Seyeon Park

The inhibitory mechanism of curcumin and its derivative against β-catenin/Tcf signaling

We investigated the inhibitory mechanism of curcumin and its derivative (CHC007) against β‐catenin/T‐cell factor (Tcf) signaling in various cancer cell lines. Curcumin is known to inhibit β‐catenin/Tcf transcriptional activity in HCT116 cells but not in SW620 cells. To clarify the inhibitory effect of curcumin against β‐catenin/Tcf signaling, we tested several cancer cell lines. In addition, in order to verify the inhibitory mechanism, we performed reporter gene assay, Western blot, immunoprecipitation, and electrophoretic mobility shift assay. Since inhibitors downregulated the transcriptional activity of β‐catenin/Tcf in HEK293 cells transiently transfected with S33Y mutant β‐catenin gene, whose product is not induced to be degraded by adenomatous polyposis coli–Axin–glycogen synthase kinase 3β complex, we concluded that the inhibitory mechanism was related to β‐catenin itself or downstream components. Western blot analysis suggested that no change in the amount of cytosolic and membranous β‐catenin in a cell occurred; however, nuclear β‐catenin and Tcf‐4 proteins were markedly reduced by inhibitors and this lead to the diminished association of β‐catenin with Tcf‐4 and to the reduced binding to the consensus DNA. In the present study, we demonstrate that curcumin and its derivative are excellent inhibitors of β‐catenin/Tcf signaling in all tested cancer cell lines and the reduced β‐catenin/Tcf transcriptional activity is due to the decreased nuclear β‐catenin and Tcf‐4.

Inhibition of fos–jun–DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells

The effect of dihydroguaiaretic acid (DHGA), isolated from the aryls of Myristica fragrans, on the transcription factor (fos-jun dimer) action was investigated via in vitro assay. DHGA showed an inhibitory effect against the complex formation of the fos-jun dimer and the DNA consensus sequence with an IC50 value of 0.21 micromol. Nordihydroguaiaretic acid (NDGA) and curcumin also inhibited fos-jun dimer action showing IC50 values of 7.9 and 6.9 nmol, respectively. DHGA and NDGA suppressed leukemia, lung cancer and colon cancer in an in vitro bioassay. The in vitro experiment suggested that inhibition of fos-jun-DNA complex formation could be due to the direct interference of fos-jun dimer binding onto the DNA consensus sequence by NDGA and curcumin.

Arsenic trioxide represses constitutive activation of NF-kappaB and COX-2 expression in human acute myeloid leukemia, HL-60.

It has been proposed that eukaryotic nuclear factor nuclear factor kappa‐B (NF‐κB) and cyclooxygenase‐2 (COX‐2) are implicated in the pathogenesis of many human diseases including cancer. Arsenic has been widely used in medicine in Oriental countries. Recent studies have shown that arsenic trioxide (As2O3) could induce in vitro growth inhibition and apoptosis of malignant lymphocytes, and myeloma cells. However, the molecular mechanisms by which As2O3 initiates cellular signaling toward cell death are still unclear. In the present study, the effects of As2O3 on NF‐κB and COX‐2 expression in HL‐60 cells were investigated. As2O3 suppressed DNA‐binding activity of NF‐κB composed of p65/p50 heterodimer through preventing the degradation of IκB‐α and the nuclear translocation of p65 subsequently as well as interrupting the binding of NF‐κB with their consensus sequences. Inhibitory effect of As2O3 on NF‐κB DNA activity was dependent upon intracellular glutathione (GSH) and H2O2 level, but not superoxide anion. Futhermore, we found that As2O3 also downregulated the expression of COX‐2, which has NF‐κB binding site on its promoter through repressing the NF‐κB DNA‐binding activity.

L‐ascorbic acid represses constitutive activation of NF‐κB and COX‐2 expression in human acute myeloid leukemia, HL‐60

There is increasing evidence that l‐ascorbic acid (LAA) is selectively toxic to some types of cancer cells at pharmacological concentrations, functioning as a pro‐oxidant rather than as an anti‐oxidant. However, the molecular mechanisms by which LAA initiates cellular signaling leading to cell death are still unclear. In an effort to gain insight into these mechanisms, the effects of LAA on eukaryotic transcription nuclear factor NF‐κB and cyclooxygenase‐2 (COX‐2) expression were investigated. In the present study, LAA suppressed DNA binding activity of NF‐κB, composed of a p65/p50 heterodimer, through inhibition of degradation of inhibitory κB‐α (IκB‐α) and prevention of nuclear translocation of p65. The inhibitory effect of LAA on NF‐κB activity was dependent upon glutathione levels in HL‐60 cells, as well as generation of H2O2 but not superoxide anion. LAA also downregulated the expression of COX‐2, which has a NF‐κB binding site on its promoter, through repressing NF‐κB DNA binding activity. Moreover, cotreatment of 1 μM arsenic trioxide (As2O3) with various concentrations of LAA enhanced an LAA‐induced repression of NF‐κB activity and COX‐2 expression. In conclusion, our data suggest that LAA exerts its anti‐tumor activity through downregulation of NF‐κB activity and COX‐2 expression, and these inhibitory effects can be enhanced by co‐treatment with As2O3.

