OBJECTIVE: To explore the molecular mechanism of fibroblast growth factor 8b (FGF8b) in promoting epithelial-mesenchymal transition in prostate cancer DU145 cells. METHODS: Cells were selected in three groups as follows: a block control group (DU145 cells), a negative control group [DU145 cells transfected with empty plasmid (pcDNA3.1/DU145)], and an experimental group [DU145 cells transfected with FGF8b (FGF8b/DU145)]. The activity of extracellular regulated protein kinases1/2( ERK1/2) pathway was detected by western-blot in the three groups. The FGF8b-DU145 cells and DU145 cells were cultured with PD98059 (an ERK kinase inhibitor) to observe microscopically the morphology changes within the cells. The experimental samples were also divided into four groups: FGF8b/DU145 cells cultured with 2% FBS (Group A); FGF8b/DU145 cells cultured with 2% FBS+PD98059 (50 μmol/L) (Group B); DU145 cells cultured with 2% FBS (Group C); DU145 cells cultured with FBS+PD98059 (50 μmol/L) (Group D). The expression of epithelial- mesenchymal transition (EMT) markers (E-cadherin, vimentin) were detected by western-blot analysis and the cell's mobility were detected by the Transwell chamber. RESULTS: The activity of ERK1/2 in the experimental group was significantly higher than that in the other two control groups; when ERK kinase inhibitor PD98059 was added to FGF8b/ DU145 cells, the expression of epithelial marker E-cadherin protein was significantly increased in group B compared with that in the group A (P<0.05). The expression of mesenchymal marker vimentin protein was significantly reduced in group B compared with that in group A (P<0.05). The cell migration assay suggested that cell migration was markedly decreased in group B (P<0.05) compared with that in group A. CONCLUSION EMT in prostate cancer induced by FGF8b can be mediated by ERK kinase pathway, in which mitogen-activated/extraceluer signal regulated kinase 1 (MEK1) may be a key factor. MEK1 could be an effective target in regulating the invasion and migration of prostate cancer.
Epithelial-Mesenchymal Transition ; genetics ; Fibroblast Growth Factor 8 ; genetics ; metabolism ; Flavonoids ; pharmacology ; Humans ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; physiology ; Male ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured

BACKGROUNDIncreased risk of bladder cancer has been reported in diabetic patients. This study was to investigate the roles of mitogen-activated protein kinase kinase (MEK) 1 and 2 in the regulation of human insulin- and insulin glargine-induced proliferation of human bladder cancer T24 cells.

METHODSIn the absence or presence of a selective inhibitor for MEK1 (PD98059) or a specific siRNA for MEK2 (siMEK2), with or without addition of insulin or glargine, T24 cell proliferation was evaluated by cell counting kit (CCK)-8 assay. Protein expression of MEK2, phosphorylation of ERK1/2 and Akt was analyzed by Western blotting.

RESULTST24 cell proliferation was promoted by PD98059 at 5 - 20 µmol/L, inhibited by siMEK2 at 25 - 100 nmol/L. PD98059 and siMEK2 remarkably reduced phosphorylated ERK1/2. Insulin- and glargine-induced T24 cell proliferation was enhanced by PD98059, suppressed while not blocked by siMEK2. Insulin- and glargine-induced ERK1/2 activation was blocked by PD98059 or siMEK2 treatment, whereas activation of Akt was not affected.

CONCLUSIONMEK1 inhibits while MEK2 contributes to normal and human insulin- and insulin glargine-induced human bladder cancer T24 cell proliferation.

Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flavonoids ; pharmacology ; Humans ; Insulin ; pharmacology ; Insulin Glargine ; Insulin, Long-Acting ; pharmacology ; MAP Kinase Kinase 1 ; antagonists & inhibitors ; metabolism ; MAP Kinase Kinase 2 ; genetics ; metabolism ; MAP Kinase Signaling System ; drug effects ; genetics ; Phosphorylation ; drug effects ; RNA, Small Interfering ; genetics ; physiology ; Urinary Bladder Neoplasms ; metabolism

