Plant Food Anthocyanins Induced Platelet Apoptosis Via BCL-2/BCL-XL Pathway

Abstract Background: Platelets are versatile cells and play important roles in hemostasis/thrombosis, inflammation, and atherosclerosis. The pathogenesis of cardiovascular diseases (CVDs) is linked to platelet hyperactivity which is considered an independent risk factor for CVDs. Platelets are critical for promoting the progression of CVDs, and platelet apoptosis have been reported to be involved in platelet activation. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies demonstrated that anthocyanins from plant food significantly inhibited platelet activation, adhesion, aggregation and granule secretion, as well as attenuated thrombus growth at both arterial and venous shear stresses in vitro and in vivo, however, the effects of anthocyanin on platelet apoptosis and its mechanisms have not been explored. In the present study, we examined whether anthocyanin Cyanidin-3-glucoside (Cy-3-g) affect platelet apoptosis and the BCL-2/BCL-XL intrinsic apoptotic pathway. Methods: Cy-3-g, the predominant bioactive compound of anthocyanin preparations, was obtained from Polyphenol AS Company in Norway.Purified gel-filtered platelets from healthy volunteers were incubated at 37oC for 40 minutes with different concentrations of Cy-3-g (0.5、5、50μM) or PBS buffer as a control. the activated platelets were triggered with 0.5U thrombin for 15min to induce apoptosis. Mitochondria membrane potential (Δψm) and membrane phospholipid phosphatidylserine (PS) exposure in both activated and resting platelets were assessed by flow cytometry. Cytochrome C release, activation of caspase-3, caspase-8, caspase-9, cleavage of gelsolin, the levels of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and proapoptotic BCL-2 family proteins Bax, Bak, Bad, Bid and tBid in both activated and resting platelets were measured by western blotting. Results: Cy-3-g at 5μM and 50μM directly induced significant ΔΨm dissipation in activated platelets dose dependently. Correspondingly, 50μM Cy-3-g increased cytochrome C release compared to control. The expression of pro-caspase-8 and pro-caspase-9 decreased, activation of caspase-3, caspase-8 and caspase-9 was induced in activated platelets in both 5μM and 50μM Cy-3-g groups. Both PS exposure and the cleavage of gelsolin increased in activated platelets, however these effects were only observed at Cy-3-g doses as high as 50μM. Cy-3-g did not induce the above changes in resting platelets. The intrinsic apoptotic pathway was initiated by Cy-3-g treatment in activated platelets; Cy-3-g significantly inhibited the expression of BCL-2, BCL-XL and increased the levels of Bax, Bak, Bad and Bid in activated platelets dose dependently. No significant difference was observed in resting platelets. Conclusions: Our data demonstrate for the first time that purified anthocyanin Cy-3-g directly accelerated apoptosis in activated platelets via the BCL-2/BCL-XL pathway. Anthocyanins may possess therapeutic potential for patients suffering from thrombotic conditions. Disclosures No relevant conflicts of interest to declare.

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LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00406.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. G821-G829 ◽

Cited By ~ 54

Author(s):

Wenlin Deng ◽

De-An Wang ◽

Elvira Gosmanova ◽

Leonard R. Johnson ◽

Gabor Tigyi

Keyword(s):

Epithelial Cells ◽

Cytochrome C ◽

Protein Expression ◽

Caspase 3 ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Caspase 9 ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Antiapoptotic Activity

We previously showed ( Gastroenterology 123: 206–216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through Gi-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.

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JS-K, a Novel Nitric Oxide (NO) Generator, Induces Cytochrome c Release and Caspase Activation in HL-60 Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v104.11.3415.3415 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3415-3415

Author(s):

Paul J. Shami ◽

Vidya Udupi ◽

Margaret Yu ◽

Swati Malaviya ◽

Joseph E. Saavedra ◽

...

