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Objective:To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI).Methods:Forty-two rats were divided into sham operation (Sham) group ( n = 6), model group ( n = 12), dimethyl sulfoxide (DMSO) control group ( n = 12), and PD98059 group ( n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. Results:There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. Conclusions:ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.
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OBJECTIVE：To investiga te the effects of MEK/ERK pathway specific inhibitor PD 98059 combined with paclitaxel on the proliferation and apoptosis of human gastric signet ring cell carcinoma （SRCC）cells. METHODS ：Using human SRCC KATO Ⅲ cells as object ，CCK-8 assay was used to detect cell proliferation after treated with paclitaxel ，PD98059 and two drug combination for 48 h，and the proliferation rate was calculated. Flow cytometry ，Western blotting and Transwell assay were used to detect the cell proliferation ，the expression of apoptosis related protein （Cleaved-caspase-3）and cell migration after treated with paclitaxel，PD98059 and two drug combination for 48 or 24 h. RESULTS ：After treated with paclitaxel （1 μg/mL），PD98059（5， 20，40 μmol/L）and two drug combination （1 μg/mL+5，20，40 μmol/L），the proliferation rate of cells was increased significantly in administration groups ，and the combination groups were significantly higher than paclitaxel and PD 98059 alone groups （P＜ 0.05）. After treated with paclitaxel （1 μg/mL），PD98059（5，20，40 μmol/L）and two drug combination （1 μg/mL+40 μmol/L）， early and late apoptosis rate ，the protein expression of Cleaved-caspase- 3 were significantly increased in paclitaxel group and combination group ；combination group was significantly higher than paclitaxel and PD 98059 alone group （P＜0.05）. The number of migrated cells in administration groups were reduced significantly ，and the combination group was significantly lower than paclitaxel and PD 98059 alone group （P＜0.05）. CONCLUSIONS ：Paclitaxel and PD 98059 can inhibit the proliferation and migration of human SRCC KATO Ⅲ cells，paclitaxel can also promote the apoptosis and the expression of apoptosis related protein，which may be related to the inhibition of MEK/ERK pathway. The effect of the combination of the two drugs is better than paclitaxel or PD 98059 alone.
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Objective To investigate the protective mechanism of MEK1/2 inhibitor PD98059 on ox-LDL induced injury of human umbilical vein endothelial cells (HUVEC),and its influence on the expression of LOX-1.Methods HUVEC damage models were established by using ox-LDL and were treated with PD98059 later,divided into the negative control group,the ox-LDL group,the positive control group and the PD98059+ox-LDL group.The effect of inhibition of MEK1/2 on ox-LDL induced HUVEC damage was measured.Results Compared with the negative control group,the levels in the ox-LDL group of LOX-1,pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were increased significantly,the proliferations of HUVEC and the productions of NO were decreased (P＜0.05).Compared with the ox-LDL group,the levels in the positive control group and the PD98059+ox-LDL group of pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were decreased,the proliferation of HUVEC and the production of NO were increased (P＜0.05).Conclusion PD98059 inhibit the MEK1/2 signaling pathway to suppress the ox-LDL induced damage of HUVEC by decreasing the expression of LOX-1.
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Aims: Preliminary observations showed that 75 ìM PD98059 had a long-term effect on growth of maize seedlings. To verify and systemically analyze the effects of 75 ìM PD98059 on growth of maize seedlings, we designed and conducted this experiment. Methodology: We recorded and analyzed the effects of 75 ìM PD98059 on growth of maize seedling during the first fourteen days. The growth traits were observed. The length, fresh weight, and dry weight of shoots or roots, and the root/shoot ratio of fourteen-day old seedlings were measured. Cutting analysis was conducted to analyze the effects of PD98059 on shoot growth. Results: The shoot and root length of control showed about 1.38- and 1.5-fold longer than that of PD98059-treated seedlings, respectively. The shoot and root fresh weight of PD98059-treated seedlings declined to 80% and 79.4% of the control, respectively. The shoot and root dry weight of PD98059-treated seedlings declined to 68.3% and 69.8% of the control, respectively. PD98059 also decreased the length, fresh weight, and dry weight of cuttings. Conclusion: PD98059 had a negative effect on the growth of maize seedlings and this effect was overall on both shoots and roots. The effect of PD98059 on shoot growth seemed to be not due to detrimental effects of PD98059 on roots.
