Several Features Of Apoptosis Research Articles

The Curcumin Analogue, EF-24, Triggers p38 MAPK-Mediated Apoptotic Cell Death via Inducing PP2A-Modulated ERK Deactivation in Human Acute Myeloid Leukemia Cells.

Curcumin (CUR) has a range of therapeutic benefits against cancers, but its poor solubility and low bioavailability limit its clinical use. Demethoxycurcumin (DMC) and diphenyl difluoroketone (EF-24) are natural and synthetic curcumin analogues, respectively, with better solubilities and higher anti-carcinogenic activities in various solid tumors than CUR. However, the efficacy of these analogues against non-solid tumors, particularly in acute myeloid leukemia (AML), has not been fully investigated. Herein, we observed that both DMC and EF-24 significantly decrease the proportion of viable AML cells including HL-60, U937, and MV4-11, harboring different NRAS and Fms-like tyrosine kinase 3 (FLT3) statuses, and that EF-24 has a lower half maximal inhibitory concentration (IC50) than DMC. We found that EF-24 treatment induces several features of apoptosis, including an increase in the sub-G1 population, phosphatidylserine (PS) externalization, and significant activation of extrinsic proapoptotic signaling such as caspase-8 and -3 activation. Mechanistically, p38 mitogen-activated protein kinase (MAPK) activation is critical for EF-24-triggered apoptosis via activating protein phosphatase 2A (PP2A) to attenuate extracellular-regulated protein kinase (ERK) activities in HL-60 AML cells. In the clinic, patients with AML expressing high level of PP2A have the most favorable prognoses compared to various solid tumors. Taken together, our results indicate that EF-24 is a potential therapeutic agent for treating AML, especially for cancer types that lose the function of the PP2A tumor suppressor.

Bee Venom Induces Apoptosis and Inhibits Expression of Cyclooxygenase-2 mRNA in Human Lung Cancer Cell Line NCI-H1299

To investigate whether bee venom (BV) induces apoptosis, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling assay, 4,6-diamidino-2-phenylindole staining, flow cytometric analysis, and DNA fragmentation assay were performed on NCI-H1299 lung cancer cells treated with BV. Through morphological and biochemical analyses, it was demonstrated that NCI-H1299 cells treated with BV exhibit several features of apoptosis. In addition, reverse transcription-polymerase chain reaction and prostaglandin E2 (PGE2) immunoassay were performed to verify whether BV possesses an inhibitory effect on the expression of cyclooxygenase (COX) and PGE2 synthesis. Expression of COX-2 mRNA and synthesis of PGE2 were inhibited by BV. These results suggest the possibility that BV may exert an anti-tumor effect on human lung cancer.

Glabridin induces apoptosis and cell cycle arrest in oral cancer cells through the JNK1/2 signaling pathway.

Glabridin, a flavonoid extracted from licorice (Glycyrrhiza glabra), possesses various biological properties, including anticancer activities. However, the effect of glabridin on oral cancer cell apoptosis and the underlying molecular mechanisms has not been elucidated. In this study, we demonstrated that glabridin treatment significantly inhibits cell proliferation in human oral cancer SCC-9 and SAS cell lines. Flow cytometric assays demonstrated that glabridin induced several features of apoptosis, such as sub-G1 phase cell increase and phosphatidylserine externalization. Furthermore, glabridin induced apoptosis dose-dependently in SCC-9 cells through caspase-3, -8, and -9 activation and poly (ADP-ribose) polymerase cleavage. Moreover, glabridin increased the phosphorylation of the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK) pathways in a dose-dependent manner. Moreover, the inhibition of the JNK1/2 inhibitor significantly reversed the glabridin-induced activation of the caspase pathway. In conclusion, our findings suggest that glabridin induces oral cancer cell apoptosis through the JNK1/2 pathway and is a potential therapeutic agent for oral cancer.

