benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and thiazolyl-blue

The objective was to determine the effects of growth factor treatment on dental pulp cell sensitivity to toxicity of 2 composite restoration materials, Flow Line and Durafill VS, and a calcium hydroxide pulp capping material, Dycal.. Toxicity of the dental materials to cultures of primary dental pulp cells was determined by the MTT metabolism assay. The ability of 6 different growth factors to influence the toxicity was tested.. A 24-hour exposure to either Flow Line or Durafill VS caused approximately 40% cell death, whereas Dycal exposure caused approximately 80% cell death. The toxicity of Flow Line and Durafill VS was mediated by oxidative stress. Four of the growth factors tested (bone morphogenetic protein [BMP]-2, BMP-7, epidermal growth factor [EGF], and transforming growth factor [TGF]-beta) decreased the basal MTT values while making the cells resistant to Flow Line and Durafill VS toxicity except BMP-2, which made the cells more sensitive to Flow Line. Treatment with fibroblast growth factor-2 caused no change in basal MTT metabolism, prevented the toxicity of Durafill VS, but increased the toxicity of Flow Line. Treatment with insulin-like growth factor-I (IGF-I) increased basal MTT metabolism and made the cells resistant to Flow Line and Durafill VS toxicity. None of the growth factors made the cells resistant to Dycal toxicity.. The results indicated that growth factors can be used to alter the sensitivity of dental pulp cells to commonly used restoration materials. The growth factors BMP-7, EGF, TGF-beta, and IGF-I provided the best profile of effects, making the cells resistant to both Flow Line and Durafill VS toxicity.

Topics: Adult; Amino Acid Chloromethyl Ketones; Antioxidants; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Calcium Hydroxide; Caspase Inhibitors; Cell Death; Cell Survival; Cells, Cultured; Chromans; Coloring Agents; Composite Resins; Dental Materials; Dental Pulp; Drug Tolerance; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Materials Testing; Minerals; Oxidative Stress; Tetrazolium Salts; Thiazoles; Time Factors; Transforming Growth Factor beta

In the present study, we investigated the effects of 3-oxoolean-12-en-27-oic acid (3-OA) isolated from the underground parts of Aceriphyllum rossii (Saxifragaceae) on the viability and apoptosis of HL-60 human promyelocytic leukemia cells, and the mechanisms underlying its action. 3-OA-treated HL-60 cells and HeLa human cervix adenocarcinoma cells displayed several apoptotic features, such as, DNA fragmentation, DNA laddering by agarose gel electrophoresis, and hypodiploid DNA contents by flow cytometry, and 3-OA also caused the activations of caspase-8, -9 and -3. Pretreatment with z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed 3-OA-induced DNA ladder formation and hypodiploid DNA contents, thereby implicating the caspase cascade in the apoptotic process. In addition, z-IETD-fmk (a caspase-8 inhibitor) and z-DEVD-fmk (a caspase-3 inhibitor) also completely neutralized the apoptotic effect of 3-OA in HL-60 cells. Furthermore, 3-OA increased Fas-related protein contents and the mRNA expressions of Fas ligand (FasL), Fas, and Fas-associated death domain (FADD). Preincubation with anti-Fas or anti-FasL blocking antibodies completely prevented 3-OA-induced apoptosis. Taken together, these results suggest that 3-oxoolean-12-en-27-oic acid induces apoptosis by activating caspase-8 via FasL-stimulated death receptor signaling.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 8; Cell Line, Transformed; Dose-Response Relationship, Drug; Fas Ligand Protein; Fas-Associated Death Domain Protein; HL-60 Cells; Humans; Neuroprotective Agents; RNA, Messenger; Saxifragaceae; Serine Endopeptidases; Tetrazolium Salts; Thiazoles; Time Factors; Triterpenes

We have investigated the signaling of OX(1) receptors to cell death using Chinese hamster ovary cells as a model system. OX(1) receptor stimulation with orexin-A caused a delayed cell death independently of cytosolic Ca(2+) elevation. The classical mitogen-activated protein kinase (MAPK) pathways, ERK and p38, were strongly activated by orexin-A. p38 was essential for induction of cell death, whereas the ERK pathway appeared protective. A pathway often implicated in the p38-mediated cell death, activation of p53, did not mediate the cell death, as there was no stabilization of p53 or increase in p53-dependent transcriptional activity, and dominant-negative p53 constructs did not inhibit cell demise. Under basal conditions, orexin-A-induced cell death was associated with compact chromatin condensation and it required de novo gene transcription and protein synthesis, the classical hallmarks of programmed (apoptotic) cell death. However, though the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethyl ketone (Z-VAD-fmk) fully inhibited the caspase activity, it did not rescue the cells from orexin-A-induced death. In the presence of Z-VAD-fmk, orexin-A-induced cell death was still dependent on p38 and de novo protein synthesis, but it no longer required gene transcription. Thus, caspase inhibition causes activation of alternative, gene transcription-independent death pathway. In summary, the present study points out mechanisms for orexin receptor-mediated cell death and adds to our general understanding of the role of G-protein-coupled receptor signaling in cell death by suggesting a pathway from G-protein-coupled receptors to cell death via p38 mitogen-/stress-activated protein kinase independent of p53 and caspase activation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Blotting, Western; Bromodeoxyuridine; Calcium; Caspases; Cell Proliferation; CHO Cells; Chromatin; Coloring Agents; Cricetinae; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Genetic Vectors; Humans; Indoles; Maleimides; MAP Kinase Signaling System; Orexin Receptors; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Tetrazolium Salts; Thiazoles; Time Factors; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53