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Repeated administration of psychostimulants such as cocaine leads to the development of behavioral sensitization. Extracellular signal-Regulated Kinase (ERK), an enzyme important for long-term neuronal plasticity, has been implicated in such effects of these drugs. Although the nucleus accumbens (NAcc) is the site mediating the expression of behavioral sensitization by drugs of abuse, the precise role of ERK activation in this site has not been determined. In this study we demonstrate that blockade of ERK phosphorylation in the NAcc by a single bilateral microinjections of PD98059 (0.5 or 2.0micro g/side), or U0126 (0.1 or 1.0microg/side), into this site dose-dependently inhibited the expression of cocaine-induced behavioral sensitization when measured at day 7 following 6 consecutive daily cocaine injections (15 mg/kg, i.p.). Acute microinjection of either vehicle or PD98059 alone produced no different locomotor activity compared to saline control. Further, microinjection of PD98059 (2.0micro g/side) in the NAcc specifically lowered cocaine-induced increase of ERK phosphorylation levels in this site, while unaffecting p-38 protein levels. These results indicate that ERK activation in the NAcc is necessary for the expression of cocaine-induced behavioral sensitization, and further suggest that repeated cocaine evokes neuronal plasticity involving ERK pathway in this site leading to long-lasting behavioral changes.
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Animals , Rats , Butadienes , Cocaine , Flavonoids , MAP Kinase Signaling System , Microinjections , Motor Activity , Negotiating , Neuronal Plasticity , Nitriles , Nucleus Accumbens , Phosphorylation , Phosphotransferases , Illicit DrugsABSTRACT
Objective: To investigate the role of extracellular signal-regulated protein kinase (ERK) in estrogen-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7 and the related mechanisms. Methods: Estrogen receptor-positive breast cancer cell line MCF-7 was used in our study. The effects of 17β-E2 on the proliferation of MCF-7 cells was investigated by MTT assay to determine the optimal concentration of 17β-E2 for the following experiment. The effect of PD98059 on 17β-E2-induced proliferation of MCF-7 cells was measured by MTT assay to determine the intermediate concentration of PD98059. The cell cycle was analyzed by flow cytometry and telomerase activity was determined by Telomerase repeat amplification protocol PCR (TRAP-PCR) silver staining. The expression of wild-type p53 and phosphorylated ERK1/2 protein was determined by Western blotting and the expression of wild-type p53 mRNA was detected by RT-PCR. Results: ERK phosphorylation inhibitor PD98059 inhibited the proliferation of MCF-7 cells treated with 17β-E2 in a time- and dose-dependent manner(P<0.01). The intermediate concentrations of PD98059 were 89.28 μmol/L for 24 h, 39.81 μmol/L for 48 h and 21.87 μmol/L for 72 h. Treatment with 20 μmol/L PD98059 for 48 h reversed the promoting effect of 17β-estradiol on the cell cycle transformation of MCF-7, increasing the number of G1 phase cells and decreasing the number of S and M phase cells (P<0.01), inhibited the enhancing effect of 17β-E2 on the telomerase activity of MCF-7 cells(P<0.05), increased the protein expression level and genetic transcription of wild-type p53 (P<0.01), and decreased the expression of p-ERK1/2 protein(P <0.01). Conclusion: ERK plays an important role in 17β-E2-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7, which might be related to the changes of genetic transcription of wild type p53 and telomerase activity.
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Objective:To explore the mechanisms of ERK in affecting estrogen receptor signaling pathway by investigating the changes of the expression levels of nuclear receptor co-activatorPCAF protein and wild-type P53 protein and their gene transcription in the process of estrogen promoting transformation of cell cycle and resisting apoptosis of breast cancer cell MCF-7 after inhibitting ERK. Methods:Estrogen receptor-positive breast cancer cells MCF-7 were divided into 17?-estradiol treatment group,17 ?-E2 + ERK inhibitor PD98059 treatment group,and the control group. The apoptosis of cell and cycle were analyzed by flow cytometry. The expression of phosphorylated ERK1/2 and PCAF protein and wild-type p53 were detected by Western blot.The expression of pcaf mRNA and wild-type p53 mRNA were detected by RT-PCR. Results:With phosphorylation inhibitor of ERK PD98059,the effect of 17?-estradiol in resisting apoptosis and prmoting transformation of MCF-7 cell cycle was reversed,and the rate of early apoptosis of MCF-7 cell was raised(P0.05).The protein expression level and gene transcription of wild-type P53 were reinforced (P
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Summary: To determine the biological effects of extracelluar signal regulated kinase (ERK) specific inhibitor PD98059 on pancreatic stellate cells (PSCs) activated by platelet-derived factor-BB (PDGF-BB), cultured rat PSCs were co-incubated at 37 ℃ for 24 h with 25 ng/ml PDGF-BB and different doses of PD98059 (ranging from 5 ng/ml to 40 ng/ml). Expression of pERK1 protein was detected by Western blot and collagen α1 (Ⅰ) mRNA was tested by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that there were statistical differences in the expression of pERK1 protein in all groups. Expression of pERK1 protein was up-regulated in the group treated by PDGF-BB, and gradually down-regulated in the other groups treated by PD98059 of different doses. An excellent positive correlation was revealed between the inhibitory effect and PD98059 concentrations. It was also observed that the expression of collagen α1 (Ⅰ) mRNA had similar response to pERK1. The level of collagen α1 (Ⅰ) mRNA was the highest in the PDGF-BB group, and gradually reduced in the other groups treated by PD98059 of different doses. It is concluded that expression of pERK1 protein and collagen α1 (Ⅰ) mRNA was up-regulated in rat PSCs activated by PDGF-BB. Meanwhile, PD98059 could inhibit PSCs activation mediated by PDGF. It is suggested that ERK1 protein plays an important role on PSCs activation mediated by PDGF signal pathway.
