Escherichia coli ; chemistry ; cytology ; Fluorine Radioisotopes ; MAP Kinase Kinase 5 ; chemistry ; metabolism ; MAP Kinase Kinase Kinase 3 ; chemistry ; metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Interaction Mapping ; methods ; Protein Interaction Maps

OBJECTIVETo investigate the MEK and ERK expression and their relationship with clinicopathological parameters in human breast carcinoma, and the effect of preoperative chemotherapy on MEK and ERK protein expression.

METHODSSamples were obtained from 56 patients with breast carcinoma and 8 patients with benign tumors. Sixteen of the 56 patients received preoperative chemotherapy. Western blot and immunohistochemistry were used to measure the expression of MEK1, MEK2 and ERK1, ERK2 protein.

RESULTSMEK2 and ERK1, ERK2 protein levels were increased in breast carcinoma tissue compared with those in adjacent normal tissues (t = 7.244, 5.959, 3.735, P < 0.01) and benign tumors (t = 2.206, P < 0.05). The levels of MEK1 were decreased. The expression of MEK2 protein in ER negative patients was higher than that in ER positive ones. MEK2 protein levels were lower in patients who received preoperative chemotherapy than in those who did not.

CONCLUSIONOverexpression of MEK-ERK may play an important role in the development of human breast carcinoma. MEK and ERK protein expressions are inhibited by preoperative chemotherapy.

Adult ; Aged ; Blotting, Western ; Breast Neoplasms ; diagnosis ; enzymology ; metabolism ; Female ; Humans ; Immunohistochemistry ; MAP Kinase Kinase 1 ; MAP Kinase Kinase 2 ; MAP Kinase Signaling System ; physiology ; Middle Aged ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Prognosis ; Protein Kinases ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.
Mice ; Animals ; Alocasia/metabolism* ; MAP Kinase Signaling System ; Caspase 3/metabolism* ; Apoptosis ; RNA, Messenger/metabolism*

OBJECTIVETo study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.

METHODSChinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies.

RESULTSPower-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK.

CONCLUSIONPower-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.

Animals ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Activation ; radiation effects ; Lung ; enzymology ; radiation effects ; MAP Kinase Kinase 3 ; metabolism ; MAP Kinase Kinase 6 ; metabolism ; Magnetics ; p38 Mitogen-Activated Protein Kinases ; metabolism ; radiation effects

AIMTo assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 mitogen-activated protein kinases (MAPK) in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation.

METHODSImmunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism.

RESULTSThe abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 (CK18), a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2.

CONCLUSIONThe activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Animals ; Cryptorchidism ; enzymology ; pathology ; Disease Models, Animal ; Enzyme Activation ; Immunohistochemistry ; MAP Kinase Kinase 4 ; metabolism ; Macaca mulatta ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Scrotum ; enzymology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the characteristic of the signal transduction in BEAS cells induced by the crocidolite fibers.

METHODSThe human respiratory airway epithelial cells BEAS-2B were cultured in vitro. The final 100 microg/ml crocidolite concentration and lOnM of epidermal growth factor were cocultured with BEAS-2B cells for 30 minutes and 120 minutes. Phosphorylated ERKl/2 and MEKl/2 were detected by Western Blotting using specific antibodies.

RESULTSA rapid phosphorylation expression of ERK1/2 (molecular weight at 44 kD and 42 kD, also called as p44 and p42) was observed by treatment of the BEAS-2B cells with 100 microg/ml crocidolite or 100 ng/ml EGF (the proven activator of the ERK signaling pathway) at 30 minutes. This phosphorylation could be still detected by incubation the cells at 2 hours. However no expression was changed for the total ERKl/2 expression at 30 minutes or 120 minutes. Treatment of BEAS cells with 100 microg/ml crocidolite fiber or 100 ng/ml EGF led to the rapid increased phosphorylation of MEK1/2 at 30 minutes; similarly, the overexpression of MEK1/2 could last 2 hours.