Mitotic catastrophe and cell death induced by depletion of centrosomal proteins

Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, γ-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Autophagy precedes apoptosis during the remodeling of silkworm larval midgut

Although several features of apoptosis and autophagy have been reported in the larval organs of Lepidoptera during metamorphosis, solid experimental evidence for autophagy is still lacking. Moreover, the role of the two processes and the nature of their relationship are still cryptic. In this study, we perform a cellular, biochemical and molecular analysis of the degeneration process that occurs in the larval midgut of Bombyx mori during larval-adult transformation, with the aim to analyze autophagy and apoptosis in cells that die under physiological conditions. We demonstrate that larval midgut degradation is due to the concerted action of the two mechanisms, which occur at different times and have different functions. Autophagy is activated from the wandering stage and reaches a high level of activity during the spinning and prepupal stages, as demonstrated by specific autophagic markers. Our data show that the process of autophagy can recycle molecules from the degenerating cells and supply nutrients to the animal during the non-feeding period. Apoptosis intervenes later. In fact, although genes encoding caspases are transcribed at the end of the larval period, the activity of these proteases is not appreciable until the second day of spinning and apoptotic features are observable from prepupal phase. The abundance of apoptotic features during the pupal phase, when the majority of the cells die, indicates that apoptosis is actually responsible for cell death and for the disappearance of larval midgut cells.

Synthesis, and biological evaluation of 2-(4-aminophenyl)benzothiazole derivatives as photosensitizing agents

Photodynamic therapy (PDT) employing exogenous photosensitizers is currently being approved for treatment of basal cell carcinoma (BCC). 2-(4-Aminophenyl)benzothiazoles (6) consist of chromophoric structure and absorb light in the UVA (315-400 nm). These results encouraged us to design and synthesize a diversity of 2-phenylbenzothiazoles (6). Studies on the apoptotic mechanism involved in photosensitive effects induced by UVA-activated 6 in BCC cells are carried out in the present article. 6-UVA-treated cells displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, and activation of caspase-3. 6-UVA induced a decrease in mitochondrial membrane potential (Deltapsi(mt)) and ATP via enhanced ROS generation and promoted phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK expression. These results suggest that 6-UVA elicits photosensitive effects in mitochondria processes which involve ERK and p38 activation, and ultimately lead to BCC cell apoptosis.

Towards a pharmacological neuro-protectant: can anesthesia deliver?

Transient global cerebral ischemic injury (tGCI) continues to be a significant cause of death and disability. Global cerebral ischemia usually results from complex medical conditions, including cardiac arrest, circulatory shock, and birth asphyxia or iatrogenic interventions, such as circulatory arrest for cardiovascular surgery. The pathophysiology of global cerebral ischemia is complex. Experimental studies have identified a number of potential therapeutic treatments. Some of these treatments have targeted the initial metabolic events following brain ischemia, including energy failure, calcium ion flux, free radical generation, and the attenuation of excitotoxicity. These harmful mechanisms have been targeted with pharmacological interventions in experimental models. However, none of these promising pharmacological therapies have been successfully translated into clinical use. Such ‘‘easy to administer’’ therapies could be stocked in crash carts for administration to patients once the heart has been restarted. By contrast, physiological therapies, such as therapeutic hypothermia, have gained success in both experimental and clinical settings. This type of therapy is difficult to administer and involves extensive infrastructure, resources, and personnel. However, even therapeutic hypothermia may not be applicable across all human populations. Pre-conditioning is a strategy that involves increasing the brain’s tolerance to ischemia by exposing the brain to minor ischemic insults or pharmacological agents prior to the occurrence of a major ischemic event. While anesthetic preconditioning has shown initial promise in protecting the brain from ischemic events in pre-clinical studies, to date, there is no convincing clinical epidemiology supporting this practice. The pre-clinical science regarding the potential of anesthetic preconditioning is far from clear. This is illustrated in a recent article by Codaccioni et al., who report a transient but non-sustained improvement in neurocognitive testing in a rodent model of focal brain ischemia. In reality, this may simply be reflective of and/or specific to the particular model employed in their experiments. Any potential neuroprotective effects of anesthetic preconditioning depend on the anesthetic agent used to precondition, the species studied, the model of ischemia, the age and sex of the animal, the duration of preconditioning, and the interval between preconditioning and ischemic insult. In other words, it is not clear whether this will translate into a universally applicable therapy for tGCI. Anesthetic preconditioning is one of several strategies that prepare the brain to anticipate or react to the ischemic insult. Examples of other strategies used to precondition the brain include toxins, temperature, pharmaceuticals, and ischemia itself, with the latter showing most promise. The common theme of these strategies is to unbalance several key cell signal transcriptional programs in favour of cell survival, rather than any single metabolic pathway or target protein. Neuronal death after tGCI is complex. Characteristic lesions in vulnerable regions of the brain (such as the hippocampus and the striatum) appear from 12 h to 3 days after resuscitation and continue to evolve in the following weeks to months. Most cells that die do so by programmed cell death (PCD), as opposed to necrosis. This type of neuronal death has several features of apoptosis, with the cysteine-dependent aspartate-directed proteases family (the caspases) featuring strongly as executioners of PCD. Caspases normally exist in an inactivated state (pro-caspases), but under stressful conditions, cleavage (activation) D. R. Doherty, MBBCh (&) Department of Anesthesiology, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, ON K1H 8L1, Canada e-mail: ddoherty@cheo.on.ca

