Ean-Jeong Seo

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

The probiotic Escherichia coli strain Nissle 1917 (EcN) is one of the few probiotics licensed as a medication in several countries. Best documented is its effectiveness in keeping patients suffering from ulcerative colitis (UC) in remission. This might be due to its ability to induce the production of human β-defensin 2 (HBD2) in a flagellin-dependent way in intestinal epithelial cells. In contrast to ulcerative colitis, for Crohns disease (CD) convincing evidence is lacking that EcN might be clinically effective, most likely due to the genetically based inability of sufficient defensin production in CD patients. As a first step in the development of an alternative approach for the treatment of CD patients, EcN strains were constructed which were able to produce human α-defensin 5 (HD5) or β-defensin 2 (HBD2). For that purpose, codon-optimized defensin genes encoding either the proform with the signal sequence of human α-defensin 5 (HD5) or the gene encoding HBD2 with or without the signal sequence were cloned in an expression vector plasmid under the control of the T7 promoter. Synthesis of the encoded defensins was shown by Western blots after induction of expression and lysis of the recombinant EcN strains. Recombinant mature HBD2 with an N-terminal His-tag could be purified by Ni-column chromatography and showed antimicrobial activity against E. coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes. In a second approach, that part of the HBD2 gene which encodes mature HBD2 was fused with the yebF gene. The resulting fusion protein YebFMHBD2 was secreted from the encoding EcN mutant strain after induction of expression. Presence of YebFMHBD2 in the medium was not the result of leakage from the bacterial cells, as demonstrated in the spent culture supernatant by Western blots specific for β-galactosidase and maltose-binding protein. The dialyzed and concentrated culture supernatant inhibited the growth of E. coli, S. enterica serovar Typhimurium and L. monocytogenes in radial diffusion assays as well as in liquid culture. This demonstrates EcN to be a suitable probiotic E. coli strain for the production of certain defensins.

Interaction of antihistaminic drugs with human translationally controlled tumor protein (TCTP) as novel approach for differentiation therapy

Translationally controlled tumor protein (TCTP) represents an exquisite target for cancer differentiation therapy, because it was most strikingly down-regulated in tumor reversion experiments. Since TCTP is identical with the histamine releasing factor, antihistamic drugs may inhibit TCTP. Indeed, antihistaminics, such as promethazine, thioridazine, perphemazine and chlorpromazine reveal antiproliferative effects. The aim of this investigation was to study antihistaminic drugs as new TCTP inhibitors to inhibit tumor growth. Levomepromazine and buclizine showed higher in silico binding affinities to TCTP among 12 different antihistaminic compounds including the control drugs, promethazine and hydroxyzine by using Autodock4 and AutodockTools-1.5.7.rc1. Recombinant human TCTP was codon-optimized, expressed in E. coli and purified by chitin affinity chromatography. For experimental validation of in silico data, we applied microscale thermophoresis. Levomepromazine bound with a Kd of 57.2 μM (p < 0.01) and buclizine with a Kd of 433μM (p < 0.01) to recombinant TCTP. Both drugs inhibited MCF-7 breast cancer cell growth in resazurin assays. TCTP expression was down-regulated after treatment with the two drugs. Cell cycle was arrested in the G1 phase without apoptosis as confirmed by the expression of cell cycle and apoptosis-regulating proteins. Annexin V-PI staining and Trypan blue exclusion assay supported that the two drugs are cytostatic rather than cytotoxic. Induction of differentiation with two drugs was detected by the increased appearance of lipid droplets. In conclusion, levomepromazine and buclizine inhibited cancer cell growth by binding to TCTP and induction of cell differentiation. These compounds may serve as lead compounds for cancer differentiation therapy.

Antiangiogenic Activity and Pharmacogenomics of Medicinal Plants from Traditional Korean Medicine

