Jung-Il Chae

Epigallocatechin Gallate Suppresses Lung Cancer Cell Growth through Ras-GTPase-Activating Protein SH3 Domain-Binding Protein 1

Green tea is a highly popular beverage globally. Green tea contains a number of polyphenol compounds referred to as catechins, and (−)-epigallocatechin gallate (EGCG) is believed to be the major biologically active compound found in green tea. EGCG has been reported to suppress lung cancer, but the molecular mechanisms of the inhibitory effects of EGCG are not clear. We found that EGCG interacted with the Ras–GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) with high binding affinity (Kd = 0.4 μmol/L). We also showed that EGCG suppressed anchorage-independent growth of H1299 and CL13 lung cancer cells, which contain an abundance of the G3BP1 protein. EGCG was much less effective in suppressing anchorage-independent growth of H460 lung cancer cells, which express much lower levels of G3BP1. Knockdown shG3BP1-transfected H1299 cells exhibited substantially decreased proliferation and anchorage-independent growth. shG3BP1 H1299 cells were resistant to the inhibitory effects of EGCG on growth and colony formation compared with shMock-transfected H1299 cells. EGCG interfered with the interaction of G3BP1 and the Ras–GTPase-activating protein and further suppressed the activation of Ras. Additional results revealed that EGCG effectively attenuated G3BP1 downstream signaling, including extracellular signal-regulated kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, in wild-type H1299 and shMock H1299 cells but had little effect on H460 or shG3BP1 H1299 cells. Overall, these results strongly indicate that EGCG suppresses lung tumorigenesis through its binding with G3BP1. Cancer Prev Res; 3(5); 670–9. ©2010 AACR.

Mitochondrial dynamics modulate the expression of pro‐inflammatory mediators in microglial cells

Over‐activation of microglia cells in the brain contributes to neurodegenerative processes promoted by the production of various neurotoxic factors including pro‐inflammatory cytokines and nitric oxide. Recently, accumulating evidence has suggested that mitochondrial dynamics are an important constituent of cellular quality control and function. However, the role of mitochondrial dynamics in microglial activation is still largely unknown. In this study, we determined whether mitochondrial dynamics are associated with the production of pro‐inflammatory mediators in lipopolysaccharide (LPS)‐stimulated immortalization of murine microglial cells (BV‐2) by a v‐raf/v‐myc carrying retrovirus (J2). Excessive mitochondrial fission was observed in lentivirus‐transfected BV‐2 cells stably expressing DsRed2‐mito following LPS stimulation. Furthermore, mitochondrial localization of dynamin‐related protein 1 (Drp1) (a key regulator of mitochondrial fission) was increased and accompanied by de‐phosphorylation of Ser637 in Drp1. Interestingly, inhibition of LPS‐induced mitochondrial fission and reactive oxygen species (ROS) generation by Mdivi‐1 and Drp1 knock‐down attenuated the production of pro‐inflammatory mediators via reduced nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and mitogen‐activated protein kinase (MAPK) signaling. Our results demonstrated for the first time that mitochondrial fission regulates mitochondrial ROS production in activated microglial cells and influences the expression of pro‐inflammatory mediators through the activation of NF‐κB and MAPK. We therefore suggest that mitochondrial dynamics may be essential for understanding pro‐inflammatory mediator expression in activated microglial cells. This could represent a new therapeutic approach for preventing neurodegenerative diseases.

Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos

Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.

Natural diterpenes from coffee, cafestol and kahweol induce apoptosis through regulation of specificity protein 1 expression in human malignant pleural mesothelioma.