Simultaneous induction of apoptotic, autophagic, and necrosis-like cell death by monoclonal antibodies recognizing chicken transferrin receptor

Programmed cell death (PCD) is categorized as apoptotic, autophagic, or necrosis-like. Although the possibility that plural (two or three) death signals could be induced by a given stimulus has been reported, the precise mechanisms regulating PCD are not well understood. Recently, we have obtained two anti-chicken transferrin receptor (TfR) monoclonal antibodies (mAbs; D18 and D19) inducing a unique cell death. Although the cell death had several features of apoptosis, autophagic and necrosis-like morphological alterations were simultaneously observed in electron microphotographs. In addition to cells with condensed chromatin and an intact plasma membrane (apoptotic cells), cells having many vacuoles in the cytoplasm (autophagic cells), and enlarged cells with ruptured plasma membranes (necrosis-like cells) were observed in DT40 cells treated with the mAbs, however, the latter two types of dead cells were not detected upon treatment with staurosporine, a typical apoptosis inducer. In autophagic cells, numerous membrane-bound vesicles occupying most of the cytoplasmic space, which frequently contained electron-dense materials from cytoplasmic fragments and organelles, were observed. The simultaneous induction of multiple death signals from a stimulus via the TfR is of great interest to those researching cell death. In addition, activation of caspases was observed in DT40 cells treated with D19, however, the cell death was not inhibited with z-VAD-fmk, a pan-caspase inhibitor, suggesting that at least in part, a caspase-independent pathway is involved in the TfR-mediated cell death.

DC-81-Indole Conjugate Agent Induces Mitochondria Mediated Apoptosis in Human Melanoma A375 Cells

DC-81, an antitumor antibiotic produced by the Streptomyces species, belongs to pyrrolo[2,1-c] [1,4]benzodiazepine (PBD), which are potent inhibitors of nucleic acid synthesis. We previously reported an efficient synthesis of PBD hybrids linked with indole carboxylates. This is the first demonstration on the mechanism of the anticancer effect of PBD hybrid (IN6CPBD) agent on human melanoma A375 cells. IN6CPBD-treated cells exhibited higher cytotoxicity than DC-81 and displayed several features of apoptosis, including an increase in the sub-G1 population, a significantly increased annexin V binding, a degradation of caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage. Because degradative changes associated with apoptosis are often preceded by the disruption of mitochondrial function, the assessment of mitochondrial function in IN6CPBD-treated cells is worthy of investigation. Our data revealed that treatment of A375 cells with IN6CPBD resulted in the loss of mitochondrial membrane potential (DeltaPsimt), a decrease in intracellular pH (pHi), a reduction of ATP synthesis, increased reactive oxygen species (ROS) generation, and cytochrome c release. Collectively, our studies indicate that IN6CPBD induces apoptosis in A375 cells through a mitochondrial dysfunction pathway, leading to caspase-3 substrate PARP cleavage and subsequent apoptotic cell death.

Saucernetin-7 Isolated from Saururus chinensis Induces Caspase-Dependent Apoptosis in Human Promyelocytic Leukemia HL-60 Cells

In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.

CARDIOTOXIN III INDUCES APOPTOSIS IN K562 CELLS THROUGH A MITOCHONDRIAL‐MEDIATED PATHWAY

1. Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells. 2. Cardiotoxin III was found to inhibit the growth of K562 cells in a time- and dose-dependent manner, with an IC(50) value of 1.7 mug/mL, and displayed several features of apoptosis, including apoptotic body formation, an increase in the sub-G(1) population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. 3. Investigation of the mechanism of CTX III-induced apoptosis revealed that treatment of K562 cells with CTX III resulted in the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into the cytosol and activation of caspase-9 and caspase-3 and the subsequent cleavage of the caspase-3 substrate PARP; however, CTX III did not generate reactive oxygen species (ROS). 4. Taken together, the results indicate that CTX III induces apoptosis in K562 cells through an ROS-independent mitochondrial dysfunction pathway.

