OBJECTIVETo study the expressions and clinical significances of p-extracellular regulated kinase(P-ERK)1/2 and matrix metalloproteinase-9(MMP-9)in cervical squamous cell carcinoma.

METHODSThe expressions of P-ERK1/2 and MMP-9 in 30 cases with chronic cervicitis, 45 cases with cervical intraepithelial neoplasia (CIN), and 58 cases with cervical squamous cell carcinoma were detected by immunohistochemical method.

RESULTSThe positive rates of P-ERK1/2 and MMP-9 in chronic cervicitis, CIN, and cervical squamous cell carcinoma were 0 and 0, 28.9% and 24.4%, 77.6% and 65.5%, respectively, showing significant differences among these three groups (χ(2)= 54.393,p=0.003;χ(2)=40.968,p=0.005). The positive rates of P-ERK1/2 and MMP-9 in patients at clinical stages 2-3, at G3, with lymphatic metastasis, or with a tumor diameter greater than 4 cm were significantly higher than those at clinical stage 1(p=0.015,p=0.002), at G1-G2(p=0.013,p=0.017), without lymphatic metastasis (p=0.017,p=0.021), or with a tumor diameter less or equal than 4 cm in cervical squamous cell carcinoma(p=0.008,p=0.004). There was a positive correlation between P-ERK1/2 and MMP-9 in cervical squamous cell carcinoma (χ(2)=8.955,p=0.006).

CONCLUSIONSThe expressions of P-ERK1/2 and MMP-9 increase gradually with the progression of cervical squamous cell carcinoma. The over expressions of P-ERK1/2 and MMP-9 may promote the infiltration of cervical squamous cell carcinoma and lymphatic metastasis, druing which these two enzymes may exert their effects in a synergistic manner.

Carcinoma, Squamous Cell ; enzymology ; pathology ; Female ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Uterine Cervical Neoplasms ; enzymology ; pathology

OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-kappa B activation and NF-kappa B is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-kappa B activation.
Animals ; Cell Line ; Enzyme Activation/drug effects ; Enzyme Induction/drug effects ; Matrix Metalloproteinase 9/*biosynthesis ; Mice ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; NF-kappa B/*metabolism ; Oligodeoxyribonucleotides/*pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 9/antagonists & inhibitors/*metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism

OBJECTIVETo investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism.

METHODSThe human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting.

RESULTSS. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells.

CONCLUSIONSS. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Anthracenes ; pharmacology ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Mitogen-Activated Protein Kinase 8 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 9 ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Signal Transduction ; physiology ; Staphylococcus aureus ; physiology ; U937 Cells ; bcl-2-Associated X Protein ; metabolism

The aim of this study was to investigate the apoptosis-inducing effect of gambogic acid (GA) on Jurkat cells and its underlying signaling pathway. Apoptosis induced by GA and some inhibitors was assayed by Annexin V/PI doubling staining. The levels of caspase 3, caspase 8 and caspase 9 activated in living Jurkat cells were measured by flow cytometry. The expressions of caspase 3, caspase 9, p-JNK and P38 were detected by Western blot. The results showed that GA induced apoptosis of Jurkat cells in a dose-dependent manner. The positive cell number of activated caspase 3, caspase 8, caspase 9 and the levels of activated caspase 3, caspase 9, p-JNK, P38 increased after Jurkat cells were treated with GA. ROS, CaMKII, caspase 3, caspase 9, MAPKK, JNK1/2 and P38 inhibitors had some significant effect on GA-induced apoptosis. ROS, CaMKII, MAPKK, JNK1/2 and P38 inhibitors decreased the levels of activated caspase 3, caspase 9 by GA.ROS, CaMKII, MAPKK, JNK1/2 inhibitors decreased the levels of p-JNK by GA. ROS, CaMKII, MAPKK, P38 inhibitors decreased the levels of P38 by GA. It is concluded that GA induced apoptosis of Jurkat cells by activated caspases through activating of ROS-CaMKII-MAPKK-JNK/P38 pathway.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Jurkat Cells ; MAP Kinase Signaling System ; drug effects ; Xanthones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVE: To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity. METHODS: Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. RESULTS: DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05). CONCLUSION DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.
Apoptosis ; Caspase 3 ; Caspase 9/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Doxycycline/pharmacology* ; Humans ; Mitogen-Activated Protein Kinase Kinases/pharmacology* ; Multiple Myeloma

OBJECTIVETo investigate the expressions of mitogen-activated protein kinases (MAPKs) and matrix metalloproteinases (MMPs) in apoptotic human T cell lymphoma Jurkat cells induced by curcumin in vitro and explore the possible molecular mechanisms of curcumin-induced apoptosis.

METHODSJurkat cells were treated with different concentrations of curcumin, and the cell proliferation and cell cycle changes were detected by MTT assay and flow cytometry, respectively. Western blotting and gelatin zymography were employed to examine the protein expression levels of MAPKs and MMPs activity in the exposed cells.

RESULTSCurcumin inhibited the proliferation of Jurkat cells in a time- and dose-dependent manner, and caused cell cycle arrest in G0/G1 phase. Treatment of Jurkat cells with 25, 50, and 75 µmol/L curcumin resulted in a concentration-dependent increase of JNK and p-JNK expressions (P<0.01) without significantly affecting the expressions of ERK1/2 and P38 MAPK or the activity of MMP-2 and MMP-9.

CONCLUSIONCurcumin within the concentration range of 6.25-25.00 µmol/L can induce apoptosis and cell cycle arrest of Jurkat cells, the mechanism of which might involve the activation of JNK pathway but not the MMPs.

Apoptosis ; Cell Cycle ; Cell Division ; Cell Proliferation ; Curcumin ; pharmacology ; Flow Cytometry ; Humans ; Jurkat Cells ; MAP Kinase Signaling System ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDC-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in cerebral ischemia. Although the mechanistic basis for this activation of JNK1/2 is uncertain, oxidative stress may play a role. The purpose of this study was to investigate whether the activation of JNK1/2 is associated with the production of endogenous nitric oxide (NO).

METHODSIschemia and reperfusion (I/R) was induced by cerebral four-vessel occlusion. Sprague-Dawley (SD) rats were divided into 6 groups: sham group, I/R group, neuronal nitric oxide synthase (nNOS) inhibitor (7-nitroindazole, 7-NI) given group, inducible nitric oxide synthase (iNOS) inhibitor (2-amino-5,6-dihydro-methylthiazine, AMT) given group, sodium chloride control group, and 1% dimethyl sulfoxide (DMSO) control group. The levels of protein expression and phospho-JNK1/2 were detected by Western blotting and the survival hippocampus neurons in CA1 zone were observed by cresyl violet staining.

RESULTSThe study illustrated two peaks of JNK1/2 activation occurred at 30 minutes and 3 days during reperfusion. 7-NI inhibited JNK1/2 activation during the early reperfusion, whereas AMT preferably attenuated JNK1/2 activation during the later reperfusion. Administration of 7-NI and AMT can decrease I/R-induced neuronal loss in hippocampal CA1 region.

CONCLUSIONJNK1/2 activation is associated with endogenous NO in response to ischemic insult.

Animals ; Blotting, Western ; Brain Ischemia ; enzymology ; Enzyme Inhibitors ; Hippocampus ; cytology ; metabolism ; Indazoles ; pharmacology ; Male ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Neurons ; cytology ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; Phosphorylation ; drug effects ; Rats ; Rats, Sprague-Dawley

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.
Acid Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Bone Resorption ; Cells, Cultured ; Cnidium ; chemistry ; Coumarins ; isolation & purification ; pharmacology ; Gene Expression ; Isoenzymes ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Osteoclasts ; metabolism ; pathology ; Osteoprotegerin ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; RANK Ligand ; metabolism ; Rabbits ; Seeds ; chemistry ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase

OBJECTIVETo investigate the effects of phosphorylated mitogen-activated protein kinases (MAPK), including the phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), the phosphorylated protein p38 (p-p38), the phosphorylated c-Jun N-terminal kinase (p-JNK), on phosgene inhalation-induced lung injury and its relationship with matrix metalloproteinase 9 (MMP-9).

METHODSAccording to the random number table, 30 male Wistar rats were divided into air control group (C), phosgene inhalation group (P), PD98059 (specific inhibitor of ERK1/2) group, SB203580 (specific inhibitor of p38) group, and SP600125 (specific inhibitor of JNK) group, with 6 rats in each group. The number of neutrophils in the bronchoalveolar lavage fluid (BALF) was counted and the lung wet-dry ratio (W/D) was examined. The serum levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-8 were determined with ELISA. The protein expressions of p-ERK1/2, p-p38, p-JNK, and MMP-9 in lung tissue were detected with Western blotting. The mRNA level of MMP-9 in lung tissue was detected with real-time fluorescence quantitative PCR. Data were processed with one-way analysis of variance (among groups) and SNK method (paired comparison).

RESULTSCompared with those of group C [respectively (2.0 ± 0.7)×10(4) /mL and 3.7 ± 0.6], the number of neutrophils and W/D of group P [respectively (10.7 ± 1.4)×10(4) /mL and 7.6 ± 0.4] were increased. The number of neutrophils in group SB203580 and group SP600125 was respectively (8.3 ± 1.1)×10(4), (7.9 ± 1.3)×10(4)/mL, with W/D respectively 6.1 ± 1.4, 6.1 ± 0.9, all of which decreased as compared with those of group P (with P values all below 0.01). Compared with those of group C, the levels of TNF-a, IL-1β, IL-6, and IL-8 of group P were increased, but decreased in group SB203580 and group SP600125 compared with that of group P, though still higher than those of group C, and the differences were statistically significant (P < 0.05 or P<0.01). Protein quantities of p-p38 and p-JNK were higher in group P (respectively 1.19 ± 0.22 and 1.43 ± 0.14) than in group C (respectively 0.76 ± 0.06 and 0.74 ± 0.05). Compared with those of group P, the protein levels of p-ERK1/2 (0.47 ± 0.05) in group PD98059, p-p38 (0.88 ± 0.07) in group SB203580, and p-JNK (0.91 ± 0.07) in group SP600125 were significantly reduced (P < 0.05 or P < 0.01). The protein and mRNA levels of MMP-9 were higher in group P (respectively 2.23 ± 0.18 and 4.93 ± 0.12) than in group C (respectively 1.26 ± 0.14 and 1.80 ± 0.03). The protein and mRNA levels of MMP-9 in group SB203580 (respectively 1.58 ± 0.14 and 2.96 ± 0.09) and group SP600125 (respectively 1.55 ± 0.30 and 3.00 ± 0.13) were lower than those in group P (P < 0.05 or P < 0.01).

CONCLUSIONSThe phosgene inhalation can activate the MAPK signaling protein pathway by increasing expressions of p-p38 and p-JNK, which lead to an up-regulation of MMP-9, and this may contribute to the phosgene inhalation-induced lung injury.

Animals ; Burns, Inhalation ; enzymology ; Cytokines ; metabolism ; Disease Models, Animal ; Flavonoids ; pharmacology ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Phosgene ; Phosphorylation ; Pyridines ; pharmacology ; Rats ; Rats, Wistar