Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study, we investigated the protective mechanism of quercetin (QUE) and its glycosides, rutin (RUT) and quercitrin (QUI), on reactive oxygen species (ROS)-dependent (H(2)O(2)) and -independent (chemical anoxia) cell death in rat glioma C6 cells. Induction of HO-1 protein expression was detected in QUE- but not RUT- or QUI-treated C6 cells, and this was prevented by cycloheximide and actinomycin D. Incubation of C6 cells with QUE, but not RUT or QUI, protected C6 cells from H(2)O(2)- and chemical anoxia-induced cytotoxicity according to the MTT and LDH release assays. Apoptotic characteristics including chromatin condensation, DNA ladders, and hypodiploid cells appeared in H(2)O(2)-and chemical anoxia-treated C6 cells, and those events were significantly suppressed by adding QUE (but not RUT or QUI). Increases in caspase 3, 8, and 9 enzyme activities with decreases in pro-PARP and pro-caspase 3 protein levels and an increase in cleaved D4-GDI protein were identified in H(2)O(2)-and chemical anoxia-treated C6 cells, and these were blocked by the addition of QUE, but not by RUT or QUI. Intracellular peroxide levels increased with H(2)O(2) and decreased with chemical anoxia, and the addition of QUE reduced the intracellular peroxide levels induced by H(2)O(2). Results of an anti-DPPH radical assay showed that QUE, RUT, and QUI dose-dependently inhibited the production of DPPH radicals in vitro; however, QUE (but not RUT or QUI) prevention of DNA damage induced by OH radicals was identified with a plasmid digestion assay. Increases in phosphorylated ERK and p53 protein expressions were detected in H(2)O(2)- but not chemical anoxia-treated C6 cells, and the addition of QUE significantly blocked H(2)O(2)-induced phosphorylated ERK and p53 protein expressions. Adding the HO-1 inhibitors, SnPP, CoPP, and ZnPP, reversed the protective effect of QUE against H(2)O(2)- and chemical anoxia-induced cell death according to the MTT assay and morphological observations. Additionally, QUE exhibited inhibitory effects on LPS/TPA-induced transformation in accordance with a decrease in MMP-9 enzyme activity and iNOS protein expression in C6 cells. Taken together, the results of this study suggest that QUE exhibits an inhibitory effect on both ROS-dependent and -independent cell death, and induction of HO-1 protein expression is involved.
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PMID:Quercetin inhibition of ROS-dependent and -independent apoptosis in rat glioma C6 cells. 1664 78

The aim of this study was to analyze cell viability and expression of apoptotic-related signaling proteins in MCF-7 breast cancer cells induced by combinations of ultrasound, the anticancer drug 5-fluorouracil (5-FU) and the ultrasound contrast agent Optison. MCF-7 cells were treated with 5-FU and sonicated at the frequency of 3.0 MHz and intensity of 3.0 W/cm2 for 1 min in the presence of Optison. The cells were analyzed for lactate dehydrogenase (LDH) release (a measure of cytotoxicity) and cell proliferation (by MTT assays). The LDH/MTT ratio was used for assessment of cell death. Expression of the apoptotic-related proteins, Bax and p27kip1, as well as phosphorylated forms of ERK and Akt proteins was assessed by Western blot analysis. We demonstrate that, immediately after treatment, cell death was most dependent on Optison; however, 24 h after treatment, cell death was more dependent on 5-FU. Ultrasound duty cycle increased cell death associated with either Optison or 5-FU. Furthermore, we show that treatment with 5-FU and ultrasound increased the levels of the Bax and p27kip1 proteins, but the addition of Optison appears to suppress apoptotic protein expression.
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PMID:Effect of 5-fluorouracil, Optison and ultrasound on MCF-7 cell viability. 1667 34

This study examined the effect of wild-type Smad3 gene on the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the complexes of pcDNA3. 0-Myc-Smad3 or pcDNA3. 0-Myc-Smad3deltaC and Lipofectamine reagent. Immunofluorescence staining was performed to evaluate the c-Myc signal in MSCs. The cell proliferation was detected by MTT method. To clarify the osteoblastic characteristics in stably transfected MSCs, alkaline phosphatase (ALP) mRNA and core binding factor alpha1 (Cbfa1) mRNA were investigated by RT-PCR, and ALP activity and mineralization were examined by p-nitrophenolphosphate method and alizarin red staining respectively. PD98059, a specific inhibitor of the ERK signaling pathway, was used to determine the role of ERK in Smad3-MSCs osteoblastic differentiation. c-Myc signal was detected in Smad3-MSCs and Smad3 deltaC-MSCs. The proliferation of Smad3-MSCs was slower than that of Smad3deltaC-MSCs or V-MSCs. The relative levels of ALP mRNA and Cbfal mRNA in Smad3-MSCs, as well as ALP activity and mineralization, were markedly higher than those in Smad3deltaC-MSCs or V-MSCs. Although ALP activity and mineralization were slightly lower in Smad3-MSCs treated with PD98059 than in those without PD98059 treatment, no significant difference was found between them (P > 0.05). It is concluded that the wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSCs and enhance the osteoblastic differentiation of uncommitted MSCs and the maturation of committed MSCs independent of the ERK signaling pathway.
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PMID:Wild-type Smad3 gene enhances the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. 1669 23

