OBJECTIVETo investigate the possible mechanism through which Artemisinin induced apoptosis in pancreatic cell line.

METHODSColumn chromatography, thin layer chromatography (TLC) and proton NMR spectroscopy were used to purify Artemisinin. The flowcytometry was employed to detect apoptosis and reactive oxygen species (ROS).

RESULTSThe results indicated that 50% inhibiting concentration (IC50 value) for pancreatic cell line (RIN) was 45 μmol/L of Artemisinin. Artemisinin had no cytotoxic effect on the growth of peripheral blood lymphocytes. The mechanism of apoptosis was evaluated by measuring intracellular ROS. It was shown that Artemisinin-induced apoptosis occurred independently of the binding of CD95L to CD95 receptor in the RIN cells. Moreover, Artemisinin, in a dose-dependent manner, could significantly increase the level of ROS.

CONCLUSIONArtemisinin can induce apoptosis in the RIN cells via the generation of ROS and triggering the intrinsic pathway of cell death.

Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorimetry ; Flow Cytometry ; Humans ; Iron ; pharmacology ; Pancreatic Neoplasms ; enzymology ; pathology ; Propidium ; metabolism ; Proton Magnetic Resonance Spectroscopy ; Reactive Oxygen Species ; metabolism ; Time Factors ; fas Receptor ; metabolism

OBJECTIVETo investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence.

METHODSChromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA.

RESULTSMitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells.

CONCLUSIONMitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azure Stains ; Benzimidazoles ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Nucleus ; drug effects ; metabolism ; Cellular Senescence ; drug effects ; Chromatin ; metabolism ; DNA, Neoplasm ; analysis ; Dose-Response Relationship, Drug ; Enediynes ; pharmacology ; Genome, Human ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitosis ; drug effects ; Phenotype ; Propidium ; Telomerase ; metabolism ; Time Factors ; beta-Galactosidase ; metabolism

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.
Time Factors ; Rats, Sprague-Dawley ; Rats ; Propidium ; Neuroprotective Agents/*pharmacology ; In Situ Nick-End Labeling ; Hypoxia-Ischemia, Brain/*drug therapy/metabolism/pathology ; Cycloheximide/*pharmacology ; Brain Infarction/pathology/prevention & control ; Apoptosis/drug effects ; Annexin A5/metabolism ; Animals, Newborn ; Animals

Apoptosis is a programmed, physiologic mode of cell death that plays an important role in tissue homeostasis. As for the central nervous system, ischemic insults can induce pathophysiologic cascade of apoptosis in neurophils. Impairment of astroctye functions during brain ischemia can critically influence neuron survival by neuronglia interactions. We aimed to elucidate the protective effect of ketamine on apoptosis by energy deprivation in astrocytes. Ischemic insults was induced with iodoacetate/ carbonylcyanide mchlorophenylhydrazone (IAA/CCCP) 1.5 mM/ 20 micrometer or 150 micrometer/2 micrometer for 1 hr in the HTB-15 and CRL-1690 astrocytoma cells. Then these cells were reperfused with normal media or ketamine (0.1 mM) containing media for 1 hr or 24 hr. FITC-annexin-V staining and propidium iodide binding were determined by using flow cytometry. Cell size and granularity were measured by forward and side light scattering properties of flow cytometry system, respectively. An addition of keta-mine during reperfusion increased the proportion of viable cells. Ketamine alleviated cell shrinkage and increased granularity during the early period, and ameliorated cell swelling during the late reperfusion period. Ketamine may have a valuable effect on amelioration of early and late apoptosis in the astrocytoma cells, even though the exact mechanism remains to be verified.
Anesthetics, Dissociative/*pharmacology ; Annexin A5/pharmacology ; Apoptosis ; Astrocytes/metabolism ; Astrocytoma/*drug therapy/pathology ; Brain/pathology ; Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology ; Cell Line, Tumor ; Cell Size ; Cell Survival ; Central Nervous System/drug effects/pathology ; Enzyme Inhibitors/pharmacology ; Flow Cytometry/*methods ; Humans ; Indicators and Reagents/pharmacology ; Iodoacetates/pharmacology ; Ischemia/pathology ; Ketamine/metabolism/*pharmacology ; Light ; Neurons/metabolism/pathology ; Neutrophils/metabolism ; Perfusion ; Propidium/pharmacology ; Scattering, Radiation ; Time Factors ; Uncoupling Agents/pharmacology

OBJECTIVETo compare two fluorochrome staining methods for the assessment of sperm quality.

METHODSWashed sperm cells were incubated in 0, 0.15, or 15 micromol/L camptothecin (CAM), or 0.37 or 3.7 mmol/L genistein (GEN) at 37 degrees C for 4 hours. The sperm cells were analyzed for cycle-independent apoptosis and necrosis by single-stain compared with dual-stain fluorescence microscopy to contrast the relative effectiveness of these two approaches.

RESULTSThe single-stain procedure could not detect the sperm viability differences. In contrast, the dual-stain procedure identified a dosage-dependent decrease in the viability and increased necrozoospermia after topoisomerase inhibitor CAM and GEN treatments. Apoptosis was 2-fold higher with topoisomerase inhibitor treatment.

CONCLUSIONThe two topoisomerase inhibitors were associated with increased apoptosis and dosage-dependent necrosis. The data suggested that the dual-stain combination Hoechst 33342/PI was more sensitive than the single Hoechst 33342 stain analysis and permitted quantitative analysis of the apoptosis and necrosis in sperm.

Apoptosis ; drug effects ; Benzimidazoles ; Camptothecin ; pharmacology ; Cell Survival ; drug effects ; DNA ; metabolism ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fluorescent Dyes ; Genistein ; pharmacology ; Humans ; Male ; Propidium ; Sensitivity and Specificity ; Spermatozoa ; drug effects ; physiology ; Staining and Labeling ; methods