Rapid and transient intracellular oxidative stress due to novel macrosphelides trigger apoptosis via Fas/caspase-8-dependent pathway in human lymphoma U937 cells

The ability of the derivatives of macrosphelides (MS) core (simplified 16-membered core structure of natural MS) to induce apoptosis in human lymphoma U937 cells was investigated. Of the five compounds examined, MS core with ketones at 8 and 14 positions (MS5) showed the highest potency to induce apoptosis, while another, MS3 with one ketone, was minimal potent. MS5 was found to induce apoptosis in the U937 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis. MS5 treated cells showed increase in intracellular reactive oxygen species (ROS), glutathione depletion, Bid activation and lipid peroxidation. Pretreatment of cells with pancaspase inhibitor resulted in the complete inhibition of MS5-induced apoptosis. N-Acetyl-l-cysteine (NAC) pretreatment resulted in the increase in glutathione concentration, reduction of intracellular ROS, complete inhibition of DNA fragmentation, mitochondrial membrane potential (MMP) collapse, Fas externalization and caspase-8 activation. Furthermore, MS5-induced oxidative stress also triggered transient increase in intracellular calcium ion ([Ca2+]i) concentration which was completely inhibited by NAC. Pretreatment with an intracellular Ca2+ chelator, BAPTA-AM reduced MS5-induced DNA fragmentation and caspase-8 activation while it has marginal effects on MMP collapse. Taken together our present data showed that a rapid increase in intracellular ROS by MS5 triggers apoptosis via the Fas/caspase-8-mediated mitochondrial pathway suggesting that the presence of diketone makes the compound more potent to induce apoptosis. These characteristics of MS5 will make it useful for therapeutic applications of targeted apoptosis.     

Characterization of N,N,-dimethyl-D-erythro-sphingosine-induced apoptosis and signaling in U937 cells: independence of sphingosine kinase inhibition

In the present study, we studied N,N-dimethyl-D-erythro-sphingosine (DMS)-induced cell death and its signaling mechanism in U937 human monocytes. We found that DMS induced cell death in a concentration-dependent manner, while sphingosine 1-phosphate did not. DMS also induced DNA fragmentation, nuclear disruption, and cytochrome c release from mitochondria in a concentration- and time-dependent manner, implying apoptotic cell death. DMS was found to increase mitochondrial membrane potential (MMP) immediately after addition of DMS and to decrease MMP at 2h after addition. However, sphingosine kinase inhibitors and PKC inhibitors did not induce cell death in U937 cells, a result that appears to exclude sphingosine kinase and PKC as target molecules of DMS in the cell death induction process. Furthermore, DMS modulated the activity of several signaling molecules. DMS induced activation of JNK and p38 MAP kinase, while it decreased the activity of ERK and Akt kinase. However, decrease of MMP, inhibition of JNK, p38 MAP kinase, ERK, or Akt with specific inhibitors could not mimic the DMS-induced cell death, implying multiple concerted processes are involved in DMS-induced cell death. In summary, DMS induced apoptotic cell death via modulation of MMP, JNK, p38 MAP kinase, ERK, and Akt kinase, but not through inhibition of sphingosine kinase or PKC in U937 cells.     

Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 μg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2α and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells. Jin and Song contributed equally to this article.     

Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.     

MEK�CERK-dependent multiple caspase activation by mitochondrial proapoptotic Bcl-2 family proteins is essential for heavy ion irradiation-induced glioma cell death

Recently developed heavy ion irradiation therapy using a carbon beam (CB) against systemic malignancy has numerous advantages. However, the clinical results of CB therapy against glioblastoma still have room for improvement. Therefore, we tried to clarify the molecular mechanism of CB-induced glioma cell death. T98G and U251 human glioblastoma cell lines were irradiated by CB, and caspase-dependent apoptosis was induced in both cell lines in a dose-dependent manner. Knockdown of Bax (BCL-2-associated X protein) and Bak (BCL-2-associated killer) and overexpression of Bcl-2 or Bcl-xl (B-cell lymphoma-extra large) showed the involvement of Bcl-2 family proteins upstream of caspase activation, including caspase-8, in CB-induced glioma cell death. We also detected the activation of extracellular signal-regulated kinase (ERK) and the knockdown of ERK regulator mitogen-activated protein kinase kinase (MEK)1/2 or overexpression of a dominant-negative (DN) ERK inhibited CB-induced glioma cell death upstream of the mitochondria. In addition, application of MEK-specific inhibitors for defined periods showed that the recovery of activation of ERK between 2 and 36 h after irradiation is essential for CB-induced glioma cell death. Furthermore, MEK inhibitors or overexpression of a DN ERK failed to significantly inhibit X-ray-induced T98G and U251 cell death. These results suggested that the MEK–ERK cascade has a crucial role in CB-induced glioma cell death, which is known to have a limited contribution to X-ray-induced glioma cell death.     

Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

Apoptosis induction by epigallocatechin gallate involves its binding to Fas

Epigallocatechin gallate (EGCG) is known to induce apoptosis in various types of tumor cells, but the precise mechanism by which EGCG induces apoptosis remains to be elucidated. The Fas-Fas ligand system is one of the major pathways operating in the apoptotic cascade. The aim of this study was to examine the possibility that EGCG-binding to Fas triggers the Fas-mediated apoptosis. The EGCG treatment of human monocytic leukemia U937 cells resulted in elevation of caspase 8 activity and fragmentation of caspase 8. The DNA ladder formation caused by the EGCG treatment was inhibited by the caspase 8 inhibitor. These findings suggested the involvement of the Fas-mediated cascade in the EGCG-induced apoptosis in U937 cells. Affinity chromatography revealed the binding between EGCG and Fas. Thus, the results suggest that EGCG-binding to Fas, presumably on the cell surface, triggers the Fas-mediated apoptosis in U937 cells.     

Apoptotic effect of eugenol in human colon cancer cell lines

Eugenol, a natural compound available in honey and various plants extracts including cloves and Magnoliae flos, is exploited for various medicinal applications. Since most of the drugs used in the cancer are apoptotic inducers, the apoptotic effect and anticancer mechanism of eugenol were investigated against colon cancer cells. Antiproliferative effect was estimated using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay]. Earlier events like MMP (mitochondrial membrane potential), thiol depletion and lipid layer break were measured by using flow cytometry. Apoptosis was evaluated using PI (propidium iodide) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay and DNA fragmentation assay. MTT assay signified the antiproliferative nature of eugenol against the tested colon cancer cells. PI staining indicated increasing accumulation of cells at sub-G1-phase. Eugenol treatment resulted in reduction of intracellular non-protein thiols and increase in the earlier lipid layer break. Further events like dissipation of MMP and generation of ROS (reactive oxygen species) were accompanied in the eugenol-induced apoptosis. Augmented ROS generation resulted in the DNA fragmentation of treated cells as shown by DNA fragmentation and TUNEL assay. Further activation of PARP (polyadenosine diphosphate-ribose polymerase), p53 and caspase-3 were observed in Western blot analyses. Our results demonstrated molecular mechanism of eugenol-induced apoptosis in human colon cancer cells. This research will further enhance eugenol as a potential chemopreventive agent against colon cancer.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

Mechanism of apoptosis induced by a newly synthesized derivative of macrosphelides with a thiazole side chain

The apoptosis-inducing ability of hybrid compounds composed of macrosphelide and thiazole-containing side chain of epothilones was investigated. Among the tested series of hybrid compounds the one containing thiazole side chain at C15 (MSt-2) showed the maximum potency to induce apoptosis, while another containing thiazole side chain at C3 (MSt-6) was less potent. MSt-2 was found to induce apoptosis in human lymphoma (U937) cells in a dose- and time-dependent manner as confirmed by DNA fragmentation analysis. MSt-2 treated cells showed rapid reactive oxygen species (ROS) formation and c-Jun N-terminal kinase (JNK) activation. Furthermore, significant activation of extrinsic pathway as evident by Fas expression and caspase-8 activation was also observed. MSt-2-mediated decreased expression of Bid is an important event for cross talk between intrinsic and extrinsic signaling. N-acetyl-l-cysteine pre-treatment rescued cells from MSt-2-induced ROS formation, mitochondrial membrane potential (Δψ_m) loss, Fas expression, caspase-8 and -3 activation and DNA fragmentation. Moreover, antioxidant enzymes catalase and/or superoxide dismutase conjugated with polyethylene glycol also inhibit MSt-2-induced ROS formation, apoptosis and Δψ_m loss suggesting thereby pro-oxidant effects of MSt-2. Furthermore, JNK and pan-caspase inhibitors also protect cells from MSt-2-induced apoptosis. In addition to this, MSt-2 was found to be more potent in human colon carcinoma (HCT116) and human gastric cancer (AGS) cells while it has no effect on human normal dermal fibroblast. The important structure-activity relationship observed in the current study which makes MSt-2 more potent than MSt-6 is the position of thiazole side chain and stereochemistry of position 3 in chemical structure. In short the results of our study demonstrate that MSt-2-induced rapid ROS generation and mitochondrial dysfunction in cells trigger events responsible for mitochondria-dependent apoptosis pathway.     

