acid and a decrease in that of stearic acid were noted. The pattern of fatty acyl moieties on carbon atoms 1 and 2 of the glycerol backbone of phospholipids was unchanged in nodular tissue compared to normal liver. The amount of dolichol was significantly higher in microsomes and mitochondria, but not in lysosomes, and the relative amounts of longer polyisoprenoid compounds were increased in the liver nodules. The relative concentration of esterified dolichol was decreased and an enrichment in saturated fatty acids in this fraction could be observed. The cholesterol concentration was found to be lower in microsomes, but was unchanged in mitochondria and lysosomes, and the normally low concentration of cholesteryl esters was elevated somewhat in microsomes and lysosomes. The ubiquinone content of liver nodular mitochondria was unchanged, but increased 7-fold in microsomes and 2-fold in lysosomes. The alterations found in the lipid composition of liver nodules are significant and have functional implications in many cellular processes of proposed importance for the carcinogenic process, i.e., protein glycosylation cholesterogenesis, regulation of the mevalonate pathway, cellular oxidation-reduction state, and resistance to oxidative stress.

Development of liver tumors in transforming growth factor alpha transgenic mice.

We studied the development of liver tumors in male transforming growth factor alpha (TGF-alpha) transgenic mice of the CD1 strain and examined the expression of the transgene by immunohistochemistry and in situ hybridization. Livers of 4-5-week-old transgenic mice contained areas of centribobular hypertrophy with low glucose-6-phosphatase activity. These areas progressively expanded, and hypertrophy and dysplasia became generalized in livers of mice at 10-12 months of age. The expression of the transgene, determined by either immunohistochemistry or in situ hybridization, was uneven in animals that were 10 weeks old or older. The positive hepatocytes formed patches with a predominant centrilobular distribution. We studied a total of 23 liver tumors (7 hepatocellular carcinomas and 16 adenomas) obtained from 11 mice at 13-15 months of age and from one 7-month-old animal which received zinc sulfate to induce the transgene. The carcinomas were well differentiated tumors, without glucose-6-phosphatase or gamma-glutamyltranspeptidase activity, that developed from the dysplastic parenchyma and occasionally within an adenoma. In all carcinomas and in 56% of the adenomas there was overexpression of the transgene in relationship to the surrounding tissue. The majority of the tumors that overexpressed TGF-alpha were alpha-fetoprotein positive, while alpha-fetoprotein staining was not detected in tumors (all adenomas) that did not show excessive transgene expression. We conclude that TGF-alpha functions as a promoter of liver carcinogenesis through its effect as an autocrine inducer of hepatocyte proliferation. Further, the data indicate that TGF-alpha overexpression may favor tumor progression.

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.

TuAg.1 is the liver isoform of the rat colon tumor-associated antigen pE4 and a member of the immunoglobulin-like supergene family.

TuAg.1 is a tumor-associated membrane glycoprotein first identified in rat hepatocellular carcinoma by monoclonal antibodies (mAbs) 324.5 and 324.9. This oncofetal antigen is also expressed by hepatocytes in cell culture but not normal adult hepatocytes in vivo. Affinity chromatography and preparative continuous elution slab-gel electrophoresis were used to separate TuAg.1 from co-purified actin and immunoglobulin. TuAg.1 was recovered as a series of bands Mr 82,000-90,000, which were pooled and subjected to CNBr digestion for primary amino acid sequence analysis. Computer database analysis of TuAg.1 peptide sequence revealed homology to the rat colon carcinoma-associated antigen pE4, a member of the immunoglobulin gene superfamily. Oligonucleotide primers derived from sequences shared by TuAg.1 and pE4 were used in reverse transcription-PCR to amplify tumor-specific products corresponding to TuAg.1 cDNA. Northern blot analysis with one of these products confirmed the oncofetal expression of transcripts related to TuAg.1/pE4 and indicated an RNA species of different size expressed only in normal liver. Identity between TuAg.1 and pE4 was further confirmed by immunochemical analysis with mAb 324.5 and mAb E4. Both antibodies were reactive with the same protein on transplantable hepatocellular carcinoma AS30D but recognized different epitopes. The reactivity of human tumor cells with mAb 324.5 and 324.9 indicates the presence of a related TuAg.1 molecule expressed in human neoplasia as well.

Dose-response relationships for chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a rat tumor promotion model: quantification and immunolocalization of CYP1A1 and CYP1A2 in the liver.

