[Show abstract][Hide abstract] ABSTRACT: Objective:
To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer.
The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis.
The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed.
Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.
Chinese Journal of Integrative Medicine 08/2014; DOI:10.1007/s11655-014-1789-8 · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice.
We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells.
Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice.
CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.
[Show abstract][Hide abstract] ABSTRACT: Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR mass spectrometry, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, 6 proteins are associated with energy stress-induced, 9 proteins with inflammatory stress-induced, and 14 proteins with ER stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a JNK-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomic approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus casei extract (LBX) has been reported to prevent gastric cancer, but the underlying mechanism remains unclear. The proliferation and cell death of gastric cancer KATO3 cells were examined after treatment with LBX for various times and at various doses. LBX inhibited the growth of gastric cancer cells and induced apoptosis by inactivating NF-κB promoter activity. Apoptosis induced by LBX, however, is not directly associated with the intrinsic mitochondrial pathway. Immunoblot analysis revealed that LBX decreased the expressions of NF-κB and IκB. The reduced NF-κB levels led to the decreased phosphorylation of mTOR signaling components, such as PI3K, Akt, and (p70)S6 kinase. These results showed for the first time that LBX induced apoptosis in gastric cancer cells by inhibiting NF-κB and mTOR-mediated signaling.
Integrative Cancer Therapies 04/2012; 12(2). DOI:10.1177/1534735412442380 · 2.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis.
[Show abstract][Hide abstract] ABSTRACT: Administration of mountain ginseng (MG) extract can restore advanced cancer to a normal state. To elucidate the mechanism by which MG extract prevents the progression of lung cancer, the processes of proliferation and death of lung cancer cells (A549) were examined after treatment with MG extract. Butanol-extracted MG (BX-MG) showed a high inhibitory effect (IC(50) = 2 mg/ml) by attenuating proliferation and inducing apoptosis in lung cancer cells. By HPLC-UV analysis of BX-MG, ginsenosides, Rb1 was identified as the most abundant ginsenoside, followed by Rg1, Re, Rc and Rb2. BX-MG induced caspase-3 dependent apoptosis by inhibiting NF-κB. In addition, BX-MG activated p53 and p21, resulting in the attenuated proliferation of A549 cells. Reduced activity of the NF-κB promoter and increased activity of the p53 promoter indicate that BX-MG regulates apoptosis at the level of transcription in lung cancer cells. Furthermore, BX-MG blocked the nuclear translocation of RelA and the associated reduction in surviving. These results suggest that BX-MG inhibits lung cancer cell growth by activating tumor suppressors and inhibiting nuclear translocation of NF-κB.
The American Journal of Chinese Medicine 01/2012; 40(1):187-202. DOI:10.1142/S0192415X12500152 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seeing is believing: When a bait protein is fused to protein kinase C (PKCδ), the proteins can interact and cotranslocate from the cytoplasm to the plasma membrane (see picture; imaging with red and green fluorescent proteins (RFP and GFP)). In contrast, when the bait-target interaction is inhibited, only the bait protein is translocated to the plasma membrane. This method was applied to several protein pairs.
[Show abstract][Hide abstract] ABSTRACT: Heterogeneity of ginsenosides is a remarkable and significant issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. This study describes ginsenoside profile variations in Panax ginseng C.A. Meyer cultivated in Korea using an HPLC-UV coupled to ESI-MS for characterization and quantification of ginsenosides contained in the root of ginseng. The [M-H]− ion was observed for ginsenoside standards (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3 and Rh2) and 190 different ginseng extracts. The individual variations in the P. ginseng population were classified into three types, based on the expression pattern of six core ginsenosides containing Ra1/Ra2 and malonyl-Ra1/Ra2 isomers. The variations in the six core ginsenosides, which are defined as pattern indicators, play a key role in discriminating between three subtypes of standard P. ginseng species. In addition, cross-correlation analysis of the identified ginsenosides indicates that the specific ginsenosides, including pattern-indicating components, are in part possibly synthesized via common ginsenoside biosynthesis pathways. Taken together, these findings provide a basis for identifying standard P. ginseng landraces.
PROCESS BIOCHEMISTRY 01/2011; 46(1-46):258-264. DOI:10.1016/j.procbio.2010.08.020 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.
[Show abstract][Hide abstract] ABSTRACT: Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.
[Show abstract][Hide abstract] ABSTRACT: In this paper, we report a simple method for spatially confining Bacillus subtilis (BS) spores into semi-three dimensional, non-biofouling microwells by using biospecific (such as biotin-streptavidin) interactions. Non-biofouling poly(ethylene glycol) (PEG)-based microwells were fabricated by employing a process of capillary molding on a glass slide. The biospecific interactions between biotinylated BS spores and streptavidin led to the selective deposition of BS spores onto the bottom of the microwells of which presented streptavidin. The viability of the patterned spores was confirmed by the induction of germination. Bacterial spores were found to maintain extreme robustness until they were exposed to favorable conditions. This work suggests that the use of bacterial spore-based sensors would increase the shelf-life (such as long-term storage and stability) of cell-based sensors.