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive cancer with a very poor prognosis. Several clinical studies such as immunotherapy, gene therapy and molecular targeting agents have been tried for treatment of malignant mesothelioma, however, there is no application for effective clinical treatment. Coffee has various biological functions such as anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. The therapeutic activities of the bioactive compounds in coffee was sugested to influence intracellular signaling of MPM. Regarding to the cancer-related functions, In this study, suppression of Sp1 protein level followed by induction of MSTO-211H cell apoptosis by cafestol and kahweol were investigated in oreder to determine Sp1s potential as a significant target for human MPM therapy as well.MethodsCells were treated separately with final concentration of cafestol and kahweol and the results were analyzed by MTS assay, DAPI staining, PI staining, luciferase assay, RT-PCR, and immunoblotting.ResultsViability of MSTO-211H and H28 cells were decreased, and apoptotic cell death was increased in MSTO-211H as a result of cafestol and kahweol treatment. Cafestol and kahweol increased Sub-G1 population and nuclear condensation in MSTO-211H cells. Roles of Sp1 in cell proliferation and apoptosis of the MSTO-211H cells by the Sp1 inhibitor of Mithramycin A were previously confirmed. Cafestol and kahweol significantly suppressed Sp1 protein levels. Kahweol slightly attenuated Sp1 mRNA, while Cafestol did not affect in MSTO-211H cells. Cafestol and kahweol modulated the promoter activity and protein expression level of the Sp1 regulatory genes including Cyclin D1, Mcl-1, and Survivin in mesothelioma cells. Apoptosis signaling cascade was activated by cleavages of Bid, Caspase-3, and PARP with cafestol and by upregulation of Bax, and downregulation of Bcl-xl by kahweol.ConclusionsSp1 can be a novel molecular target of cafestol and kahweol in human MPM.

Proteomic Analysis of the Extraembryonic Tissue from Cloned Porcine Embryos

Cloned animals developed from somatic cell nuclear transfer (SCNT) embryos are useful resources for agricultural and medical applications. However, the birth rate in the cloned animals is very low, and the cloned animals that have survived show various developmental defects. In this report, we present the morphology and differentially regulated proteins in the extraembryonic tissue from SCNT embryos to understand the molecular nature of the tissue. We examined 26-day-old SCNT porcine embryos at which the sonogram can first detect pregnancy. The extraembryonic tissue from SCNT embryos was abnormally small compared with the control. In the proteomic analysis with the SCNT extraembryonic tissue, 39 proteins were identified as differentially regulated proteins. Among up-regulated proteins, Annexins and Hsp27 were found. They are closely related to the processes of apoptosis. Among down-regulated proteins, Peroxiredoxins and anaerobic glycolytic enzymes were identified. In the Western blot analysis, antioxidant enzymes and the antiapoptotic Bcl-2 protein were down-regulated, and caspases were up-regulated. In the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with the placenta from SCNT embryos, apoptotic trophoblasts were observed. These results demonstrate that a major reason for the low birth rate of cloned animals is due to abnormal apoptosis in the extraembryonic tissue during early pregnancy.

Hispidulin attenuates bone resorption and osteoclastogenesis via the RANKL-induced NF-κB and NFATc1 pathways

Hispidulin, a flavonoid that is known to have anti-inflammatory and anti-oxidant effects, attenuates osteoclastogenesis and bone resorption. To investigate the molecular mechanism of its inhibitory effect on osteoclastogenesis, we employed the receptor activator of the nuclear factor κB (NF-κB) ligand (RANKL)-induced murine monocyte/macrophage RAW 264.7 cells and bone marrow-derived macrophages (BMMs) for osteoclastic differentiation in vitro. The inhibitory effect on in vitro osteoclastogenesis was evaluated by counting the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and by measuring the expression levels of osteoclast-specific genes such as matrix metalloproteinase 9 (MMP9), TRAP and cathepsin K. Similarly, hispidulin significantly inhibited osteoclast activity in RAW 264.7 cell as well as stimulated the ALP activity of MC3T3E1 cells. Furthermore, the in vivo suppressive effect on bone loss was assessed quantitatively in a lipopolysaccharide (LPS)-induced mouse model using microcomputational tomography (μCT) and histochemical analyses. Hispidulin was found to inhibit RANKL-induced activation of Jun N-terminal kinase (JNK) and p38, in addition to NF-κB in vitro experiment. Additionally, hispidulin decreased NFATc1 transcriptional activity in RANKL-induced osteoclastogenesis. This study identifies hispidulin as a potent inhibitor of osteoclastogenesis and bone resorption and provides evidence for its therapeutic potential to treat diseases involving abnormal bone lysis.

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells

BackgroundStudies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.ResultsFrom hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a γ-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.ConclusionOur results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.

Comparative Analysis of Human Mesenchymal Stem Cells Derived From Bone Marrow, Placenta, and Adipose Tissue as Sources of Cell Therapy.

