cytochrome-c-t and thiazolyl-blue

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Topics: Cloning, Molecular; Cytochromes c; Enzyme Assays; Escherichia coli; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Fungal Proteins; Fusarium; Gene Expression; Genetic Vectors; Histidine; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; NADPH-Ferrihemoprotein Reductase; Oligopeptides; Oxidation-Reduction; Recombinant Fusion Proteins; Substrate Specificity; Temperature; Tetrazolium Salts; Thiazoles; Trichothecenes

Wasabia japonica (wasabi) has been shown to exhibit properties of detoxification, anti-inflammation and the induction of apoptosis in cancer cells. This study aimed to investigate the molecular mechanism of the cytotoxicity of wasabi extract (WE) in colon cancer cells to evaluate the potential of wasabi as a functional food for chemoprevention.. Colo 205 cells were treated with different doses of WE, and the cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Apoptosis and autophagy were detected by 4',6-diamidino-2-phenylindole, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-yanine iodide and staining for acidic vascular organelles (AVOs), along with Western blotting.. The results demonstrated that WE induced the extrinsic pathway and mitochondrial death machinery through the activation of TNF-α, Fas-L, caspases, truncated Bid and cytochrome C. WE also induced autophagy by decreasing the phosphorylation of Akt and mTOR and promoting the expression of microtubule-associated protein 1 light chain 3-II and AVO formation. An in vivo xenograft model verified that tumor growth was delayed by WE treatment.. Our studies revealed that WE exhibits anti-colon cancer properties through the induction of apoptosis and autophagy. These results provide support for the application of WE as a chemopreventive functional food and as a prospective treatment of colon cancer.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Apoptosis; Autophagy; BH3 Interacting Domain Death Agonist Protein; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cytochromes c; Humans; Indoles; Mice; Mice, Inbred BALB C; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Phosphorylation; Plant Extracts; Proto-Oncogene Proteins c-akt; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Wasabia; Xenograft Model Antitumor Assays

To determine if dioscin protects cardiac cells from ischemia/reperfusion (I/R) injury by preventing apoptosis.Cardiac H9c2 cells were subjected to simulated I/R. Cell viability was evaluated by the methyl thiazolyl tetrazolium (MTT) colorimetry assay. Reactive oxygen species (ROS) were detected with dichlorodihydrofluorescein (DCF). Apoptosis was evaluated with flow cytometric assay. Rhodamine 123 (Rho123) was used to measure mitochondrial membrane potential (ΔΨm). ELISA was used to detect cytochrome c (Cyt-c) release from mitochondria to the cytosol. Bax and Bcl-2 mRNA expressions were measured with RT-PCR.Dioscin reduced cell death and lactate dehydrogenase (LDH) release in cells subjected to I/R. I/R induced apoptosis and cytochrome c release from mitochondria to the cytosol and this was prevented by dioscin. In support, dioscin decreased Bax but increased Bcl-2 mRNA expression. Dioscin prevented I/R induced dissipation of ΔΨm. Finally, dioscin increased superoxide dismutase (SOD) expression but reduced intracellular ROS and malondialdehyde (MDA) levels.Dioscin protects H9c2 cells from H/R injury by modulating the mitochondrial apoptotic pathway through attenuation of oxidative stress.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Cardiovascular Agents; Cell Line; Coloring Agents; Cytochromes c; Diosgenin; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; L-Lactate Dehydrogenase; Malondialdehyde; Membrane Potential, Mitochondrial; Mitochondria; Myocardium; Myocytes, Cardiac; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase; Tetrazolium Salts; Thiazoles

Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

Topics: Animals; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cytochromes c; DNA Primers; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondria; Nicotinamide N-Methyltransferase; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles

A series of 7-amino- and 7-acetamidoquinoline-5,8-diones with aryl substituents at the 2-position were synthesized, characterized, and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1) -directed antitumor agents. The synthesis of lavendamycin analogues is illustrated. Metabolism studies demonstrated that 7-amino analogues were generally better substrates for NQO1 than 7-amido analogues, as were compounds with smaller heteroaromatic substituents at the C-2 position. Surprisingly, only two compounds, 7-acetamido-2-(8'-quinolinyl)quinoline-5,8-dione (11) and 7-amino-2-(2-pyridinyl)quinoline-5,8-dione (23), showed selective cytotoxicity toward the NQO1-expressing MDA468-NQ16 breast cancer cells versus the NQO1-null MDA468-WT cells. For all other compounds, NQO1 protected against quinoline-5,8-dione cytotoxicity. Compound 22 showed potent activity against human breast cancer cells expressing or not expressing NQO1, with respective IC50 values of 190 nM and 140 nM and a low NQO1-mediated reduction rate, which suggests that the mode of action of 22 differs from that of lavendamycin and involves an unidentified target(s).

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Coloring Agents; Cytochromes c; Drug Screening Assays, Antitumor; Electrochemistry; Humans; Indicators and Reagents; Magnetic Resonance Spectroscopy; Microwaves; Models, Molecular; NAD(P)H Dehydrogenase (Quinone); Oxygen Consumption; Quinolines; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles

Cisplatin remains one of the most effective current chemotherapeutic agents; however, metal complexes synthesis has increased in order to produce new anti-neoplastic drugs with DNA binding and apoptotic activities in tumor cells and less toxicity for patients. In this study, we evaluated the cytotoxic activity of a novel copper(II) complex (LQM402) against cervical cancer cell lines and found that LQM402 exhibited selective cytotoxicity against HeLa and Ca Ski cells. FITC-annexin assay and DNA fragmentation indicated that apoptosis could be involved in HeLa cell death. Caspase 3/7 and cytochrome c analysis by immunoblotting suggest the intrinsic pathway. LQM402 is a lipid peroxidation inductor according to TBARS production. Additionally, the Ames and micronucleus tests demonstrated non-genotoxic activity for this compound in Salmonella typhimurium and CD1 mice, respectively. Therefore, LQM402 may be a promising and safe anti-cervical cancer compound.

Topics: Animals; Annexins; Apoptosis; Brain Chemistry; Caspase 3; Caspase 7; Cell Line, Tumor; Coloring Agents; Coordination Complexes; Copper; Cytochromes c; Cytosol; DNA Fragmentation; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; HeLa Cells; Humans; In Situ Nick-End Labeling; Lipid Peroxidation; Mice; Micronucleus Tests; Mutagenicity Tests; Mutagens; Oxidants; Phosphatidylserines; Rats; Salmonella typhimurium; Tetrazolium Salts; Thiazoles; Uterine Cervical Neoplasms

Magnolol, a small-molecule hydroxylated biphenol, isolated from the root and stem bark of Magnolia officinalis, has been shown to possess antiproliferative effect on various cancer cell lines. In the current study, we found that magnolol potently inhibited proliferation and induced apoptosis in MCF-7 human breast cancer cells. Further mechanistic studies revealed that induction of apoptosis is associated with cell cycle arrest at G2/M phase, increased generation of reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP), release of cytochrome c (Cyto c) and apoptosis inducing factor (AIF) from mitochondria to cytosol, upregulation of Bax, p21 and p53, and down-regulation of Bcl-2, cyclin B1 and cyclin-dependent kinase 1 (CDK1). Our findings indicated that magnolol induced apoptosis in MCF-7 cells via the intrinsic pathway with release of AIF from mitochondrial and G2/M phase arrest pathway. Therefore, magnolol might be a potential lead compound in the therapy of breast cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Biphenyl Compounds; Bisbenzimidazole; Breast Neoplasms; Caspases; Cell Division; Cell Line, Tumor; Cell Proliferation; Coloring Agents; Cytochromes c; Female; Flow Cytometry; G2 Phase; Genes, bcl-2; Humans; Indicators and Reagents; Lignans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

Hirsutanol A is a novel sesquiterpene compound purified from fungus Chondrostereum sp. in Sarcophyton tortuosum. Our previous studies had demonstrated that hirsutanol A exhibited potent cytotoxic effect on many kinds of cancer cell lines. In the current study, the antitumor activity of hirsutanol A and its molecular mechanisms were investigated.. Hirsutanol A induced growth inhibition and apoptotic cell death of human colon cancer SW620 cells and human breast cancer MDA-MB-231cells were determined using MTT assay and flow cytometry assay, respectively. The effect of hirsutanol A on intrinsic ROS level and change in mitochondrial membrane potential (△ψm) of different cell lines were also measured by flow cytometry assay. The function of JNK was compromised by JNK siRNA or JNK inhibitor SP600125. The expression of cytochrome c, p-JNK, p-c-Jun after treatment with hirsutanol A were detected by Western blot analysis. Finally, the in vivo anti-tumor effect of hirsutanol A was examined in human cancer cell SW620 xenograft model.. The results showed that hirsutanol A significantly induced apoptosis, mitochondrial-independent increase of Reactive Oxygen Species (ROS) level, change of mitochondrial membrane potential, release of cytochrome c in human cancer cells. Preventing increase of ROS level using the potent antioxidant N-acetyl-L-cysteine (NAC) markedly decreased hirsutanol A-induced apoptosis. In addition, JNK signaling pathway was activated by hirsutanol A through elevating ROS level. Blockade of JNK signaling pathway by JNK specific inhibitor SP600125 enhanced apoptosis and hirsutanol A-induced ROS accumulation. Also, hirsutanol A exhibited antitumor activity in human cancer cell SW620 xenograft model.. These data suggested that hirsutanol A inhibited tumor growth through triggering ROS production and apoptosis.

