Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells

We evaluated the antitumor activity of tocotrienol (T3) on human hepatoma Hep3B cells. At first, we examined the effect of T3 on the proliferation of human hepatoma Hep3B cells and found that gamma-T3 inhibited cell proliferation at lower concentrations and shorter treatment times than alpha-T3. Then, we examined the effect of gamma-T3 apoptosis induction and found that gamma-T3 induced poly (ADP-ribose) polymerase (PARP) cleavage and stimulated a rise in caspase-3 activity. In addition, gamma-T3 stimulated a rise in caspase-8 and caspase-9 activities. We also found that gamma-T3-induced apoptotic cell death was accompanied by up-regulation of Bax and a rise in the fragments of Bid and caspase-8. These data indicate that gamma-T3 induced apoptosis in Hep3B cells and that caspase-8 and caspase-9 were involved in apoptosis induction. Moreover, these results suggest that Bax and Bid regulated apoptosis induction by gamma-T3.     

Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in Hep G2 Hepatocelluar Carcinoma Cells

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Hepatocyte growth factor enhances protein phosphatase Cdc25A inhibitor compound 5-induced hepatoma cell growth inhibition via Akt-mediated MAPK pathway

We have previously shown that Compound 5 (Cpd 5), an inhibitor of protein phosphatase Cdc25A, inhibits Hep3B human hepatoma cell growth. We now show that hepatocyte growth factor (HGF), a hepatocyte growth stimulant, can strongly enhance Cpd 5-induced growth inhibition in Hep3B cells, and this enhancement in cell growth inhibition is correlated with a much stronger ERK phosphorylation when compared to cells treated with Cpd 5 or HGF separately. We found that HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition were mediated by Akt (protein kinase B) pathway, since combination HGF/Cpd 5 treatment of Hep3B cells inhibited Akt phosphorylation at Ser-473 and its kinase activity, which led to the suppression of Raf-1 phosphorylation at Ser-259. The suppression of Raf-1 Ser-259 phosphorylation caused the induction of Raf-1 kinase activity, as well as hyper-ERK phosphorylation. Transient transfection of Hep3B cells with dominant negative Akt c-DNA further enhanced both Cpd 5- and HGF/Cpd 5-induced ERK phosphorylation, while over-expression of wild-type Akt c-DNA diminished their effects. In contrast, HGF antagonized the growth inhibitory actions of Cpd 5 on normal rat hepatocytes, thus showing a selective effect on tumor cells compared to normal cells. Our data suggest that Akt kinase negatively regulates MAPK activity at the Akt-Raf level. Suppression of Akt activity by either combination HGF/Cpd 5 treatment or by dominant negative Akt c-DNA transfection antagonizes the Akt inhibitory effect on Raf-1, resulting in an enhancement of Cpd 5-induced MAPK activation and cell growth inhibition.     

Design, synthesis and cytotoxic effect of hydroxy- and 3-alkylaminopropoxy-9,10-anthraquinone derivatives

In previous paper, we have reported the synthesis and the cytotoxic effect of 1,3-dihydroxy-9,10-anthraquinone derivatives. For further design of more potent compounds, a new series of 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinones and 3-(3-alkylaminopropoxy)-9,10-anthraquinones have been synthesized. The cytotoxicity of synthetic compounds were evaluated against human Hep G2, Hep 3B and HT-29 cells. Almost all compounds indicated significant inhibitory activity against Hep G2, Hep 3B and HT-29 cell lines in vitro. Compound 5 exhibited selective cytotoxicity against Hep G2 in a concentration-dependent manner with ED50 value of 1.23 +/- 0.05 microM. Structure-activity analysis revealed that most of the 1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-anthraquinone showed stronger cytotoxic effects than those of 1-hydroxy-3- or 3-(3-alkylaminopropoxy)-9,10-anthraquinones against Hep 3B cell line in vitro. A sub-G1 cell stage and DNA fragmentation in MCF-7 cells were significantly observed after 72 h incubation with selective compound 16. The results show that 16 causes cell death by apoptosis.     

Imaging and mapping heparin-binding sites on single fibronectin molecules with atomic force microscopy

Fibronectin is composed of multiple homologous repeats and contains many functional domains. Two major heparin-binding domains have previously been identified: the Hep I site near the amino terminus and the Hep II site near the carboxyl terminus. The Hep II site has been considered the high-affinity heparin-binding site based on studies of fibronectin fragments. However, few studies have been carried out on heparin binding by intact fibronectin. We imaged single fibronectin molecules as well as heparin-coated gold particles bound to whole dimeric plasma fibronectin molecules with tapping mode atomic force microscopy. We observed heparin-gold particles preferentially bound at two locations that correspond to the Hep I and Hep II sites. Quantitative analysis of images of individual fibronectin-heparin-gold complexes showed that almost twice as many heparin-gold particles bound to the N-terminal Hep I site compared to the Hep II site. In contrast to previous findings with fibronectin fragments, these results suggest that the Hep I site has a binding affinity higher than or comparable to the Hep II site in the intact fibronectin molecule.     