Nicotine induces apoptosis in TM3 mouse Leydig cells

To investigate whether nicotine-induced testicular toxicity involves the induction of apoptosis in Leydig cells. Cell study using cells of the TM3 cell line derived from mouse Leydig cells. Academic research laboratory. Morphological and biochemical analyses for the detection of apoptosis. The effect of nicotine on the occurrence of apoptosis was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4,6-diamidino-2-phenylindole staining, terminal deoxynuclotidyl transferase-mediated dUTP nick end labeling assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction, caspase-3 enzyme assay, and Western blot analysis. Nicotine treatment exhibits several features of apoptosis in mouse Leydig cells. TM3 cells treated with nicotine exhibit several features of apoptosis. It was also shown that nicotine increases the mRNA level of bax and decreases that of bcl-2. In addition, nicotine enhanced the expression of the activated form of caspase-3 and caspase-3 enzyme activity. Nicotine appears to activate specific intracellular death-related pathways, probably by bax-dependent activation of caspase-3, inducing apoptosis in Leydig cells. Thus, nicotine-induced apoptosis of Leydig cells might be one of the important mechanisms behind nicotine-related urogenital disorders in men.

Induction of apoptosis in human leukemia K562 cells by cardiotoxin III

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III was found to inhibit the growth of K562 cells in a time-and dose-dependent manner with IC 50 value of 1.7 μg/ml, and it displayed several features of apoptosis including apoptotic body formation, increase of sub G 1 population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. Investigation of the mechanism of CTXIII - induced apoptosis revealed that the treatment of K562 cells with CTX III resulted in the activation of caspase-9, caspase-3 and subsequent cleavage of its substrate PARP and that CTXIII was also associated with an early release of cytochrome c from the mitochondria. These results suggest that CTX III may induce apoptosis through a mitochondria- and caspase-dependent mechanism.

.BETA.-D-Xylopyranosyl-(1.RAR.3)-.BETA.-D-glucuronopyranosyl Echinocystic Acid Isolated from the Roots of Codonopsis lanceolata Induces Caspase-Dependent Apoptosis in Human Acute Promyelocytic Leukemia HL-60 Cells

We previously demonstrated that beta-D-xylopyranosyl-(1-->3)-beta-D-glucuronopyranosyl echinocystic acid (codonoposide 1c), a biologically active compound isolated from the roots of Codonopsis lanceolata, is cytotoxic to cancer cells. In the present study, we investigated the effects of codonoposide 1c on the induction of apoptosis, and its putative action pathway in HL-60 human promyelocytic leukemia cells. Codonoposide 1c-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, formation of DNA ladders by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. We observed that codonoposide 1c caused activation of caspase-8, caspase-9, and caspase-3. A broad caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-3 inhibitor (z-DEVD-fmk) almost completely suppressed codonoposide 1c-induced DNA fragmentation. We further found that codonoposide 1c induces mitochondrial translocation of Bid from cytosol, reduction of cytosolic Bax, and cytochrome c release from mitochondria. Interestingly, codonoposide 1c also triggered the mitochondrial release of Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) into cytosol, and a reduction in X-linked inhibitor of apoptosis protein (XIAP). Taken together, our data indicate that codonoposide 1c is a potent inducer of apoptosis and facilates its activity via Bid cleavage and translocation to mitochondria, Bax reduction in cytosol, release of cytochrome c and Smac/DIABLO into the cytosol, and subsequently caspase activation, providing a potential mechanism for the cytotoxic activity of codonoposide 1c.

Cardiotoxin III induces apoptosis in T24 cells via reactive oxygen species‐independent mitochondrial death pathway

AbstractCardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III inhibited the growth of T24 cells in a time‐ and dose‐dependent manner with an IC50value of 1.7 µg/mL and displayed several features of apoptosis including apoptotic body formation, increase of sub G1 population, DNA fragmentation, and poly (ADP‐ribose) polymerase (PARP) cleavage. Using apoptosis analysis, measurement of reactive oxygen species (ROS) and assessment of mitochondrial membrane potentials (ΔΨm), CTX III was found to be a potent inducer of apoptosis, transducing apoptotic signals via a decrease in mitochondrial membrane potential (ΔΨm) and release of cytochrome c from mitochondria into cytosol. However, CTX III did not generate reactive oxygen species (ROS). Taken together, the present data indicate that CTX III induces apoptosis in T24 cells through an ROS‐independent mitochondrial dysfunction pathway and resultant cytochrome c release. This is the first report on the mechanism of the anticancer effect of CTX III on T24 cells. Drug Dev. Res. 63:219–224, 2004. © 2004 Wiley‐Liss, Inc.