CONCLUSIONThe crocidolite induces the MAPK (ERK1/2 and MEK1/2) phosphorylation within a shorter time. It indicates that the MAPKs signals are involved in the process of crocidolite induced damage.

Asbestos, Crocidolite ; toxicity ; Bronchi ; cytology ; Cells, Cultured ; Epidermal Growth Factor ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Humans ; MAP Kinase Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation

Both interleukin (IL)-12, an important cytokine skewing the immune response towards a Th1 cytokine profiles, and tumor necrosis factor (TNF)-alpha, are thought to be critical factors in defenses against mycobacteria. In this study, we evaluated the roles of phosphatidylinositol 3-kinase (PI 3-K), and extracellular signal-regulated kinase (ERK) 1/2 pathways in the expression of IL-12 in human monocyte-derived macrophages (MDMs) after stimulation with Mycobacterium tuberculosis H37Rv (M. tbc) or the Triton X-114 solublized proteins (TSP) of M. tbc. Both M. tbc and TSP rapidly phosphorylated ERK 1/2, and Akt in human MDMs. Inhibition of PI 3-K-Akt pathway by specific inhibitors (LY294002 and wortmannin) dramatically increased M. tbc- or TSP-induced IL-12 p40 and p35 mRNA and IL-12 production. In addition, blockade of ERK 1/2 pathway by specific inhibitors (PD98059 and U0126) significantly increased the mRNA levels and cytokine production in M. tbc- or TSP-treated MDMs. On the contrary, M. tbc- or TSP-induced TNF-a production was significantly depressed in human MDMs by pretreatment with inhibitors of PI 3-K or ERK pathways. The M. tbc or TSP stimulation decreased ERK 1/2 phosphorylation by 70% in the presence of wortmannin or LY294002, suggesting that some cross-talk between the PI 3-K-Akt and mitogen-activated protein kinase kinase (MEK)-ERK pathways may be operating in human monocytes during mycobacterial infection. PI 3-K activity is partially required for the M. tbc- or TSP-induced ERK 1/2 phosphorylation. Collectively, these data suggest that the PI 3-K and ERK 1/2 pathways play a central role in the negative regulation of IL-12, but not TNF-a, production by M. tbc.
Humans* ; Interleukin-12* ; Interleukins ; Macrophages* ; MAP Kinase Signaling System ; Monocytes ; Mycobacterium tuberculosis ; Neptune ; Phosphatidylinositol 3-Kinase* ; Phosphatidylinositols* ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; RNA, Messenger ; Tumor Necrosis Factor-alpha

OBJECTIVES: This study aimed to demonstrate the activities and phosphorylation changes induced by acute ethanol treatment and withdrawal conditions in the intracellular signal transduction molecules [such as extracellular signal-regulated kinase (ERK), glycogen synthase kinase 3beta (GSK3beta), and Akt] of the SH-SY5Y neuroblastoma cell line. METHODS: The acute treatment exposed SH-SY5Y cells to 100 mM ethanol, and we took samples 30 minutes, 60 minutes, and 24 hours after initiating this treatment. After 24 hours' continuous ethanol treatment, we initiated ethanol withdrawal, taking samples at 30 minutes and 60 minutes. We assayed the kinase phosphorylations via an immunoblot analysis using phosphorspecific antibodies, quantified by optical densitometry. RESULTS: Ethanol treatment induced a transient increase in phosphorylation of GSK3beta and Akt at 30 minutes but failed to change the phosphorylation level of ERK. Ethanol withdrawal induced a transient ERK phosphorylation increase at 30 minutes, but it had no effect on the phosphorylation of GSK3beta or Akt. CONCLUSION: The results indicate that the ethanol-induced cellular response includes the ERK, GSK3beta, and Akt systems. In particular, the ERK pathway may play a role in the acute withdrawal response. This also suggests that a relatively short exposure to ethanol, such as the 24-hour exposure in this study, can induce functional adaptation within a cell.
Antibodies ; Cell Line ; Densitometry ; Ethanol ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinases ; Humans ; MAP Kinase Signaling System ; Neuroblastoma ; Phosphorylation ; Phosphotransferases ; Signal Transduction

OBJECTIVETo investigate the effect of ERK1/2 inhibitor AZD8330 on human Burkitt's lymphoma cell line Raji cells and its mechanism.