Calcitonin (CT), a polypeptide hormone, plays important roles in a variety of physiological processes. CT has been used clinically to treat osteoporosis and humoral hypercalcemia of malignancy. In order to clarify the pharmacological effects of CT in the kidney, we identified potential downstream genes induced by CT in the renal cells. Using a cDNA subtraction hybridization method, we identified connective tissue growth factor (CTGF) as a CT-induced gene in the porcine renal cell line, LLC-PK1. Furthermore, we found that CT-mediated induction of the gene was not inhibited by cycloheximide, which suggests that CTGF gene was not induced by an increased synthesis of regulating proteins. Therefore, CTGF is an immediate early gene. We further demonstrated that the regulation of CTGF gene expression by CT involved the ERK1/2 pathway, because PD98059, a MEK1 inhibitor, partially inhibited the mRNA expression of CTGF induced by CT. CT-induced CTGF protein expression was also observed in vivo. Our present findings suggest that CT induces the transcription of CTGF through ERK1/2 phosphorylation. We also identified twelve other genes induced by CT that, like CTGF, were related to wound healing. These results suggest that CT may have an effect on renal differentiation and wound healing in the kidney.
Animals ; Calcitonin/*pharmacology ; Cell Line ; Connective Tissue Growth Factor/*genetics/metabolism ; Female ; Kidney Tubules, Proximal/*enzymology/metabolism ; *MAP Kinase Signaling System ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinase 1/*metabolism ; Mitogen-Activated Protein Kinase 3/*metabolism ; Phosphorylation ; Swine

Calcitonin (CT), a polypeptide hormone, plays important roles in a variety of physiological processes. CT has been used clinically to treat osteoporosis and humoral hypercalcemia of malignancy. In order to clarify the pharmacological effects of CT in the kidney, we identified potential downstream genes induced by CT in the renal cells. Using a cDNA subtraction hybridization method, we identified connective tissue growth factor (CTGF) as a CT-induced gene in the porcine renal cell line, LLC-PK1. Furthermore, we found that CT-mediated induction of the gene was not inhibited by cycloheximide, which suggests that CTGF gene was not induced by an increased synthesis of regulating proteins. Therefore, CTGF is an immediate early gene. We further demonstrated that the regulation of CTGF gene expression by CT involved the ERK1/2 pathway, because PD98059, a MEK1 inhibitor, partially inhibited the mRNA expression of CTGF induced by CT. CT-induced CTGF protein expression was also observed in vivo. Our present findings suggest that CT induces the transcription of CTGF through ERK1/2 phosphorylation. We also identified twelve other genes induced by CT that, like CTGF, were related to wound healing. These results suggest that CT may have an effect on renal differentiation and wound healing in the kidney.
Animals ; Calcitonin/*pharmacology ; Cell Line ; Connective Tissue Growth Factor/*genetics/metabolism ; Female ; Kidney Tubules, Proximal/*enzymology/metabolism ; *MAP Kinase Signaling System ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinase 1/*metabolism ; Mitogen-Activated Protein Kinase 3/*metabolism ; Phosphorylation ; Swine

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser
A549 Cells ; Animals ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Focal Adhesion Kinase 1/metabolism* ; Humans ; Lung Neoplasms/pathology* ; MAP Kinase Signaling System ; Mice ; Mitogen-Activated Protein Kinase 7/metabolism* ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins/metabolism*

The object was to study the effect of ouabain on Jurkat cells and its possible mechanism. The effect of ouabain of low concentration on Jurkat cells was confirmed by MTT, while c-myc gene transcription was measured by RT-PCR, and the phosphorylation of MAPK (ERK1/2) as well as the expression of c-myc gene was tested by Western blot respectively. The results showed that ouabain at low concentration could induce the proliferation of Jurkat in a time-and dose-dependent manner. At the same time, the phosphorylation of MAPK (ERK1/2) and the expression of c-myc gene was enhanced. In conclusion, ouabain stimulates the intracellular MAPK signal pathway by acting on the Na, K-ATPase, and thus induce the proliferation of Jurkat cells, in which the regulation of c-myc gene expression may be involved.
Blotting, Western ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Jurkat Cells ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; physiology ; Ouabain ; pharmacology ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. Aeruginosa.
Animals ; Caenorhabditis elegans ; enzymology ; genetics ; immunology ; microbiology ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Disease Resistance ; Enzyme Activation ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; Membrane Proteins ; deficiency ; genetics ; metabolism ; Mutation ; Pseudomonas Infections ; enzymology ; Pseudomonas aeruginosa ; physiology ; RNA Interference ; p38 Mitogen-Activated Protein Kinases ; metabolism

