Glutathione export during apoptosis requires functional multidrug resistance-associated proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.     

Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function

Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance–associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.     

Iodination of verapamil for a stronger induction of death,through GSH efflux,of cancer cells overexpressing MRP1

The multidrug resistance protein 1 (MRP1), involved in multidrug resistance (MDR) of cancer cells, was found to be modulated by verapamil, through stimulation of GSH transport, leading to apoptosis of MRP1-overexpressing cells. In this study, various iodinated derivatives of verapamil were synthesized, including iodination on the B ring, known to be involved in verapamil cardiotoxicity, and assayed for the stimulation of GSH efflux by MRP1. The iodination, for nearly all compounds, led to a higher stimulation of GSH efflux. However, determination of concomitant cytotoxicity is also important for selecting the best compound, which was found to be 10-fold more potent than verapamil. This will then allow us to design original anti-cancer compounds which could specifically kill the resistant cancer cells.     

Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1

Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.     

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux

Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.     

SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis

Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH](i), nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH](i) can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH](i) in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and alpha-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

Programmed Cell Death in 2′,3′-dideoxycytidine-resistant Human Monoblastoid U937 Cells

2′,3′-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O₂ and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c_50%=258±24 μM HOCl) and bimane-S-glutathione (B-SG, c_50%=125±16 μM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH]_oxidized=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg²⁺-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c_50%=478±36 μM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis

Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic volume decrease (AVD), is necessary for the progression of apoptotic cell death, we sought to evaluate the relationship between GSH transport and ionic homeostasis during Fas ligand (FasL)-induced apoptosis in Jurkat cells. GSH depletion in response to FasL was paralleled by distinct degrees of AVD identified by differences in cellular forward scatter and electronic impedance analysis. Inhibition of GSH efflux prevented AVD, K+ loss, and the activation of two distinct ionic conductances, mediated by Kv1.3 and outward rectifying Cl- channels. Reciprocally, stimulation of GSH loss accelerated the loss of K+, AVD, and consequently the progression of the execution phase of apoptosis. Although high extracellular K+ inhibited FasL-induced apoptosis, GSH depletion was largely independent of K+ loss. These results suggest that deregulation of GSH and ionic homeostasis converge in the regulation of apoptosis in lymphoid cells.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H₂O₂ was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA₂ was blocked by a selective inhibitor of Lp-PLA₂, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me'' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

The MRP1-mediated effluxes of arsenic and antimony do not require arsenic-glutathione and antimony-glutathione complex formation

Arsenic trioxide is an effective treatment for acute promyelocytic leukemia, but resistance to metalloïd salts is found in humans. Using atomic absorption spectroscopy, we have measured the rate of uptake of arsenic trioxide and of antimony tartrate in GLC4 and GLC4/ADR cells overexpressing MRP1 and the rate of their MRP1-mediated effluxes as a function of the intracellular GSH concentration. In sensitive cells, after 1 h, a pseudosteady state is reached where intra- and extracellular concentrations of metalloid are the same. This precludes the formation, at short term, of complexes between arsenic or antimony with GSH. In resistant cells reduced intracellular accumulation of arsenic (or antimony), reflecting an increased rate of arsenic (or antimony) efflux from the cells, is observed. No efflux of the metalloid is observed in GSH depleted cells. The two metalloïds and GSH are pumped out by MRP1 with the same efficiency. Moreover for the three compounds 50% of the efflux is inhibited by 2 M MK571. This led us to suggest that As- and Sb-containing species could be cotransported with GSH.     

Intracellular redox changes during apoptosis

In the current paradigm for apoptotic cell death, the activity of a family of proteases related to interleukin 1-beta converting enzyme (ICE) orchestrates the multiple downstream events (such as cell shrinkage and chromatin degradation) that comprise apoptosis. A variety of stimuli can induce this type of cell death. One of the most reproducible inducers is mild oxidative stress, although it is unclear how an oxidative stimulus activates ICE-like proteases. Oxidative modification of proteins and lipids have also been observed in cells undergoing apoptosis in response to non-oxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. However, attempts to consistently detect a requirement for reactive oxygen species in apoptosis have been inconclusive. Recent experiments revealing that apoptosis is typically accompanied by a depletion of intracellular reduced glutathione (GSH) are also discussed. In JURKATT lymphocytes treated with antibodies to the Fas/APO-1 surface receptor, this depletion results from an accelerated efflux of the reduced thiol rather than any intracellular oxidation. As GSH is the most abundant cytosolic reductant, we propose that its efflux may provide a non-oxidative mechanism by which the reducing environment of apoptotic cells is lost. An increase in oxidative damage to proteins and lipids would then result even in the absence of an increase in the production of oxidants. This may explain the seemingly contradictory findings that increased oxidative stress is not required for apoptosis even though antioxidants often inhibit the process and peroxidised products accumulate in apoptotic cells.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Ceefourin-1, a MRP4/ABCC4 inhibitor,induces apoptosis in AML cells enhanced by histamine

BackgroundCeefourin-1 is a specific MRP4/ABCC4 inhibitor with potential antileukemic activity. In this study, we evaluate the ability of ceefourin-1 alone or in combination with histamine, an approved antileukemic agent, to induce cell differentiation or apoptosis in human acute myeloid leukemic cells. We also examine ceefourin-1 toxicity in mice.MethodsU937, HL-60, and KG1a cells were used as models for human acute myeloid leukemia. Cyclic AMP efflux was estimated by measuring intracellular and extracellular cAMP levels. Cell differentiation was assessed by levels of CD14 and CD11b by FACS, and CD88 by western blot, and by cell morphology. Apoptosis was evaluated by cleavage of caspase-3 and PARP by western blot, and by annexin V binding assay. Subacute toxicity study of ceefourin-1 was carried out in BALB/c mice.ResultsCeefourin-1 inhibits cAMP exclusion in AML cells and promotes intracellular signaling via CREB. Ceefourin-1 leads AML cells to apoptosis and histamine potentiates this effect, without evidence of cell differentiation. Intraperitoneal administration of ceefourin-1 shows no important alterations in mice blood parameters, hepatic, and renal functions, nor signs of histologic damage.ConclusionsThese results show that ceefourin-1 promotes apoptosis in AML cells that is enhanced by histamine.General significance:This work indicates that ceefourin-1 represents a promising molecule that could be used alone or in combination with histamine for in vivo evaluation in acute myeloid leukemia malignancies.