METHODSRaji cells were treated with different concentrations of AZD8330. CCK-8 was used to detect the cell viability. The apoptosis rate of Raji cells was detected by flow cytometry using Annexin V/PI-staining. Real-time PCR was used to assess the expression of Bcl-2, Bcl-xl, caspase-3 and VEGF genes. The protein expression level of Bcl-2, Bcl-xl, caspase-3 and p-ERK1/2 was tested with Western blot.

RESULTSThe cell survival rate decreased to(62.09±0.86)%,(50.06±1.33)% and (39.13±2.34)% respectively after cells were treated with AZD8330 at 1.00 μmol/L in vitro for 24 h, 48 h and 72 h, and statistically significant differences were observed in groups with different time of treatment(P<0.05). Apoptosis of cells treated with AZD8330 at 0.10, 1.00, 10.00 μmol/L in vitro for 24 h, 48 h and 72 h was analyzed, and the statistically significant differences were observed in groups of different time and concentration treatment (P<0.05). AZD8330 induced Raji cell apoptosis and upregulated expression of Bcl-2, Bcl-xl, VEFG and decreased the expression of caspase-3 in a dose and time dependent manner, and statistically significant differences were observed in groups of different time and concentration treatment (P<0.05). At the same time, the Bcl-2, Bcl-xl and p-ERK1/2 proteins expression is suppressed obviously, but the expression of caspase-3 protein increased.

CONCLUSIONAZD8330 induces cell apoptosis by down-regulating the activation of ERK1/2 signal transduction pathway in Burkitt's lymphoma cell line Raji cells in a dose and time dependent manner.

Apoptosis ; Burkitt Lymphoma ; Caspase 3 ; Cell Line, Tumor ; Dihydropyridines ; Flow Cytometry ; Humans ; MAP Kinase Signaling System ; Protein Kinase Inhibitors ; Real-Time Polymerase Chain Reaction

OBJECTIVETo explore the effect of extracellular signal regulated kinase 1/2 (ERK1/2) on edaravone (EDA)-triggered protection against myocardial toxicity induced by isoprenaline (ISO) in H9c2 myocardial cells (H9c2 cells).

METHODSH9c2 cells were exposed to ISO at different concentrations to establish a cardiac toxicity model induced by persistent excitation of β1 receptor. EDA was added before ISO as a pretreatment. PD-98059, an ERK1/2 inhibitor, was administered 1 h prior to EDA to inhibit the phosphorylation of ERK1/2. Cell viability was measured using cell counter kit (CCK-8). The expressions of p-ERK1/2 and t-ERK1/2 were tested by Western blotting. Mitochondrial membrane potential (MMP) was detected by Rhodamine123 (Rh123) staining and photofluorography.

RESULTSExposure of H9c2 cells to 80 µmol/L ISO for 24 h down-regulated ERK1/2 phosphorylation and repressed MMP. Pretreatment with 10-40 µmol/L EDA for 1 h inhibited ISO-induced myocardial toxicity and pretreatment of 40 µmol/L EDA partially rescued ERK1/2 phosphorylation and MMP level. PD-98059 abolished cardiac protection of EDA, leading to myocardial toxicity and MMP loss.

CONCLUSIONEDA can protect H9c2 cells against myocardial injury induced by ISO by suppressing ISO-triggered inhibition of ERK1/2 activation.

Animals ; Antipyrine ; analogs & derivatives ; pharmacology ; Cell Line ; Flavonoids ; pharmacology ; Isoproterenol ; toxicity ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Phosphorylation ; Rats