c-Jun N-terminal kinase (JNK) is activated during hepatic reperfusion, and JNK inhibitors are known to protect other major organs from ischemia-reperfusion (I/R) injury. We attempted to determine the effect of SP600125, a JNK inhibitor, on hepatic I/R injury using a partial ischemia model in mice. Compared to a vehicle-treated group, the SP600125-treated group showed a greater increase in serum ALT levels 24 h after reperfusion with more severe parenchymal destruction and leukocyte infiltration. Similarly, tissue myeloperoxidase and malondialdehyde levels were higher in the SP600125-treated group, and chemokine expression was also higher in the SP600125-treated group. These data, which are contradictory to previous results, indicate that JNK inhibition by SP600125 may be harmful in hepatic I/R injury. Therefore, care must be taken when investigating the therapeutic use of JNK inhibitors in hepatic I/R injury, especially in the context of the effects of JNK inhibition on inflammatory infiltration.
Reperfusion Injury/*drug therapy ; Oxidative Stress/drug effects ; Mice, Inbred C57BL ; Mice ; Matrix Metalloproteinase 9/metabolism ; Male ; MAP Kinase Kinase 4/*antagonists & inhibitors ; Liver/cytology/*drug effects/*injuries ; Chemokines/metabolism ; Anthracenes/*pharmacology ; Animals

To investigate Bax translocation from cytosol into mitochondria induced by staurosporine (STS) in GFP-Bax-tagged MCF7 stable cell line, the viability was measured by MTT method. Bax translocation from cytosol into mitochondria was investigated under the fluorescence microscope. The dose-effect and time-course relationships were also observed and the percentage of GFP-Bax punctuate cells were calculated. Immunofluoresence method was used to observe Bax translocation to mitochondria, Cyt-c release from mitochondria and Annexin V label. The TMRE assay was used to investigate membrane pertential (Deltapsim) and function of mitochondria. Western blotting was used to observe the mechanism of apoptosis induced by STS. The results showed that STS can induce Bax translocation from cytoplasm to mitochondria, Cyt-c release from mitochondria and Annexin V label. The Western blotting analysis presented the inhibitory effect on apoptosis induced by STS of SP600125 which is a specific JNK inhibitor. The study revealed the mechanism of STS induced apoptosis associated with JNK activated pathway.
Anthracenes ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Cytosol ; metabolism ; Humans ; MAP Kinase Kinase 4 ; antagonists & inhibitors ; Membrane Potentials ; drug effects ; Mitochondria ; metabolism ; Protein Transport ; drug effects ; Staurosporine ; pharmacology ; bcl-2-Associated X Protein ; metabolism

AIMTo explore possible signal transmission way through which ginsenoside Rg1 protect cells from MPP(+)-induced apoptosis.

METHODSThe apoptosis of SHSY5Y induced by 1-methyl-4-phenylpyridinium (MPP+) was observed by AO-EB staining. Flow cytometry was used to quantitate the reactive oxygen species (ROS). Western Blotting was used to detect the c-jun NH2-terminal kinase (JNK) activity in SHSY5Y cells. Immunocytochemistry staining was used to detect cleaved Caspase-3 positive cells.

RESULTSMPP+ was shown to induce apoptosis in SHSY5Y cells. The percentage of apoptotic SHSY5Y cells induced by MPP+ was obviously lower in those groups pretreated with 10 mumol.L-1 Rg1 or 2.5 mmol.L-1 N-acetylcysyteine (NAC). It showed more ROS in MPP+ groups than in control. JNK activity increased with time within 72 hours in 1 mmol.L-1 MPP+ group. Simultaneously, it showed decrease of ROS, less activity of JNK and lower expression of cleaved Caspase-3 in 10 mumol.L-1 Rg1 and 2.5 mmol.L-1 NAC pretreated groups compared with groups treated with MPP+ only.

CONCLUSIONRg1 protects against MPP(+)-induced apoptosis in SHSY5Y cells and the effect might be attributed to its removal of ROS, inhibition of the activity of JNK and expression of cleaved Caspase-3.

1-Methyl-4-phenylpyridinium ; antagonists & inhibitors ; pharmacology ; Apoptosis ; drug effects ; Caspases ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ginsenosides ; isolation & purification ; pharmacology ; Humans ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 4 ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Neuroblastoma ; pathology ; Neuroprotective Agents ; pharmacology ; Panax ; chemistry ; Reactive Oxygen Species ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo investigate the effects of mitogen activated protein kinase (MAPK) signal transduction pathways on heat shock protein 70 (HSP70) gene expression in endothelial cells exposed to benzo(a)pryene (BaP).

METHODSPorcine aortic endothelial cells were pre-treated or by PD98059 (10 micro mol/L) or SB203580 (20 micro mol/L) for 1 hour, then treated with different concentrations of BaP (0, 0.1, 0.5, 1.0, 5.0 and 10.0 micro mol/L) for 24 hours respectively;Expression levels of three phosphorylated MAPKs [extracellular signal regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38] and HSP70 were determined by Western-blot.

RESULTSThe three phosphorylated MAPKs expressional levels especially p-ERK1 had different extents of changes with dose-response relationship under BaP exposure. BaP inhibited the expression of HSP70, which significantly decreased in medium and high dose group (>or= 1.0 micro mol/L) but did not decrease in control group (P < 0.05). Although the inhibitor of ERK (PD98059) could partly weaken the inhibited effects of BaP on HSP70 expression, HSP70 expression levels of endothelial cells pre-treated with PD98059 were still significantly lower than that of control cells (P < 0.05).

CONCLUSIONERK1 pathway might play some roles in HSP70 gene expression in endothelial cells exposed to BaP, and other unknown signal pathways might also have some effects on this process.

Animals ; Benzo(a)pyrene ; toxicity ; Blotting, Western ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; HSP70 Heat-Shock Proteins ; analysis ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 4 ; Mitogen-Activated Protein Kinase Kinases ; analysis ; Mitogen-Activated Protein Kinases ; analysis ; antagonists & inhibitors ; Pyridines ; pharmacology ; Signal Transduction ; physiology ; Swine ; p38 Mitogen-Activated Protein Kinases

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal thatgammagammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.
Anthracenes/pharmacology ; Apoptosis/drug effects/*genetics ; Carbazoles/pharmacology ; Cell Cycle/drug effects/genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/*biosynthesis/genetics ; Cytosol/drug effects/enzymology/ultrastructure ; DNA Damage/drug effects/genetics ; Doxorubicin/pharmacology ; Histones/*metabolism ; Humans ; Indole Alkaloids/pharmacology ; MAP Kinase Kinase 4/antagonists & inhibitors ; Nerve Growth Factor/antagonists & inhibitors/metabolism ; Phosphorylation/drug effects ; Protein Binding ; *Protein Transport/drug effects/genetics ; Receptor, trkA/antagonists & inhibitors/*genetics/metabolism ; Signal Transduction ; Transfection