Topics: Agaricales; Animals; Apoptosis; Cell Death; Cell Line, Tumor; Cytochromes c; Cytosol; Flow Cytometry; Humans; Membrane Potentials; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Neoplasm Transplantation; Reactive Oxygen Species; Sesquiterpenes; Signal Transduction; Tetrazolium Salts; Thiazoles

Hydroxyapatite nanoparticles (nano-HAP) have been reported to cause inflammatory reactions. Here, we aimed to compare the effects of four types of nano-HAP with different nanocrystal morphologies (short rod-like, long rod-like, spherical or needle-shaped crystals) and sizes (10-20, 10-30 or 20-40 nm) on growth inhibition and apoptosis in primary cultured rat osteoblasts. The osteoblasts was treated with the four types of nano-HAP at various concentrations (20, 40, 60, 80 or 100 mg l⁻¹). The nano-HAP specific surface area was detected using the Brunauer, Emmet and Teller method. The cell growth rate was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; apoptotic alterations and the level of reactive oxygen species in osteoblasts were measured using flow cytometry; and the amounts of apoptotic p53 and cytochrome c proteins were measured using western blotting. We observed that all four types of nano-HAP inhibited the growth of osteoblasts in a dose-dependent manner. These nano-HAP significantly induced apoptosis in osteoblasts. Nano-HAP with smaller specific surface areas induced lower apoptosis rates. The needle-shaped and the short rod-like particles induced greater cellular injury than the spherical and long rod-like particles, respectively. The increased apoptosis rates were accompanied by increased p53 and cytochrome c expression. These findings indicate that nano-HAP inhibit the activity of osteoblasts and also induce the apoptosis of osteoblasts in vitro. These findings also suggest that the nano-HAP-induced apoptotic pathway is mediated by a mitochondrial-dependent pathway. Moreover, the sizes, morphologies and concentrations of nano-HAP have significant effects on the apoptotic level.

Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Cell Proliferation; Cell Survival; Cells, Cultured; Coloring Agents; Crystallization; Cytochromes c; Dose-Response Relationship, Drug; Durapatite; Nanoparticles; Osteoblasts; Oxidative Stress; Particle Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in regulating the adaptive response to hypoxia. HIF-1α is stabilised during hypoxia and, after dimerisation with hypoxia-inducible factor 1β (HIF-1β), triggers the expression of various genes involved in cell cycle control and energy metabolism associated with cell survival. However, HIF-1α also regulates the expression of proapoptotic genes. The aim of this study was to ascertain the influence of HIF-1α on neurotoxicity evoked by hypoxia in rat cortical neurons. We found that mild hypoxia induces time-dependent neuronal death involving free radical production, mitochondrial depolarisation, cytochrome c release and caspase-3 activation. Lentivirus-mediated HIF-1α knockdown markedly strengthened all of these effects during the initial 24h of hypoxia, which suggests that HIF-1α plays a neuroprotective role in hypoxia-mediated neuronal death. After this initial period, the protective actions of HIF-1α disappeared over the course of the hypoxia-mediated HIF-1α stabilisation. Moreover, lentiviral-mediated overexpression of HIF-1α increased lactate dehydrogenase (LDH) A, one of the target genes for HIF-1α, but did not show protective actions on hypoxia-mediated neuronal death, indicating that the level of endogenous HIF-1α stabilisation achieved during hypoxia was already the maximum required for HIF-1α transcription activities. These results indicate that HIF-1α is neuroprotective in the early phases of hypoxia.

Topics: Animals; Animals, Newborn; Cell Death; Cells, Cultured; Cerebral Cortex; Cyclooxygenase 1; Cytochromes c; Dose-Response Relationship, Drug; Free Radical Scavengers; Gene Expression Regulation; Glutathione; Green Fluorescent Proteins; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Membrane Proteins; Metalloporphyrins; Neurons; Oxygen; Phenanthridines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA Interference; Tetrazolium Salts; Thiazoles; Time Factors

Epidemiological data have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer's disease (AD). Phytoestrogens have been proposed as potential alternatives to ERT. The aim of the present study was to assess the neuroprotective effects of puerarin, a phytoestrogen isolated from Pueraria lobata, against the toxicity of beta-amyloid (Aβ) in relation to the mitochondria-mediated cell death process, and to elucidate the role the activation of Akt and modulation of the pro- and antiapoptotic proteins in puerarin-induced neuroprotection. The present study shows that puerarin afforded protection against Aβ-induced toxicity through inhibiting apoptosis in PC12 cells. This result was also confirmed by the activated caspase-3 assay. P-Akt, Bcl-2 and p-Bad expression increased after pretreatment with puerarin in PC12 cells exposed to Aβ(25-35), whereas Bax expression and cytochrome c release decreased. Interestingly, these effects of puerarin against Aβ(25-35) insult were abolished by wortmannin, an inhibitor of PI3K phosphorylation. These findings suggest that puerarin prevent Aβ-induced neurotoxicity through inhibiting neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD.

Topics: Amyloid beta-Peptides; Animals; Annexin A5; Apoptosis; bcl-2-Associated X Protein; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Expression Regulation; Isoflavones; Neuroprotective Agents; Oncogene Protein v-akt; PC12 Cells; Peptide Fragments; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles

Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs).. Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed.. Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage.. These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Culture Techniques; Cell Nucleus; Cell Survival; Coloring Agents; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fluoresceins; Fluorescent Dyes; Gingiva; Humans; In Situ Nick-End Labeling; Indoles; JNK Mitogen-Activated Protein Kinases; Nicotine; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Time Factors

Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 μM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 μM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 μM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

Topics: Antioxidants; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Dopamine; Dose-Response Relationship, Drug; Glutathione; Humans; Neuroblastoma; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue; Ubiquinone

Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 2; Caspase Inhibitors; Comet Assay; Cytochromes c; DNA Damage; DNA Topoisomerases, Type II; Enzyme Inhibitors; Flow Cytometry; Glutathione; Goniothalamus; Humans; Jurkat Cells; Oligopeptides; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Pyrones; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Thiazoles

Altered glial function may contribute to the initiation or progression of neuronal death in neurodegenerative diseases. Thus, modulation of astrocyte death may be essential for preventing pathological processes in the CNS. In recent years, metabotropic glutamate receptor (mGluR) activation has emerged as a key target for neuroprotection. We investigated the effect of subtype 3 mGluR (mGluR3) activation on nitric oxide (NO)-induced astroglial death. A mGluR3 selective agonist, LY379268, reduced inducible NO synthase expression and NO release induced by bacterial lipopolysaccharide and interferon-gamma in cultured rat astrocytes. In turn, a NO donor (diethylenetriamine/NO) induced apoptotic-like death in cultured astrocytes, which showed apoptotic morphology and DNA fragmentation, but no caspase 3 activation. LY379268 prevented astrocyte death induced by NO exposure, which correlates with a reduction in: phosphatidylserine externalization, p53 and Bax activation and mitochondrial permeability. The reported effects of LY379268 were prevented by the mGluR3 antagonist (s)-alpha-ethylglutamic acid. All together, these findings show the protective effect of mGluR3 activation on astroglial death and provide further evidence of a role of these receptors in preventing CNS injury triggered by several inflammatory processes associated with dysregulated NO production.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acids; Animals; Animals, Newborn; Astrocytes; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cell Death; Cells, Cultured; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Gene Expression Regulation; In Situ Nick-End Labeling; Interferon-gamma; Neurotransmitter Agents; Nitric Oxide; Nitric Oxide Donors; Phosphatidylserines; Phosphodiesterase Inhibitors; Polyamines; Polysaccharides; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Tetrazolium Salts; Thiazoles