Keyword(s):

Cytochrome C ◽

Caspase 3 ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Caspase 8 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Dose Dependent Fashion ◽

Acute Myelogenous ◽

Dose Dependent

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity. We have previously shown that JS-K induces apoptosis in HL-60 cells by a caspase dependent mechanism (Molecular Cancer Therapeutics2:409-417,2003). The purpose of this study was to determine the pathway through which JS-K induces apoptosis. Western blot analysis showed that treatment of HL-60 cells with JS-K (0 – 1 μM) for 6 hours results in release of Cytochrome c from mitochondria in a dose dependent fashion. Treatment with JS-K resulted in a dose dependent activation of Caspase 9. Sixteen and 24 hours after exposure to 1 μM JS-K, Caspase 9 activity was induced by 393 ± 93% and 237 ± 13% of control, respectively (p = 0.03 at the 24 hours time point). Treatment with JS-K resulted in a dose dependent activation of Caspase 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 3 activity was 208 ± 3.4 % of control (p = 0.02). Treatment with JS-K also resulted in a dose dependent activation of Caspase 8, but to a lesser extent than Caspase 9 and 3. Twenty four hours after exposure to 1 μM JS-K, Caspase 8 activity was 144 ± 5.3 % of control (p = 0.04). We conclude that JS-K activates the intrinsic pathway of apoptosis in leukemia cells by inducing the release of Cytochrome c from mitochondria. (NO1-CO-12400).

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Differential Expression of Markers of Apoptosis in Platelets From Children with Acute Versus Chronic Immune Thrombocytopenia

Blood ◽

10.1182/blood.v120.21.1092.1092 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1092-1092

Author(s):

Markus Schmugge ◽

Jeanine Winkler ◽

Sabine Kroiss ◽

Margaret L. Rand ◽

Oliver Speer

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Caspase 3 ◽

Pediatric Patients ◽

Caspase 8 ◽

Caspase 9 ◽

Platelet Apoptosis ◽

Chronic Itp ◽

Acute Itp

Abstract Abstract 1092 Immune thrombocytopenia (ITP) is a common hematologic disorder in children that can lead to severe bleeding symptoms. In most children with ITP, platelet counts return to normal after weeks to months (acute ITP), however, in about 10–20% of patients, the low platelet counts persist for 12 months or longer (chronic ITP). No biological markers have been identified to predict the duration and/or severity of ITP. We have previously reported enhanced platelet apoptosis at the time of diagnosis of ITP in pediatric patients that was ameliorated after intravenous immunoglobulin (IVIg) (Winkler et al, Br J Haematol 2012;156:508–15). We have now investigated differences in the expression of markers of apoptosis in platelets from children with acute vs. chronic ITP. 23 pediatric patients with acute ITP were investigated and compared to 10 children with chronic ITP. In addition, from the initial group of acute ITP, 6 children developed chronic ITP and initial- and follow up results were compared. Markers of apoptosis, including activated caspase-3, caspase-8 and caspase-9, phosphatidylserine (PS) exposure, dissipation of the mitochondrial inner membrane potential (ΔYm), as well as microparticle formation, were analyzed by flow cytometry. At ITP diagnosis, the mean platelet count was 4×109/L (range: 1–14×109/L) and the proportions of platelets with activated caspase-3 (median, range) (20.4%, 1.4–64%, n=23), caspase-8 (16.7%, 1.0 – 42.7%, n=12) and caspase-9 (13.1%, 5 – 59.6%, n=12) were increased. While a higher mean platelet count was found in 10 children with chronic ITP (25×109/L, 4–60G/l), the proportions of platelets with activated caspase-3 (2.6%, 0.3–11.6%), caspase-8 (5.6%, 0.3–12.6%) and caspase-9 (4.3%, 0.3–15.6%) were significantly lower compared to children at diagnosis of acute ITP, but still higher compared to healthy controls (0.95%, 0 – 5.9%; 0.7%, 0.04 – 2.3% and 0.4%, 0.03 – 2.16%, respectively; n = 11) and children with thrombocytopenia due to chemotherapy (1.3%, 0.1 – 4.6%; 1.8%, 0.9 – 3.8%; and 1.8%, 0.6 – 2.9%, respectively; n = 11). Among the 6 children (26%) who developed chronic ITP from the initial cohort of 23 children, a mean platelet count of 29 (3–67×109/L) at >12 months after initial presentation was found. Except for one, none of the children with chronic ITP presented with bleeding symptoms; the median bleeding score was 2.5 (range: 1–3) at diagnosis and 1 (range: 0–2.5) at follow up during chronic ITP. In 5 of the children who developed chronic ITP, caspase activation was studied at diagnosis and at follow up >12 months after. In all of them, the proportions of platelets with activated caspase-3 (1.6%, 0.3–3.3%), caspase-8 (4.8%, 0.3–6.3%) and caspase-9 (4.1%, 0.3–7%) were found to be significantly lower at follow up compared to the time at diagnosis. In conclusion, although platelet apoptosis is enhanced at the time of diagnosis of pediatric ITP, this is not observed in platelets from patients with chronic ITP to the same degree. Further studies are needed to investigate other markers of apoptosis in platelets in the course of acute and chronic ITP. Disclosures: No relevant conflicts of interest to declare.