In vivo antitumour activity of amphiphilic silicon(IV) phthalocyanine with axially ligated rhodamine B

We explore the possible cellular cytotoxic activity of an amphiphilic silicon(IV) phthalocyanine with axially ligated rhodamine B under ambient light experimental environment as well as its in vivo antitumour potential using Hep3B hepatoma cell model. After loading into the Hep3B hepatoma cells, induction of cellular cytotoxicity and cell cycle arrest were detected. Strong growth inhibition of tumour xenograft together with significant tumour necrosis and limited toxicological effects exerted on the nude mice could be identified.     

Construction of three different recombinant scorpion fusion proteins with bifunctional activity

This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). The fusion proteins were constructed in the form of chimeric toxins, aiming to obtain bifunctional analgesic and antitumor activity. The fusion proteins consisted of luteinizing hormone-releasing hormone (LHRH), three different types of flexible linkers (L1, Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met; L2, Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser; L3, Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Ser-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser), and BmKAGAP. The genes coding three fusion proteins were cloned and expressed in E. coli in soluble form. Following two successive column chromatographic separations, purified fusion proteins were obtained. These fusion proteins exhibited analgesic activity in mice and were cytotoxic to a hepatocellular carcinoma cell line Hep3B.     

Variations in gamma-glutamyl transpeptidase glycosylation and kinetic parameters in cultured liver cells.

In rat hepatocytes; the tumorigenic rat liver cell line ARL-16; and the human hepatoma line, Hep G2, 50% of the total gamma-glutamyl transpeptidase (GGT) activity was bound by a Concanavalin-A Sepharose 4B column, calling for alpha-methylmannoside elution (Peak I). Non-binding GGT was distributed between a rapidly eluting Peak II and a slightly retained Peak III. The Km for gamma-glutamyl-p-nitroanalide for either hydrolysis or transpeptidation, or glutathione (GSH) transpeptidation did not vary with peak number or cell type. The GSH hydrolysis Km was essentially constant in Peak I and II GGT. Peak III GGT exhibited a lower Km for GSH hydrolysis with Hep G2 Peak III GGT being the lowest. Peak III GGT increased to 50% of the GGT activity in Hep G2 cells cultured with GSH as the sole cysteine source.     

Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway

Transforming growth factor-beta (TGF-beta) is a potent inducer of apoptosis in Hep 3B cells. This work investigated how hepatitis B virus X protein (HBx) affects TGF-beta-induced apoptosis. Trypan blue exclusion and colony formation assays revealed that HBx increased the ID(50) toward TGF-beta. In the presence of HBx, TGF-beta-induced DNA laddering was decreased, indicating that HBx had the ability to block TGF-beta-induced apoptosis. Furthermore, HBx did not alter the expression levels of type I and type II TGF-beta receptors. HBx did not affect TGF-beta-induced activation of promoter activities of the plasminogen activator inhibitor-1 (PAI-1) gene. These results indicate that HBx interferes with only a subset of TGF-beta activity. In the presence of phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin or LY294002, the HBx-mediated inhibitory effect on TGF-beta-induced apoptosis was alleviated. In addition, the tyrosine phosphorylation levels of the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI 3-kinase) and PI 3-kinase activity were elevated in stable clones with HBx expression. Transactivation-deficient mutants of HBx lost their ability to inhibit TGF-beta-induced apoptosis. Phosphorylation of the p85 subunit of PI 3-kinase and Akt, a downstream target of PI 3-kinase, was not observed in stable clones with transactivation-deficient HBx mutant's expression. Thus, the anti-apoptotic effect of HBx against TGF-beta can be mediated through the activation of the PI 3-kinase signaling pathway, and the transactivation function of HBx is required for its anti-apoptosis activity.     

Identification of dipeptidyl peptidase IV as a protein shared by the plasma membrane of hepatocytes and liver biomatrix

The histotypic organization of liver parenchyma involves specific intercellular contacts and interaction of hepatocytes with supporting biomatrix. Evidence from this laboratory identified a peptide (Hep105, apparent Mr 105 000) that is shared by the plasma membrane of rat hepatocytes and rat liver biomatrix. This report identifies Hep105 as a peptide component of dipeptidyl peptidase IV (DPPIV; EC 3.4.14.-). A monoclonal antibody (MAb 236.3) was shown to immunoprecipitate DPPIV from non-ionic detergent extracts of surface-labeled 125I hepatocytes. The immunoprecipitate contained two 125I-labeled peptides: Hep105 and a peptide of apparent Mr 150000 (Hep150). Proteolysis of 125I-labeled Hep105 and Hep150 by Staphylococcus aureus V8 protease yielded essentially identical patterns of 125I-labeled peptide degradation products, indicating that Hep105 and Hep150 are structurally related. Only Hep150 exhibited DPPIV activity on transblot analysis, an observation that is consistent with the interpretation that it is the monomeric form of the enzyme. Heating (100 degrees C, 5 min) of purified Hep150 in the presence of sodium dodecylsulfate (SDS) resulted in its conversion to Hep105 and the disappearance of any demonstrable enzymatic activity. 3H-labeled diisopropyl fluorophosphate was incorporated into Hep105, indicating that Hep105 contains the active site for DPPIV. Purified rat liver biomatrix was shown to possess significant DPPIV activity. Taken together, these data indicate that Hep105 s a peptide component of DPPIV.     