Increasing evidence suggests that oxidative stress (OS)-induced pancreatic beta-cell impairments is involved in diabetes and diabetic complications. Our group has recently synthesized two multifunctional nontoxic, lipophilic, iron-chelating drugs, 5-{N-methyl-N-propargylaminomethyl}-8-hydroxyquinoline (M30) and 5-{4-propargylpiperazin-1-ylmethyl}-8-hydroxyquinoline (HLA20), for the treatment of various OS-mediated pathogeneses. These compounds contain the N-propargylamine cytoprotective moiety of the antiparkinsonian drug rasagiline (Azilect) and the iron-complexing component 8-hydroxyquinoline. The aim of this research was to evaluate the protective effect of the multifunctional iron-chelating drugs on rat insulin-producing pancreatic beta-cells (INS-1E and RINm) against OS-induced cytotoxicity. We found that M30 and HLA20 markedly and dose-dependently inhibited H(2)O(2)-induced cytotoxicity, associated with decreased intracellular reactive oxygen species formation and increased catalase activity. In accordance, the catalase inhibitor 3-amino-1,2,4-triazol blocked the protective action of M30 against H(2)O(2)-induced damage. Both compounds significantly increased the levels of the iron-responsive protein transferrin receptor indicating their iron-chelating effect. Further mechanistic studies showed that M30 and HLA20 attenuated H(2)O(2)-induced mitochondrial membrane potential loss, decreased the release of cytochrome c into the cytoplasm, and inhibited the activation of caspase-3, suggesting that these drugs may produce cytoprotective effects via the preservation of mitochondrial function. These results indicate that the novel drugs, M30 and HLA20 display significant cytoprotective activity against OS-induced cytotoxicity in insulin producing beta-cells, which might be of therapeutic use in the treatment of diabetes mellitus.

Topics: Animals; Antioxidants; Apoptosis; Blotting, Western; Caspase 3; Catalase; Cell Line; Cell Survival; Coloring Agents; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Hydrogen Peroxide; Hydroxyquinolines; Insulin-Secreting Cells; Iron Chelating Agents; Membrane Potentials; Mitochondria; Monoamine Oxidase Inhibitors; Oxidants; Oxidative Stress; Piperazines; Rats; Signal Transduction; Tetrazolium Salts; Thiazoles

Antimycin A (AMA) treatment of cells blocks mitochondrial electron transport chain, and leads to elevated ROS generation, thereby causing damage to mtDNA, proteins and lipids, along with mitochondrial membrane depolarization, release of pro-apoptotic proteins into the cytoplasm, and induction of apoptosis. Prevention of such oxidative cellular damage by the aqueous extract of Phyllanthus amarus has been investigated in this study. The extract demonstrated significant potential in mitigating H(2)O(2)-induced membrane damage along with considerable recession in AMA-governed mitochondrial protein and lipid degradation in Hep3B cells. 8-OHdG analysis of mtDNA damage revealed substantial protective potential of the extract against mtDNA damage. SQ-PCR of selected mtDNA sequences confirmed the potential of the extract to alleviate levels of mtDNA damage. FACS analysis with JC-1 fluorescent dye established significant escalation of mitochondrial membrane potential by the extract in AMA-treated cells. Extract treatment resulted in a distinct decline in the degrees of AMA-induced release of cytochrome c and AIF into the cytoplasm along with consequent pacification of apoptosis. All protective efficiencies of the extract reported in this study were found to hold strong and significant (P<0.05) positive correlation to its total phenolic contents, thereby proving that polyphenolic constituents of P. amarus aqueous extract mitigate oxidative stress-induced cellular degeneration and aging.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Anti-Bacterial Agents; Antimycin A; Apoptosis; Apoptosis Inducing Factor; Bromodeoxyuridine; Cell Line; Coloring Agents; Cytochromes c; Deoxyguanosine; DNA, Mitochondrial; Flow Cytometry; Humans; In Vitro Techniques; Indicators and Reagents; Lipid Peroxidation; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Phenols; Phyllanthus; Plant Extracts; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Autoradiography; Benzoquinones; Caspase 3; Cell Line, Tumor; Copper; Cytochromes c; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; NAD; Neuroblastoma; Phenanthrolines; Phosphorus Isotopes; Salsoline Alkaloids; Tetrahydroisoquinolines; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine 3-Monooxygenase

Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11-O-hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor gamma activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c, cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo, where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.

Topics: Analysis of Variance; Animals; Annexin A5; Antineoplastic Agents; bcl-2-Associated X Protein; Brain Neoplasms; Bromodeoxyuridine; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Glioma; Humans; Ligands; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Pioglitazone; PPAR gamma; Proto-Oncogene Proteins c-bcl-2; Rats; Retinoid X Receptor gamma; Tetrazolium Salts; Thiazoles; Thiazolidinediones; Transfection; Tretinoin; Tumor Stem Cell Assay

Mitochondrial calcium (Ca(2+)) dyshomeostasis constitutes a critical step in the metabolic crossroads leading to cell death. Therefore, we have studied here whether 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; CGP), a blocker of the mitochondrial Na(+)/Ca(2+)-exchanger (mNCX), protects against veratridine-elicited chromaffin cell death, a model suitable to study cell death associated with Ca(2+) overload. Veratridine produced a concentration-dependent cell death, measured as lactate dehydrogenase released into the medium after a 24-h incubation period. CGP rescued cells from veratridine-elicited death in a concentration-dependent manner; its EC(50) was approximately 10 microM, and 20 to 30 microM caused near 100% cytoprotection. If preincubated for 30 min and washed out for 3 min before adding veratridine, CGP still afforded significant cytoprotection. At 30 microM, CGP blocked the veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release. At this concentration, CGP also inhibited the Na(+) and Ca(2+) currents by 50 to 60% and the veratridine-elicited oscillations of cytosolic Ca(2+). This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX.

Topics: Animals; Calcium Channels; Calcium Signaling; Cattle; Cell Death; Cells, Cultured; Chromaffin Cells; Clonazepam; Coloring Agents; Cytochromes c; L-Lactate Dehydrogenase; Membrane Potentials; Mitochondria; Patch-Clamp Techniques; Reactive Oxygen Species; Sodium Channels; Sodium-Calcium Exchanger; Tetrazolium Salts; Thiazepines; Thiazoles; Veratridine

Nitric oxide (NO) is a major factor contributing to the loss of neurons in ischemic stroke, demyelinating diseases, and other neurodegenerative disorders. NO not only functions as a direct neurotoxin, but also combines with superoxide (O(2)(-)) by a diffusion-controlled reaction to form peroxynitrite (ONOO(-)), a species that contributes to oxidative signaling and cellular apoptosis. However, the mechanism by which ONOO(-) induces apoptosis remains unclear, although subsequent formation of reactive oxygen species (ROS) has been suggested. The aim of this study was to further investigate the triggers of the apoptotic pathway using O(2)(-) scavenging with light irradiation to block ONOO(-) production. Antiapoptotic effects of light irradiation in sodium nitroprusside (SNP)-treated SH-SY5Y cells were assayed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation, flow cytometry, Western blot, and caspase activity assays. In addition, NO, total ROS, O(2)(-), and ONOO(-) levels were measured to observe changes in NO and its possible involvement in radical induction. Cell survival was reduced to approximately 40% of control levels by SNP treatment, and this reduction was increased to 60% by low-level light irradiation. Apoptotic cells were observed in the SNP-treated group, but the frequency of these was reduced in the irradiation group. NO, O(2)(-), total ROS, and ONOO(-) levels were increased after SNP treatment, but O(2)(-), total ROS, and ONOO(-) levels were decreased after irradiation, despite the high NO concentration induced by SNP treatment. Cytochrome c was released from mitochondria of SNP-treated SH-SY5Y cells, but not of irradiated cells, resulting in a decrease in caspase-3 and -9 activity in SNP-treated cells. Finally, these results show that 635-nm irradiation, by promoting the scavenging of O(2)(-), protected against neuronal death through blocking the mitochondrial apoptotic pathway induced by ONOO(-) synthesis.