This study was aimed to investigate the effect of intercellular adhesion molecule-1 (ICAM-1) on the migration in vitro of the murine mesenchymal stem cells (MSC) and its related mechanisms. The migration ability of murine MSC (C3H10T1/2), ICAM-1 transfected MSC (C3H10T1/2-MIGR1-ICAM-1) and empty vector-transfected MSC (C3H10T 1/2-MIGR1) were assayed in vitro by using the transwell system. Briefly, the cells were seeded on the membrane with 8 µm aperture and the fetal bovine serum was used as the chemotactic agent to induce MSC migration. The transmigrated cells were stained by crystal purple as well as DAPI for 8 h and 12 h respectively. The absolute cell numbers were counted and the migration rates of MSC were evaluated in each group. To explore the potential mechanisms which control the migration of MSC, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the transwell system and the alteration of the MSC migration ability were evaluated at 12 h. The results showed that the migration ability at 8 h and 12 h of the ICAM-1-transfected MSC increased. Both absolute cell number and migration rate of MSC were significantly up-regulated by ICAM-1. Furthermore, the promoting effect of ICAM-1 on migration was partially suppressed by the inhibition of JNK/SAPK pathway. The transmigrated cell numbers and the migration rate decreased with the addition of JNK inhibitor II. However, the ICAM-1 promoting migration of MSC was not suppressed by the inhibitors for ERK/MAPK and p38/MAPK pathway did not work in the present study. It is concluded that ICAM-1 can induce mouse MSC migration in vitro, and the promoting effect is partially dependent on the activation of JNK/SAPK pathway.
Animals ; Cell Line ; Cell Movement ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; MAP Kinase Signaling System ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Transfection

OBJECTIVE: To determine the association between activity of BRAF and mitogen-activated protein/ extracellular signal-regulated kinase kinase (MEK) / extracellular signal-regulated kinase (ERK) signal pathway in papillary thyroid cancer and its mechanism. METHODS: We collected the clinical data and blood samples from 73 cases of papillary thyroid cancer and another 16 cases of benign thyroid gland tumor, and detected the expression of rat sarcoma (RAS), BRAF, MEK1/2, and ERK1/2 in all tumor specimens and benign thyroid tissues with immunohistochemistry and Western blot. RESULTS: The expression of RAS, BRAF, pMEK1/2, and pERK1/2 protein in papillary thyroid cancer tissues was higher than those in the benign thyroid tissues(P<0.05 or P<0.01). The expression of RAS, BRAF, MEK1/2, and ERK1/2 was associated with the tumor size, the lymph node metastasis, and the clinical stage of papillary thyroid cancer(P<0.05 or P<0.01). CONCLUSION The expression of RAS, BRAF, pMEK1/2, and pERK1/2 is associated with the pathogenesis, the lymph node metastasis, and the clinical stage of papillary thyroid cancer. The MEK/ERK signaling pathway may be activated by BRAF in papillary thyroid cancer.
Adult ; Aged ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; MAP Kinase Signaling System ; Male ; Middle Aged ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Proto-Oncogene Proteins B-raf ; genetics ; metabolism ; Thyroid Neoplasms ; genetics ; metabolism ; pathology ; Young Adult ; ras Proteins ; genetics ; metabolism

The purpose of this study was to investigate the effects of Celecoxib on the proliferation of the FLT3-ITD positive and negative acute myeloid leukemia cells and its mechanism. The proliferation inhibition effect of Celecoxib with different doses on the FLT3-ITD positive cells MV4-11 and the FLT3-ITD negative K562 cells was detected by CCK-8 method, the cell apoptosis was determined by flow cytometry, and the MEK, Mcl-1, pAKT expression was tested by Western blot. The results showed that Celecoxib inhibited the proliferation of both MV4-11 and K562 cells, but the IC50 for MV4-11 was (29.14 ± 2.4) µmol/L, which was significantly lower than that of K562 cells (39.84 ± 1.0) µmol/L (P < 0.05); The induced apoptosis rate of Celecoxib at 20-80 µmol/L on MV4-11 was not observed, but there was apparent influence on K562 at the same concentration. Western blot showed that Celecoxib down-regulated the expression of MEK and Mcl-1 but did not change the expression of pAKT obviously on MV4-11 cells, while the expression of Mcl-1 was reduced a little, but no obvious change were found in the expression of MEK and pAKT on K562 cells. It is concluded that the Celecoxib can inhibit the proliferation of FLT3-ITD positive AML cells distinctly, and the potential mechanism may be related to the inhibition of the MEK/Mcl-1 signaling pathway.
Apoptosis ; drug effects ; Celecoxib ; Cell Proliferation ; drug effects ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute ; drug therapy ; metabolism ; pathology ; MAP Kinase Kinase 1 ; genetics ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; Proto-Oncogene Proteins c-akt ; genetics ; Pyrazoles ; pharmacology ; Signal Transduction ; Sulfonamides ; pharmacology ; fms-Like Tyrosine Kinase 3 ; genetics