[show abstract][hide abstract] ABSTRACT: Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoosusceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAPtagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.
Journal of Microbiology and Biotechnology 01/2013; 23(1):22-8. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.
[show abstract][hide abstract] ABSTRACT: This study demonstrated that the surface crystallinity of carbon nanostructures is an additional independent factor that should be considered for the inhibition of cancer proliferation without activating reactive oxygen species (ROS). In addition, cytotoxic evaluation of both proliferating cancer cells and fully differentiated nerve cells (i.e. non-proliferative) showed selective cytotoxicity: single-walled and highly crystalline carbon nanostructures aggressively inhibited the proliferation of glioma cancer cells, but exhibited no notable cytotoxicity effects on differentiated nerve cells. Although single-wall carbon nanotubes have been shown to elicit potent proinflammatory responses by means of trigger ROS, our results demonstrated that highly crystalline carbon structures can be utilized as a selective antiproliferative agent against brain tumor cells without increasing the ROS level and without significant cytotoxic effects to adjacent nerve cells.
[show abstract][hide abstract] ABSTRACT: Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.
PLoS ONE 01/2012; 7(2):e29100. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.
[show abstract][hide abstract] ABSTRACT: Transcriptional factor nuclear factor-kappaB (NF-κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70-interacting protein (CHIP) is a U-box-type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF-κB pathway in the invasion of MDA-MB-231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA-MB-231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF-κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor-associated factor 2 (TRAF2), an upstream key player in the NF-κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG-132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF-7 cells, cell invasion significantly increased in conjunction with enhanced NF-κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF-κB-mediated cell invasion via the down-regulation of TRAF2.
Journal of Cellular Biochemistry 07/2011; 112(12):3612-20. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.
Journal of Cellular Biochemistry 05/2011; 112(9):2454-62. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of human MutY homolog (hMYH) on the activation of checkpoint proteins in response to hydroxyurea (HU) and ultraviolet (UV) treatment was investigated in hMYH-disrupted HEK293 cells. hMYH-disrupted cells decreased the phosphorylation of Chk1 upon HU or UV treatment and increased the phosphorylation of Cdk2 and the amount of Cdc25A, but not Cdc25C. In siMYH-transfected cells, the increased rate of phosphorylated Chk1 upon HU or UV treatment was lower than that in siGFP-transfected cells, meaning that hMYH was involved in the activation mechanism of Chk1 upon DNA damage. The phosphorylation of ataxia telangiectasia and Rad3- related protein (ATR) upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells. Co-immunoprecipitation experiments showed that hMYH was immunoprecipitated by anti-ATR. These results suggest that hMYH may interact with ATR and function as a mediator of Chk1 phosphorylation in response to DNA damage.
[show abstract][hide abstract] ABSTRACT: We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein-protein interactions in cells.
Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1-Glu164), Dronpa C-terminal: DC (Gly165-Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)×2] was introduced to enhance Dronpa complementation by hHus1-hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region.
Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1-LDN and DCL-hRad1 expression vectors, but not with hHus1-LDN or DCL-hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH-LDN and DCL-hHus1 expression vectors or hMYH-LDN and hHus1-LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH-LDN and DCL-hHus1, showed almost identical photoswitching activity as that of native Dronpa.
These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein-protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein-protein interaction.
Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging 04/2010; 12(5):468-78. · 2.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of 7-O-butyl naringenin (BN), a chemically synthesized derivative of naringenin, was tested on the proliferation of human breast
cancer MCF-7 cells. BN inhibited the proliferation of MCF-7 cells in dosedependent manner (IC50: 67.5±2.1 μM), resulting in an increase in the sub-G1 phase cell population. BN induced the generation of intracellular reactive
oxygen species (ROS), which were reduced by pretreatment with N-acetylcysteine (NAC). BN also increased the phosphorylation of stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/JNK1/2), c-Jun, and p38. However, the phosphorylation of extracellular-regulated kinase 1/2 (Erk1/2)
was decreased in BN-treated cells. Pretreatment of cells with the specific inhibitors SP600125 and SB203580 diminished the
BN-induced activation of SAPK/JNK1/2 and p38, respectively. These results indicate that the BN-induced cytotoxicity of MCF-7
cells is mediated by the generation of ROS as well as through the p38, SAPK/JNK1/2, and c-Jun activation signaling pathways.
BN may therefore possess chemotherapeutic potential as an anti-proliferative agent.
