Jeong-Heon Ko

Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism

Our previous studies have clearly shown that the angiogenic enzymes, matrix metalloproteinase (MMP) ‐2/9, are directly involved in human hepatic tumorigenesis and metastasis and suggest that the MMP‐2/9 inhibitors, which have dual inhibitory activi¬ties on enzyme activity and transcription, represent the best candidates for achieving tumor regression. Many anti‐cancer drugs have strong cellular cytotoxicity and side effects, indicating that strong anti‐cancer drugs that have no or minimal cytotoxicity and side effects need to be developed. The specific aim of the present study was to develop powerful anti‐cancer drugs with specific tumor regression and anti‐metastatic potential having the dual inhibitory activities of specific MMP‐2 and ‐9 enzyme activities and gene transcription at the molecular level. Caffeic acid (CA), a strong and selective MMP‐9 activity and transcription inhibitor, was isolated from the plant Euonymus alatus and its derivative, caffeic acid phenethyl ester (CAPE), was synthesized. CA and CAPE selectively inhibited MMP‐2 and ‐9 but not ‐1, ‐3, ‐7, or cathepsin K. Treatment of HepG2 cells with CA (100 μg/mL) and CAPE (5 μg/mL) suppressed phorbol 12‐myristate 13‐acetate (PMA) ‐in¬duced MMP‐9 expression by inhibiting the function of NF‐κB, but not AP‐1. We confirmed that CA and CAPE suppressed the growth of HepG2 tumor xenografts in nude mice in vivo. The subcutaneous and oral administrations of CA and CAPE significantly reduced the liver metastasis. These results confirm the therapeutic potential of the compounds and suggest that the antimetastatic and anti‐tumor effects of CA and CAPE are mediated through the selective suppression of MMP‐9 enzyme activity and transcriptional down‐regulation by the dual inhibition of NF‐κB as well as MMP‐9 catalytic activity.—Chung, T.‐W., Moon, S.‐K., Chang, Y.‐C., Ko, J.‐H., Lee, Y.‐C., Cho, G., Kim, S.‐H., Kim, J.‐G., Kim, C.‐H. Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEBJ. 18, 1670–1681 (2004)

Enhanced expression of matrix metalloproteinase-9 by hepatitis B virus infection in liver cells

The hepatitis B virus (HBV) is a major cause of human liver disease, including hepatocellular carcinoma (HCC). The prognosis for HCC is largely dependent on the clinicopathological characteristics regarding invasion and metastasis. Enhanced matrix metalloproteinase-9 (MMP-9) expression has been implicated as playing an important role in metastasis and invasion of HCC. However, the relationship between HBV infection and MMP-9 expression in HCC is currently poorly understood. We report here on a study of the levels of MMP-9 and MMP-2 expression in human fetal liver tissue, rat liver tissue, and Chang, HepG2, and Hep3B cells by gelatin zymography. Among these sources, Hep3B cells, which contain the integrated hepatitis B viral genome, continuously secrete the hepatitis B viral surface antigen, and express HBV genomic RNA, expressed high levels of proMMP-9, and a small amount of active MMP-9 was detected in Hep3B cells as assayed by zymography. We investigated the issue of whether HBV infection affects MMP-9 expression, which is known to play an important role in HCC invasion and metastasis. As a first step, human fetal hepatocyte (HFH) and HepG2 (HCC origin, HBV not detected) cells were subjected to infection with HBV, and the resulting infected cells successfully established are hereafter referred to as HFH-T2 and HepG2-HBV. The expression of MMP-9 was upregulated by the infected HBV in HFH-T2 and HepG2-HBV cells, as assayed by zymography, Northern blot, and Western blot analysis, and small amounts of active MMP-9 were detected in HFH-T2 and HepG2-HBV cells as assayed by zymography. The activation of the immature proMMP-9 to the mature MMP-9 could be induced by plasmin treatment. The activation of proMMP-9 was increased to a greater extent with plasmin treatment than without plasmin in HFH-T2 and HepG2-HBV cells but the addition of recombinant TIMP-1 inhibited the activation of proMMP-9. Finally, the addition of plasmin to the invasion assay using Matrigel resulted in an increase in invasiveness of HFH-T2 and HepG2-HBV cells, as well as MMP-9 activation, but the treatment with TIMP-1 inhibited the invasiveness of HFH-T2 and HepG2-HBV cells as well as MMP-9 activation. We conclude from these findings that HBV infection of hepatocytes and HepG2 cells affected the upregulation of MMP-9 expression and MMP-9 activation and, thus, increased the invasion potential by plasmin. To our knowledge, this is a first report showing that an HBV infection is linked to the upregulation of MMP-9 in HCC.

