Apoptosis ; Calcineurin ; metabolism ; Chymotrypsin ; metabolism ; Humans ; Lysosomes ; enzymology ; Mitochondria ; metabolism ; pathology ; Mitochondrial Dynamics ; Neuroblastoma ; metabolism ; pathology

Caffeine is one of the most widely consumed neuroactive drugs, coming mostly from everyday beverages such as coffee and tea. To investigate whether caffeine induces apoptosis in the central nervous system, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, flow cytometric analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with caffeine at concentrations as high as 10 mM exhibited several characteristics of apoptosis. In addition, caffeine was shown to increase the caspase-3 activity. These results suggest that high-dose of caffeine induces apoptosis in human neuroblastoma cells, probably by increasing the caspase-3 enzyme activity.
Apoptosis/*drug effects ; Caffeine/*toxicity ; Caspase 3 ; Caspases/metabolism ; Cell Cycle/drug effects ; Cell Survival/drug effects ; Central Nervous System/cytology/*drug effects ; DNA Fragmentation ; Humans ; Neuroblastoma/enzymology/pathology ; Tumor Cells, Cultured

OBJECTIVETo study MMP-2, MMP-9, TIMP-2 and TIMP-1 expression, and their association to invasion and metastasis of neuroblastoma (NB).

METHODSThe staining status was compared of MMP-2, MMP-9, TIMP-2 and TIMP-1 in cryostat sections of tumor tissue in 35 NB patients by immunohistochemistry.

RESULTSStrong expression of MMP-2 was detected only in 2 patients with early stage NB (group A without metastasis), but in 9 and 10 respectively with advanced stage NB (group B with local metastasis and group C with distant metastasis) (compared to group A, P < 0.01). Strong MMP-9 staining was found in 4, 8 and 11 patients for group A, B and C patients (group A vs group C, P < 0.05). The expression of TIMP-2 was the strongest in 4 group A patients, but it decreased with progression of the disease. There was no statistical difference in TIMP-1 expression among the three groups of patients.

CONCLUSIONMMP-2, MMP-9 expression may be related to metastasis and progression of neuroblastoma, while TIMP-2 may have an inhibitory effect.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local ; Neuroblastoma ; enzymology ; metabolism ; secondary ; Retroperitoneal Neoplasms ; enzymology ; metabolism ; pathology ; Testicular Neoplasms ; enzymology ; metabolism ; secondary ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

Alcohol influences the neuroadaptation of brain cells where receptors and enzymes like protein kinase C (PKC) exist. Naltrexone acts on opioid receptors. However, other mechanisms of action remain unknown. We prepared SH-SY5Y neuroblastoma cells, and fed them with 150 mM ethanol for 72 h followed by treatment with naltrexone for 24 h. We performed microarray analysis and reverse transcriptase-polymerase chain reaction. Our results showed that PKC epsilon increased 1.90 times and showed an overall decreasing pattern as time increased. Phosphorylated ERK also increased 2.0 times according to the change of PKC epsilon. Integrin alpha7 increased 2.32 times and showed an increasing pattern as time increased. In conclusion, naltrexone influences PKC epsilon neuronal signaling system and endothelial adhesion molecule integrin alpha7 in addition to the well-known opioid system.
Antigens, CD/*metabolism ; Cell Line, Tumor ; Comparative Study ; DNA, Complementary/genetics ; Humans ; Integrin alpha Chains/*metabolism ; Naltrexone/*pharmacology ; *Neuroblastoma/enzymology/metabolism/pathology ; Oligonucleotide Array Sequence Analysis ; Protein Kinase C-epsilon/*metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo study the effect of gamma-interferon (IFNgamma), tumor necrosis factor related apoptosis inducing ligand (TRAIL), and cisplatin or etoposide induced apoptosis in human neuroblastoma cell line SH-SY5Y and its possible molecular mechanisms.

METHODSThe expressions of Caspase 8 mRNA and protein were detected with RT-PCR and Western blot analysis. The effects of IFN-gamma, TRAIL, IFNgamma + TRAIL, IFN-gamma + Caspase 8 inhibitor + TRAIL, IFNgamma + cisplatin + TRAIL, and IFNgamma + etoposide + TRAIL on the growth and apoptosis of SH-SY5Y cells were detected with the methods of MTT and flow cytometry. The relative Caspase 8 activity was measured with colorimetric assay.

RESULTSCaspase 8 was undetectable in SH-SY5Y cells but an increased expression of Caspase 8 mRNA and protein was found after treatment with IFNgamma. SH-SY5Y cells themselves were not sensitive to TRAIL, but those expressing Caspase 8 after treatment with IFNgamma were. The killing effect of TRAIL on SH-SY5Y cells expressing Caspase 8 was depressed by Caspase 8 inhibitor. Cisplatin and etoposide could enhance the sensitivity of TRAIL on SH-SY5Y cells. The relative Caspase 8 activity of SH-SY5Y cells in IFN-gamma + TRAIL group was significantly higher than those of control group, IFN-gamma group, TRAIL group, and inhibitor group (P < 0.01). There was no significant difference among IFN-gamma + TRAIL group, IFNgamma + cisplatin + TRAIL group, and IFNgamma + etoposide + TRAIL group.

CONCLUSIONSIFNgamma could sensitize SH-SY5Y cells to TRAIL-induced apoptosis and this may be realized by the up-regulation of Caspase 8. Cisplatin and etoposide could enhance the killing effect of TRAIL on SH-SY5Y cells.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 8 ; genetics ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cisplatin ; pharmacology ; Etoposide ; pharmacology ; Humans ; Interferon-gamma ; pharmacology ; Neuroblastoma ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology