Loss Of Membrane Asymmetry Research Articles

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, “crossmember” helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

Stimulation of calcium influx and CK1α by NF-κB antagonist [6]-Gingerol reprograms red blood cell longevity.

Chemotherapy-induced anemia (CIA) is a major obstacle in cancer management. Although the mechanisms governing CIA are poorly understood, recent efforts have identified suicidal erythrocyte (red blood cell, RBC) death as a possible cause of CIA. [6]-Gingerol (GNG), a polyphenol extracted from Zingiber officinale plant, exhibits a wide array of biological activities including antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, and anticancer activities, in vitro and in vivo. However, the potential toxicity of GNG to human RBCs remains unexplored. RBCs from heparinized blood were isolated by centrifugation and exposed to antitumor concentrations (10-100µM) of GNG for 24hr at 37°C. Hemolysis was calculated from hemoglobin leakage in the supernatant (λmax =405nm), while cytofluorometric analysis of eryptosis employed Annexin-V-FITC to detect phosphatidylserine (PS) exposure, forward scatter (FSC) to estimate cell volume, Fluo4/AM to measure calcium activity, and H2 DCFDA to assess oxidative stress. Moreover, zVAD(OMe)-FMK, SB203580, necrostatin-2, staurosporin, and D4476 were used to identify signaling pathways responsive to GNG. GNG induced significant hemolysis at 100µM, independently of extracellular calcium, and increased Annexin-V-FITC fluorescence that was thoroughly abrogated without extracellular calcium. GNG also enhanced Fluo4 fluorescence and reduced FSC, but had no significant effect on DCF fluorescence. Importantly, the presence of D4476 significantly attenuated GNG-induced hemolysis. In conclusion, GNG stimulates premature RBC death characterized by loss of membrane asymmetry, elevated cytosolic calcium, cell shrinkage, and casein kinase 1α activation. Blocking the activity of calcium channels or CK1α may, therefore, ameliorate the toxic effects of GNG on RBCs. PRACTICAL APPLICATIONS: This report presents a safety assessment of GNG as a chemotherapeutic agent and highlights the novel toxicity of GNG to human RBCs. Our findings provide novel insights that may lead to more efficient utilization of GNG in chemotherapy. Specifically, our data revealed the involvement of calcium channels and casein kinase 1α in mediating GNG-induced premature RBC death, and, therefore, inverse agonists or inhibitors of either pathway may be used as pharmaceutical adjuvants to attenuate the toxic effects of GNG.

Loss of membrane asymmetry alters the interactions of erythrocytes with engineered silica nanoparticles.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.

Pathogen reduction with riboflavin and ultraviolet light induces a quasi-apoptotic state in blood leukocytes.

Alloimmunization to platelet-rich plasma (PRP) transfusions can cause adverse reactions such as platelet refractoriness or transplant rejection. Pathogen reduction treatment with ultraviolet light and riboflavin (UV + R) of allogeneic PRP was shown to reduce allogeneic antibody responses and confer partial antigen-specific immune tolerance to subsequent transfusions in mice. Studies have shown that UV + R was effective at both rapidly killing donor white blood cells (WBCs) and reducing their ability to stimulate an allogeneic response in vitro. However, the manner in which UV + R induces WBC death and its associated role in the immune response to treated PRP is unknown. This study evaluates whether UV + R causes WBC apoptosis by examining phosphatidylserine exposure on the plasma membrane, membrane asymmetry, caspase activity, and chromatin condensation by flow cytometry. The immunogenicity of WBCs killed with UV + R versus apoptotic or necrotic pathways was also examined in vivo. WBCs after UV + R exhibited early apoptotic-like characteristics including phosphatidylserine exposure on the outer leaflet of the plasma membrane and loss of membrane asymmetry, but unlike canonical apoptotic cells, caspase activity and chromatin condensation were not apparent. However, in vivo studies demonstrated, unlike untreated or necrotic WBCs, both apoptotic WBCs and UV + R-treated WBCs failed to prime alloantibody responses to subsequent untreated transfusions. Overall, the mechanism of WBC death following UV + R treatment shares some membrane characteristics of early apoptosis but is distinct from classic apoptosis. Despite these differences, UV + R-treated and apoptotic WBCs both offer some protection from alloimmunization.