Higher intracellular levels of uridinemonophosphate under nitrogen‐limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium Species

Changes of intracellular nucleotide levels and their stimulatory effects on curdlan synthesis in Agrobacterium species were investigated under different culture conditions. Under nitrogen-limited conditions where curdlan synthesis was stimulated, intracellular levels of UMP were as high as 87 and those of AMP were 78 nmol/mg of cellular protein, while those under nitrogen-sufficient conditions were lower than 45 nmol/mg-protein. The levels of other nucleotides such as UDP, UTP, UDP-glucose, ADP, ATP, and ADP-glucose were lower than 30 nmol/mg-protein under both nitrogen-limited and sufficient conditions. The time profiles of curdlan synthesis and cellular nucleotide levels showed that curdlan synthesis had a positive relationship with intracellular levels of UMP and AMP. After the ammonium concentration in the medium fell below 0.1 g/L, intracellular levels of UMP and AMP increased, followed by curdlan synthesis. However, no significant changes in the specific activities of UMP kinase, UDP kinase, and UDP-glucose pyrophosphorylase were observed during cultivation. In vitro enzyme reactions for the synthesis of UDP-glucose, which serve as a precursor for curdlan synthesis, demonstrated that the synthesis of UDP-glucose increased with the increase of UMP concentration. In contrast, AMP had no effect on UDP-glucose synthesis at all. Addition of UMP in the medium increased the curdlan synthesis, whereas curdlan synthesis was inhibited in the presence of AMP. From these results, we concluded that only the higher intracellular UMP levels caused by nitrogen limitation in the medium enhance the metabolic flux of curdlan synthesis by promoting cellular UDP-glucose synthesis.

Heat Shock 70‐kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation

The cell‐surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC‐specific cell‐surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz‐hES1, and selected 26 MAbs that were able to bind to Miz‐hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz‐hES1 cells, either. Of these, MAb 20‐202S (IgG1, κ) immunoprecipitated a cell‐surface protein of 72‐kDa from the lysate of biotin‐labeled Miz‐hES1 cells, which was identified to be heat shock 70‐kDa protein 8 isoform 1 (HSPA8) by quadrupole time‐of‐flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz‐hES4, Miz‐hES6, and HSF6. Two‐color flow cytometric analysis of Miz‐hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage‐specific embryonic antigen 3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐81. Flow cytometric and Western blot analyses using various cells showed that MAb 20‐202S specifically bound to the HSPA8 protein on the surface of Miz‐hES1, contrary to other anti‐HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz‐hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20‐202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell‐surface marker for undifferentiated hESCs.

A water-extract of the Korean traditional formulation Geiji-Bokryung-Hwan reduces atherosclerosis and hypercholesteremia in cholesterol-fed rabbits

Geiji-Bokryung-Hwan (GBH), a drug preparation consisting of five herbs of Cinnamomi Ramulus (Geiji), Poria Cocos (Bokryun), Mountan Cortex Radicis (Mokdanpi), Paeoniae Radix (Jakyak) and Persicae Semen (Doin), is a traditional Korean herbal medicine that is widely used in the treatment of atherosclerosis-related disorders. A water extract of GBH was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and inhibit low-density lipoprotein (LDL) oxidation more effectively than probucol, a well-known commercially available antioxidant. In order to evaluate the anti-atherogenic potential of this medication, New Zealand White (NZW) rabbits were fed a normal diet for 12 weeks, a high cholesterol diet, a high cholesterol diet containing 1% probucol or a high cholesterol diet containing 5% water-soluble extract of GBH. Both GBH and probucol reduced plasma cholesterol levels. LDLs from the GBH-treated group were more resistant to Cu(2+)-induced oxidation and contained more vitamin E than LDLs from the high cholesterol diet group. Endothelial damage, determined at week 6, was reduced by 55% in the GBH group (P<0.01). GBH treatment reduced an atherosclerotic area in the abdominal aorta by 58% (P<0.05) and cholesterol deposition in the thoracic aorta by 55% (P<0.05). The severity of atherosclerosis in the GBH group was significantly reduced after an adjustment using cholesterol exposure as an index of the cholesterol-lowering effect. On the other hand, diet-induced hyperlipidemic rabbits were given water extract of GBH in doses of 50 (Group B) and 200 mg/kg (Group C) and compared with controls (Group A). At 40 days after intervention in groups A, B and C, total and LDL cholesterol levels were significantly lowered (P<0.01). LDL/high density lipoprotein (HDL) ratio was also significantly decreased (P<0.01). This study concludes that the reduction in atherosclerosis by GBH relies not only on its cholesterol-lowering effect but also more heavily on its antioxidant potential, which prevents endothelial damage and inhibits LDL oxidative modification in hypercholesterolemic animals.

