Suppression of apoptosis is one of the hallmarks of carcinogenesis. Tumor cells endure apoptotic pressure by overexpressing several antiapoptotic proteins, and FLICE inhibitory protein (FLIP) is one of the important antiapoptotic proteins that have been shown to be overexpressed in various primary tumor cells. FLIP has two death-effector domains in tandem, mimicking the prodomain of procaspase-8. It is recruited to Fadd in death-inducing signaling complex, thereby preventing the activation of procaspase-8. To date, three isoforms of human cytosolic FLIP (c-FLIP) and six viral homologs (v-FLIP) have been identified. Recently, the crystal structure of v-FLIP MC159 was determined for the first time as an atomic-detail FLIP structure, which revealed that two death effector domains are packed tightly against each other mainly through conserved hydrophobic interactions. The overexpression of c-FLIP in tumor cells has been shown to be the determinant of the tumor's resistance to death ligands such as FasL and TRAIL. It has also been shown that the down-regulation of c-FLIP results in sensitizing resistant tumor cells. Therefore, the agents directly targeting c-FLIP at mRNA and protein levels are expected to be developed in near future and tested for the potential as a new class of anti-cancer drugs.
Apoptosis ; CASP8 and FADD-Like Apoptosis Regulating Protein/antagonists & inhibitors/chemistry/*metabolism ; Caspases/antagonists & inhibitors/metabolism ; Humans ; Neoplasms/*metabolism/pathology/*therapy ; Signal Transduction

BACKGROUNDNeuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis.

METHODSThe expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated.

RESULTSA converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously.

CONCLUSIONThere is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process.

Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Female ; Humans ; Immunohistochemistry ; Infant ; Infant, Newborn ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; analysis ; physiology ; Neoplasm Proteins ; Neuroblastoma ; chemistry ; pathology

AIMTo study the mechanism of evodiamine-induced growth inhibition of HeLa cells.

METHODSHeLa cells viability and the effect of caspase inhibitors on evodiamine-induced apoptosis were measured by crystal violet assay. Changes in cellular morphology were observed by phase-contrast microscopy. Apoptosis-specific nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis.

RESULTSEvodiamine was found to inhibit HeLa cell growth in dose- and time-dependent manners. Caspase-3 inhibitor, z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk) was shown to partially inhibit evodiamine-induced apoptosis. However, caspase-1 inhibitor, Ac-Tyr-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-cmk), did not antagonize evodiamine induced cell death.

CONCLUSIONEvodiamine suppresses the growth of HeLa cells in vitro by apoptosis. Evodiamine-induced apoptosis is partially dependent on caspase-3 pathway in HeLa cells. Other apoptotic pathways might be also related to the induction of apoptosis by evodiamine.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Caspase 3 ; Caspases ; metabolism ; Evodia ; chemistry ; Fruit ; chemistry ; HeLa Cells ; Humans ; Plant Extracts ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Quinazolines ; isolation & purification ; pharmacology ; Signal Transduction ; drug effects

AIMTo study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis.

METHODSCell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis.

RESULTSDracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules.

CONCLUSIONDracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; Arecaceae ; chemistry ; Benzopyrans ; isolation & purification ; pharmacology ; Caspase Inhibitors ; Caspases ; metabolism ; Cell Line, Tumor ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; HeLa Cells ; Humans ; Plants, Medicinal ; chemistry ; Proteins ; metabolism

OBJECTIVETo in vitro compare the induction of extracts of Stellera chamaejasme ESC, ESC-1 and ESC-2 on NCI-H157 cell apoptotic.

METHODThe apoptosis rate was inspected by flow cytometry; caspase-3, 8, 9 activities was measured by spectrophotometry. Fas, Fas-L, TNF-alpha, Trail-R, Cyto-C, Smac/diablo protein expressions of apoptosis pathway was observed by Elisa method.

RESULTCompared with the control group, ESC, ESC-1, ESC-2 can significantly improve the apoptosis rate of NCI-H157 cell. ESC significantly improved cells caspase-3, 8 activity, ESC-2 can significantly improve the activity of caspase-3, 8, 9. ESC, ESC-1, ESC-2 significantly increased Fas expression and ESC significantly increased Fas/Fas-L ratio. ESC, ESC-1, ESC-2 significantly increased TNF-alpha protein expression. ESC-1 significantly lowered TRAIL-R expression. ESC, ESC-1, ESC-2 had no significant effect on Cyto-C. ESC-1, ESC-2 significantly reduced Smac protein expression.

CONCLUSIONThe apoptotic effect induced by ESCs may be related to the regulation of death receptor pathway proteins. Induction mechanisms of ESCs were so complicated that it may have a two-way regulatory effect. Its induction in apoptosis is a result from comprehensive regulation and control.

Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Neoplasms ; chemistry ; drug therapy ; pathology ; Plant Extracts ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; analysis ; Thymelaeaceae ; chemistry ; Tumor Necrosis Factor-alpha ; analysis ; fas Receptor ; analysis

Apoptosis ; drug effects ; Caspases ; physiology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; pathology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Plant Extracts ; pharmacology ; Proanthocyanidins ; pharmacology ; Seeds ; chemistry ; Vitis ; chemistry

Vimentin is an intermediate filament that regulates cell attachment and subcellular organization. In this study, vimentin filaments were morphologically altered, and its soluble subunits were rapidly reduced via cadmium chloride treatment. Cadmium chloride stimulated three major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and led apoptotic pathway via caspase-9 and caspase-3 activations. In order to determine whether MAPKs were involved in this cadmium-induced soluble vimentin disappearance, we applied MAPK- specific inhibitors (PD98059, SP600125, SB203580). These inhibitors did not abolish the cadmium-induced soluble vimentin disappearance. Caspase and proteosome degradation pathway were also not involved in soluble vimentin disappearance. When we observed vimentin levels in soluble and insoluble fractions, soluble vimentin subunits shifted to an insoluble fraction. As we discovered that heat- shock protein 27 (HSP27) was colocalized and physically associated with vimentin in unstressed cells, the roles of HSP27 with regard to vimentin were assessed. HSP27-overexpressing cells prevented morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the cadmium-treated cells. Moreover, HSP27 antisense oligonucleotide augmented these cadmium-induced changes in vimentin. These findings indicate that HSP27 prevents disruption of the vimentin intermediate filament networks and soluble vimentin disappearance, by virtue of its physical interaction with vimentin in cadmium-treated SK-N-SH cells.
Cadmium/*pharmacology ; Caspases/metabolism ; Cell Line ; Heat-Shock Proteins/*metabolism ; Humans ; Mitogen-Activated Protein Kinases/metabolism ; Protein Binding/drug effects ; Protein Subunits/chemistry/metabolism ; Solubility/drug effects ; Vimentin/*chemistry/*metabolism

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H₂O₂ stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O₂ ^•- levels and construct an O₂ ^•- reaction network. To elucidate the consequences of increased O₂ ^•- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca²⁺ levels, mitochondrial Ca²⁺ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS Results from a modified microchip demonstrated that 1 mmol/L H₂O₂ induced a rapid increase in cellular O₂ ^•- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca²⁺ and mitochondrial Ca²⁺ by confocal laser scanning microscopy and confirmed that Ca²⁺ stores in the endoplasmic reticulum were the primary source of H₂O₂-induced cytosolic Ca²⁺ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca²⁺ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
Apoptosis ; Calcium/metabolism* ; Calcium Signaling ; Caspases/metabolism* ; Cell Line, Tumor ; Cell Lineage ; Cytosol/metabolism* ; Glutathione/metabolism* ; Humans ; Hydrogen Peroxide/chemistry* ; Mitochondria/metabolism* ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Superoxides/chemistry*

Tanshinone II-A is a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza BUNGE, a traditional herbal medicine that is known to induce antiinflammatory, anti-oxidative and cytotoxic activity. We have examined cellular effects of Tanshione II-A on HL60 human promyelocytic leukemic cells and K562 human erythroleukemic cells. Tanshione II-A induced a dose- and time-dependent DNA fragmentation into the multiples of 180 bp and specific proteolytic cleavage of poly(ADP-ribose) polymerase in both cell lines. PI-staining and flow cytometry analysis of K562 cells following Tanshione II-A treatment showed an increase of the cells possessing hypodiploid DNA indicative of apoptotic state of cells. Caspase-3 activity was significantly increased during Tanshinone II-A treatment of both HL60 and K562 cells, whereas caspase-1 activity was not changed. These results suggest that Tanshione II-A induced HL60 and K562 cellular apoptosis that may be associated with the selective members of caspase family. Copyright 2000 Academic Press.
Antineoplastic Agents, Phytogenic/pharmacology* ; Antineoplastic Agents, Phytogenic/chemistry ; Apoptosis/physiology* ; Caspases/metabolism* ; Caspases/drug effects* ; Cell Cycle/drug effects ; DNA Fragmentation/drug effects ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal/pharmacology* ; Drugs, Chinese Herbal/chemistry ; Enzyme Activation/drug effects ; HL-60 Cells/pathology ; HL-60 Cells/metabolism ; HL-60 Cells/drug effects ; Human ; Lamiaceae/chemistry ; Leukemia/pathology* ; Leukemia/metabolism ; Leukemia/drug therapy ; Leukemia, Erythroblastic, Acute/pathology ; Leukemia, Erythroblastic, Acute/metabolism ; Leukemia, Erythroblastic, Acute/drug therapy ; Phenanthrenes/pharmacology* ; Phenanthrenes/chemistry ; Tumor Cells, Cultured

Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.
Alkaline Phosphatase/pharmacology ; Animal ; Brain/metabolism ; Calpain/metabolism ; Carrier Proteins/*chemistry ; Caspases/metabolism ; Cattle ; Cell-Free System ; Clathrin/*chemistry ; Electrophoresis, Polyacrylamide Gel ; Glutathione Transferase/metabolism ; Lipids/chemistry ; Membrane Proteins/*chemistry ; Phosphorylation ; Protein Binding ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Protein Transport ; Recombinant Proteins/chemistry/metabolism ; Reticulocytes/metabolism ; Support, Non-U.S. Gov't ; Translation, Genetic ; src Homology Domains