Changes in Membrane Ceramide Pools in Rat Soleus Muscle in Response to Short-Term Disuse.

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET)

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1–5 mM (0.02%–0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

Structure-dependent induction of apoptosis by hepatotoxic pyrrolizidine alkaloids in the human hepatoma cell line HepaRG: Single versus repeated exposure

Pyrrolizidine alkaloids (PA) are secondary plant compounds. PA intoxication in humans causes severe acute and chronic hepatotoxicity. However, the molecular mechanisms of PA hepatotoxicity in humans are not well understood yet.Therefore, we investigated cell death parameters in human HepaRG cells following either single (24 h) or repeated dose treatment (14 d) with structurally different PA of the retronecine (echimidine, senecionine), heliotridine (heliotrine), and otonecine type (senkirkine). After 24 h of exposure only retronecine-type PA were cytotoxic in HepaRG cells and induced apoptosis indicated by a loss of membrane asymmetry, disruption of the mitochondrial membrane potential, and increased pro-caspase and PARP cleavage. In contrast, after 14 d all four PA exerted the aforementioned effects. Furthermore, the apoptotic events caspase 3, 8 and 9 activation as well as nuclear condensation and DNA fragmentation were only detected for the retronecine-type PA after single exposure (6 h).Overall, our studies revealed a time- and structure-dependent apoptosis after PA exposure, suggesting that retronecine-type PA seem to be more potent apoptosis inducers than heliotridine- or otonecine-type PA. Furthermore, our results suggest that PA-induced apoptosis in HepaRG cells occur most probably by involving both, the extrinsic death receptor pathway as well as the intrinsic mitochondrial pathway.

Status of Membrane Asymmetry in Erythrocytes: Role of Spectrin.

Spectrin-based proteinaceous membrane skeletal network has been found to be implicated in membrane disorders like hereditary spherocytosis (HS). HS greatly affects eryptosis via loss of membrane asymmetry which is seen to be the case in haemoglobin disorders like thalassemia and sickle cell disease as well. The biological implications of the status of membrane asymmetry are strongly correlated to spectrin interactions with aminophospholipids, e.g. PE and PS. Fluorescence and X-ray reflectivity (XRR) measurements of spectrin interactions with small unilamellar vesicles (SUVs) and cushioned bilayers of phospholipids, respectively, were studied. Both the XRR and fluorescence measurements led to the characterization of spectrin orientation on the surface of lipid bilayer of phosphatidylcholine (PC) and PC/aminophospholipid mixed membrane systems showing formation of a uniform layer of spectrin on top of the mixed phospholipid bilayer. Fluorescence studies show that spectrin interacts with PC and phosphatidylethanolamine (PE)/phosphatidylserine (PS) membranes with binding dissociation constants (Kd) in the nanomolar range indicating the role of spectrin in the maintenance of the overall membrane asymmetry of erythrocytes.

Erythrocyte and platelet proteomics in hematological disorders.

Erythrocytes undergo ineffective erythropoesis, hemolysis, and premature eryptosis in sickle cell disease and thalassemia. Abnormal hemoglobin variants associated with hemoglobinopathy lead to vesiculation, membrane instability, and loss of membrane asymmetry with exposal of phosphatidylserine. This potentiates thrombin generation resulting in activation of the coagulation cascade responsible for subclinical phenotypes. Platelet activation also results in the release of microparticles, which express and transfer functional receptors from platelet membrane, playing key roles in vascular reactivity and activation of intracellular signaling pathways. Over the last decade, proteomics had proven to be an important field of research in studies of blood and blood diseases. Blood cells and its fluidic components have been proven to be easy systems for studying differential expressions of proteins in hematological diseases encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies. Proteomic studies of erythrocytes and platelets reported from several groups have highlighted various factors that intersect the signaling networks in these anucleate systems. In this review, we have elaborated on the current scenario of anucleate blood cell proteomes in normal and diseased individuals and the cross-talk between the two major constituent cell types of circulating blood.