Obesity has been recognized as a risk factor for breast cancer. Adipocyte-derived leptin may play as a paracrine regulator on the growth of breast cancer cells. Expression of both leptin and its OB-Rb receptor was detected in human breast cancer ZR-75-1 cells and further induced by leptin, suggesting that both expression and message mediation of leptin were autoregulated by itself. With cell counting and MTT assay, we had observed leptin stimulated ZR-75-1 growth in dose- and time-dependent manners. To study what steps of cell cycle progression leptin may involve in, we analyzed cell-cycle profile with flow cytometric analysis, mRNA and protein expressions of four cell-cycle regulators with RT-PCR and Western blotting analysis. Under the treatment of leptin, the G1 arrest of cells was reduced accompanied with up-regulation of G1 phase-specific cyclin D1 and proto-oncogene c-Myc, but down-regulation of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and tumor suppressor p53. Furthermore, JAK2 inhibitor AG490, PI3K/Akt inhibitor Wortmannin, and MEK/ERK1/2 inhibitor PD98059 were efficiently prevented leptin-promoted cell growth. Effect of cooperation between leptin and estrogen on ZR-75-1 growth had been observed. Collectively, the results showed that the proliferative effect of leptin on ZR-75-1 was associated with the up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21(WAF1/CIP1) plausibly through a hypothesized JAK2-PI3K/Akt-MEK/ERK pathway. The leptin- and OB-Rb-expressing capability of ZR-75-1 created a possible autocrine control of leptin, in which signal could be effectively amplified by itself, on cell growth.
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PMID:Leptin-induced growth of human ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21WAF1/CIP1. 1675 79

Mixed lineage leukemia (MLL) rearrangements occur in 80% of infants and 5% of older children with acute lymphoblastic leukemia (ALL). These cases have a poor prognosis with current therapy. The FLT3 kinase is overexpressed and constitutively activated in MLL-rearranged ALL cells. The FLT3 inhibitor CEP-701 selectively kills these cells, but is unlikely to be curative if used as monotherapy. To identify potentially synergistic combination strategies, we studied CEP-701 and six standard chemotherapeutic agents in three sequences of exposure (S1: chemotherapy followed by CEP-701, S2: simultaneous exposure to both; and S3: CEP-701 followed by chemotherapy) using MLL-rearranged ALL cell lines and patient bone marrow samples. MTT cytotoxicity and annexin V binding apoptosis assays were used to assess antileukemic effects. Combination indices (CI) were calculated for each combination (CI<0.9 - synergistic; CI 0.9-1.1 - additive; CI>1.1 - antagonistic). A striking pattern of sequence-dependent synergy was observed: S1 was markedly synergistic (mean CI=0.59+/-0.10), S2 was additive (mean CI=0.99+/-0.09) and S3 was antagonistic (mean CI=1.23+/-0.10). The sequence dependence is attributable to the effect of CEP-701 on cell cycle kinetics, and is mediated specifically by FLT3 inhibition, as these effects are not seen in control cells without activated FLT3.
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PMID:Combinations of the FLT3 inhibitor CEP-701 and chemotherapy synergistically kill infant and childhood MLL-rearranged ALL cells in a sequence-dependent manner. 1676 Oct 17

