Inhibition of proliferation of breast cancer cells by down-regulation of cyclin d1 and survivin protein expression by erk inhibitor u0126 / 中国药理学通报

AimTo investigate the inhibitory effect of ERK signaling pathway inhibitor UO126 on breast cancer cell proliferation and explore its specific regulatory mechanism. Methods Cultured human breast cancer cell MCF-7, MDA-MB231, the cell were divided into different groups and intervened. MTT was used to measure the cell proliferation; Flow cytometry was employed to test cell cycle and cell apoptosis; Western blot was applied to test p-ERK/ERK, cyclin D1, survivin and cleaved caspase-3 protein expression. Results The results of MTT showed that the inhibitory rate of MCF-7 and MDA-MB231 cells increased significantly after U0126 intervention for 24 h and 48 h(P < 0.01). Cell cycle and apoptosis were detected by MCF-7 and MDA-MB231 cells treated with U0126 for 24 h. The proportion of cells in G0/G1 phase was significantly higher than that in control group, and the proportion of cells in S and G2 phase decreased(P < 0.05). The apoptotic rate in intervention group was significantly higher than that in non-intervention group, and the difference was significant(P < 0.01); U0126 treatment of human breast cancer cells could block ERK phosphorylation, increase cyclin D1 protein expression, inhibit survivin and up-regulate cleaved caspase3 expression, which were significantly different from control group(P < 0.05). Conclusions U0126 blocks ERK signaling pathway in MCF-7, MDA-MB231, down-regulates the cyclin D1, and blocks cell cycle in G0/G1 phase, then it inhibits the expression of survivin and increases cleaved caspase-3, promotes cell apoptosis, and inhibits the proliferation of breast cancer cell MCF-7, MDA-MB231.

Role of Baicalein combined with U0126 resisting human bladder carcinoma cell in vitro and mechanism research / 中国免疫学杂志

Objective:To investigate the role of Baicalein combined with U0126 resisting human bladder cancer T-24 cells in vitro and mechanism.Methods: T-24 cells were dealt with Baicalein combined with U0126,flow cytometry was used to detect cell cycle and cell apoptosis,microscope to count cell number,TUNEL method to detects cell apoptosis index,and Real time quantitative PCR and Western blot to measure extracellular signal regulating kinase 1/2 (ERK1/2), CyclinD1, GSK-3β and AKT RNA level, protein level of T-24 cells respectively.Effect of Baicalein and U0126 on apoptosis and proliferation of bladder cancer cell was analyzed.Results: Cell apoptosis rate was significantly increased after T-24 cells dealt with various concentrations of Baicalein.Cell proportion of G0/G1 phase was significantly increased,while cell percentage of S phase was obviously decreased and cell count was decreased,after T-24 cells were dealt with Baicalein for 24 h.After T-24 cells were dealt with Baicalein combined with U0126 for 24 h,cell proportion of S phase was evidently decreased.T-24 cells were dealt with Baicalein or U0126 obviously promoted cell apoptosis,which was more obvious with Baicalein combined with U0126.Phosphorylation level of GSK-3β,ERK1/2,and AKT was significantly reduced and expression of ERK1/2 and CyclinD1 mRNA was evidently lower after Baicalein or U0126 or Baicalein combined with U0126,and combined application had more remarkable effect.Conclusion: Baicalein and U0126 can induce apoptosis of T-24 cells,increase cell proportion in G0/G1 phase,reduce cell proportion of S phase,and Baicalein combined with U0126 effect has more remarkable effect.

Anti-cancer by baicalein combined with U0126 on human breast cancer in vitro / 中国免疫学杂志

Objective:To investigate the effects and mechanisms of anti-cancer by bacailein combined with U0126 on human breast cancer in vitro. Methods: The human breast cancer cell line MCF-7 was treated by baicalein,U0126 and baicalein combined with U0126 respectively. CCK8 assay measured cell proliferation of MCF-7;flow cytometry tested the cell cycle and apoptosis of MCF-7;microscopy observed the amount;TUNEL assay evaluated the apoptosis of MCF-7;Western blot detected the protein level of proliferation and apoptosis related protein;scratch assay measured the ability of migration. Results: Human breast cancer cell line MCF-7 was treated by baicalein or U0126 at different concentration for 24 h, CCK8 assay suggested that both of them can dramatically inhibit MCF-7 proliferation in a dose-dependent way (P<0. 05). Compared to the blank and DMSO groups,the human breast cancer cell line MCF-7 was treated with baicalein for 24 h,the cellular rate at G0-G1 phase increased a lot (91%) (P<0. 05),while the cellular rate at S phase reduced dramatically (P<0. 05),cell apoptosis increased dramatically by microscopy and TUNEL assay(P<0. 05),the level of ERK1/2,CyclinD1 and JNK reduced quickly (P<0. 05). Compared to the baicalein group,MCF-7 was treated by baicalein combined with U0126,the cellular rate at S phase decreased remarkably (P<0. 05),apoptosis was much obvious (P<0. 05),the phosphorylation level of ERK1/2 and JNK reduced a lot (P<0. 05),and the proliferation accelerator CyclinD1 highly decreased (P<0. 05);the scratch assay demonstrated that cell migration was dramatically inhibited when MCF-7 was treated by 20 μmol/L baicalein ( P<0. 05 ) . Conclusion:Both of baicalein and U0126 can inhibit the proliferation and migration,induce the apoptosis of human breast cancer cell line MCF-7 through decreasing the level of ERK, JNK and CyclinD1. Baicalein and U0126 can provide some novel avenues to treat breast cancer in clinic.