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Objective To investigate the effects of MEK1 specific inhibitor PD98059 on oxaliplatin-treated colorectal cancer cells and the potential mechanism. Methods Cell proliferation was assessed by MTT assay after transfecting MEK1 active plasmid into LoVo cells. LoVo cells were treated with oxaliplatin or PD98059, and the proliferation was assessed by MTT assay. PUMA expression and ERK activity were determined by Western blot. Apoptosis was assessed by Hoechst 33258 dye after PUMA expression was suppressed. Results Increasing activity of ERK enhanced the proliferation of LoVo cells. The activity of ERK was suppressed by oxaliplatin. PD98059 and oxaliplatin decreased the proliferation rate of LoVo cells synergistically. PUMA expression increased after PD98059 and oxaliplatin treatment. The suppression of PUMA expression by stably transfecting PUMA anti-sense vector decreased apoptosis induced by oxaliplatin and PD98059. Conclusion PD98059 enhances the effects of oxaliplatin on colorectal cancer cells mediated by PUMA expression.
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Aim To study IL-8 expression of U937 cells directly induced by Pseudomonas aeruginosa(PA)and its mechanism through the mitogen-activated protein kinase pathways.Methods The expressions of IL-8 mRNA in human monocytic leukemia cell lines(U937 cells)infected by Pseudomonas aeruginosa which were in differently differentiated and its protein secretion in the supernatant of culture medium were examined by the methods of RT-PCR and ELISA respectively.And these were then to be compared with those inhibited by MAPK inhibitors.Results Pseudomonas aeruginosa was able to induce IL-8 mRNA expression and their protein secretion both in U937 cells and in PMA differentiated U937 cells and had marked time and concentration dependent relations.Pretreament of U937 cells with specific inhibitors of ERK1/2(PD98059),the kinase that activated ERK1/2,p38(SB203580)could significantly diminished the PA-induced IL-8 production(P
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PURPOSE: Extracellular signal-regulated kinase (ERK), which is part of the mitogen-activated protin kinase cascade, opposes initiation of the apoptotic cell death which is programmed by diverse cytotoxic stimuli. In this regard, the inhibition of ERK may be useful in improving the therapeutic efficacy of established anticancer agents. MATERIALS AND METHODS: Murine hepatocarcinoma, HCa-I is known to be highly radioresistant with a TCD50 (radiation dose yield in 50% cure) of more than 80 Gy. Various anticancer drugs have been found to enhance the radioresponse of this particular tumor but none were successful. The objective of this study was to explore whether the selective inhibition of MEK could potentiate the antitumor efficacy of radiation in vivo, particularly in the case of radioresistant tumor. C3H/HeJ mice bearing 7.5~8 mm HCa-I, were treated with PD98059 (intratumoral injection of 0.16 microgram in 50 microliter). RESULTS: Downregulation of ERK by PD98059 was most prominent 1h after the treatment. In the tumor growth delay assay, the drug was found to increase the effect of the tumor radioresponse with an enhancement factor (EF) of 1.6 and 1.87. Combined treatment of 25 Gy radiation with PD98059 significantly increased radiation induced apoptosis. The peak apoptotic index (number of apoptotic nuclei in 1000 nuclei X100) was 1.2% in the case of radiation treatment alone, 0.9% in the case of drug treatment alone and 4.9%, 5.3% in the combination treatment group. An analysis of apoptosis regulating molecules with Western blotting showed upregulation of p53, p21WAF1/CIP1 and Bcl-XS in the combination treatment group as compared to their levels in either the radiation alone or drug alone treatment groups. The level of other molecules such as Bcl-XL, Bax and Bcl-2 were changed to a lesser extent. CONCLUSION: The selective inhibition of MEK in combination with radiation therapy may have potential benefit in cancer treatment.