Electrical Immunosensor Based on a Submicron-gap Interdigitated Electrode and Gold Enhancement

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations).

Comparative effect of prunus persica L. BATSCH-water extract and tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) on concentration of extracellular acetylcholine in the rat hippocampus

Prunus persica L. BATSCH seed-water extract (PPE) has been used in the treatment of the degenerative disorders, such as hypermenorrhea and dysmenorrhea, in Taiwan, China, Japan and Korea. In this study, the effects of oral administration of PPE on the extracellular acetylcholine concentration in the hippocampus of rats were evaluated, and compared to that of tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride), a well-known and centrally acting acetylcholinesterase (AChE) inhibitor, which had been developed for the treatment of Alzheimers disease. We measured the inhibition of brain AChE. PPE at 2.5g/kg and tacrine at 5mg/kg showed significant effects for more than 6h. At these doses, the maximum increases were observed at about 1.5h after administration of PPE, and at about 2h with tacrine, and were 454 and 412% of the pre-level, respectively. The results suggest that oral administration of PPE and tacrine increases acetylcholine concentration in the synaptic cleft of the hippocampus mostly through AChE inhibition, and that PPE has a potent and long-lasting effect on the central cholinergic system.

Membrane type sialidase inhibits the megakaryocytic differentiation of human leukemia K562 cells

The membrane type sialidase (Neu3) has been suggested to participate in cell growth, migration and differentiation. To determine whether a Neu3 is able to modulate megakaryocytic differentiation of K562 cells, we studied the functional significance of human Neu3 induced by phorbol 12-myristate 13-acetate (PMA). Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) indicated that the induction of hST3Gal V, which synthesizes ganglioside GM3 and reduction of Neu3 by PMA, are linked for the expression of differentiation marker protein, CD41b surface antigen. To elucidate the mechanism underlying the down-regulation of the CD41b surface antigen expression when Neu3 gene is expressed in PMA-treated cells, we characterized the Neu3-mediated signaling pathway. Neu3 overexpression inhibited the PMA-induced ERK1/2 and p38 MAPK phosphorylation in the K562 cells. Down-regulation of expression of CD41b surface antigen was dependent on expression of Neu3 gene. However, a Neu3 inhibitor Neu5Ac2en induced morphological changes, showing megakaryocytic differentiation of K562 cells, with expression of CD41b surface antigen, while a specific glucosylceramide synthase inhibitor PDMP inhibited megakaryocytic differentiation of K562 cells. The molecular mechanisms involved in Neu3-involved inhibition of CD41b surface antigen expression in K562 cells have been suggested: the Neu3 degrades membrane sialic acids and the resulting signaling pathway of the PKC/ERKs/p38 MAPK is down-regulated, causing a decrease in CD41b surface antigen expression and inhibition of megakaryocytic differentiation of K562 cells.

Bombycis corpus extract (BCE) protects hippocampal neurons against excitatory amino acid-induced neurotoxicity

Bombycis corpus (BC) or Bombyx Batryticatus, a batryticated silkworm and white-stiff silkworm, is a drug consisting of the dried larva of silkworm, Mobyz mori L., dead and stiffened due to the infection of Beauveria (Bals.) Vuill. In a previous paper (Kim et al., Pharmacol. Res., 43, 12–16, 2001), BC was shown to protect amyloid-β-induced cytotoxicity. In the present study, we have found that BCE can prevent or reduce the neurotoxic actions in the hippocampus of the glutamate agonists N-methyl-D-aspartic acid (NMDA) in vitro or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid in vitro. Pre-treatment with BCE (0, 1, 2, 5, and 10 μg/ml for 6–8 h) protected primary hippocampal cultures from embryonic day 18 (E18) embryos against NMDA-induced toxicity (0.1, 1, 10, and 50 mM/ml). BCE added either with NMDA (1 mM) or 1 h later had lesser, but still significant, protective actions. BCE also reduced NMDA-induced toxicity (1 mM). BCE (10 μg/ml) protected cultured neurons against the neurotoxic actions of either AMPA (25 μM) or kainic acid (1 mM) as well. Because the release of glutamate has been implicated in the neural damage after cerebral ischemia and other neural insults, these results suggest that BCE may contribute significantly to protect human brain to such damage.

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display.

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.