The cardiotoxicity of adriamycin limits its clinical use as a powerful drug for solid tumors and malignant hematological disease. Although the precise mechanism by which it causes cardiac damage is not yet known, it has been suggested that apoptosis is the principal process in adriamycin-induced cardiomyopathy, which involves DNA fragmentation, cytochrome C release, and caspase activation. However, there has been no direct evidence for the critical involvement of caspase-3 in adriamycin-induced apoptosis. To determine the requirements for the activation of caspase-3 in adriamycin-treated cardiac cells, the effect of a caspase inhibitor on the survival of and apoptotic changes in H9c2 cells was examined. Exposure of H9c2 cells to adriamycin resulted in a time- and dose-dependent cell death, and the cleavage of pro-caspase-3 and of the nuclear protein poly (ADP'ribose) polymerase (PARP). However, neither the reduction of cell viability nor the characteristic morphological changes induced by adriamycin were prevented by pretreatment with the general caspase inhibitor z-VAD.FMK. In contrast, caspase inhibition effectively blocked the apoptosis induced by H202 in H9c2 cells, as determined by an MTT assay or microscopy. We also observed that p53 expression was increased by adriamycin, and this increase was not affected by the inhibition of caspase activity, suggesting a role for p53 in adriamycin-induced caspase-independent apoptosis in cardiac toxicity.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen Peroxide; Myocytes, Cardiac; Poly(ADP-ribose) Polymerases; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Suppressor Protein p53; Up-Regulation

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Genes, Dominant; Humans; In Situ Nick-End Labeling; MAP Kinase Kinase 1; Microscopy, Fluorescence; Mitochondria; Mitogen-Activated Protein Kinase 3; Models, Biological; Phosphatidylinositol 3-Kinases; Plasmids; Protein Denaturation; Protein Folding; Protein Kinases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Subcellular Fractions; Tetrazolium Salts; Thapsigargin; Thiazoles; Time Factors; TOR Serine-Threonine Kinases; Transcription, Genetic; Tunicamycin; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

The clinical benefit of suicide gene therapy of tumors has been marginal, mostly due to the low gene transfer efficiency in vivo. The death-inducing ligand, TRAIL, effectively kills many tumor cell types, while sparing most normal tissues. We hypothesized that TRAIL may enhance HSV thymidine kinase/ganciclovir (TK/GCV) gene therapy of tumor cells by augmenting both target and bystander cell kill. Human SH-EP neuroblastoma cells expressing TK as well as bystander cells were effectively killed by apoptosis, and their clonogenicity was ablated following GCV. Human TRAIL enhanced TK/GCV-induced cell death and decreased clonogenicity of TK-expressing cells and also of bystander cells. Cooperation between TRAIL and TK/GCV depended both on caspase activation and on mitochondrial apoptogenic function because both the broad-spectrum caspase inhibitor zVAD.fmk and overexpression of Bcl-2 decreased enhancement of cell kill by TRAIL. Facilitation of TRAIL signalling by up-regulation of TRAIL receptors did not contribute to enhancement because cell surface expression of the agonistic TRAIL receptors 1 and 2 was not increased by TK/GCV. In conclusion, the concerted activation of caspases and the mitochondrial amplification of caspase activation by TK/GCV may explain the cooperative effect of TK/GCV and TRAIL on the kill of neuroblastoma cells. Because combined treatment also augmented the bystander cell kill, the addition of TRAIL may increase the efficacy of TK/GCV gene therapy of neuroblastoma.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Antiviral Agents; Apoptosis; Apoptosis Regulatory Proteins; Caspase Inhibitors; Caspases; Colony-Forming Units Assay; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Ganciclovir; Genetic Therapy; Genetic Vectors; Herpesvirus 1, Human; Humans; Membrane Glycoproteins; Neuroblastoma; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Tetrazolium Salts; Thiazoles; Thymidine Kinase; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The cytokine hepatocyte growth factor/scatter factor (HGF/SF) has been found to protect a variety of epithelial and cancer cell types against cytotoxicity and apoptosis induced by DNA damage, but the specific apoptotic signaling events and the levels at which they are blocked by HGF/SF have not been identified. We found that treatment of MDA-MB-453 human breast cancer cells with adriamycin (also known as doxorubicin, a DNA topoisomerase IIalpha inhibitor) induced a series of time-dependent events, including the mitochondrial release of cytochrome c and apoptosis-inducing factor, mitochondrial membrane depolarization, activation of a set of caspases (caspase-9, -3, -7, -2, and -8), cleavage of poly(ADP-ribose) polymerase (PARP), and up-regulation of expression of the Fas ligand. All of these events were blocked by preincubation of the cells with HGF/SF. In contrast, the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone blocked some of these events (e.g. caspase-3 activation and PARP cleavage) but did not block cytochrome c release or mitochondrial depolarization. These findings suggest that HGF/SF functions, in part, upstream of the mitochondria to block mitochondrial apoptosis signaling, prevent activation of multiple caspases, and protect breast cancer cells against apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspase 3; Caspases; Coloring Agents; Cysteine Proteinase Inhibitors; Cytochrome c Group; Doxorubicin; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; Flavoproteins; Hepatocyte Growth Factor; Humans; Membrane Glycoproteins; Membrane Potentials; Membrane Proteins; Mitochondria; Recombinant Proteins; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Cells, Cultured; Up-Regulation