Biochemical evidence for Ca2+-independent functional activation of hPLSCR1 at low pH

Human phospholipid scramblase 1 (hPLSCR1) is a Ca2+-dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry. It has been reported that hPLSCR1 exhibits Ca2+- independent activity at low pH. However, the conformational changes induced at low pH leading to functional activation are not known. Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1. Tryptophan fluorescence measurements showed a decrease in Ca2+-binding affinity at low pH, although not at pH 5.5. Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the β-sheet content of the protein. At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH. The effect occurred at the promoter level. The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis. Based on these results, we conclude that the mechanisms of Ca2+- and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression.

Role of Microparticles in Hemostasis and Thrombosis

Abstract SCI-10 Microparticles (MPs) (also known as or microvesicles) are submicron plasma membrane vesicles released from activated or apoptotic cells. Current estimates indicate that MPs are ∼200 nm in diameter. They are formed as membrane blebs and have phosphatidylserine exposed on the surface due to a loss of membrane asymmetry. MPs containing tissue factor (TF) are highly procoagulant. We recently developed an assay to measure TF activity of MPs isolated from plasma. We found that healthy individuals contain low levels of TF+ MPs, but levels are increased in many pathological conditions associated with thrombosis, such as sepsis, cardiovascular disease, sickle cell disease, diabetes mellitus, and cancer. We have analyzed the role of TF+ MPs in various murine models of thrombosis and in different patient populations. Work in collaboration with Bruce and Barbara Furie demonstrated that leukocyte-derived TF+ MPs contributed to microvascular thrombosis in a model of laser injured cremaster arterioles using healthy mice. Further studies have been performed using different murine models of disease. Arterial thrombosis is mostly caused by rupture of atherosclerotic plaques and leads to myocardial infarction and stroke. In hypercholesterolemic LDLR−/− mice we observed increased levels of hematopoietic cell-derived TF+ MPs that were associated with a prothrombotic state. These TF+ MPs may enhance thrombosis after plaque rupture. Indeed, we observed more fibrin deposition in the laser injury model of thrombosis in hypercholesterolemic mice than controls. Importantly, we found that simvastatin reduced TF expression in mononuclear cells and levels of TF+ MPs in the plasma in these hypercholesterolemic mice. Venous thrombosis occurs without significant damage to the vessel wall and is associated with embolization of clot fragments that can block pulmonary arteries. Venous clots appear to form due to changes in the vessel wall, stasis, and/or increased coagulability of the blood. We have analyzed levels of TF+ MPs in murine cancer models and cancer patient samples. We and other have found that tumors release TF+ MPs into the circulation. In one study, we found that pancreatic patients with the highest levels of MP TF activity develop thrombosis. These results suggest that TF+ MPs may enhance venous thrombosis in cancer patients by binding to activated endothelium. Further studies are needed to determine if levels of TF+ MPs in the circulation can be used to identify patients at risk for thrombosis. Disclosures: Mackman: Merck: Consultancy; Daiichi: Consultancy; Daiichi: Research Funding.

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

INHIBITION OF CASPASE-LIKE ACTIVITIES PREVENTS THE APPEARANCE OF REACTIVE OXYGEN SPECIES AND DARK-INDUCED APOPTOSIS IN THE UNICELLULAR CHLOROPHYTE DUNALIELLA TERTIOLECTA(1).