Nitric oxide-induced cell death of cerebrocortical murine astrocytes is mediated through p53- and Bax-dependent pathways.

We have investigated the mechanism by which nitric oxide (NO) induces the death of mouse astrocytes. We show that NO (from donor diethylenetriamine-NO adduct) induces death with several features of apoptosis, including chromatin condensation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, Bax translocation to the mitochondria and cytochrome c release, but no caspase activation or nuclear fragmentation is observed. Nitric oxide also elevates p53 expression, causing a concomitant increase in p53 serine 18 phosphorylation and p53 translocation from the cytoplasm to the nucleus. Activation of Bax and p53 is important for NO-induced apoptosis-like cell death because Bax- or p53-deficient astrocytes are much more resistant than wild-type cells to the same NO treatment. We further demonstrate that LY294002-sensitive kinases are responsible for controlling serine 18 phosphorylation of p53, thereby regulating the pro-apoptotic activity of p53 in astrocytes. While apoptosis is suppressed in the presence of LY294002, however, death by necrosis is increased, suggesting that LY294002-sensitive kinases additionally suppress a latent necrotic response to NO. We conclude that NO-induced death in astrocytes is mediated by p53- and Bax-dependent mechanisms, although full manifestation of apoptosis is aborted by concomitant inhibition of caspase activation. More generally, our data suggest that apoptotic mediators should be evaluated as the cause of cell death even in cases where a full apoptotic phenotype is lacking.

Bee Venom Induces Apoptosis and Inhibits Expression of Cyclooxygenase-2 mRNA in Human Lung Cancer Cell Line NCI-H1299

To investigate whether bee venom (BV) induces apoptosis, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling assay, 4,6-diamidino-2-phenylindole staining, flow cytometric analysis, and DNA fragmentation assay were performed on NCI-H1299 lung cancer cells treated with BV. Through morphological and biochemical analyses, it was demonstrated that NCI-H1299 cells treated with BV exhibit several features of apoptosis. In addition, reverse transcription-polymerase chain reaction and prostaglandin E(2) (PGE(2)) immunoassay were performed to verify whether BV possesses an inhibitory effect on the expression of cyclooxygenase (COX) and PGE(2 )synthesis. Expression of COX-2 mRNA and synthesis of PGE(2) were inhibited by BV. These results suggest the possibility that BV may exert an anti-tumor effect on human lung cancer.

Amiodarone induces apoptosis in L-132 human lung epithelial cell line

To investigate whether amiodarone induces apoptosis in cells of the L-132 human lung epithelial cell line, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, 4,6-diamidino-2-phenylindole staining, DNA fragmentation assay, reverse transcription-polymerase chain reaction, and casapse-3 enzyme assay were performed. Through morphological and biochemical analyses, it was demonstrated that L-132 cells treated with amiodarone exhibit several features of apoptosis. In addition, it was shown that amiodarone increases the mRNA levels of bax and caspase-3. Based on the results, amiodarone appears to activate specific intracellular death-related pathways, including possibly the bax-dependent caspase-3 activation pathway, and thus induce apoptosis in human lung epithelial cells.

3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity.

3-Hydroxykynurenine (3-HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3-HK. Of the tested kynurenine pathway metabolites, only 3-HK, and to a lesser extent 3-hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3-HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3-HK-induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3-HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3-HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3-HK, depending on transporter-mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.

EGF-R dependent regulation of keratinocyte survival.

Tissue organization and maintenance within multicellular organisms is in part dependent on the ability of cells to undergo programmed cell death or apoptosis. Conversely, disruption of cell death pathways often is associated with tumor development. At present, the molecular control of apoptosis in epithelial cells is poorly understood. Here we describe evidence linking epidermal growth factor-receptor (EGF-R) activation to survival of normal human keratinocytes in culture. Inhibition of EGF-R activation by an anti-EGF-R antagonistic monoclonal antibody (mAb 425), followed by detachment of keratinocytes from the substratum, induced extensive death with several features of apoptosis in keratinocyte cultures. Other, non-epithelial normal human cells including melanocytes and fibroblasts, did not show this effect. Similar to EGF-R blockade by mAb 425, inhibition of the EGF-R tyrosine kinase activity using tyrphostin AG1478 resulted in lack of attachment and extensive cell death upon passaging. Attachment to keratinocyte-derived ECM partially resuced mAb 425-treated keratinocytes from cell death, indicating that adhesion-dependent and EGF-R-dependent signal transduction pathways serve partially overlapping but not redundant roles in supporting keratinocyte survival.