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Dexamethasone-induced apoptosis of thymocytes: role of glucocorticoid receptor–associated Src kinase and caspase-8 activation

Blood ◽

10.1182/blood-2002-06-1779 ◽

2003 ◽

Vol 101 (2) ◽

pp. 585-593 ◽

Cited By ~ 83

Author(s):

Maria Cristina Marchetti ◽

Barbara Di Marco ◽

Grazia Cifone ◽

Graziella Migliorati ◽

Carlo Riccardi

Keyword(s):

Cytochrome C ◽

Glucocorticoid Receptor ◽

Caspase 3 ◽

Kinase Inhibitor ◽

Src Kinase ◽

Caspase 8 ◽

Caspase 9 ◽

Cytochrome C Release ◽

Sequence Of Events ◽

Induced Apoptosis

Glucocorticoid hormones (GCHs) regulate normal and neoplastic lymphocyte development by exerting antiproliferative and/or apoptotic effects. We have previously shown that dexamethasone (DEX)–activated thymocyte apoptosis requires a sequence of events including interaction with the glucocorticoid receptor (GR), phosphatidylinositol-specific phospholipase C (PI-PLC), and acidic sphingomyelinase (aSMase) activation. We analyzed the mechanisms of GCH-activated apoptosis by focusing on GR-associated Src kinase, cytochrome c release, and caspase-8, -9, and -3 activation. We show here that PI-PLC binds to GR-associated Src kinase, as indicated by coimmunoprecipitation experiments. Moreover, DEX treatment induces PI-PLC phosphorylation and activation. DEX-induced PI-PLC phosphorylation, activation, and apoptosis are inhibited by PP1, a Src kinase inhibitor, thus suggesting that Src-mediated PI-PLC activation is involved in DEX-induced apoptosis. Caspase-9, -8, and -3 activation and cytochrome c release can be detected 1 to 2 hours after DEX treatment. Caspase-9 inhibition does not counter cytochrome crelease, caspase-8 and caspase-3 activation, and apoptosis. Caspase-8 inhibition counters cytochrome c release, caspase-9 and caspase-3 activation, and apoptosis, thus suggesting that caspase-8 inhibitor can directly inhibit caspase-9 and/or that DEX-induced caspase-8 activation is upstream to mitochondria and can regulate caspase-3 directly or through cytochrome c release and the consequent caspase-9/caspase-3 activation. DEX-induced caspase-8 activation, like ceramide-induced caspase-8 activation, correlates with the formation of Fas-associated death domain protein (FADD)/caspase-8 complex. Caspase-8 activation is countered by the inhibition of macromolecular synthesis and of Src kinase, PI-PLC, and aSMase activation, suggesting it is downstream in the DEX-activated apoptotic pathway of thymocytes.

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Delineation of the Molecular Mechanisms of Francisella tularensis-Induced Apoptosis in Murine Macrophages

Infection and Immunity ◽

10.1128/iai.71.8.4642-4646.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4642-4646 ◽

Cited By ~ 60

Author(s):

Xin-He Lai ◽

Anders Sjöstedt

Keyword(s):

Francisella Tularensis ◽

Mitochondrial Membrane ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Live Vaccine Strain ◽

Intrinsic Apoptotic Pathway ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Murine Macrophages ◽

Induced Apoptosis

ABSTRACT Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection with F. tularensis strain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochrome c into the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, the F. tularensis infection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.