Topics: Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Humans; Light; Mitochondria; Neurons; Nitric Oxide; Nitroprusside; Peroxynitrous Acid; Superoxides; Tetrazolium Salts; Thiazoles

The proteasome inhibitor model of Parkinson's disease (PD) appears to reproduce many of the important behavioural, imaging, pathological and biochemical features of the human disease. However, the mechanisms involved in the lactacystin-induced, mitochondria-mediated apoptotic pathway remain poorly defined.. We have used lactacystin as a specific inhibitor of the 20S proteasome in the dopaminergic neuroblastoma cell line SH-SY5Y. We over-expressed a green fluorescent protein (GFP)-Bax fusion protein in these cells to study localization of Bax. Free radical scavengers were used to assess the role of reactive oxygen species (ROS) in these pathways.. Lactacystin triggered a concentration-dependent increase in cell death mediated by the mitochondrial apoptotic pathway, and induced a change in mitochondrial membrane permeability accompanied by cytochrome c release. The participation of Bax protein was more critical than the formation of the permeability transition pore in mitochondria. GFP-Bax over-expression demonstrated Bax redistribution from the cytosol to mitochondria after the addition of lactacystin. ROS, but not p38 mitogen-activated protein kinase, participated in lactacystin-induced mitochondrial Bax translocation. Lactacystin disrupted the intracellular redox state by increasing ROS production and depleting endogenous antioxidant systems such as glutathione (GSH). Pharmacological depletion of GSH, using L-buthionine sulphoxide, potentiated lactacystin-induced cell death. Lactacystin sensitized neuroblastoma cells to oxidative damage, induced by subtoxic concentrations of 6-hydroxydopamine.. The lactacystin-induced, mitochondrial-mediated apoptotic pathway involved interactions between ROS, GSH and Bax. Lactacystin could constitute a potential factor in the development of sporadic PD.

Topics: Acetylcysteine; Apoptosis; bcl-2-Associated X Protein; Cell Death; Cell Line, Tumor; Cell Survival; Coloring Agents; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria, Liver; Neuroblastoma; Reactive Oxygen Species; Recombinant Fusion Proteins; Tetrazolium Salts; Thiazoles

Parkinson disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been proposed that dysfunction of the ubiquitin proteasome system plays an important role in the pathogenesis of Parkinson disease, but the mechanisms underlying ubiquitin proteasome system-related neuron degeneration are unknown. Here, we demonstrate that the proteasome inhibitor lactacystin induces phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, the release of cytochrome c, activation of both caspase-9 and caspase-3, and sequential apoptosis of dopaminergic neurons in vitro. Most of these effects can be attenuated by the JNK inhibitor SP600125. Furthermore, infusion of lactacystin in rats in vivo also leads to phosphorylation of JNK before nigral neuron loss; chronic administration of SP600125 also blocks this loss. These results indicate that JNK is involved in proteasome inhibition-induced dopaminergic neuron degeneration through caspase-3-mediated apoptotic pathways, suggesting that this kinase may be a therapeutic target for the prevention of substantia nigra pars compacta degeneration in Parkinson disease patients.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Line; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; Cytochromes c; Dopamine; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Male; Nerve Degeneration; Neurons; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Substantia Nigra; Tetrazolium Salts; Thiazoles

Total saponin of Solanum lyratum Thunb (TSSLT), a species of natural biologically active substances isolated from Solanum lyratum Thunb, possesses various bioactivities. It has been proposed that the induction of apoptosis may be the basis of its antitumor activity. However, the molecular mechanism underlying the total saponin-induced apoptotic process remains unknown. In the present study, we describe the anti-proliferative effect of TSSLT on human cervical cancer cells (Hela). The TSSLT induced apoptosis of Hela in a time-dependent manner with an IC50 for cell viability of 6 microg/ml. The TSSLT-induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-like activities, poly (ADP-ribose) polymerase (PARP) cleavage and release of cytochrome c (cyt c) into cytosol. TSSLT activated various caspases such as caspase-3, -8, and -9 (like) activities but not caspase-1 like activity. The cell death was completely prevented by the pan caspase inhibitor benzyloxy carbonyl-Val-Ala-Asp- fluoromethyl-ketone (Z-VAD-FMK). More than 80% cell survival was observed in the presence of a caspase-3 inhibitor. In addition, treatment with TSSLT induced the increase of Bax:Bcl-2 ratio in Hela cells. These results suggest that the induction of apoptosis by TSSLT involves multiple pathways antigen including death receptor and mitochondrial pathway and strongly suggest that the mitochondrial pathway was mediated by low expression of Bcl-2 and upregulation of Bax, release of cyt c and subsequent activation of caspase-3 followed by down stream events leading to apoptotic cell death.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cell Death; Cell Proliferation; Cytochromes c; DNA Fragmentation; Flow Cytometry; HeLa Cells; Humans; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Saponins; Signal Transduction; Solanum; Tetrazolium Salts; Thiazoles

In our previous study, Abrus agglutinin showed antitumor activity both native and heat-denatured condition in mouse model. The purpose of this study is to explore the presence of anticancer peptide in agglutinin, and to elucidate the mechanism of its activity in vitro. A tryptic digested Abrus agglutinin peptide fractions obtained from 10-kDa molecular weight cut off membrane permeate (10 kMPP), was found to have selective antiproliferative activity (1-10 microg/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with dose of 100 microg/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed nuclear fragmentation and condensation with appearance of the sub-G 0/G1 peak indicative of apoptosis. Furthermore, the peptide fraction induced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3, finally leads to DNA fragmentation, and the hallmark of apoptosis. LC-MS/MS analysis reflected the molecular masses of peptides in 10 kMPP were in the range of 500 Da to 3000 Da. The significant antitumor activity of 10 kMPP deserves further laboratory and in vivo experimentation.

Topics: Abrus; Agglutinins; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; HeLa Cells; Humans; Indicators and Reagents; Membrane Potentials; Mitochondria; Molecular Weight; Peptides; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Tumor Stem Cell Assay

Bidens alba has been used for healing cuts, injuries, swellings, hypertension, jaundice, and diabetes in some countries. However, the effect of B. alba on human cancer remains poorly understood. The goal of this study was to investigate whether B. alba protein-extract could have an anticancer property against human colorectal cancer. The human colorectal cancer SW 480 cells treated with the protein-extract of B. alba would cause marked DNA damages and apoptosis-related cellular morphologies. Treatment with 225 microg/ml B. alba protein-extract also led to the SW480 cells to produce readily intracellular reactive oxygen species (ROS) after 1h of treatment and last to 24 h. The intracellular glutathione (GSH) depletion occurred after 12-24h of treatment. The treatment of the protein-extract would also caused mitochondrial transmembrane potential (DeltaPsi(m)) to decrease and cytosolic cytochrome c to increase. The caspase 3/7 activities were activated from 3 to 6 h after the treatment. The percentages of apoptosis induced by the protein-extract of B. alba decreased 26.4%, 10.1%, and 29.4% when the SW 480 cells were pretreated with Vitamin C, N-acetylcysteine, and Boc-Asp(OMe)-fmk, respectively. Taken together, we demonstrated for the first time that the protein-extract of B. alba could induce apoptosis that was related to the ROS production and GSH depletion in human colorectal cancer. The protein-extract of B. alba might have therapeutic value against the human colorectal cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bidens; Caspases; Cell Count; Cell Cycle; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cytochromes c; Cytosol; Flow Cytometry; Glutathione; Humans; In Situ Nick-End Labeling; Indicators and Reagents; Mitochondria; Plant Extracts; Plant Proteins; Reactive Oxygen Species; Superoxide Dismutase; Tetrazolium Salts; Thiazoles

Upper gastrointestinal adenocarcinomas are a common cause of cancer-related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining on tumor tissue microarrays (TMA) were used to study the expression of AURKA in upper gastrointestinal adenocarcinomas. To investigate the biological and signaling impact of AURKA, the authors used multiple in vitro assays that included 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling), cytochrome C release, flow cytometry, luciferase reporter, and Western blot analysis.. Frequent overexpression of AURKA transcript in upper gastrointestinal adenocarcinomas was detected compared with normal samples (47%; P= .001). The immunohistochemical analysis of 130 tumors demonstrated moderate-to-strong immunostaining of AURKA in >50% of upper gastrointestinal adenocarcinomas. By using camptothecin as a drug-induced apoptosis in vitro model, the authors demonstrated that the expression of AURKA provided protection against apoptosis to gastrointestinal cancer cells (AGS and RKO) (P= .006) and RIE-1 primary intestinal epithelial cells (P= .001). The AURKA overexpression mediated an increase in phosphorylation of AKT(Ser473) with an increase in HDM2 level. The shRNA-knockdown of AKT in AURKA-overexpressing cells reversed this effect and showed a significant increase in the p53 protein level, indicating a possible nexus of AURKA/AKT/p53. Indeed, overexpression of AURKA led to a remarkable reduction in the transcription activity of p53, with subsequent reductions in transcript and protein levels of its downstream proapoptotic transcription targets (p21, BAX, NOXA, and PUMA).. Study results indicated that AURKA provides potent antiapoptotic properties to gastrointestinal cells by regulating levels of p53 through the AKT/HDM2 axis.

Topics: Adenocarcinoma; Apoptosis; Aurora Kinase A; Aurora Kinases; Biomarkers, Tumor; Blotting, Western; Camptothecin; Coloring Agents; Cytochromes c; Enzyme Inhibitors; Esophageal Neoplasms; Flow Cytometry; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Luciferases; Luminescent Agents; Polymerase Chain Reaction; Protein Array Analysis; Protein Serine-Threonine Kinases; Stomach Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Suppressor Protein p53

Berberine, a main component of Coptidis Rhizoma, has been extensively studied and is known to exhibit multiple pharmacologic activities. In this study, we investigated whether the combination of berberine and cisplatin exhibited significant cytotoxicity in HeLa cells. Apoptosis was evaluated based on DNA fragmentation and cytofluorometrically with the annexin-V/propidium iodide labeling method. Combined treatment with berberine and cisplatin acted in concert to induce loss of mitochondrial membrane potential (Delta Psi m), release of cytochrome-c from mitochondria, and decreased expression of antiapoptotic Bcl-2, Bcl-x/L, resulting in activation of caspases and apoptosis. Further study showed that cell death induced by the combined treatment was associated with increased reactive oxygen species generation and lipid peroxidation. Moreover, we discovered that the combined treatment-induced apoptosis was mediated by the activation of the caspase cascade. These results indicated that the potential of cytotoxicity mediated through the mitochondria-caspase pathway is primarily involved in the combined treatment-induced apoptosis.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Caspases; Cisplatin; Cytochromes c; DNA Fragmentation; Drug Synergism; Enzyme Activation; HeLa Cells; Humans; Indicators and Reagents; Lipid Peroxidation; Membrane Potentials; Mitochondria; Reactive Oxygen Species; Signal Transduction; Tetrazolium Salts; Thiazoles

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Catechin; Cytochromes c; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; PC12 Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Time Factors; Voltage-Dependent Anion Channels

The cytokine tumour necrosis factor-alpha (TNFalpha) has been implicated in the pathogenesis of neurodegenerative conditions as well as in the establishment of neural networks during development. This study investigated the in vitro effects of TNFalpha on embryonic dopaminergic neurons of the ventral mesencephalon. TNFalpha treatment enhanced the number of dopaminergic neurons in cultures derived from E12.5 mice embryos in a dose-dependent manner. In order to achieve this effect TNFalpha signalled via NF-kappaB. This enhancement in cell number was found to be due to TNFalpha promoting the differentiation of dopaminergic neurons rather than to an increase in cell survival. In contrast, TNFalpha-treated cultures derived from E14 or E16 mice demonstrated a decrease in dopaminergic neurons, and this loss was negated by pharmacological inhibition of caspases. The data presented demonstrate that during embryonic development, dopaminergic ventral mesencephalic neurons switch their in vitro response to TNFalpha from neurotrophic to neurotoxic. This is the first report of a population of neurons exhibiting this switch in TNFalpha responsiveness during neurodevelopment.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cytochromes c; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Female; Floxuridine; Glial Fibrillary Acidic Protein; Mesencephalon; Mice; Mice, Inbred C57BL; Neurons; Peptides, Cyclic; Pregnancy; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Ceramides; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme Activation; Glutathione; HL-60 Cells; Humans; Immunoblotting; Indicators and Reagents; Mitochondria; Reactive Oxygen Species; Signal Transduction; Spectrophotometry, Ultraviolet; Tetrazolium Salts; Thiazoles

Cordyceps militaris is a traditional herbal ingredient frequently used for tonic and medicinal purposes in eastern Asia. The hot water extract of its cultivated fruiting bodies demonstrated a potent cytotoxic effect against the proliferation of the human premyelocytic leukemia cell HL-60, with an IC50 of 0.8 mg/ml for a 12-h treatment. It induced the characteristic apoptotic symptoms in the HL-60 cells, including DNA fragmentation and chromatin condensation, occurring within 12-16 h of treatment at a dose of 1 mg/ml. The activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase were detected during the course of apoptosis induction. These results indicate that the hot water extract of Cordyceps militaris fruiting bodies inhibited cancer cell proliferation by inducing cell apoptosis through the activation of caspase-3, and that the Cordyceps militaris extract may therefore have therapeutic potential against human leukemia.

Topics: Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Proliferation; Cell Survival; Cordyceps; Cytochromes c; DNA Fragmentation; DNA, Neoplasm; Electrophoresis; Enzyme Activation; HL-60 Cells; Humans; Poly(ADP-ribose) Polymerases; Tetrazolium Salts; Thiazoles

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. Depending on the exposure conditions, Cd may cause necrosis or apoptosis. Oxidative stress is believed to participate in Cd toxicity but the molecular signaling responsible for Cd-induced apoptosis in non-malignant liver cells still needs to be clarified. Therefore we have studied apoptosis in primary cultures of rat hepatocytes incubated with low levels of Cd for short exposure times. Studies of nuclear morphology, chromatin condensation, and oligonucleosomal DNA fragmentation demonstrate that 1-5 microM Cd induces apoptosis as early as 6-12 h with minor effects on MTT activity. A concomitant time- and concentration-dependent increase in caspase-9 and -3 activities was observed, whereas Cd did not affect caspase-8 activity as much, suggesting a minor role of the death-receptor pathway. Significant release of cytochrome c into the cytosol demonstrated the involvement of a mitochondrial-dependent apoptotic pathway. However, cell pre-treatment with caspase inhibitors (Z-VAD-fmk or Ac-DEVD-CHO) did not prevent apoptosis. Increases in the cytosolic levels of the mitochondrial apoptosis-inducing factor (AIF) were also observed: kinetics of cytochrome c and AIF release were similar. These results show that Cd-induced apoptosis in rat hepatocytes is time- and concentration-dependent. The early apoptotic events involved mitochondrial-dependent pathways but not necessarily caspase-dependent signaling.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Bisbenzimidazole; Cadmium; Caspase 3; Caspases; Cell Separation; Cell Survival; Cells, Cultured; Chromatin; Cytochromes c; Cytosol; DNA; DNA Fragmentation; Hepatocytes; Male; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetrazolium Salts; Thiazoles

Several studies have shown that violacein, a purple pigment extracted from Chromobacterium violaceum, is capable to induce apoptosis in a variety of cancer cells, including those leukemia cell lines. Herein, we examined the effects of violacein on reactive oxygen species (ROS) production during the apoptotic colon cancer cell death. We demonstrate that violacein mediates ROS production followed by activation of Caspase-3, release of cytochrome c, and calcium release to citosol in Caco-2 cells. Moreover, presence of ROS scavengers such as N-acetyl-cysteine (NAC) diminishes ROS cytotoxicity induced by violacein in Caco-2 cells, indicating that violacein mediates cellular critical mechanisms in the triggering of apoptotic tumor cell death. These data also imply that violacein-induced ROS are collectively key mediators of mitochondrial membrane collapse, leading to cytochrome c release, and culminating in tumor apoptosis. Unlike in Caco-2 cells, violacein was incapable of increasing ROS levels in HT29 cells, suggesting the existence of violacein cell-type specific mechanisms. Those findings bring light to the violacein cytotoxic mechanism studies, indicating that oxidative stress play a role in the violacein-induced cytotoxicity.