Food science and biotechnology 01/2010; 19(3):717-724. · 0.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quercetin is a low molecular weight flavonoid found in dietary fruits and vegetables. Quercetin, like other flavonoids, has demonstrated neuroprotective effects in vitro and in vivo. However, relatively little is known about how quercetin achieves its neuroprotective abilities. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is one of several excitatory receptors, which play an important role in postsynaptic neurotransmission. Over-stimulation of ionotropic glutamate receptor including AMPA receptors is closely associated with excitatory neurotoxicities. In the present study, we investigated the effects of quercetin on the glutamate-induced inward current (IGlu) in Xenopus oocytes that heterologously express human AMPA receptor and stargazin, an auxiliary subunit of AMPA receptor. IGlu was measured using the two-electrode voltage clamp technique. In oocytes injected with cRNAs coding AMPA receptor (GluR1) and stargazin, quercetin inhibited IGlu in a reversible and concentration-dependent manner. The IC50 was 84.9+/-15.0 microM. Quercetin action on IGlu was attenuated by increasing glutamate concentration, and was membrane holding potential-dependent. These results show a possibility that quercetin interacts with AMPA receptor, which was heterologously expressed in Xenopus oocytes and that quercetin action on IGlu of AMPA receptor could be one of contributions of quercetin-mediated neuroprotections.
[show abstract][hide abstract] ABSTRACT: Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.
DNA repair 08/2009; 8(10):1190-200. · 4.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human ribosomal protein S3 (hRpS3) is a small ribosomal subunit showing apurinic/apyrimidinic (AP) lyase activity and has been suggested to play a role in the cellular DNA-damage response pathway. However, the functional interactions between hRpS3 and other base excision repair (BER) DNA glycosylases have not been reported. We identified, for the first time, the interaction between hRpS3 and human uracil-DNA glycosylase (hUNG) and investigated the functional consequences of this interaction. hRpS3 was shown to interact with hUNG in co-immunoprecipitation assay using transiently transfected HEK293 cells and GST pull-down assay using microbial expression systems. In an assay using a 5'-end-radiolabeled 39-mer oligonucleotide duplex containing a U/G mismatch, hRpS3 dramatically stimulated the uracil-excision activity of hUNG, whereas hRpS3 alone had no cleavage activity. Pre-incubation of hRpS3 with the U/G mismatch containing DNA duplex also increased the hUNG uracil-excision activity; however, hRpS3 did not increase the DNA binding activity of hUNG in a trapping assay of hUNG and the U/G mismatch containing DNA duplex using UV cross-linking. hRpS3 has been suggested to stimulate the uracil-excision activity of hUNG by enhancing its dissociation from AP sites and increasing its turn-over rate. The disruption of hRpS3 by small-interfering RNA (siRNA-hRpS3) transfection reduced the uracil-excision activity preserved in cell extracts, whereas the supplement of purified hRpS3 retained uracil-excision activity. These results strongly suggest that hRpS3 may be involved in the uracil-excision pathway, probably by participating in the DNA repair mechanism to remove uracil generated by the deamination of cytosine in DNA, and by preventing C/G-->T/A transition mutations.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2008; 648(1-2):54-64. · 3.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to study the currently unknown cellular signaling pathways of Ca(v)3.1 T-type Ca(2+) channels (Ca(v)3.1 channels), we performed a yeast two-hybrid screening using intracellular domains of Ca(v)3.1 alpha1 subunit as bait. After screening the human brain cDNA library, several proteins, including RanBPM, were identified as interacting with Ca(v)3.1 channels. RanBPM was found to bind to the cytoplasmic intracellular loop between transmembrane domains I and II of Ca(v)3.1 channels. Using whole-cell patch-clamp techniques, we found that Ca(v)3.1 currents were increased by the expression of RanBPM in HEK293/Ca(v)3.1 cells. We next examined whether RanBPM affected the biophysical properties and plasma membrane expression of Ca(v)3.1 channels. Furthermore, we showed that the PKC activator inhibited Ca(v)3.1 currents, an effect that was abolished by the expression of RanBPM. These results suggest that RanBPM could be a key regulator of Ca(v)3.1 channel-mediated signaling pathways.
Biochemical and Biophysical Research Communications 10/2008; 378(1):15-20. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quercetin, one of the flavonoids, is a compound of low molecular weight found in various plants and shows a wide range of diverse neuropharmacological actions. In fruits and vegetables, quercetin exists as monomer- (quercetin-3-O-rhamnoside) (Rham1), dimer- (Rutin), or trimer-glycosides [quercetin-3-(2G-rhamnosylrutinoside)] (Rham2) at carbon-3. In the previous studies, we demonstrated that quercetin inhibits both glycine and 5-hydroxytryptamine type 3, (5-HT3A) receptor channel activities expressed in Xenopus oocytes. However, the effects of quercetin glycosides on glycine and 5-HT3A receptor channel activities are not well known. In the present study, we investigated the effects of quercetin glycosides on the human glycine alpha1 receptor and mouse 5-HT3A receptor channel activities expressed in Xenopus oocytes using a two-electrode voltage clamp technique. In oocytes expressing glycine or 5-HT3A receptors, quercetin- or its glycosides-induced inhibitions on glycine- (IGly) and 5-HT-induced current (I5-HT) were dose-dependent and reversible. Applications of quercetin and its glycosides inhibited IGly in order of quercetin>Rutin> or =Rham1>Rham2. Applications of quercetin and its glycosides inhibited I5-HT in order of Rham2> or =quercetin>Rutin=Rham1. The inhibitions of IGly by quercetin glycosides were non-competitive and voltage-sensitive, whereas the inhibitions of I5-HT by quercetin glycosides were competitive and voltage-insensitive manners. These results also indicate that quercetin glycosides might regulate the human glycine alpha1 and mouse 5-HT3A receptors with differential manners.
[show abstract][hide abstract] ABSTRACT: Although flavonoids, which are both qualitatively and quantitatively one of the largest groups of natural products, exhibit a variety of beneficial health effects, the exact molecular mechanism of the cellular activities is still not fully explained and there currently exists a lack of evidence for any relationship between the structure-activity relationship and apoptosis-inducing activity. In order to determine the importance of the OH group or substitution of the 5 or carbon-7 in the diphenylpropane skeleton of flavonoids, we originally synthesized several modified naringenin derivatives, including 7-O-benzyl naringenin (KUF-1) and 7-O-(MeO-L-Leu-D-Pro-carbonylmethyl) naringenin (KUF-7). Treatment with KUF-1 or KUF-7 resulted in significant apoptosis-inducing effects concomitant with chromatin condensation, caspase activation, and intracellular ROS production. Our data indicate that originally synthesized naringenin derivatives, KUF-1 and KUF-7 differentially regulate the apoptosis of A549 cells via intracellular ROS production coupled with the concomitant activation of the caspase cascade signaling pathway, thereby implying that hydroxylation or substitution at Carbon-7 is critical for the apoptosis-inducing activity of flavonoids.
[show abstract][hide abstract] ABSTRACT: We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.
Journal of Microbiology and Biotechnology 11/2007; 17(10):1607-15. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Quercetin, one of the flavonoids, is a compound of low molecular weight found in fruits and vegetables. Besides its antioxidative effect, quercetin also shows a wide range of diverse neuropharmacological actions. However, the cellular mechanisms of quercetin's actions, especially on ligand-gated ion channels and synaptic transmissions, are not well studied. We investigated the effect of quercetin on the human glycine alpha1 receptor channel expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Application of quercetin reversibly inhibited glycine-induced current (I(Gly)). Quercetin's inhibition depends on its dose, with an IC(50) of 21.5+/-.2 microM. The inhibition was sensitive to membrane voltages. Site-directed mutations of S267 to S267Y but not S267A, S267F, S267G, S267K, S267L and S267T at transmembrane domain 2 (TM2) nearly abolished quercetin-induced inhibition of I(Gly). In contrast, in site-directed mutant receptors such as S267 to S267I, S267R and S267V, quercetin enhanced I(Gly) compared to the wild-type receptor. The EC(50) was 22.6+/-1.4, 25.5+/-4.2, and 14.5+/-3.1 microM for S267I, S267R and S267V, respectively. These results indicate that quercetin might regulate the human glycine alpha(1) receptor via interaction with amino acid residue alpha267 and that alpha267 plays a key role in determining the regulatory consequences of the human glycine alpha1 receptor by quercetin.
Brain Research 09/2007; 1161:1-10. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.
Molecules and Cells 09/2007; 24(1):95-104. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The molecular mechanisms of genistein-induced apoptosis of human breast cancer MCF-7 cells were investigated. Genistein showed 50% cell growth inhibition at IC50=27.5+/-0.8 micromol/l in 24 h incubation under 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay conditions. Genistein is known to express both cell growth activity at nanomolar concentrations and anti-cell growth activity at micromolar concentrations. It was found that genistein at 100 micromol/l concentration effectively induced apoptosis of MCF-7 cells in 24 h. Genistein-induced apoptosis involved activation of calpain, caspase 7 and poly(ADP ribose) polymerase. Dantrolene, an inhibitor of Ca release from the endoplasmic reticulum, inhibited genistein-induced activation of calpain and caspase 7, in addition to effectively negating genistein-induced apoptosis. MCF-7 cells treated with genistein also showed increased phosphorylation of p38 mitogen-activated protein kinase, whereas no effect was observed for extracellular signal-regulating kinase 1/2. Phosphorylation of apoptosis signaling kinase 1, an upstream regulator of p38 mitogen-activated protein kinase, was also increased by genistein treatment. Genistein-induced phosphorylation of apoptosis signaling kinase 1 and p38 mitogen-activated protein kinase was diminished by the presence of dantrolene. These results suggest that genistein-induced apoptosis in MCF-7 cells is mediated through calpain-caspase 7 and apoptosis signaling kinase 1-p38 mitogen-activated protein kinase activation cascades that involve Ca release from the endoplasmic reticulum.