When the chlorophyte alga Dunaliella tertiolecta Butcher is placed in darkness, a form of programmed cell death with many similarities to apoptosis is induced, including the induction of caspase-like proteases. Many uncertainties about the regulation and mediators that participate in the process remain. To examine the relationship between caspase-like activities and different apoptotic events (i.e., phosphatidylserine [PS] translocation), increases in membrane permeability and numbers of dead cells revealed by SYTOX-green staining, and the generation of reactive oxygen species (ROS), we used the broad-range caspase inhibitor Boc-D-FMK to block the activity of the whole class of caspase-like proteins simultaneously. In the presence of the inhibitor, ROS were not produced, and cells did not die. Loss of membrane asymmetry, indicated by external labeling of PS by annexin V, was apparent at midstages of light deprivation, although it did not conform to the typical pattern for PS exposure observed in metazoans or vascular plants, which occurs at early stages of the apoptotic event. Thus, we have evidence for a link between ROS and cell death involving caspase-like enzymes in an alga. The fact that caspase-like inhibitors prevent not only cell death, but also ROS and loss of cell membrane integrity and asymmetry, suggests that caspase-like proteases might have regulatory roles early in cell death, in addition to dismantling functions.

Porous red cell ultrastructure and loss of membrane asymmetry in a novel case of hemolytic anemia

Transmission electron microscopic study revealed large pores on the erythrocyte ghost membranes, disrupted cytoskeleton and microcytosis of circulating erythrocytes in a novel case of hemolytic anemia. Greater loss of phosphatidylserine (PS) asymmetry was observed in younger erythrocytes compared with the aged ones in contrast to the normal red cells. Levels of sialylated glycoconjugates, such as glycophorin, measured by the binding of wheat germ agglutinin, showed greater loss upon aging. Such drastic loss of PS asymmetry leads to faster eryptosis, mediated by shedding of glycophorin-containing microvesicles leaving highly PS-exposed erythrocytes accessible to the phagocytes.

Induction of apoptotic cell death by a ceramide analog in PC-3 prostate cancer cells

Ceramide analogs are potential chemotherapeutic agents. We report that a ceramide analog induces apoptosis in human prostate cancer cells. The ceramide analog induced cell death through an apoptotic mechanism, which was demonstrated by DNA fragmentation, the cleavage of poly ADP ribose polymerase (PARP), and a loss of membrane asymmetry. Treating the cells with ceramide analog resulted in the release of various proapoptotic mitochondrial proteins including cytochrome c and Smac/DIBLO into the cytosol, and a decrease in the mitochondrial membrane potential. In addition, the ceramide analog decreased the phospho-Akt and phospho-Bad levels. The expression of the antiapoptotic Bcl-2 decreased slightly with increasing Bax to Bcl-2 ratio. These results suggest that the ceramide analog induces apoptosis by regulating multiple signaling pathways that involve the mitochondrial pathway.

The Bcl-2 Family Protein Inhibitor, ABT-263, Promotes Apoptotic-Like Response in Isolated Platelets.

Platelets are relatively short-lived, anucleated cells derived from megakaryocytes that are essential for proper hemostasis. Although they lack a nucleus, platelets possess a highly organized structure with multiple cellular organelles including mitochondria and a number of specific granules. Upon recruitment to sites of endothelial injury, platelet activation stimulates their adhesion, aggregation and release of granule components leading to coagulation. Many of the cellular changes observed during platelet activation and platelet senescence resemble those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, cell shrinkage and microparticle formation (analogous to membrane blebbing). ABT-263 is an orally bioavailable Bcl-2 family protein inhibitor currently in clinical development. This compound is a potent antagonist of the anti-apoptotic proteins Bcl-xL, Bcl-2 and Bcl-w and induces apoptosis in cancer cells dependent on these proteins for survival. ABT-263 induces a unique thrombocytopenia in multiple species that is characterized by a rapid clearance of circulating platelets with recovery to normal platelet counts within several days after treatment cessation. To elucidate the mechanisms underlying this thrombocytopenia, a series of in vitro experiments were performed using freshly isolated platelets comparing ABT-263, its enantiomer (which has significantly lower activity against Bcl-2 family proteins) and known platelet activators. ABT-263, but not the enantiomer control, reduced the viability of dog and human platelets and activated key apoptotic processes, including cytochrome c release, caspase 3 activation, and PS externalization. ABT-263 did not induce aggregation of isolated platelets and did not induce surface expression of GPIIb/IIIa that is essential for platelet adhesion to fibrinogen during clot formation. While ABT-263 had no effect on markers of lysosomal- or alpha-granule release, increases in a marker of dense granule release were apparent at high concentrations. Taken together these data suggest that ABT-263 induces an apoptotic-like response in platelets that is distinct from platelet activation. Furthermore, the lack of effect of the enantiomer control compound suggests this effect is mediated through inhibition of antiapoptotic Bcl-2 family members. Loss of membrane asymmetry and PS externalization has been suggested to be the primary signal for the recognition and clearance of senescent platelets by phagocytes of the reticuloendothelial system. Induction of apoptosis in platelets by Bcl-2 family protein inhibitors such as ABT-263 may coopt this process inducing a premature platelet senescence resulting in a novel type of thrombocytopenia, in vivo.