Topics: Apoptosis; Caco-2 Cells; Calcium; Caspase 3; Cell Death; Cell Survival; Colonic Neoplasms; Cytochromes c; Enzyme Activation; HT29 Cells; Humans; Indicators and Reagents; Indoles; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22 microM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.

Topics: Apoptosis; Apoptosis Inducing Factor; Camptothecin; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytoplasm; DNA Fragmentation; Enzyme Inhibitors; Hepatocytes; Humans; Membrane Potential, Mitochondrial; Mitochondria; Tetrazolium Salts; Thiazoles

The effects of diallyl disulfide (DADS), a garlic-derived compound, on the viability of neuronal cells and cell signals, including phosphatidylinositol 3-kinase (PI3K)/Akt, glycogen synthase kinase-3 (GSK-3), cytochrome c, caspase-3, and poly(ADP-ribose) polymerase (PARP), were investigated in PC12 cells neuronally differentiated by nerve growth factor. To evaluate the toxicity of DADS itself, nPC12 cells were treated with several concentrations of DADS, and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue stain revealed that the viability was not affected by low concentration of DADS, up to 20 microM, but it was decreased at higher than this concentration. The levels of free radicals and membrane lipid peroxidation were significantly increased in nPC12 cells when treated with more than 50 microM DADS, and treatment of PC12 cells with 100 microM DADS killed the cells by inhibiting PI3K/Akt and by promoting activation of GSK-3 and caspase-3, release of cytochrome c, and cleavage of PARP. To evaluate the protective effects of low concentration of DADS on oxidative stress-injured nPC12 cells, the viability of the cells (pretreated with DADS for 2 h vs. not pretreated) was evaluated 24 h after exposure to 100 microM H2O2 for 30 min. Compared to the cells treated with 100 microM H2O2 only, pretreatment of the cells with 20 microM DADS before exposure to 100 microM H2O2 increased the viability and induced activation of PI3K and Akt, inactivation of GSK-3, and inhibition of cytochrome c release, caspase-3 activation, and PARP cleavage. These results indicate that low concentration of DADS has neuroprotective effects by activating PI3K/Akt and by inhibiting GSK-3 activation, cytochrome c release, caspase-3 activation, and PARP cleavage, whereas high concentration is rather cytotoxic. Therefore, some specific optimum concentration of DADS may be a new potential therapeutic strategy for oxidative stress injured in vitro model of neurodegenerative diseases.

Topics: Allyl Compounds; Animals; Antihypertensive Agents; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Count; Cell Differentiation; Cell Survival; Chromones; Cytochromes c; Disulfides; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Fluoresceins; Gene Expression Regulation; Glycogen Synthase Kinase 3; Hydrogen Peroxide; Indoles; Membrane Glycoproteins; Morpholines; Neurons; Oxidative Stress; PC12 Cells; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Tetrazolium Salts; Thiazoles; Thiobarbituric Acid Reactive Substances; Trypan Blue

While agents targeting estrogen receptors are most effective in adjuvant therapy for human breast cancers expressing estrogen receptors after surgery, breast cancers lacking estrogen receptor are clinically serious, because they are highly malignant and exhibit resistance to the usual anti-cancer drugs, including estrogen receptor-antagonists and DNA breaking agents. Here, we found that MX-1, a human breast cancer cell line lacking estrogen receptors, exhibited higher AP-1 activity and expressed higher levels of c-Jun, c-Fos, and Fra-1 when compared with conventional estrogen receptor-positive human breast cancer cell lines. The prenylphenol antibiotic ascochlorin suppressed the AP-1 activity of MX-1 cells, and selectively killed MX-1 cells, partly due to induction of apoptosis. Our results suggest that AP-1 is an effective clinical target molecule for the treatment of estrogen receptor-negative human breast cancer.

Topics: Alkenes; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Line, Tumor; Cell Nucleus; Cell Survival; Coloring Agents; Cytochromes c; DNA Damage; Dose-Response Relationship, Drug; Genes, Reporter; Humans; Luciferases; Phenols; Plasmids; Receptors, Estrogen; Tetrazolium Salts; Thiazoles

Despite major advances, multiple myeloma (MM) remains an incurable malignancy. Recently we have found that disease stabilization was achieved in 64% of patients with advanced MM treated with the farnesyltransferase inhibitor R115777 (Zarnestra) in a phase 2 clinical trial. In order to enhance R115777 antitumor activity in MM, we examined the combination of this novel agent with other anticancer drugs in MM cell lines. In this study, R115777 was found to synergize with paclitaxel and docetaxel, but not with other chemotherapy agents, including doxorubicin, 5-fluorouracil, cisplastin, melphalan, mitoxantrone, and dexamethasone. R115777 synergized with paclitaxel to inhibit MM cell proliferation and to induce apoptosis. Synergism in the induction of apoptosis was accompanied by increase in cytochrome c release and caspase-3 activation. Furthermore, flow cytometry analysis also showed that paclitaxel and R115777 synergized to induce G(2)/M cell-cycle arrest. Importantly, synergism was observed in taxane- and R115777-resistant MM cells. In the human severe combined immunodeficient (SCID-hu) bone model of myeloma growth, the ability of paclitaxel to inhibit tumor growth in vivo was enhanced by R115777. Combination of paclitaxel or docetaxel with R115777 in the treatment of MM cells from patients with multiple myeloma was more beneficial than treatment with single agents. Our results provide the basis for combination therapy clinical trials with paclitaxel or docetaxel with R115777 in MM patients.

Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Marrow Cells; Caspase 3; Caspases; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Proliferation; Cisplatin; Clinical Trials as Topic; Cytochromes c; Deoxycytidine; Docetaxel; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Enzyme Activation; Farnesyltranstransferase; Flow Cytometry; Fluorouracil; G2 Phase; Gemcitabine; Homozygote; Humans; In Situ Nick-End Labeling; Inhibitory Concentration 50; Mice; Mice, SCID; Mitosis; Multiple Myeloma; Paclitaxel; Quinolones; Taxoids; Tetrazolium Salts; Thiazoles

Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 microM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

Topics: Antifungal Agents; Apoptosis; Blotting, Western; Brain Neoplasms; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cytochromes c; Humans; L-Lactate Dehydrogenase; Microscopy, Fluorescence; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Subcellular Fractions; Tetrazolium Salts; Thiazoles; Thimerosal

Point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase-3 (GSK-3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimer's disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol-3-kinase (PI3-K)/Akt and GSK-3 pathway as well as the caspase-3 pathway, VSC4.1 motoneuron cells were transfected with G93A- or A4V-mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24 h after neuronal differentiation, their viability and intracellular signals, including PI3-K/Akt, GSK-3, heat shock transcription factor-1 (HSTF-1), cytochrome c, caspase-3 and poly(ADP-ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK-3beta-mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK-3beta inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK-3beta inhibitor increased their viability by activating HSTF-1 and by reducing cytochrome c release, caspase-3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3-K/Akt and GSK-3beta. These results suggest that the G93A or A4V mutations inhibit PI3-K/Akt and activate GSK-3beta and caspase-3, thus becoming vulnerable to oxidative stress, and that the GSK-3beta-mediated cell death mechanism is important in G93A and A4V cell death.

Topics: Alanine; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Differentiation; Cell Line, Tumor; Cell Survival; Collagen Type XI; Cytochromes c; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Heat-Shock Proteins; Humans; Hybrid Cells; Indoles; Motor Neurons; Mutagenesis; Neuroblastoma; Phosphatidylinositol 3-Kinases; Point Mutation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Trypan Blue; Valine

Pancreatic cancer is characterised by a highly malignant phenotype with a marked resistance to conventional therapies and to apoptotic activators. Here, we demonstrate that sodium butyrate (NaBt), an inhibitor of histone deacetylases, sensitises human pancreatic cancer cell lines to both mitochondria- and Fas-mediated apoptosis. The analysis of anti-apoptotic and pro-apoptotic members of the Bcl-2 family in untreated pancreatic cancer cell lines shows a generalised low expression of Bcl-2 and a strong expression of Bcl-xL. NaBt treatment results in a marked down-regulation of Bcl-xL expression, mitochondrial membrane depolarization, cytochrome c release from mitochondria, activation of caspase-9 and -3 and apoptosis induction. Furthermore, NaBt sensitises pancreatic cancer cells to Fas-mediated apoptosis as well. In fact, the combined treatment with NaBt and the agonistic antibody anti-Fas (CH11) is able to induce apoptosis at an early time, in which neither NaBt nor CH11 alone induce apoptosis. Down-regulation of FLIP and activation of caspase-8 allow apoptosis to occur. These findings suggest that sodium butyrate could represent a good candidate for the development of new therapeutic strategies aimed at improving chemotherapy and immunotherapy in pancreatic cancer.

Topics: Apoptosis; bcl-X Protein; Blotting, Western; Butyrates; Caspases; Cell Line, Tumor; Cytochromes c; DNA Primers; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Mitochondrial Membranes; Pancreatic Neoplasms; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles

Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(-)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(-) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cisplatin; Cytochromes c; fas Receptor; Gene Transfer Techniques; Genes, p53; Genetic Therapy; Humans; Inhibitory Concentration 50; Liposomes; Models, Statistical; Necrosis; Oligonucleotides, Antisense; Phosphatidylethanolamines; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles; Transfection; Transferrin; Tumor Suppressor Protein p53

Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2).. Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1.. Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers).. Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.

Topics: Anesthetics, Inhalation; Animals; Animals, Newborn; Apoptosis; Biotransformation; Caspases; Cell Line; Cell Separation; Cytochromes c; Dogs; Genes, bcl-2; Hydrogen Peroxide; Hypoxia; In Situ Nick-End Labeling; Inflammation; Isoflurane; L-Lactate Dehydrogenase; Male; Myocytes, Cardiac; Neutrophil Activation; Neutrophils; Oxidation-Reduction; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Tetrazolium Salts; Thiazoles

In contrast to the anti-apoptotic action of nitric oxide (NO) on endothelial cells, NO exerts a pro-apoptotic effect on vascular smooth muscle cells (VSMCs). This study was designed to elucidate the mechanism underlying NO-induced apoptosis in rat VSMCs.. (1) Using the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay and fluorescence activated cell sorter (FACS) analyses, apoptosis of rat VSMCs were confirmed after exposure to sodium nitroprusside (SNP) (0.5 to 4 mmol/l), an exogenous NO donor. The effects of SNP were blocked by hemoglobin. (2) A universal caspase inhibitor, z-VAD-fmk, dose-dependently inhibited NO-induced apoptosis. VSMCs degraded Ac-DEVD-pNA rather than Ac-WHED-pNA after exposure to SNP, which suggested that the activation of caspase 3 rather than caspase 1 was involved in the process. Immunoblot analysis confirmed the activation of caspase-3. (3) Exposure to SNP induced the release of cytochrome c from the mitochondria to the cytosol, which was detected by immunoblot analysis of mitochondrial and cytosol fractions. (4) SNP exposure increased the ratio of Bax/Bcl-2 protein expression twofold by immunoblot analysis.. The mechanism of NO-induced apoptosis in rat VSMCs involves an increase in the ratio of Bax/Bcl-2 gene expression, which leads to the release of cytochrome c from the mitochondria to the cytosol, finally activating caspase-3 and resultant apoptosis.

Topics: Animals; Aorta, Thoracic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspases; Cell Count; Cell Survival; Cells, Cultured; Cytochromes c; Cytoplasm; Enzyme Activation; Flow Cytometry; Gene Expression; Genes, bcl-2; In Situ Nick-End Labeling; Male; Mitochondria, Muscle; Muscle, Smooth, Vascular; Nitric Oxide; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Thiazoles

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP+ that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 microM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.

Topics: Allopurinol; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cells, Cultured; Cerebellum; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Female; Herbicides; In Situ Nick-End Labeling; Male; Mitochondria; Models, Biological; Neurons; Paraquat; Piperazines; Rats; Tetrazolium Salts; Thiazoles; Time Factors; Vitamin E

The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-x(L), and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Benzamides; Blotting, Western; Cell Line; Cell Line, Tumor; Coloring Agents; Cytochromes c; DNA-Binding Proteins; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Flavoproteins; Flow Cytometry; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; K562 Cells; Membrane Potentials; Membrane Proteins; Milk Proteins; Mitochondria; Mutation; Piperazines; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; src-Family Kinases; STAT5 Transcription Factor; Tetrazolium Salts; Thiazoles; Trans-Activators; Transfection; Up-Regulation

YCT is a semipurified extract from Cratoxylum cochinchinense that has antioxidant properties and contains mostly mangiferin. We show here that YCT is selectively toxic to certain cell types and investigate the mechanisms of this toxicity in Jurkat T cells. By flow cytometric analyses, we show that YCT causes intense oxidative stress and a rise in cytosolic Ca(2+). This is followed by a rise in mitochondrial Ca(2+), release of cytochrome c, collapse of Deltapsi(m), a fall in ATP levels, and eventually cell death. The mechanism(s) of intense oxidative stress may involve a plasma membrane redox system, as cell death is inhibited by potassium ferricyanide. Cell death has some features of apoptosis (propidium iodide staining, externalization of phosphatidylserine, limited caspase-3 and -9 activities), but there was no internucleosomal DNA fragmentation.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Apoptosis; Blotting, Western; Calcium; Caspase 3; Caspase 9; Caspases; Cell Adhesion; Cell Line; Cell Membrane; Cell Survival; Clusiaceae; Coloring Agents; Cytochromes c; DNA Fragmentation; Dogs; Ferricyanides; Flow Cytometry; Glutathione; HL-60 Cells; Humans; Jurkat Cells; Lymphocytes; Membrane Potentials; Mitochondria; Models, Biological; Nucleosomes; Oxidation-Reduction; Oxidative Stress; PC12 Cells; Phosphatidylserines; Plant Extracts; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

Topics: Acetylcysteine; Animals; Apoptosis; Azathioprine; Caspase 3; Caspases; Cell Survival; Cytochromes c; DNA; DNA Fragmentation; Glutathione; Hepatocytes; JNK Mitogen-Activated Protein Kinases; Male; Mitochondria, Liver; Mitogen-Activated Protein Kinase 3; Necrosis; Oxygen Consumption; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Stress, Physiological; Superoxides; Tetrazolium Salts; Thiazoles; Thymidine; Tritium

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5'- and 3'-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells (P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.

Topics: Adenoviridae; Animals; Aspartic Acid; Blotting, Northern; Brain; Cells, Cultured; Coloring Agents; Cytochromes c; Digitonin; DNA, Complementary; Excitatory Amino Acid Transporter 1; Genetic Vectors; Glutamic Acid; Hypoxia; Immunoblotting; Immunohistochemistry; Malates; Microscopy, Fluorescence; Mitochondria; Mitochondria, Heart; Myocardium; Open Reading Frames; Rats; Rats, Inbred WKY; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Subcellular Fractions; Tetrazolium Salts; Thiazoles; Transfection

This study investigated whether nicastrin can induce apoptotic cell death in SK-N-MC cells. MTT assays revealed the transfected cells expressing mutant nicastrin, compared with those expressing wild nicastrin or the control vector, showing significantly increased cell death. The mutant nicastrin transfectants were also observed to induce cytosolic cytochrome c release from the mitochondria, and Bax protein expression in response, to increased cell death. These observations suggested that nicastrin, as well as the APP and PS proteins, were also involved in the upregulated Bax mediated neuroblastoma cell death and the release of cytochrome c in the neuroblastoma.

Topics: Amyloid Precursor Protein Secretases; bcl-2-Associated X Protein; Blotting, Western; Cell Death; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Mutational Analysis; Gene Expression Regulation; Humans; Membrane Glycoproteins; Mitochondria; Mutagenesis, Site-Directed; Mutation; Neuroblastoma; Polymerase Chain Reaction; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles; Transfection

Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF.