The effect of inhibition of MEK/ERK pathway on the behaviors of autistic rats / 重庆医学

Objective To explore the effect of inhibitor of MEK/ERK pathway on the behaviors of autistic rats .Methods Autistic rats were made by intraperitoneal injection of sodium valproate (VPA) after pregnancy for 12 .5 days .After VPA injec-tion ,pregnant rats were treated with U0126 via oral at 400 μg/kg dose per day until weaning .Young rats were divided to 4 groups :control group ,VPA group ,U0126 group ,VPA combined U0126 group .The social interaction and behaviors of young rats were e-valuated at 35 days after bornning .The levels of MEK and phosphorylated ERK in brain tissues were investigated by Western blot . Results The autistic rat mode was prepared successfully .Compared with control rats ,the rats treated with VPA showed low the social interaction ,long moving time in central area and reducing standing times .Treatment with U0126 alone didn′t change the so-cial interaction and behaviors of young rats ,but VPA combined U0126 group could improve VPA-induced autistic-like behaviors . Western blot results show that compared with the control group ,the rats treated with VPA could enhance the prefrontal cortex of rats ,the hippocampus and cerebellum in the organization of MEK and ERK phosphorylation level ;while VPA combined U0126 group could inhibit the brain tissue of MEK and ERK phosphorylation level .Conclusion U0126 can improve the model rats of au-tism disorders behavior ,the mechanism may be related to the inhibition of MEK/ERK signaling pathway in the brain .

Modulation of Human Cardiac Progenitors via Hypoxia-ERK Circuit Improves their Functional Bioactivities

Recent accumulating studies have reported that hypoxic preconditioning during ex vivo expansion enhanced the self-renewal or differentiation of various stem cells and provide an important strategy for the adequate modulation of oxygen in culture conditions, which might increase the functional bioactivity of these cells for cardiac regeneration. In this study, we proposed a novel priming protocol to increase the functional bioactivity of cardiac progenitor cells (CPCs) for the treatment of cardiac regeneration. Firstly, patient-derived c-kit+ CPCs isolated from the atrium of human hearts by enzymatic digestion and secondly, pivotal target molecules identified their differentiation into specific cell lineages. We observed that hCPCs, in response to hypoxia, strongly activated ERK phosphorylation in ex vivo culture conditioning. Interestingly, pre-treatment with an ERK inhibitor, U0126, significantly enhanced cellular proliferation and tubular formation capacities of CPCs. Furthermore, we observed that hCPCs efficiently maintained the expression of the c-kit, a typical stem cell marker of CPCs, under both hypoxic conditioning and ERK inhibition. We also show that hCPCs, after preconditioning of both hypoxic and ERK inhibition, are capable of differentiating into smooth muscle cells (SMCs) and cardiomyocytes (CMs), but not endothelial cells (ECs), as demonstrated by the strong expression of alpha-SMA, Nkx2.5, and cTnT, respectively. From our results, we conclude that the functional bioactivity of patient-derived hCPCs and their ability to differentiate into SMCs and CMs can be efficiently increased under specifically defined culture conditions such as short-term hypoxic preconditioning and ERK inhibition.

Blockade of ERK Phosphorylation in the Nucleus Accumbens Inhibits the Expression of Cocaine-induced Behavioral Sensitization in Rats

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.

Influence of coxsackie-adenovirus receptor expression on infection efficiency of adenovirus vector in gene therapy of hepatocellular carcinoma / 中国肿瘤生物治疗杂志

Objective: To explore the relationship between coxsackie-adenovirus receptor(CAR)expression and the infection efficiency of adenovirus vector in gene therapy for hepatocellular carcinoma through regulating CAR expression on cells surface via inhibition of the Raf/MEK/ERK pathway.Methods: Western blotting analysis was used to examine CAR expression in hepatocellular carcinoma cells(SMMC-7721 and HepG_2)before and after treatment with U0126,inhibitor of Raf/MEK/ERK signal transduetion.SMMC-7721 and HepG_2 were infected by a non-replicating,E1 A-deleted adenovirus expressing EGFP(Ad-GFP).FACS was used to analyze the infection efficiency of Ad before and after U0126 treatment. Results: The expression of CAR on cell surface had an increasing tendency after treatment with U0126.FACS analysis showed significantly increased infectivity of cells treated with the MEK inhibitor U0126 compared with untreated cells: SMMC-7721,(71.65?6.21)%→(86.54?5.70)%,HepG_2,(77.53?4.62)%→(87.06?2.83)%,when infected with Ad-GFP at the same MOI(10 MOI).Conclusion: The inhibition of Raf/MEK/ERK pathway by U0126 may up-regulate the expression of CAR in some hepatocellular carcinoma cells,which subsequently enhances the susceptibility of adenovirus infection to target cells.

Effect of ERK pathway inhibitor U0126 on cell cycle of leukemia K562 cell lines / 第三军医大学学报

Objective To study the mechanism of extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 on cell cycle of K562 cell lines. Methods The mRNA and protein expressions of ERK, Cyclin D2, Cyclin E in and P27 K562 cell lines after 10 ?mol/L U0126 treatment for 12, 24 and 72 h were detected by RT-PCR and Western blot, respectively. The cell cycle was determined by flow cytometry. Results The mRNA and protein expressions of ERK, Cyclin D2 and Cyclin E in K562 cell lines after U0126 treatment were decreased, while those of P27 were increased. The percentage of cells in G0/G1 phase was increased and that in S phase was decreased. All data mentioned above showed significant difference between the K562 cell lines before and after U0126 treatment(P