The mechanisms responsible for the braod spectrum of effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are not entirely clear but seem to involve an initial interaction with the Ah receptor. A major uncertainty in risk assessment for TCDD is the lack of adequate dose-response relationships following chronic exposure to TCDD. Induction of cytochrome P-450 enzymes (CYP1A1 and CYP1A2) is one of the most sensitive responses to TCDD and its structural analogues. We have used a two-stage model for hepatocarcinogenesis in female Sprague-Dawley rats to evaluate dose-response relationships for induction of CYP1A1 and CYP1A2 in diethylnitrosamine-initiated as well as in noninitiated rats. After initiation with a single dose of diethylnitrosamine, TCDD was administered biweekly by p.o. gavage at doses equivalent to 3.5, 10.7, 35.7, and 125 ng/kg/day for 30 weeks. CYP1A1 and CYP1A2 concentrations were quantified in hepatic microsomes by radioimmunoassay and localized in hepatic tissue slices by immunohistochemical techniques. Radioimmunoassay data revealed a maximum induction of 200-fold for CYP1A1 and 10-fold for CYP1A2 and there were no statistically significant differences between initiated and noninitiated rats. Induction at the lowest dose (3.5 ng/kg/day) was 20-fold for CYP1A1 and 3-fold for CYP1A2. Mathematical analysis indicates that the best fit of the induction data are inconsistent with a threshold for this response. There was a linear relationship between administered dose and TCDD liver concentration over the entire dose range of the study. This indicates that induction of CYP1A2 does not significantly alter the distribution of TCDD in our chronic dosing regimen. Immunolocalization of CYP1A1 and CYP1A2 revealed the same localization and induction pattern for both isozymes in the cytoplasm of hepatocytes. However, the hepatic distribution pattern was not uniform with the most intense staining observed around central veins. These studies help to clarify dose-response relationships for dioxin-mediated effects and demonstrate different sensitivity of hepatocytes to the effects of TCDD.

P-glycoprotein expression during tumor progression in the rat liver.

P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines. Overexpression of Pgp has been detected in clinical cancer samples of many histological types. The basis and biological significance of such increases in Pgp expression are not well understood. In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis. An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas. The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions. In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions. A particularly striking finding was the consistent expression of Pgp in the lung metastases. These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.

Structural differences between complex-type Asn-linked glycan chains of glycoproteins in rat hepatocytes and Zajdela hepatoma cells.

Using serial lectin-affinity chromatography, glycosidase digestion, and NMR and methylation analysis, the structures of complex N-linked glycan chains (M(r) range 2000-3500) of rat hepatocytes and poorly differentiated chemically transformed Zajdela ascites hepatoma cells were determined and compared. The results revealed considerable differences between the two cell types: (i) hepatoma cells only expressed tri- and/or tetra-antennary complex N-linked glycan chains, whereas hepatocytes displayed large amounts of bi-antennary N-linked structures and smaller amounts of tri-/tetra-antennary structures; (ii) 20% of the glycan chains in hepatoma cells contained a bisecting GlcNAc residue which was beta (1,4)-linked to the beta-mannosyl residue of the core and was not detected in the hepatocytes; (iii) hepatoma cells expressed a high proportion of the fucosylated or not GlNAc beta (1,6) Man alpha 1-->branch, whereas hepatocytes only contained a little of this branch; (iv) hepatoma cells, but not hepatocytes, exhibited a repeating (Gal beta(1,4) GlcNAc beta (1,3)) sequence characteristic of poly-N-acetyllactosaminoglycans. These glycans were capped by both alpha-galactosyl and sialyl residues; (v) The alpha (2,3)/alpha (2,6)-linkage ratio of sialic acid was significantly higher in hepatoma cells (4/1 vs. 2/1 in hepatocytes); (vi) Only hepatocytes expressed an unusual structure in which a sialyl residue was alpha (2,6)-linked to a GlcNAc residue located within a NeuAc alpha (2,3) Gal beta (1,3) GlcNAc branch which was beta (1,4)-linked to Man alpha 1,3-->. The differences between these complex N-linked glycan chains in hepatocytes and hepatoma cells seem to be both quantitative and qualitative, since some glycan structures were only present in one cell type.

Growth-associated changes in fatty acid compositions of nuclear phospholipids of liver cells.