Caffeine induces matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 down‐regulation in human leukemia U937 cells via Ca2+/ROS‐mediated suppression of ERK/c‐fos pathway and activation of p38 MAPK/c‐jun pathway

Caffeine attenuated invasion of human leukemia U937 cells with characteristic of decreased protein expression and mRNA levels of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9. Down‐regulation of MMP‐2 and MMP‐9 in U937 cells was abrogated by abolishment of caffeine‐elicited increase in intracellular Ca²⁺ concentration and ROS generation. Pretreatment with BAPTA‐AM (Ca²⁺ chelator) and N‐acetylcysteine (ROS scavenger) abolished caffeine‐induced ERK inactivation and p38 MPAK activation. Moreover, caffeine treatment led to MAPK phosphatase‐1 (MKP‐1) down‐regulation and protein phosphatase 2A catalytic subunit (PP2Ac) up‐regulation, which were involved in cross‐talk between p38 MAPK and ERK. Transfection of constitutively active MEK1 or pretreatment with SB202190 (p38 MAPK inhibitor) restored MMP‐2 and MMP‐9 protein expression in caffeine‐treated cells. Caffeine treatment repressed ERK‐mediated c‐Fos phosphorylation but evoked p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun by siRNA reflected that c‐Fos counteracted the effect of c‐Jun on MMP‐2/MMP‐9 down‐regulation. Taken together, our data indicate that MMP‐2/MMP‐9 down‐regulation in caffeine‐treated U937 cells is elicited by Ca²⁺/ROS‐mediated suppression of ERK/c‐Fos pathway and activation of p38 MAPK/c‐Jun pathway. J. Cell. Physiol. 224: 775–785, 2010. © 2010 Wiley‐Liss, Inc.     

Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.     

Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937

Background

Agaricus blazei Murrill (ABM) has been shown to exhibit immunostimulatory and anti-cancer activities; however, its mechanism of action is poorly understood. We recently found that the diffusible fraction of hot-water extract of ABM exhibits anti-tumor activity toward leukemic cells, and identified it as agaritine, a hydrazine-containing compound. In the present study, we examined the morphological and cytochemical effects of agaritine on U937 cells to elucidate the tumoricidal mechanism of agaritine.

Methods

Surface expression of phosphatidylserine (evaluated by annexin V binding), Fas antigen, DNA cleavage using TUNEL staining, changes in caspase activities and cytochrome c release, before and after treatment with agaritine, were examined using U937 cells.

Results

Nuclear damage, DNA fragmentation, was observed by Wright–Giemsa, TUNEL staining and agarose gel electrophoresis when U937 cells were incubated with 10 μg/mL of agaritine for 48 h. Flow cytometric analysis indicated that agaritine augments the proportion of annexin V-positive U937 cells without significant change in Fas antigen expression. Activities of caspase-3, -8 and -9 were gradually increased after the addition of agaritine. In the presence of caspase-3 or granzyme B inhibitor, except for the caspase-8 inhibitor, annexin V expression was significantly decreased, suggesting that mainly caspase-3 and -9 participate in the apoptotic pathway. Furthermore, cytochrome c release was detected by western blotting analysis after agaritine treatment.

Conclusions

These results strongly suggest that the ABM constituent agaritine moderately induces apoptosis in U937 leukemic cells via caspase activation through cytochrome c release from mitochondria.

General significance

This is the first report suggesting that the anti-tumor effect of agaritine is mediated through apoptosis. The present results might provide helpful suggestions for the design of anti-tumor drugs toward leukemia patients.     