Genetic Deletion of NAD(P)H:Quinone Oxidoreductase 1 Abrogates Activation of Nuclear Factor-κB, IκBα Kinase, c-Jun N-terminal Kinase, Akt, p38, and p44/42 Mitogen-activated Protein Kinases and Potentiates Apoptosis

The NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II enzyme that reduces and detoxifies quinones and their derivatives. Although overexpressed in tumor cells, the NQO1 has been linked with the suppression of carcinogenesis, and the effect of NQO1 on tumor necrosis factor (TNF), a cytokine that mediates tumorigenesis through proliferation, invasion, angiogenesis, and metastasis of tumors, is currently unknown. The purpose of our study was to determine the role of NQO1 in TNF cell signaling by using keratinocytes derived from wild-type and NQO1 gene-deleted mice. TNF induced nuclear factor (NF)-kappaB activation in wild-type but not in NQO1-deleted cells. The treatment of wild-type cells with dicoumarol, a known inhibitor of NQO1, also abolished TNF-induced NF-kappaB activation. NF-kappaB activation induced by lipopolysaccharide, phorbol ester, and cigarette smoke, was also abolished in NQO1-deleted cells. The suppression of NF-kappaB activation was mediated through the inhibition of IkappaBalpha kinase activation, IkappaBalpha phosphorylation, and IkappaBalpha degradation. Further, the deletion of NQO1 abolished TNF-induced c-Jun N-terminal kinase, Akt, p38, and p44/p42 mitogen-activated protein kinase activation. TNF also induced the expression of various NF-kappaB-regulated gene products involved in cell proliferation, antiapoptosis, and invasion in wild-type NQO1 keratinocytes but not in NQO1-deleted cells. The suppression of these antiapoptotic gene products increased TNF-induced apoptosis in NQO1-deleted cells. We also found that TNF activated NQO1, and NQO1-specific small interfering RNA abolished the TNF-induced NQO1 activity and NF-kappaB activation. Overall, our results indicate that NQO1 plays a pivotal role in signaling activated by TNF and other inflammatory stimuli and that its suppression is a potential therapeutic strategy to inhibit the proliferation, survival, invasion, and metastasis of tumor cells.

Plumbagin (5-Hydroxy-2-methyl-1,4-naphthoquinone) Suppresses NF-κB Activation and NF-κB-regulated Gene Products Through Modulation of p65 and IκBα Kinase Activation, Leading to Potentiation of Apoptosis Induced by Cytokine and Chemotherapeutic Agents

Plumbagin, derived from the medicinal plant Plumbago zeylanica, modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-kappaB, suggesting plumbagin might affect the NF-kappaB activation pathway. We found that plumbagin inhibited NF-kappaB activation induced by TNF, and other carcinogens and inflammatory stimuli (e.g. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1beta, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-kappaB activation in certain tumor cells. The suppression of NF-kappaB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappaB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-beta, and the p65 subunit of NF-kappaB. Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol both in vitro and in vivo. However, plumbagin did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine 38 mutated to serine. Plumbagin down-regulated the expression of NF-kappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion. Overall, our results indicate that plumbagin is a potent inhibitor of the NF-kappaB activation pathway that leads to suppression of NF-kappaB-regulated gene products. This may explain its cell growth modulatory, anticarcinogenic, and radiosensitizing effects previously described.