Synthesis, characterization and in vitro anti-tumor activities of matrine derivatives

Nineteen previously unreported matrine derivatives were synthesized and characterized using elemental analysis, infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and mass spectrometry. Target compounds 6a-6l and 7a-7c showed stronger inhibitory activities than matrine in the in vitro antitumor tests and inhibited the growth of the Hep7402, B16-F10, A549, and TW03 cell lines. In addition, compound 6i exhibited a potent antitumor activity similar to that of colchicine.     

Proapoptotic activity of NAG-1 is cell type specific and not related to COX-2 expression

Non-steroidal anti-inflammatory drugs (NSAIDs) activated gene (NAG-1) is a newly identified member of the transforming growth factor-β (TGF-β) superfamily. Members of the TGF-β family are multifunctional growth factors, and the nature of their effects depends on the cellular context and cell type. NAG-1 has antitumorigenic and proapoptotic activities in colon and gastric cancer cells lacking endogenous cyclooxgenase-2 (COX-2) expression. In contrast, COX-2 overexpression is related to antiapoptotic activity. The purpose of this study is to evaluate the proapoptotic activity of NAG-1 according to COX-2 expression and cell type. NAG-1 cDNA was transfected in SNU668 cells with endogenous COX-2 expression, SNU601 cells with forced COX-2 expression and Hep3B hepatocellular carcinoma cells. SNU668 cells with ectopic expression of NAG-1 showed markedly elevated subG1 population, induced death receptor-4 (DR-4) and DR-5, and revealed smaller active fragments of caspase-3. Forced COX-2 expression in SNU601 cells did not inhibit apoptosis caused by NAG-1 expression. Sulindac sulfide caused apoptosis, and induced expression of DR-5 and NAG-1 in Hep3B cells. However, Hep3B cells ectopically expressing NAG-1 did not cause apoptosis, and smaller active fragments of caspase-3 and an 85 kDa band of poly ADP-ribose polymerase (PARP) did not appear in the transfected cells, either. This study suggests that proapoptotic activity of NAG-1 is cell type specific and not related to COX-2 expression.     

Cell death signal by glycine- and proline-rich plant glycoprotein is transferred from cytochrome c and nuclear factor kappa B to caspase 3 in Hep3B cells

This study was carried out to investigate the apoptotic effects of glycine- and proline-rich glycoprotein [Solanum nigrum Linne (SNL) glycoprotein, 150-kDa] isolated from SNL, which has been used as an antipyretic and anticancer agent in Korean herbal medicine. We found that SNL glycoprotein has obviously cytotoxic and apoptotic effects at 80 microg/ml of SNL glycoprotein for 4 h in Hep3B cells (hepatocellular carcinoma cells). In mitochondria-mediated apoptosis pathway, SNL glycoprotein has abilities to stimulate release of mitochondrial cytochrome c, activations of caspase-9 and caspase-3, cleavage of poly(ADP-ribose)polymerase and production of intracellular reactive oxygen species in Hep3B cells. In nuclear factor-kappa B (NF-kappaB)-mediated apoptosis pathway, the results showed that SNL glycoprotein dose-dependently blocked DNA binding activity of NF-kappaB, activity of inducible nitric oxide synthase (iNOS) and production of inducible nitric oxide (NO). Interestingly, pyrrolidine dithiocarbamate (for NF-kappaB inhibitor) and Nomega-nitro-l-arginine methylester hydrochloride (for NO inhibitor) effectively stimulated the caspase-3 activation and induced apoptosis in Hep3B cells. These results indicate that SNL glycoprotein transfers its cell death signal from cytochrome c to caspase 3 by inhibiting NF-kappaB and iNOS activation in Hep3B cells. Here, we speculate that SNL glycoprotein is one of the chemotherapeutic agents to modulate mitochondria-mediated apoptosis signals in Hep3B cells.     

HIF-1alpha-prolyl hydroxylase: molecular target of nitric oxide in the hypoxic signal transduction pathway

We have investigated inhibitory mechanisms of hypoxic activation of HIF-1alpha by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1alpha activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel-Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1alpha, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl(2) or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl(2)-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl(2) was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.