The role of caspase-3 (CPP32) protease in the molecular pathways of genistein-induced cell death in TM4 cells was investigated. Fluorescence microscopy with Hoechst-33258-PI nuclear stain was used to distinguish between apoptosis and necrosis pathways of cell death. The viability of the test cells was assessed with both the trypan blue exclusion and MTT tetrazolium (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetralzolium bromide, 2.5 mg/mL) assays. Caspase-3 enzymatic activity was determined using CasPASE Apoptosis Assay Kit. The overall results from all the data demonstrated that: i) genistein exerts dose- and time-dependent effects on TM4 testis cells; ii) apoptosis is induced by lower concentrations of genistein and necrosis induced by higher concentrations of genistein; iii) genistein induced activation caspase-3 enzymatic activity; iv) genistein-induction of apoptosis and necrosis was significantly inhibited by the caspase-3 inhibitor, z-DEV-FMK; v) sodium azide induced necrosis without activation of CPP32 enzymatic activity, and induction of apoptosis; and vi) genistein-induced apoptosis was associated with activation of CPP32 enzymatic activity in the cells. The overall results indicate a strong evidence of caspase-3 (CPP332) mediation in the molecular pathways of genistein-induced apoptosis in testicular cells. Apoptosis is the physiologically programmed cell death in which intrinsic mechanisms participate in the death of the cell, in contrast to necrosis, which induces inflammatory response in the affected cell. The fact that the chemopreventive role of several cancer drugs is due to induction of apoptosis augments the biotherapeutic potential of genistein for the treatment of malignant diseases including prostate and testicular cancers. It is therefore inevitable that identification of the apoptotic pathways and the points at which regulation occurs could be instrumental in the design of genistein biotherapy for such diseases.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Membrane Permeability; Enzyme Inhibitors; Genistein; Leydig Cells; Male; Microscopy, Fluorescence; Necrosis; Protein Kinase Inhibitors; Sertoli Cells; Tetrazolium Salts; Thiazoles; Trypan Blue

Neuronal loss is a salient feature of prion diseases. However, its cause and mechanism, particularly its relationship with the accumulation and precipitation of the pathogenic, protease-resistant isoform PrP(Sc) of the cellular prion protein PrP(C), are still an enigma. Several studies suggest that neuronal loss could occur through a process of programmed cell death, which is consistent with the lack of inflammation in these conditions. By analogy with the pathological events occurring during the development of Alzheimer's disease, controversies still exist regarding the relationship between amyloidogenesis, prion aggregation, and neuronal loss. We recently demonstrated that a prion protein fragment (118-135) displayed membrane-destabilizing properties and was able to induce, in a nonfibrillar form, the fusion of unilamellar liposomes. To unravel the mechanism of prion protein neurotoxicity, we characterize the effects of the human Pr[118-135] peptide on rat cortical neurons. We demonstrate that low concentrations of the Pr[118-135] peptide, in a nonfibrillar form, induce a time- and dose- dependent apoptotic cell death, including caspase activation, DNA condensation, and fragmentation. This toxicity might involve oxidative stress, because antioxidant molecules, such as probucol and propyl gallate, protect neurons against prion peptide toxicity. By contrast, a nonfusogenic variant Pr[118-135, 0 degrees ] peptide, which displays the same amino acid composition but several amino acid permutations, is not toxic to cortical neurons, which emphasizes the critical role of the fusogenic properties of the prion peptide in its neurotoxicity. Taken together, our results suggest that the interaction between the Pr[118-135] peptide and the plasma membrane of neurons might represent an early event in a cascade leading to neurodegeneration.

Topics: Amino Acid Chloromethyl Ketones; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Caspase Inhibitors; Caspases; Cell Nucleus; Cell Survival; Cells, Cultured; Cerebral Cortex; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; L-Lactate Dehydrogenase; Membrane Fusion; Neurons; Peptide Fragments; Prions; Rats; Rats, Wistar; Tetrazolium Salts; Thiazoles