Aim. In the present study, we investigated the antiangiogenic properties of 59 plants used in traditional Korean medicine. Selected phytochemicals were investigated in more detail for their modes of action. Methods. A modified chicken-chorioallantoic-membrane (CAM) assay using quail eggs was applied to test for antiangiogenic effects of plant extracts. A molecular docking in silico approached the binding of plant constituents to the vascular endothelial growth factor receptors 1 and 2 (VEGFR1, VEGFR2). Microarray-based mRNA expression profiling was employed to correlate the 50% inhibition concentrations (IC50) of a panel of 60 NCI cell lines to these phytochemicals. Results. Extracts from Acer mono leaves, Reynoutria sachalniensis fruits, Cinnamomum japonicum stems, Eurya japonica leaves, Adenophora racemosa whole plant, Caryopteris incana leaves-stems, and Schisandra chinensis stems inhibited angiogenesis more than 50% in quail eggs. Selected phytochemicals from Korean plants were analyzed in more detail using microarray-based mRNA expression profiles and molecular docking to VEGFR1 and VEGFR2. These results indicate multifactorial modes of action of these natural products. Conclusion. The antiangiogenic activity of plants used in traditional Korean medicine implicates their possible application for diseases where inhibition of blood vessel formation is desired, for example, cancer, macular degeneration, diabetic retinopathy and others.

Phytochemicals as inhibitors of NF-κB for treatment of Alzheimer’s disease

&NA; Alzheimers disease (AD) is the most prevalent form of dementia. The exact pathophysiology of this disease remains incompletely understood and safe and effective therapies are required. AD is highly correlated with neuroinflammation and oxidative stress in brain causing neuronal loss. Nuclear factor of activated B‐cells (NF‐&kgr;B) is involved in physiological inflammatory processes and thus representing a promising target for inflammation‐based AD therapy. Phytochemicals are able to interfere with the NF‐&kgr;B pathway. They inhibit the phosphorylation or the ubiquitination of signaling molecules, and thus, inhibit the degradation of I&kgr;B. The translocation of NF‐&kgr;B to the nucleus and subsequent transcription of pro‐inflammatory cytokines are inhibited by the actions of phytochemicals. Additionally, natural compounds preventing the interaction of NF‐&kgr;B can block NF‐&kgr;Bs transcriptional activity by inhibiting its binding to target DNA. Many polyphenols including curcumin, resveratrol, pterostilbene, punicalagin, macranthoin G, salidroside, 4‐O‐methylhonokiol, lycopene, genistein, obovatol and gallic acid were reported as potent NF‐&kgr;B inhibitors for AD treatment. Several alkaloids such as galantamine, glaucocalyxin B, tetrandrine, berberine, oridonin, anatabine have been shown anti‐inflammatory effects in AD models in vitro as well as in vivo. Besides, vitamins, tanshinone IIA, artemisinin, dihydroasparagusic acid, geniposide, xanthoceraside, l‐theranine, 1,8‐cineole and paeoniflorin were described as promising NF‐&kgr;B inhibitors. In conclusion, natural products from plants represent interesting candidates for AD treatment. They may qualify as promising compounds for the development of derivatives providing enhanced pharmacological features.

Cytotoxicity and Pharmacogenomics of Medicinal Plants from Traditional Korean Medicine

Aim. The present study was designed to investigate the cytotoxicity of a panel of 280 Korean medicinal plants belonging to 73 families and 198 species against human CCRF-CEM leukemia cells. Selected phytochemicals were investigated in more detail for their mode of action. Methods. The resazurin assay was used to determine cytotoxicity of the plant extracts. Microarray-based mRNA expression profiling, COMPARE, and hierarchical cluster analyses were applied to identify which genes correlate with sensitivity or resistance to selected phytochemicals of the Korean plants. Results. The results of the resazurin assay showed that cytotoxicity extracts tested at 10 μg/mL from 13 samples inhibited proliferation more than 50% (IC50 < 10 μg/mL) and the most active plants are Sedum middendorffianum (15.33%) and Lycoris radiata (17.61%). Out of 13 selected phytochemicals from these plants, hopeaphenol and deoxynarciclasine were the most cytotoxic ones. Genes from various functional groups (transcriptional or translational regulation, signal transduction, cellular proliferation, intracellular trafficking, RNA metabolism, endoplasmic/sarcoplasmic reticulum function, etc.) were significantly correlated with response of tumor cell lines to these two compounds. Conclusion. The results provide evidence on the possible use of selected Korean medicinal plants and chemical constituents derived from them for the treatment of tumors.

Pharmacogenomics of Scopoletin in Tumor Cells.