Inhibition of AP‐1 transcription activator induces myc‐dependent apoptosis in HL60 cells

Transcriptional activation of AP‐1 is intricately involved in cell proliferation and transformation. The natural product, nordihydroguaiaretic acid (NDGA) shows an inhibitory effect on the binding of jun/AP‐1 protein to the AP‐1 site in 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated HL60 cells. The NDGA inhibits the auto‐regulated de novo synthesis of c‐jun mRNA in TPA‐stimulated HL60 cells. Our data also determine that this compound induces proliferation inhibition and apoptosis in human leukemia HL60 cells. To obtain information on the functional role of the AP‐1 inhibition by NDGA in apoptosis signaling, the effects of pharmacological inhibition of AP‐1 binding on c‐myc, p53, and bax protein level were determined. Our results indicate that treatment of cells with NDGA enhances c‐myc, p53, and bax protein levels. To rule out the possibility that NDGA will induce apoptosis because of the effects on proteins other than AP‐1, we investigated the effect of another AP‐1 inhibitor, SP600125, which is specific to Jun‐N‐terminal kinase. SP600125 decreased not only the phosphorylation level of jun protein but also AP‐1/DNA binding activity. Also, apoptosis was observed to be induced by SP600125, concomitant with the increase in c‐myc, p53, and bax protein level. In addition, apoptosis induced by both AP‐1 inhibitors was accompanied by the activation of a downstream apoptotic cascade such as caspase 9, caspase 3, and poly[ADP‐ribose]polymerase (PARP). When the cells were treated with NDGA or SP600125 in the presence of antisense c‐myc oligonucleotides, apoptosis was not observed and an increase of c‐myc, p53, and bax proteins was not manifested. All these results show that the inhibition of the transcription factor AP‐1 action is related with either the drug‐induced apoptosis or the drug toxicity of the HL60 cells. The apoptosis induced by AP‐1 inhibition may be dependent on c‐myc protein levels suggesting that the c‐myc protein induces apoptosis at a low level of AP‐1 binding activity. Altogether, our findings suggest that the presence of the AP‐1 signal acts as a survival factor that determines the outcome of myc‐induced proliferation or apoptosis.

Unsaturated fatty acids bind Myc–Max transcription factor and inhibit Myc–Max–DNA complex formation

Oncoprotein Myc, hetero-dimerized with Max through a b/HLH/Zip region, is a transcription factor that governs important cellular processes such as cell cycle entry, proliferation and differentiation. We found that linoleic acid, isolated from Pollen Typhae, and other unsaturated fatty acids have strong inhibitory effects on the binding of Myc-Max heterodimer to an E-box DNA site (CA(C/T)GTG). The interaction of a fatty acid with a protein dimer, not with DNA, is assumed to block the entire Myc-Max-DNA complex formation. Unsaturated fatty acids also showed cytotoxicity against a SNU16 human stomach cancer cell line and conjugated linoleic acid suppressed mRNA expression of several myc-target genes; ornithine decarboxylase, p53, cdc25a in the SNU16 cells.

Momordin I, a compound of ampelopsis radix, inhibits AP-1 activation induced by phorbol ester

Recently, we reported that momordin I from Ampelopsis radix is an activator protein-1 (AP-1) function inhibitor. Mormordin I showed both inhibition of AP-1 transcriptional activity and cellular cytotoxicity. Moreover, its inhibitory action was also identified in the animal experiments. The present study attempted to determine the mechanism of the action of momordin I more clearly. In mouse fibroblast cells, momordin I suppressed the AP-1 activity induced by phorbol 12-myristate 13-acetate (PMA), as well as blocked the de novo synthesis of AP-1 protein. Momordin I also showed cell cytotoxic activities on various cell lines and blocked the uptake of [(3)H]thymidine in cell proliferation induced by PMA. From these results, the inhibitory action of momordin I on AP-1 was clearly identified. Through the competitive binding assays, it appears that the inhibitory site of momordin I might be in the Jun/Fos dimer, and not in the DNA. Momordin I also showed the inhibitory action on the Jun/Jun homodimer, as well as on the Jun/Fos heterodimer. It also inhibited the autoregulated de novo synthesis of AP-1 by the Jun/Jun homodimer. In addition, since the truncated AP-1 used in this study only has the bZip regions of c-Jun and c-Fos, it appears that the inhibitory action site of momordin I may be the basic region of c-Jun instead of on the same region of c-Fos.

Performance analysis of queueing strategies for multiple priority calls in multiservice personal communications services

Supporting multiple priority calls, we propose the queueing strategies which efficiently manage the queue to reduce the blocking probability with multiclass calls in multiservice personal communications services (MPCS). The two queueing schemes are proposed and are shown the analytic model with (n+1) class calls. Numerical results demonstrate that the two proposed schemes show the improved performance compared to the previous scheme with two types of traffic in terms of the blocking probability. The proposed schemes are flexibly adaptable to MPCS.

Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways.