benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and epigallocatechin-gallate

Previous studies have shown that light impinging on the retina in situ has the capacity to kill neuronal and non-neuronal cells in vitro by interacting directly with mitochondrial constituents. A number of fluorophores are associated with mitochondria which can potentially absorb different wave-lengths of light, including cytochrome oxidase. The aim of the present study was to compare the death mechanism of a light insult to RGC-5 cells in culture with that of sodium azide. Sodium azide's main toxic action is in inhibiting the function of cytochrome oxidase in the mitochondrial electron transport chain. Our studies showed that light and sodium azide kill RGC-5 cells via different mechanisms although some similarities do occur. Both inducers of cell death caused the generation of reactive oxygen species (ROS), the expression of phosphatidylserine, the breakdown of DNA and the activation of p38 MAPK, resulting in its translocation from the nucleus to the cytoplasm. However, light-induced cell death occurs via necroptosis, in that it was inhibited by necrostatin-1 and was caspase-independent. This was not the case for sodium azide, where the death process was caspase-dependent, occurred via apoptosis and was unaffected by necrostatin-1. Moreover, light caused an activation of the apoptosis inducing factor (AIF), c-Jun, JNK and HO-1, but it did not affect alpha fodrin or caspase-3. In contrast, sodium azide caused the activation of alpha fodrin and the stimulation of caspase-3 content without influencing AIF, c-Jun, JNK or HO-1. Therefore we conclude that light does not have a specific action on cytochrome oxidase in mitochondria to cause cell death.

Topics: Amino Acid Chloromethyl Ketones; Blotting, Western; Caspase 3; Catechin; Cell Culture Techniques; Cell Death; Cell Line; Cell Nucleus; Cell Survival; DNA; Imidazoles; Immunohistochemistry; Indoles; Light; Phosphatidylserines; Reactive Oxygen Species; Retinal Ganglion Cells; Sodium Azide; Staurosporine

Alveolar bone resorption is a characteristic feature of periodontal diseases and involves removal of both the mineral and the organic constituents of the bone matrix, a process mainly carried out by multinucleated osteoclast cells. (-)-Epigallocatechin gallate, the main constituent of green tea polyphenols, has been reported to induce the apoptotic cell death of osteoclasts and to modulate caspase activation in various tumor cells. In the present study, we investigated the inhibitory effect of (-)-epigallocatechin gallate on osteoclast survival and examined if (-)-epigallocatechin gallate mediates osteoclast apoptosis via caspase activation.. The effect of (-)-epigallocatechin gallate on osteoclast survival was examined by tartrate-resistant acid phosphatase (TRAP) staining in osteoclasts differentiated from RAW 264.7 cells. In addition, we evaluated the apoptosis of osteoclasts by (-)-epigallocatechin gallate using a DNA-fragmentation assay. Involvement of caspase in (-)-epigallocatechin gallate-mediated osteoclast apoptosis was evaluated by treatment with a general caspase inhibitor, Z-VAD-FMK. Moreover, the effect of (-)-epigallocatechin gallate on the activation of caspase-3 was assessed by a colorimetric activity assay and western blotting.. (-)-Epigallocatechin gallate significantly inhibited, in a dose-dependent manner, the survival of osteoclasts differentiated from RAW 264.7 cells and induced the apoptosis of osteoclasts. Treatment with (-)-epigallocatechin gallate resulted in DNA fragmentation and induced the activation of caspase-3 in RAW 264.7 cell-derived osteoclasts. Additional treatment with Z-VAD-FMK suppressed these effects of (-)-epigallocatechin gallate.. From these findings, we could suggest that (-)-epigallocatechin gallate might prevent alveolar bone resorption by inhibiting osteoclast survival through the caspase-mediated apoptosis.

Topics: Acid Phosphatase; Alveolar Process; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Bone Resorption; Caspase Inhibitors; Caspases; Catechin; Cell Line; Cell Survival; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Isoenzymes; Mice; Osteoclasts; RANK Ligand; Tartrate-Resistant Acid Phosphatase

Green tea constituent (-) epigallocatechin-3-gallate (EGCG) has shown remarkable cancer-preventive and some cancer-therapeutic effects. This is partially because of its ability to induce apoptosis in cancer cells without affecting normal cells. Previous studies from our laboratory have shown the involvement of NF-kappa B pathway in EGCG-mediated cell-cycle deregulation and apoptosis of human epidermoid carcinoma A431 cells. Here we show the essential role of caspases in EGCG-mediated inhibition of NF-kappa B and its subsequent apoptosis. Treatment of A431 cells with EGCG (10-40 microg/ml) resulted in dose-dependent inhibition of NF-kappa B/p65, induction of DNA breaks, cleavage of poly(ADP-ribose) polymerase (PARP) and morphological changes consistent with apoptosis. EGCG treatment of cells also resulted in significant activation of caspases, as shown by the dose- and time-dependent increase in DEVDase activity, and protein expression of caspase-3, -8 and -9. EGCG-mediated caspase activation induces proteolytic cleavage of NF-kappa B/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis. EGCG-mediated induction of apoptosis was significantly blocked by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK), and moderately blocked by the specific caspase-3 inhibitor Z-DEVD-FMK. Further, pretreatment of cells with Z-VAD-FMK was found to suppress the cleavage of NF-kappa B/p65 subunit, thereby increasing nuclear translocation, DNA binding and transcriptional activity, thus protecting the cells from EGCG-induced apoptosis. Taken together, these studies for the first time demonstrate that EGCG-mediated activation of caspases is critical, at least in part, for inhibition of NF-kappa B and subsequent apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Catechin; Cell Line, Tumor; Cell Nucleus; Cell Survival; DNA; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Male; Models, Biological; NF-kappa B; Oligopeptides; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Structure, Tertiary; Time Factors; Transcription, Genetic

Epigallocatechin gallate (EGCG) is one of most famous compounds of green tea. EGCG suppresses apoptosis induced by oxidative radical stress through several mechanisms. This study was designed to investigate whether EGCG plays a cytoprotective role by activating phosphatidylinositol-3 kinase (PI3K)/Akt-dependent anti-apoptotic pathway and inhibiting glycogen synthase kinase-3 (GSK-3) activity in oxidative stressed N18D3 neural cells. N18D3 cells, mouse neuroblastoma X dorsal root ganglion hybrid cell line, were pre-treated with EGCG or z-VAD-fmk, non-selective caspase inhibitor used as a control substance, for 2 h. The N18D3 cells were then exposed to low concentration of H(2)O(2) (100 microM) for 30 min, and further incubated for 24 h. MTT (3,[4,5-dimethylthiazol]-2-yl) assay and trypan blue staining were used to identify cell viability. Immunoreactivity (IR) of PI3K, Akt, and GSK-3 beta were measured by Western blotting. MTT assay and trypan blue staining showed that EGCG and z-VAD-fmk significantly increased cell viability, and IR of PI3K, phospho-Akt and phospho-GSK-3 beta was significantly increased in the cells treated with EGCG, but not in z-VAD-fmk treated. These results imply that EGCG has neuroprotective effect by increasing PI3K/Akt-dependent anti-apoptotic signals.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Western; Caspase Inhibitors; Catechin; Cell Differentiation; Cell Line; Cell Survival; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Hydrogen Peroxide; Mice; Mice, Inbred BALB C; Neurons; Neuroprotective Agents; Oncogene Protein v-akt; Oxidants; Oxidative Stress; Phosphatidylinositol 3-Kinases; Retroviridae Proteins, Oncogenic; Signal Transduction