Drug resistance and the severe side effects of chemotherapy necessitate the development of novel anticancer drugs. Natural products are a valuable source for drug development. Scopoletin is a coumarin compound, which can be found in several Artemisia species and other plant genera. Microarray-based RNA expression profiling of the NCI cell line panel showed that cellular response of scopoletin did not correlate to the expression of ATP-binding cassette (ABC) transporters as classical drug resistance mechanisms (ABCB1, ABCB5, ABCC1, ABCG2). This was also true for the expression of the oncogene EGFR and the mutational status of the tumor suppressor gene, TP53. However, mutations in the RAS oncogenes and the slow proliferative activity in terms of cell doubling times significantly correlated with scopoletin resistance. COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression resulted in a set of 40 genes, which all harbored binding motifs in their promoter sequences for the transcription factor, NF-κB, which is known to be associated with drug resistance. RAS mutations, slow proliferative activity, and NF-κB may hamper its effectiveness. By in silico molecular docking studies, we found that scopoletin bound to NF-κB and its regulator IκB. Scopoletin activated NF-κB in a SEAP-driven NF-κB reporter cell line, indicating that NF-κB might be a resistance factor for scopoletin. In conclusion, scopoletin might serve as lead compound for drug development because of its favorable activity against tumor cells with ABC-transporter expression, although NF-κB activation may be considered as resistance factor for this compound. Further investigations are warranted to explore the full therapeutic potential of this natural product.

Synergy assessment of fixed combinations of Herba Andrographidis and Radix Eleutherococci extracts by transcriptome-wide microarray profiling.

BACKGROUND Generally accepted, but insufficiently proved, the concept of synergy is based on an assumption that combining of two biologically active substances is justified because the combination is more active and less harmful than the ingredients. HYPOTHESIS Analysis of RNA microarray of isolated neuroglia cells and the comparison the number of genes deregulated by plant extracts and their fixed herbal formulation might be a useful tool/method for assessment of synergistic and antagonistic interactions of herbal extracts in human organism. AIM The primary aim of this study was to extend a new method of assessment of synergistic and antagonistic interactions of herbal extracts in isolated human neuroglia cells when they applied in the form of fixed combinations. The secondary aim of the study was to predict possible effects of Herba Andrographidis (APE), Radix Eleutherococci (ESE) genuine extracts and their fixed combination Kan Jang (KJ) on cellular and physiological functions and associated diseases. The third task of the study was to find evidences that justify the hypothesis that these plants extracts in combination are more useful than the monodrugs. METHODS Gene expression profiling was performed on the human neuroglia cell line T98G after treatment with APE, ESE, KJ and total number of more than two fold-deregulated genes from all experiments were compared by Venn diagram. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULTS ESE and APE significantly deregulate 207 and 211 genes correspondingly; 36 deregulated genes were common for both extracts. In total of 382 deregulated genes was expected to be deregulated by their fixed combination KJ. However, it was found only 250 genes deregulated by KJ. Among these 250 genes, 111 genes were unique for the KJ combination and not affected by ESE and APE. This is presumably due to synergistic interactions of molecular networks affected by ESE and APE. Meanwhile, 170 genes deregulated by ESE, and 55 genes deregulated by APE when tested alone, were not up- or downregulated by KJ. That is the result of antagonistic integrations of ESE and APE extracts when applied in the combination. Fold change of expression of 18 common genes deregulated by APE, ESE and KJ was not additive when APE and ESE are combined in KJ herbal formula. However, a qualitative difference is observed in the fingerprint of deregulated genes of daughter substance (KJ) compared to fingerprints/signatures of deregulated genes of parent substances (APE and ESE). Specific for KJ and predictable (z-score > 2) were the effects on pathways and networks associated with infectious and chronic inflammatory disorders, namely encephalitis or neurological movement disorders. Noteworthy, Eleutherococcus alone has no effect on those networks, particularly on encephalitis network, while KJ deregulates 11 genes which have predictable inhibitory effect on infection, while APE regulates only 5 genes which are activated in encephalitis. It can be speculated that APE in combination with ESE may have better therapeutic effect, since more targets are affected. Similar suggestion is justified regarding neurological movement, which is associated with chronic inflammation, like arthritis and osteoarthrosis. Though, microarray analysis did not provide final proof that the genes induced by the KJ, APE and ESE are responsible for the physiological effects observed in humans following their oral administration. It provided insights into putative genes and directions for future research and possible implementation into practice. The most significantly affected canonical pathways deregulated by KJ and APE was interferon signaling pathway, indicating the possible effectiveness of KJ and APE in the treatment of severe sepsis, systemic lupus erythematosus and other autoimmune diseases CONCLUSION Analysis of RNA microarray data from isolated neuroglia cells and the comparison the number of genes deregulated by plant extracts and their fixed herbal formulation might be a useful tool/method for assessment of synergistic and antagonistic interactions of herbal extracts in human organism. Combination of APE and ESE in KJ formulation is most likely justified.

Cytotoxicity of Endoperoxides from the Caribbean Sponge Plakortis halichondrioides towards Sensitive and Multidrug-Resistant Leukemia Cells: Acids vs. Esters Activity Evaluation

The 6-epimer of the plakortide H acid (1), along with the endoperoxides plakortide E (2), plakortin (3), and dihydroplakortin (4) have been isolated from a sample of the Caribbean sponge Plakortis halichondrioides. To perform a comparative study on the cytotoxicity towards the drug-sensitive leukemia CCRF-CEM cell line and its multi-drug resistant subline CEM/ADR5000, the acid of plakortin, namely plakortic acid (5), as well as the esters plakortide E methyl ester (6) and 6-epi-plakortide H (7) were synthesized by hydrolysis and Steglich esterification, respectively. The data obtained showed that the acids (1, 2, 5) exhibited potent cytotoxicity towards both cell lines, whereas the esters showed no activity (6, 7) or weaker activity (3, 4) compared to their corresponding acids. Plakortic acid (5) was the most promising derivative with half maximal inhibitory concentration (IC50) values of ca. 0.20 µM for both cell lines.

Antiproliferative activity against leukemia cells of sesquiterpene lactones from the Turkish endemic plant Centaurea drabifolia subsp. detonsa

The apolar organic extract obtained from aerial parts of Centaurea drabifolia Sibth. & Sm. subsp. detonsa (Bornm.) Wagenitz, growing wild in Turkey, was investigated for the first time for its secondary metabolite composition. Seven sesquiterpene lactones belonging to the guaiane class (1-7), including the new compound 4, along with a fatty acid lactone derivative (8), were isolated. The structures of these compounds were established by spectroscopic analysis, including 2D NMR spectroscopic techniques, with the stereostructure of the new guaiane 4 determined with the help of MTPA derivatization. Cytotoxic activities of compounds 1-7 were evaluated against two cancer cell lines, namely acute lymphoblastic leukemia (CCRF-CEM) and its multidrug-resistant subline CEM/ADR5000. Results showed that aguerin B (1) and cynaropicrin (2) showed a potent activity on both cell lines revealing interesting details about the structure-activity relationships in the class of acylated guaiane sesquiterpenes.

Both Phenolic and Non-phenolic Green Tea Fractions Inhibit Migration of Cancer Cells

Green tea consumption is associated with chemoprevention of many cancer types. Fresh tea leaves are rich in polyphenolic catechins, which can constitute up to 30% of the dry leaf weight. While the polyphenols of green tea have been well investigated, it is still largely unknown, whether or not non-phenolic constituents also reveal chemopreventive and anti-metastatic effects. In this study, we investigated the effects of a fraction of green tea rich in phenolic compounds (PF), a non-phenolic fraction (NPF), which contains glyceroglycolipids (GGL), and a pure glyceroglycolipid compound isolated from the non-phenolic fraction in human cancer. Dried green tea leaves were extracted and applied to a Sephadex LH-20 column. The resazurin reduction assay was used to investigate the cytotoxicity of green tea samples toward human HepG2 hepatocellular carcinoma and normal AML12 hepatocytes cells. Gene expression profiling was performed by mRNA microarray hybridization and the microarray results were validated by RT-PCR. The scratch migration assay was used to investigate the effects of green tea samples on cell migration in vitro. The changes of microtubule dynamics were observed using fluorescence microscopy. PF and NPF were prepared from methanol extract of green tea. A GGL was isolated from NPF. All three green tea samples did not show significant cytotoxic activity up to 10 μg/mL in both HepG2 and AML12 cells, whereas cytotoxicity of the control drug doxorubicin was observed with both cell lines (IC50 on AML12: 0.024 μg/mL, IC50 on HepG2: 2.103 μg/mL). We identified three sets of genes differentially expressed upon treatment with the green tea samples. The genes were associated with cytoskeleton formation, cellular movement, and morphology. The correlation coefficients between mRNA expression values determined by microarray and RT-PCR were R = 0.94. HepG2 and U2OS cells treated with green tea extracts showed the delayed closures. Besides, the number of distinct tubulin filaments decreased upon treatment with green tea samples. We identified not only PF, but also glyceroglycolipids in NPF as contributing factors to the chemopreventive effects of green tea. Both PF and NPF of green tea inhibited cancer cell migration by the disassembly of microtubules, even though they were not cytotoxic.