epidermal-growth-factor and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

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

Cell death of neural progenitor cells is the primary problem limiting the value of neural progenitor cell-based therapy for central nervous system disorders. However, little is known about the mechanism of cell death of neural progenitor cells. In this study, we investigated the mechanisms of cell death of a multipotent cell line, MEB5, caused by deprivation of epidermal growth factor (EGF). When EGF was removed from the culture medium, the total number of viable MEB5 cells reduced, and nuclear condensation and elevation of caspase-3-like enzyme activity were observed in MEB5 cells. Treatment with a broad-range caspase inhibitor reduced cell death in a concentration-dependent manner, indicating that MEB5 cells undergo caspase-mediated apoptotic cell death caused by EGF deprivation. We also investigated the effects of glutamate receptor antagonists, antioxidants and nitric oxide synthase inhibitor on EGF deprivation-induced cell death. N-methyl-D-aspartate (NMDA) glutamate receptor antagonists, alpha-amino-3-hydrozy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor antagonist and nitric oxide synthase inhibitor failed to reduce cell death. In contrast, two antioxidants with different chemical structures reduced cell death in a concentration-dependent manner. The production of reactive oxygen species was detected in MEB5 cells after EGF deprivation by monitoring dichlorodihydrofluorescein fluorescence as a marker of reactive oxygen species-related radicals. Our results suggest that oxidative stress triggers caspase-mediated apoptosis of neural progenitor cells by trophic support deprivation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Astrocytes; Caspase 3; Caspase Inhibitors; Cell Line; Dizocilpine Maleate; Enzyme Inhibitors; Epidermal Growth Factor; Ethylenediamines; Excitatory Amino Acid Antagonists; L-Lactate Dehydrogenase; Metalloporphyrins; Mice; Multipotent Stem Cells; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Organometallic Compounds; Reactive Oxygen Species; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

Prostate cancer is one of the most frequently diagnosed cancers in males. Autocrine/paracrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors suggesting a role for EGFR in the progression of prostate cancer. The androgen-dependent prostate cancer cell line, LNCaP, expresses the EGFR as well as two additional members of the family; ErbB-2 and ErbB-3, which can be activated by neuregulin (NRG) isoforms. The effect of ErbB ligands on the viability of LNCaP cells was studied.. In the present study, we examined the effect of NRG on LNCaP cell growth and survival in the absence of androgen mimetic by the MTT assay, FACS analysis, nuclei staining, and Western blotting.. Our results demonstrate that NRG activates ErbB-2/ErbB-3 heterodimers and induces cell death of LNCaP cells. By contrast, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers and induces cell growth and survival. Interestingly, LNCaP cells treated with PI3K inhibitor underwent cell death but cells treated with both NRG and PI3K inhibitor survived as the control cells, indicating that the PI3K pathway may mediate NRG-induced cell death. NRG-induced cell death was not inhibited by the broad-spectrum caspases inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK). However, NRG-induced cell death was inhibited by type II cell death inhibitor, 3-methyladenine.. These results suggest that NRG induces type II cell death of LNCaP cells through PI3K-dependent pathway.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Autophagy; Cell Death; Cell Division; Chromones; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Ligands; Male; Morpholines; Neuregulins; Phosphorylation; Prostatic Neoplasms; Tumor Cells, Cultured; Tyrosine

Our results revealed that the blockade of epidermal growth factor receptor (EGFR) tyrosine kinase and protein kinase A (PKA) signaling pathways by specific inhibitors (PD153035 and Rp-cAMPs) leads to a synergistic inhibition of EGF- and serum-stimulated growth of human prostatic cancer cells (LNCaP, DU145 and PC3) concomitant with an arrest in the G1 phase of cellular cycle. Of particular interest, the combination of PD153035 and Rp-cAMPs also caused a more substantial apoptotic/necrotic death of these prostatic cancer cells as compared to drugs alone. Moreover, we observed that the inhibition of acidic sphingomyelinase and caspase cascades results in a marked reduction of DNA fragmentation and apoptotic death induced by PD153035, alone or in combination with Rp-cAMPs, in EGF stimulated PC3 cells. This suggests that these agents might mediate their cytotoxic effects at least in part via the ceramide generation and activation of caspase signaling pathways. N-oleoylethanolamine (OE), an inhibitor of acidic ceramidase, consistently potentiated the apoptotic effects of PD153035 in all the prostatic cancer cell lines tested. Additionally, the cellular ceramide content estimated for PC3 cells was increased after treatment with PD153035, alone or in combination, at a lower dose with OE and Rp-cAMPs. The synergistic apoptotic effect of PD153035 plus Rp-cAMPs induced in PC3 was also accompanied by a significant rate of mitochondrial membrane depolarization and release of cytochrome c into cytosol as compared to drugs alone. Combined, the results indicated that the simultaneous inhibition of EGFR and PKA signaling cascades might lead to a more massive apoptotic death of metastatic prostatic cancer cells by increasing ceramide accumulation and activating of caspase cascade of a mitochondrial dependent manner.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cell Division; Cell Separation; Ceramides; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Male; Membrane Potentials; Mitochondria; Necrosis; Prostatic Neoplasms; Protease Inhibitors; Quinazolines; Tumor Cells, Cultured