[Show abstract][Hide abstract] ABSTRACT: To determine the loading and maintenance dosage of glutathione (GSH) for patients suffering from reactive oxygen species (ROS) injury such as acute paraquat intoxication, a kinetic study of reduced GSH was performed in synchrony with that of cysteine (Cys), cystine (Cys2), and methionine (Met). Human subject's porticipitation was voluntary. The effective dose of Cys, Cys2, and Met against ROS in fibroblast cells generated by paraquat was assessed using laser scanning confocal microscopy. Both Cys and Met suppressed ROS in a dose-dependent manner at concentrations of 1-1,000 microM; the concentration required to suppress ROS by 50% was 10 microM for Cys and 50 microM for Met. Using metabolite kinetics with the assumption that Cys and Met are the metabolites of GSH, expected concentrations of Cys and Met of above 20 and 50 microM were estimated when GSH was administered at 50 mg/kg body weights every 205.4 min for Cys and 427.4 min for Met.
Journal of Korean Medical Science 11/2005; 20(5):721-6. DOI:10.3346/jkms.2005.20.5.721 · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system.
Biotechnology and Bioengineering 10/2005; 92(2):160-5. DOI:10.1002/bit.20581 · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes a versatile method for generating single cell arrays on a glass substrate, which could be applicable to any arbitrary cell types, by a combination of surface functionalization, biotinylation of cells, and microcontact printing (mCP).
[Show abstract][Hide abstract] ABSTRACT: Micropatterns of Bacillus thuringiensis spores were generated by a combination of surface chemistry, microcontact printing, and spore surface display technique. The outermost layers of B. thuringiensis spores were engineered to present enhanced green fluorescent protein (EGFP), and the biospecific interaction between biotin and streptavidin was utilized to spatially direct the EGFP-presenting spores onto the micropatterned surfaces. The viability of the micropatterned spores was confirmed by the pattern generation of vegetative cells after the germination.
Journal of the American Chemical Society 10/2004; 126(34):10512-3. DOI:10.1021/ja047894y · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The micropatterns of biological ligands (biotin and RGD peptides) were generated on a flat surface of biodegradable polymer, poly(glycolic acid) (PGA). The immobilization of biological ligands onto the surface of biodegradable polymers (especially aliphatic polyesters) is usually hampered by the absence of functionalizable groups on the polymer backbone. We demonstrate herein that PGA polymer films were modified by surface hydrolysis to introduce carboxylic acid groups on the film surfaces, which were subsequently used for patterning amine-terminated ligands by microcontact printing. Fluorescence microscopy was used to verify the pattern of biotin on the surface of the PGA films after complexation with fluorescein-conjugated streptavidin. In addition, the cellular micropatterns were obtained from micropatterns of RGD peptides on the surface-hydrolyzed PGA films.
[Show abstract][Hide abstract] ABSTRACT: Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.
Journal of biochemistry and molecular biology 04/2004; 37(2):185-91. DOI:10.5483/BMBRep.2004.37.2.185 · 2.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effectiveness of several sulfhydryl compounds in the treatment of paraquat intoxication has been previously tested based on their antioxidant ability. However, practical guidelines for their clinical use remain to be determined. As a preliminary pharmacokinetic study on sulfhydryl compounds, we attempted to establish the optimal concentration of N-acetyl-L-cysteine, glutathione, superoxide dismutase, and catalase. We measured the antioxidant effect of these antioxidants in normal pooled plasma and on intracellular reactive oxygen species (ROS) induced by paraquat. N-acetyl-L-cysteine begins to suppress the production of ROS in plasma at concentrations as low as 5 mM, with the suppression being maximal at 40 mM. In the same way, glutathione increased the total antioxidant status in plasma at concentrations of 5-40 mM in a dose-dependent manner. Complete suppression of ROS in plasma induced by exposure to 500 micro M paraquat for 40 min was observed when using 40 mM N-acetyl-L-cysteine and 5 mM glutathione. These concentrations are comparable with 50 units of catalase, which reduced ROS at concentrations of 5-100 units. Further pharmacokinetic study into the systemic administration of these antioxidants is necessary, using effective concentrations of 5-40 mM for both N-acetyl-L-cysteine and glutathione, and 1-50 units of catalase.
Journal of Korean Medical Science 11/2003; 18(5):649-54. DOI:10.3346/jkms.2003.18.5.649 · 1.27 Impact Factor