Isofraxidin,a potent reactive oxygen species (ROS) scavenger,protects human leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in p53-independent manner

Ionizing radiation (IR) leads to oxidizing events such as excessive reactive oxygen species (ROS) in the exposed cells, resulting in further oxidative damage to lipids, proteins and DNA. To screen the potential radio-protective drug, the intracellular ROS was measured in irradiated U937 cells pretreated with 80 candidate traditional herbal medicine, respectively. Isofraxidin (IF) was one possible radio-protector in these 80 drugs. This study investigated the radio-protective role of IF, a Coumarin compound, in human leukemia cell lines, for the first time. Results indicate that IF protects against IR-induced apoptosis in U937 cells in the time- and concentration- dependent manner. IF decreases IR-induced intracellular ROS generation, especially hydroxyl radicals formation, inhibits IR-induced mitochondrial membrane potential loss and reduces IR-induced high intracellular Ca²⁺ levels regardless of ER stress. IF down-regulates the expression of caspase-3, phospho-JNK, phospho-p38 and activates Bax in mitochondria. IF inhibits cytochrome c release from mitochondria to cytosol. IF also moderates IR-induced Fas externalization and caspase-8 activation. IF also exhibits significant protection against IR-induced cell death in other leukemia cell lines such as Molt-4 cells and HL60 cells regardless of p53. Taken together, the data demonstrate that IF protects leukemia cells from radiation-induced apoptosis via ROS/mitochondria pathway in a p53-independent manner.     

Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells

Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 (PP-1) and 2A (PP-2A). The phosphorylation and dephosphorylation at the serine/threonine residues on proteins play important roles in regulating gene expression, cell cycle progression, and apoptosis. In this study, phosphatase inhibitor okadaic acid induces apoptosis in U937 cells via a mechanism that appears to involve caspase 3 activation, but not modulation of Bcl-2, Bax, and Bcl-X(L) expression levels. Treatment with 20 or 40 nM okadaic acid for 24 h produced DNA fragmentation in U937 cells. This was associated with caspase 3 activation and PLC-gamma1 degradation. Okadaic acid-induced caspase 3 activation and PLC-gamma1 degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 40 nM. Moreover, PMA (phorbol myristate acetate), PKC (protein kinase C) activator, protected U937 cells from okadaic acid-induced apoptosis, abrogated okadaic acid-induced caspase 3 activation, and specifically inhibited downregulation of XIAP (X-linked inhibitor of apoptosis) by okadaic acid. PMA cotreated U937 cells exhibited less cytochrome c release and sustained expression levels of the IAP (inhibitor of apoptosis) proteins during okadaic acid-induced apoptosis. In addition, these findings indicate that PMA inhibits okadaic acid-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase 3 that is involved in the execution of apoptosis.     

The effect of alpha-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. alpha-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP+ +NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.     

Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand

Neutrophils release soluble Fas ligand (sFasL), which can induce apoptosis in certain Fas-bearing cell types (Liles WC, Kiener PA, Ledbetter JA, Aruffo A, and Klebanoff SJ. J Exp Med 184: 429-440, 1996). We hypothesized that neutrophils could induce alveolar epithelial apoptosis via release of sFasL. A549 pulmonary adenocarcinoma cells expressed surface Fas and underwent cell death (10 +/- 7% viability) and DNA fragmentation (354 +/- 98% of control cells) when incubated with agonistic CD95/Fas monoclonal antibody (P < 0.05). Coincubation with human neutrophils induced significant A549 cell death at 48 (51 +/- 9% viability; P < 0.05) and 72 h (25 +/- 10%; P < 0.05) and increased DNA fragmentation (178 +/- 42% of control cells; P < 0.05), with morphological characteristics of apoptosis. The addition of antioxidants did not inhibit apoptosis. sFasL concentrations were maximally increased in coculture medium at 24 h (4.9 +/- 0.7 ng/ml; P < 0.05). Neutrophil-induced A549 cell apoptosis was blocked by inhibitory anti-Fas (42 +/- 6% of control cells; P < 0.05) and anti-FasL monoclonal antibodies (29 +/- 3%; P < 0.05). Human neutrophils and Fas similarly affected murine primary alveolar epithelial cell bilayers, and caspase activation occurred in response to Fas exposure. We conclude that neutrophils undergoing spontaneous apoptosis induce A549 cell death and DNA fragmentation, independent of the oxidative burst, that is mediated by sFasL.