Although apoptosis has been considered the typical mechanism for physiological cell death, presently alternative mechanisms need to be considered. We previously showed that fibroblast growth factor-2 (FGF2) could act as a survival factor for neural precursor cells. To study the death mechanism activated by the absence of this growth factor, we followed the changes in cell morphology and determined cell viability by staining with several dyes after FGF2 removal from mesencephalic neural-progenitor-cell cultures. The changes observed did not correspond to those associated with apoptosis. After 48 h in the absence of FGF2, cells began to develop vacuoles in their cytoplasm, a phenotype that became very obvious 3-5 days later. Double-membrane vacuoles containing cell debris were observed. Vacuolated cells did not stain with either ethidium bromide or trypan Blue, and did not show chromatin condensations. Nonetheless, during the course of culture, vacuolated cells formed aggregates with highly condensed chromatin and detached from the plate. Neural progenitor cells grown in the presence of FGF2 did not display any of those characteristics. The vacuolated phenotype could be reversed by the addition of FGF2. Typical autophagy inhibitors such as 3-MA and LY294002 inhibited vacuole development, whereas a broad-spectrum caspase inhibitor did not. Interestingly, Bcl-2 overexpression retarded vacuole development. In conclusion, we identified a death autophagy-like mechanism activated by the lack of a specific survival factor that can be inhibited by Bcl2. We propose that anti-apoptotic Bcl2 family members are key molecules controlling death activation independently of the cell degeneration mechanism used.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Caspase Inhibitors; Cell Death; Cell Differentiation; Cell Survival; Chromones; Cycloheximide; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Immunohistochemistry; Intermediate Filament Proteins; Luminescent Proteins; Mesencephalon; Mice; Microscopy, Electron; Morpholines; Nerve Tissue Proteins; Nestin; Neurons; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stem Cells; Transformation, Genetic; Tubulin; Vacuoles

Cultured human cytotrophoblasts are more susceptible than syncytiotrophoblasts to hypoxia-induced apoptosis. Caspases are cysteine proteases that cleave cellular components to effect the apoptotic cascade. We hypothesized that cultured cytotrophoblasts exhibit a higher activity of caspases when compared with syncytiotrophoblasts. Using western analysis, we demonstrated that the pro-caspases 3, 6, 8, and 9 are expressed in cytotrophoblasts cultured for 24 h, and also, in trophoblasts cultured 72 h when syncytiotrophoblasts have formed. Importantly, we found significantly higher activity of all four caspases in trophoblasts cultured 24 h compared with cells cultured 72 h. Colchicine and DMSO, which hinder trophoblast differentiation, enhanced the activity of all four caspases in cells cultured 72 h. Conversely, caspase activity was reduced in trophoblasts cultured for 24 h in the presence of epidermal growth factor, which enhances differentiation. This effect was most pronounced on caspase 3 and was attenuated by addition of the tyrosine kinase inhibitor AG1478. We conclude that cytotrophoblasts exhibit a higher activity of caspases 3, 6, 8, and 9 when compared with the more differentiated syncytium. This may account for the higher susceptibility of cytotrophoblasts to hypoxia-induced apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Caspase Inhibitors; Caspases; Cell Differentiation; Cells, Cultured; Colchicine; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Epidermal Growth Factor; Humans; Protein Precursors; Solvents; Trophoblasts

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 7; Caspases; Cell Nucleus; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Biological; p38 Mitogen-Activated Protein Kinases; Paclitaxel; Phosphorylation; Precipitin Tests; Protein Isoforms; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; ras Proteins; Signal Transduction; Time Factors; Tumor Cells, Cultured

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Urea Nitrogen; Caspases; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; Cytokines; Depression, Chemical; Drug Administration Schedule; Epidermal Growth Factor; Humans; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Ischemia; Kidney; Male; Mice; Neoplasm Proteins; Nephritis; Peroxidase; Protein Processing, Post-Translational; Recombinant Proteins; Reperfusion Injury; RNA-Binding Proteins

Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.

Topics: Amino Acid Chloromethyl Ketones; Androstadienes; Animals; Apoptosis; Cell Survival; Chromones; Cyclic AMP; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Flavonoids; Hypoglycemic Agents; Insulin; Morpholines; Nerve Growth Factors; Neurons; Neuropeptides; Neuroprotective Agents; PC12 Cells; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-jun; Rats; Sphingosine; Thionucleotides; Wortmannin

We have examined the role of reactive oxygen species (ROS) in apoptosis induced by growth factor deprivation in primary cultures of mouse proximal tubular (MPT) cells. When confluent monolayers of MPT cells are deprived of all growth factors, the cells die by apoptosis over a 10- and 14-day period. Both epidermal growth factor (EGF) and high-dose insulin directly inhibit apoptosis of MPT cells deprived of growth factors. Growth factor deprivation results in an increase in the cellular levels of superoxide anion while apoptosis of MPT cells induced by growth factor withdrawal is inhibited by a number of antioxidants and scavengers of ROS. Growth factor deprivation also results in activation of caspase activity, which is inhibited by EGF and high-dose insulin as well as by the ROS scavengers and antioxidants that inhibit apoptosis. The cell-permeant caspase inhibitor, z-Val-Ala-Asp-CH2F (zVAD-fmk), prevents the increase in caspase activity and markedly inhibits apoptosis induced by growth factor deprivation. However, zVAD-fmk had no effect on the increased levels of superoxide associated with growth factor deprivation. Thus we provide novel evidence that ROS play an important role in mediating apoptosis associated with growth factor deprivation. ROS appear to act upstream of caspases in the apoptotic pathway. We hypothesize that oxidant stress, induced by growth factor withdrawal, represents a signaling mechanism for the default pathway of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; Flow Cytometry; Insulin-Like Growth Factor I; Kidney Tubules, Proximal; Mice; Mice, Inbred C57BL; Oxidative Stress; Reactive Oxygen Species; Superoxides