Topics: Aminopeptidases; Animals; Apoptosis; Carboxypeptidases; Cathepsin B; Cathepsin L; Cathepsins; Cell Death; Cell Line, Transformed; Cell Survival; Coloring Agents; Culture Media; Cysteine Endopeptidases; Cytochromes c; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dose-Response Relationship, Drug; Endopeptidases; Fas Ligand Protein; fas Receptor; Fibroblasts; Flow Cytometry; Golgi Apparatus; Humans; Intercellular Adhesion Molecule-1; Lymphocytes; Lysosomes; Mannosephosphates; Membrane Glycoproteins; Mice; Microscopy, Fluorescence; Mucolipidoses; Phenotype; Phosphorylation; Recombinant Proteins; Signal Transduction; Skin; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Necrosis Factor-alpha

Taxol (paclitaxel) is known to inhibit cell growth and trigger significant apoptosis in various cancer cells. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. In this study we investigated death receptors, FAS-associated death domain protein (FADD), the activation of caspases-10 and -8 as well as the downstream caspases, and reactive oxygen species (ROS) in taxol-induced apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line. Pretreating the cells with neutralizing antibodies to Fas, tumor necrosis factor (TNF)-alpha receptor 1, or TNF-related apoptosis-inducing ligand receptors (DR4 and DR5) did not affect taxol-induced apoptosis, but transfection of the cells with a dominant negative FADD plasmid resulted in inhibition of taxol-induced apoptosis, revealing that taxol induces apoptosis independently of these death receptors but dependently on FADD. Furthermore, the drug induced activation of caspases-10, -8, -6, and -3, cleaved Bcl-2, Bid, poly(ADP-ribose) polymerase, and lamin B, and down-regulated cellular levels of FLICE-like inhibitory protein (FLIP) and X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, despite the release of cytochrome c from the mitochondria in taxol-treated cells, caspase-9 was not activated. Inhibitors of caspases-8, -6, or -3 partially inhibited taxol-induced apoptosis, whereas the caspase-10 inhibitor totally abrogated this process. Taxol-induced apoptosis was also associated with decreased mitochondrial membrane potential (Deltapsim) and a significant increase in ROS generation. However, increased ROS production was not directly involved in taxol-triggered apoptosis. Therefore, these results demonstrate for the first time that taxol induces FADD-dependent apoptosis primarily through activation of caspase-10 but independently of death receptors.

Topics: Adaptor Proteins, Signal Transducing; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Caspase 10; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspases; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Survival; Coloring Agents; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Fas-Associated Death Domain Protein; Flow Cytometry; Genes, Dominant; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Models, Biological; Paclitaxel; Plasmids; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Tumor Necrosis Factor, Type I; Tetrazolium Salts; Thiazoles; Transfection

Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy.. Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting.. Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN.. These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.

Topics: Apoptosis; Bromodeoxyuridine; Caspases; Cell Hypoxia; Cell Survival; Cyclin-Dependent Kinases; Cytochromes c; DNA; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Flavonoids; G1 Phase; G2 Phase; Genes, myc; Growth Inhibitors; Humans; Hypoxia; In Situ Nick-End Labeling; Neuroblastoma; Piperidines; Proto-Oncogene Mas; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida. Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis. However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2. In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells. We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression. Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line. In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET. Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol. A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones. rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.

Topics: Apoptosis; Bisbenzimidazole; Blotting, Western; Carbon-Sulfur Lyases; Cell Line, Tumor; Cell Nucleus; Cell Survival; Coloring Agents; Cytochromes c; Cytosol; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Genetic Therapy; Humans; Lung Neoplasms; Mitochondria; Models, Biological; Oxidants; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Pseudomonas putida; Selenomethionine; Staurosporine; Tetrazolium Salts; Thiazoles; Transfection

Polyamine analogs have demonstrated considerable activity against many important solid tumor models including breast cancer. However, the precise mechanisms of antitumor activities of polyamine analogs are not entirely understood. The cytotoxicity of a newly developed polyamine analog compound, SL11144, against human breast cancer was assessed. Treatment of human breast cancer cell lines in culture with SL11144 decreased cell proliferation and induced programmed cell death in a time- and dose-dependent manner. SL11144 also profoundly inhibited the growth of MDA-MB-231 xenografts in host nude mice without overt toxic effects. Treatment of MDA-MB-435 cells with SL11144 led to the release of cytochrome c from mitochondria into cytosol, activation of caspase-3, and poly(ADP-ribose) polymerase cleavage. SL11144 decreased Bcl-2 and increased Bax protein levels in MDA-MB-231 cells. Furthermore, activator protein 1 transcriptional factor family member c-Jun was up-regulated by SL11144 in MDA-MB-435 and MDA-MB-231 cells, but not in MCF7 cells. In addition, significant inhibition of ornithine decarboxylase activity and a decrease in polyamine pools were demonstrated. These results demonstrate that the novel polyamine analog SL11144 has effective antineoplastic action against human breast cancer cells in vitro and in vivo and that multiple apoptotic mechanisms are associated with its cytotoxic effect in specific human breast cancer cell lines.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspases; Cell Death; Cell Division; Cell Line, Tumor; Cell Nucleus; Cell Survival; Chromatin; Cytochromes c; Cytoplasm; Cytosol; DNA Damage; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Models, Chemical; Neoplasm Transplantation; Nucleosomes; Poly(ADP-ribose) Polymerases; Polyamines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-jun; Tetrazolium Salts; Thiazoles; Time Factors; Up-Regulation

The intracellular signaling pathway(s) through which second messenger ceramides induce gene expression in human cells has not yet been characterized. In the present study, ceramide-induced expression of intercellular adhesion molecule-1 (ICAM-1), which requires activation of transcription factor activator protein 2 (AP-2), was found to be mediated through a mitochondrial pathway. Inhibitors of mitochondrial electron transport chain (e.g. rotenone, thenoyltrifluoroacetone, and antimycin A) reduced ceramide-induced ICAM-1 expression. Stimulation of human keratinocytes with cell-permeant ceramides at concentrations that did not induce apoptosis (no activation of caspases 3, 8, and 9 and no nucleosomal fragmentation) but caused AP-2 activation and ICAM-1 induction released cytochrome c (cyt c) from mitochondria into the cytoplasm of cells. This cyt c release was an indispensable prerequisite for effective ceramide signaling, because its inhibition by modulating the mitochondrial megachannel with bonkrekic acid or carboxyatractyloside prevented ceramide-induced AP-2 activation and ICAM-1 expression. Analysis of the interaction between cyt c and AP-2 revealed that cyt c oxidized AP-2 and that this redox regulation greatly enhanced the DNA binding capacity of AP-2. Mitochondria thus have a previously unrecognized function in signaling ceramide-induced transcription factor activation and gene regulation.

Topics: Apoptosis; Atractyloside; Blotting, Western; Cell Line; Cell Survival; Cells, Cultured; Ceramides; Coloring Agents; Cytochromes c; Cytoplasm; DNA; DNA-Binding Proteins; Electron Transport; Fibroblasts; Glutathione; HeLa Cells; Humans; Intercellular Adhesion Molecule-1; Keratinocytes; Mitochondria; Oxygen; Precipitin Tests; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factor AP-2; Transcription Factors

Apoptosis and necrosis are distinct forms of cell death that occur in response to various agents. We studied the action of N-Acetyl-D-sphingosine (C2-ceramide) or N-hexanoyl-D-sphingosine (C6-ceramide) in human hepatoma HepG2 cell line. The cells were treated in vitro for 1-24 h. Cell toxicity was evaluated by MTT assay. DNA content was estimated by gel electrophoresis and flow cytometry. Measurement of mitochondrial respiration, analysis of cytochrome c release and caspase-3 activation were assessed in order to determine if either of these events in the induction of apoptosis and/or necrosis was predominant. We have demonstrated that C2 and C6-ceramide were cytotoxic in a time and dose-dependent manner. After 24 h of treatment with 100 microM of C2 and C6 the morphology (May-Giemsa staining) of treated cells displayed an apoptotic phenotype in C6-treated cells, confirmed by a high (sub-G1 peak > 20%) proportion by flow cytometry while a necrotic morphology was observed after C2-ceramide treatment, confirmed by DNA smearing in DNA electrophoresis. After C6-ceramide incubation, the respiratory chain was functional only slightly inhibited (20%), there was production of ATP, cytochrome c release without ROS production, activation of caspase-3 and induction of apoptosis. On the contrary, C2-ceramide inhibited the respiratory chain more intensely (80%) increased significantly ROS production, which resulted in an arrest of ATP production, no cytochrome c release and absence of caspase-3 activation. Finally after complete exhaustion of intracellular ATP, mitochondrial explosion induced necrotic cell death. In conclusion, evidence suggest that mitochondrial respiratory chain function is essential for controlling the decision of the cell to enter a apoptotic or necrosis process.

Topics: Apoptosis; Caspase 3; Caspases; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Survival; Ceramides; Coloring Agents; Cytochromes c; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Electron Transport; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Flow Cytometry; Humans; Microscopy, Fluorescence; Mitochondria; Reactive Oxygen Species; Sphingosine; Tetrazolium Salts; Thiazoles; Time Factors