Various source‐derived mesenchymal stem cells (MSCs) with multipotent capabilities were considered for cell therapeutics of incurable diseases. The applicability of MSCs depends on the cellular source and on their different in vivo functions, despite having similar phenotypic and cytological characteristics. We characterized MSCs from different sources, including human bone marrow (BM), placenta (PL), and adipose tissue (AT), in terms of the phenotype, surface antigen expression, differentiation ability, proteome reference map, and blood flow recovery in a hindlimb ischemic disease model. The MSCs exhibit different differentiation potentials depending on the cellular source despite having similar phenotypic and surface antigen expression. We identified approximately 90 differentially regulated proteins. Most up‐ or down‐regulated proteins show cytoskeletal or oxidative stress, peroxiredoxin, and apoptosis roles according to their functional involvement. In addition, the PL‐MSCs retained a higher therapeutic efficacy than the BM‐ and AT‐MSCs in the hindlimb ischemic disease model. In summary, we examined differentially expressed key regulatory factors for MSCs that were obtained from several cellular sources and demonstrated their differentially expressed proteome profiles. Our results indicate that primitive PL‐MSCs have biological advantages relative to those from other sources, making PL‐MSCs a useful model for clinical applications of cell therapy. J. Cell. Biochem. 117: 1112–1125, 2016.

Proteomic analysis of apoptosis related proteins regulated by proto‐oncogene protein DEK

A nuclear phosphoprotein, DEK, is implicated in certain human diseases, such as leukemia and antoimmune disorders, and a major component of metazoan chromatin. Basically as a modulator of chromatin structure, it can involve in various DNA and RNA‐dependent processes and function as either an activator or repressor. Despite of numerous efforts to suggest the biological role of DEK, direct target proteins of DEK in different physiological status remains elusive. To investigate if DEK protein triggers the changes in certain protein networks, DEK was knocked down at both types of cell clones using siRNA expression. Here we provide a catalogue of proteome profiles in total cell lysates derived from normal HeLa and DEK knock‐down HeLa cells and a good in vitro model system for dissecting the protein networks due to this proto‐oncogenic DEK protein. In this biological context, we compared total proteome changes by the combined methods of two‐dimensional gel electrophoresis, quantitative image analysis and MALDI‐TOF MS analysis. There were a large number of targets for DEK, which were differentially expressed in DEK knock‐down cells and consisted of 58 proteins (41 up‐regulated and 17 down‐regulated) differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. In the identified 58 spots, 16% of proteins are known to be associated with apoptosis. Among others, we identified apoptosis related proteins such as Annexins, Enolase1, Lamin A, and Glutathione‐S‐transferase omega 1. These results are consistent with recent studies indicating the crucial role of DEK in apoptosis pathway. We further demonstrated by ChIP analysis that knock‐down of DEK caused hyperacetylation of histones around Prx VI promoter which is upregulated in our profile. Using immunoblotting analysis, we have demonstrated the modulation of other caspase‐dependent apoptosis related proteins by DEK knock‐down and further implicate its role in apoptosis pathway. J. Cell. Biochem. 106: 1048–1059, 2009.

Proteomic validation of multifunctional molecules in mesenchymal stem cells derived from human bone marrow, umbilical cord blood and peripheral blood.

Mesenchymal stem cells (MSCs) are one of the most attractive therapeutic resources in clinical application owing to their multipotent capability, which means that cells can differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle and marrow stroma. Depending on the cellular source, MSCs exhibit different application potentials according to their different in vivo functions, despite similar phenotypic and cytological characteristics. To understand the different molecular conditions that govern the different application or differentiation potential of each MSC according to cellular source, we generated a proteome reference map of MSCs obtained from bone marrow (BM), umbilical cord blood (CB) and peripheral blood (PB). We identified approximately 30 differentially regulated (or expressed) proteins. Most up-regulated proteins show a cytoskeletal and antioxidant or detoxification role according to their functional involvement. Additionally, these proteins are involved in the increase of cell viability, engraftment and migration in pathological conditions in vivo. In summary, we examined differentially expressed key regulatory factors of MSCs obtained from several cellular sources, demonstrated their differentially expressed proteome profiles and discussed their functional role in specific pathological conditions. With respect to the field of cell therapy, it may be particularly crucial to determine the most suitable cell sources according to target disease.