In the present study, we examined the protective mechanism of baicalein (BE) and its glycoside, baicalin (BI), on hydrogen-peroxide (H(2)O(2))-induced cell death in rat glioma C6 cells. Results of the MTT assay, LDH release assay, and morphological observation showed that H(2)O(2) addition reduced the viability of C6 cells, and this was prevented by the addition of BE but not BI. Incubation of C6 cells with BE significantly decreased the intracellular peroxide level induced by H(2)O(2) according to flow cytometric analysis using DCHF-DA as a fluorescent substrate. Suppression of H(2)O(2)-induced apoptotic events including DNA ladders, hypodiploid cells, and activation of caspases 3, 8, and, 9 by BE but not BI was identified in C6 cells. The cytotoxicity and phosphorylation of ERK proteins induced by H(2)O(2) were blocked by the ERK inhibitor PD98059. Catalase addition prevented H(2)O(2)-induced ROS production, ERKs protein phosphorylation, and cell death, and BE dose-dependently inhibited H(2)O(2)-induced ERK protein phosphorylation in C6 cells. These data suggest that ROS-scavenging activity is involved in BE prevention of H(2)O(2)-induced cell death via blocking ERKs activation. Additionally, BE but not BI induced heat shock protein 32 (HSP32; HO-1) protein expression in both time- and dose-dependent manners, but not heme oxygenase 2 (HO-2), heat shock protein 70 (HSP70), or heat shock protein 90 (HSP90) protein expression. In the absence of H(2)O(2), BE induces ERKs protein phosphorylation, and HO-1 protein expression induced by BE was blocked by the addition of cycloheximide, actinomycin D, and the ERK inhibitor PD98059. The addition of the HO inhibitor ZnPP inhibited the protective effect of BE against H(2)O(2)-induced cytotoxicity in C6 cells according to the MTT assay and apoptotic morphology under microscopic observation, accompanied by blocking the ROS-scavenging activity of BE in C6 cells. However, BE treatment was unable to protect C6 cells from C2-ceramide-induced cell death. These data indicate that BE possesses abilities to inhibit ROS-mediated cytotoxic effects through modulation of ERKs activation and induction of HO-1 protein expression. The role of HO-1 in ROS-scavenging activity of BE is proposed.
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PMID:Baicalein inhibition of oxidative-stress-induced apoptosis via modulation of ERKs activation and induction of HO-1 gene expression in rat glioma cells C6. 1681 38

Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25-50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.
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PMID:Monocyte-mediated rotenone neurotoxicity towards human neuroblastoma SH-SY5Y: role of mitogen-activated protein kinases. 1681 71

Hepatocyte growth factor (HGF) is mesenchymal-derived growth factor acting through a transmembrane tyrosine kinase receptor, c-met. HGF has multiple effects on different cells. However, its function in dentinogenesis remains unclear. In this study, the expression of HGF in human dental pulp cells (DPCs) in vitro was studied by immunostaining and RT-PCR. The effect of HGF on DPCs proliferation was determined by MTT, while its effect on cell differentiation was analyzed using ALPase activity, and further confirmed with ALP and DSPP mRNA and protein expression. Immunostaining revealed that HGF was found mainly in the cytoplasm of DPCs. RT-PCR analysis showed that both HGF and c-met were expressed from the DPCs. Exogenous addition of HGF enhanced proliferation and differentiation of DPCs by up-regulating CREB, ELK-1, and PPAR-gamma. U0126, an ERK/MAPK inhibitor, inhibited the effects of HGF on DPCs. It was concluded that HGF stimulated both proliferation and differentiation of DPCs, at least partially through the ERK/MAPK pathway.
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PMID:HGF enhanced proliferation and differentiation of dental pulp cells. 1686 Oct 72

The cDNA sequence encoding pokeweed antiviral protein-II was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-II was subcloned into the expression vector pET-28a(+) and expressed in E. coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-II existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HIV-1 integrase to some extent (IC50 = 303 microg/mL, 220 microg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 s of 93 microg/mL and 102 microg/mL, respectively. The results suggested that pokeweed antiviral protein-II is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-II.
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PMID:[Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana]. 1689 93

SU11248 is an orally available type III and V receptor tyrosine kinase inhibitor. Clinical studies have shown the efficacy of SU11248 in individuals with gastrointestinal stromal tumors (GIST); however, the molecular mechanisms by which SU11248 inhibits the proliferation of these tumor cells remains to be fully elucidated. Taking advantage of GIST-T1 cells, which possess an activating mutation in exon 11 of the c-KIT gene, we examined the medicinal action of SU11248 in GIST cells. Clonogenic and MTT assays showed that SU11248 potently inhibited the proliferation of GIST-T1 cells with IC50 of approximately 1 nM and 40 nM, respectively. SU11248 (10 or 20 nM, 48 h) activated caspase-3 and induced apoptosis of GIST-T1 cells as measured by caspase assay, annexin V staining and cleavage of poly (ADP-ribose) polymerase. Western blot analyses found that SU11248 blocked autophosphorylation of c-KIT in association with inhibition of its downstream effectors, including Akt and extracellular signal-regulated kinase, but not signal transducers and activators of transcription. Interestingly, when phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling was blocked simultaneously by either LY294002 or rapamycin, growth inhibition mediated by SU11248 was potentiated. Taken together, this study supports clinical studies of SU11248 for individuals with GIST, and the combination of SU11248 and inhibitors of 3-kinase/Akt/mammalian target of rapamycin signaling represents a promising novel treatment strategy.
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PMID:Effect of SU11248 on gastrointestinal stromal tumor-T1 cells: enhancement of growth inhibition via inhibition of 3-kinase/Akt/mammalian target of rapamycin signaling. 1691 20


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