To know the possible relationships between nuclear phospholipids and cell proliferation, we have extensively analyzed phospholipids extracted from the nuclei of rat hepatic cells at various growth states. The content of phospholipid in nuclei as well as its composition was similar among liver cells tested, i.e., the regenerating rat livers (28 h, post-hepatectomy), sham-operated or non-treated control livers, and rat ascites hepatoma, AH7974 cells. In contrast, the fatty acid compositions of phospholipids differed from each other among these cells. At the 2-position of phospholipids in the regenerating liver nuclei at 28 h after partial hepatectomy, 18:1 (oleic acid) increased transiently at the expense of 20:4 (arachidonic acid) and 22:6 (docosahexaenoic acid), compared with those in the sham-operated control nuclei. This change in fatty acid composition was commonly observed throughout all phospholipids analyzed, i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). On the other hand, the change at 1-position was rather limited: in the regenerating liver nuclei (28 h), 18:1 increased only in PC at the expense of 18:0 (stearic acid). The similar and more marked deviation at the 2-position was observed with AH7974 nuclei it contained approximately 2-times more of 18:1 in PC, PE and PI than regenerating liver nuclei (28 h), and the decreased levels of 20:4 and/or 22:6. It should be noted that there were significant differences in the fatty acid compositions of PE and PS between sham-operated and non-treated controls. So, the sham-operated rat is the appropriate control for proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Scatter factor is a glycoprotein but glycosylation is not required for its activity.

Scatter factor (SF) is a protein produced by fibroblasts, smooth muscle cells, and human placenta which scatter cohesive epithelial cell colonies and increases cellular motility. SF bound to concanavalin A and other lectins with high affinity. SF could also be stained with a glycoprotein specific stain. Incubation of producer cells (N-ras-transformed 3T3), with tunicamycin homolog A1 did not have any significant effect on the secretory activity of SF. The treatment of SF with N- and O-glycanases as well as endoglycosidase H had no effect on its activity. However, treatment of target (Madin Darby canine kidney) cells with tunicamycin A1, abolished the scattering response. These studies suggest that scatter factor is a glycoprotein, but glycosylation is not required for its secretion or activity by the producer cells; however, glycosylation of proteins in the target cells is required for SF action.

Hepatocarcinogenesis tumor grading correlated with in vivo image-guided 1H-NMR spectroscopy in a rat model.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide, the occurrence of which is unevenly distributed. Most hepatocellular carcinoma cases present late and have a poor prognosis; therefore, early diagnosis is essential to prolong survival. Differential diagnosis with magnetic resonance imaging (MRI) is difficult. We studied the feasibility of using magnetic resonance spectroscopy (MRS) at 7.0 T for the diagnosis and grading of liver tumors. An animal model of hepatocarcinogenesis was used, which allowed tumor progression from precancerous lesions to hepatocellular carcinomas. This study was focused primarily on the grading of the tumors and its correlation with the ratio between the MRS peaks arising from MRS-detected lipid hydrogens (0.9, 1.3 and 5.3 ppm) and compared to the gamma-methylene hydrogens of glutamate (Glu) and glutamine (Gln) which was used as an internal reference (2.4 ppm). The lipid methylene hydrogen (1.3 ppm) to (Glu + Gln) ratio was found to correlate with the formation of differentiated small foci and (precancerous) hepatic nodules, whereas the unsaturated olefinic lipid hydrogen (5.3 ppm) to (Glu + Gln) ratio was able to correlate with the formation of late stage tumors such as adenomas and hepatocellular carcinomas. The results of our study suggest that MRS-detected alterations in lipid metabolism can be correlated with the grading of liver tumor tissue at different stages during the carcinogenesis process.

Probucol inhibits ABCA1-mediated cellular lipid efflux.

OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of lipids from cells to lipid-poor apolipoproteins. In this article, we characterize the effect of probucol on cellular ABCA1-mediated lipid efflux. METHODS AND RESULTS: Probucol inhibited cholesterol efflux up to 80% in J774 macrophages expressing ABCA1. In Fu5AH hepatoma cells that contain scavenger receptor class B, type I, but not functional ABCA1, we observed no effect of probucol on cholesterol efflux. Probucol inhibited cholesterol efflux from normal human skin fibroblasts but not from fibroblasts from a Tangier patient. Fluorescent confocal microscopy and biotinylation assay demonstrated that in J774 cells probucol impaired the translocation of ABCA1 from intracellular compartments to the plasma membrane. Probucol also inhibited the formation of an ABCA1-linked cholesterol oxidase sensitive plasma membrane domain. Consistent with the inhibitory effect on ABCA1 translocation to the plasma membrane, probucol reduced cell surface-specific [125I]-labeled apolipoprotein-AI binding. CONCLUSIONS: We conclude that probucol is an effective inhibitor of ABCA1-mediated cholesterol efflux without influencing scavenger receptor class B type I-mediated efflux. The inhibition of ABCA1 translocation to the plasma membrane may in part explain the reported in vivo high-density lipoprotein-lowering action of probucol.