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Animals , Mice , Antineoplastic Agents , Apoptosis , Blotting, Western , Cell Death , Down-Regulation , Phosphotransferases , Radiation Dosage , Radiation, Ionizing , Up-RegulationABSTRACT
Objective:To investigate involvement of the Ras/Erk signal pathway in activation of human ??T cells stimulated by Mycobacterium tuberculosis low molecular peptide antigen(Mtb-Ag).Methods:PBMC were isolated from peripheral blood of healthy subjects and simulated in vitro with Mtb-Ag,CD3mAb or PMA and inomysin(IM).CD69 expression of total T cells and ??T cells were measured by flow cytometry at different hours after stimulation, and the number of expanded ??T cells were counted after 10 days of culture. PD98059,a specific inhibitor for Erk pathway was used to treat PBMC for 30 min before stimulation.Results:The proportions of CD69 expressing cells in total T cells and ??T cells were same as 99% at 6 h after stimulation of PBMC with PMA +IM and similar about 55% at 24 h after stimulation with CD3 mAb,whereas those at 24 h stimulation of PBMC with Mtb-Ag were 75.2% and 16.0%,respectively.CD69 expression and proliferation of ??T cells activated by Mtb-Ag were markedly inhibited by pretreatment of PBMC with PD98059.Conclusion:Mtb-Ag is a specific stimulator for activation of ??T cells, and Ras/Erk signal transduction pathway involves in the activation of ??T cells.
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AIM: The role of external-signal regulated kinaseⅠ/Ⅱ(ERKⅠ/Ⅱ) in the induction and maintenance of spinal long-term potentiation(LTP) is evaluated.METHODS: The C-fiber evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement.RESULTS:(1) PD98059(100 ?mol/L) or SL327(200(?mol/L)),a selective MEK inhibitor,completely blocked LTP induction when applied at 30 min prior to tetanic stimulation.(2) PD98059 or SL327 reversed spinal LTP in a time-dependent manner.At 15 min after LTP induction,PD98059(100 ?mol/L) or SL327(200 ?mol/L) reversed LTP completely,and at 30 min after LTP induction,the inhibitory rate of spinal LTP inhibited by PD98059 or SL327 was 62.5% and 75.0%, respectively.At 1 h after LTP induction,however,the same concentration of PD98059 or SL327 did not affect the spinal LTP.CONCLUSION: Activation of ERKⅠ/Ⅱ in spinal dorsal horn may be crucial for the induction and the early-phase maintenance of LTP of C-fiber evoked field potentials.
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The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.
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Humans , Androstadienes/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/physiology , Erythroid Precursor Cells/cytology , Erythropoiesis , Flavones/pharmacology , K562 Cells , Leukemia, Myeloid/pathology , Oligonucleotides, Antisense/pharmacology , Quinones/pharmacology , ras Proteins/metabolismABSTRACT
Aim To investigate the effect of insulin glargine and human insulin on proliferation of a human breast cancer cell line MDA-MB-231 and the role of ERK in the process.Methods MDA-MB-231 cells were incubated with insulin glargine and human insulin at different concentrations and for different time courses.A specific ERK1/2 inhibitor,PD98059,was used either alone or in combination with insulin glargine or human insulin to test the involvement of ERK pathway in cell growth.Cell proliferation was evaluated using cell counting kit-8 reagents.Cell cycle distribution was analyzed by flow cytometry.Results Both insulin glargine and human insulin dose-dependently enhanced MDA-MB-231 cell proliferation at the concentrations from 1 to 100 IU?L-1 after treatment for 96 h.At the concentration of 10 IU?L-1,both drugs promoted cell growth at 48,72,and 96 h.The percentage of S+G2/M cells was significantly increased in both insulin glargine and human insulin treated groups as compared to untreated controls.No significant difference was observed between insulin glargine and human insulin in their effects on cell proliferation and cell cycle distribution.Cell proliferation was significantly inhibited by PD98059.However,in the presence of PD98059,both drugs still promoted cell proliferation significantly as compared to untreated controls.Conclusions Insulin galrgine and human insulin similarly promote proliferation of MDA-MB-231 cells independent of ERK activation.
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Objective:To investigate the role of extracellular signal-regulated protein kinase(ERK) in estrogen-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7 and the related mechanisms.Methods: Estrogen receptor-positive breast cancer cell line MCF-7 was used in our study.The effects of 17?-E2 on the proliferation of MCF-7 cells was investigated by MTT assay to determine the optimal concentration of 17?-E2 for the following experiment.The effect of PD98059 on 17?-E2-induced proliferation of MCF-7 cells was measured by MTT assay to determine the intermediate concentration of PD98059.The cell cycle was analyzed by flow cytometry and telomerase activity was determined by Telomerase repeat amplification protocol PCR(TRAP-PCR) silver staining.The expression of wild-type p53 and phosphorylated ERK1/2 protein was determined by Western blotting and the expression of wild-type p53 mRNA was detected by RT-PCR.Results: ERK phosphorylation inhibitor PD98059 inhibited the proliferation of MCF-7 cells treated with 17?-E2 in a time-and dose-dependent manner(P