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109. TROPHOBLAST DEPORTATION IS DEPENDENT UPON CASPASES 3, 8 AND ROCK

Reproduction Fertility and Development ◽

10.1071/srb09abs109 ◽

2009 ◽

Vol 21 (9) ◽

pp. 28

Author(s):

K. J. Askelund ◽

P. Stone ◽

L. W. Chamley

Keyword(s):

Caspase 3 ◽

First Trimester ◽

Normal Pregnancy ◽

Maternal Blood ◽

Caspase 8 ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Apoptosis Pathway ◽

Villous Trophoblast

Background: Trophoblast deportation is the process whereby multinucleated fragments of the syncytiotrophoblast are shed from the placenta into the maternal blood. It is estimated that 150,000 are shed from the placenta and deported daily in normal pregnancy and that more are shed during preeclampsia1. In normal pregnancy deported trophoblasts are thought to die by apoptosis, which is also increased in villous trophoblast in preeclampsia2. However, experimental confirmation that apoptosis leads to trophoblast shedding is required and it is not clear which components of the apoptotic pathway are involved in trophoblast shedding. Objectives: To determine the effect of inhibiting caspase 3 (executioner), caspases 8 and 9 (initiators), and Rho-associated kinase (ROCK; bleb formation) on the number of trophoblasts shed from first trimester human placentae. Methods : Using an in vitro placental explant model of trophoblast deportation, first trimester placentae were cultured for 72 hours in media containing specific inhibitors of ROCK, caspases 3, 8 or 9. Trophoblasts shed from quintuple explants/inhibitor from five placentae were depleted of contaminating leucocytes and erythrocytes, labelled with trypan blue and the sizes and numbers of shed trophoblasts quantified using a Nexcelom automated counter. Results: The number of trophoblasts that were shed from the explants was significantly increased (p=0.04) when caspase 3 (2.4 fold) and caspase 8 (2.7 fold) were inhibited. There was no significant change following caspase 9 inhibition. The number of shed trophoblasts was significantly decreased when ROCK was inhibited. None of the inhibitors significantly altered the size of the shed trophoblasts. Conclusion: Our data suggest that the apoptosis pathway is involved in trophoblast shedding in vitro from first trimester placentae. That caspase 8 but not caspase 9 affected shedding suggests trophoblasts from normal placentae are induced to die via the extrinsic apoptosis pathway. Aberrant regulation of the apoptosis pathway may contribute to pregnancy pathology.

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Curcumin Inhibits Mitochondrial Injury and Apoptosis from the Early Stage in EAE Mice

Oxidative Medicine and Cellular Longevity ◽

10.1155/2014/728751 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 23

Author(s):

Jinzhou Feng ◽

Tao Tao ◽

Weiping Yan ◽

Cindy Si Chen ◽

Xinyue Qin

Keyword(s):

Neuronal Apoptosis ◽

Caspase 3 ◽

Early Stage ◽

Apoptotic Cells ◽

Intrinsic Apoptotic Pathway ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Mitochondrial Injury ◽

Early Stages ◽

Cyt C

The exact pathophysiological change concerning mitochondrial injury and oligodendrocyte apoptosis in MS and EAE model is still unknown. Whether curcumin is able to inhibit mitochondrial injury and suppress the apoptosis in the early stages of MS/EAE is still unclear. We first explored mitochondrial injury and apoptosis at different time points p.i. in C57 BL/6 EAE mice. We then explored the effects of curcumin on mitochondria and apoptosis. Results showed that mitochondrial injury can be observed 3 days p.i. Apoptosis in the spinal cord occurred 3 days p.i. and the apoptotic cells were shown to be oligodendrocytes and neuronal cells. Curcumin significantly reduced the number of apoptotic cells and inhibited the upregulation of cyt-c, caspase-9, and caspase-3 at 7 days p.i. in the EAE mice. These observations demonstrate that mitochondrial injury and oligodendrocyte/neuronal apoptosis occur in the early stages of EAE. Curcumin can inhibit apoptosis in EAE mice which maybe act through protection of mitochondrial injury and inhibition of the intrinsic apoptotic pathway.

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The Apoptosis Induction of Oleandrin on Osteosarcoma Cells through Regulating Mitochondrial- and Death Receptor-Dependent Apoptotic Pathways in Vitro

10.20944/preprints201608.0188.v1 ◽

2016 ◽

Author(s):

Yunlong Ma ◽

Bin Zhu ◽

Lei Yong ◽

Chunyu Song ◽

Xiao Liu ◽

...

Keyword(s):

Cytochrome C ◽

Cell Apoptosis ◽

Caspase 3 ◽

Treatment Time ◽

Death Receptor ◽

Caspase 8 ◽

Osteosarcoma Cells ◽

Caspase 9 ◽

Apoptotic Pathway

Our previous study has found the anti-tumor activity of oleandrin in osteosarcoma cells in vitro, but the signal transduction process of cell apoptosis induced by oleandrin is uncertain, which is explored in this study. Fluorescence staining and flow cytometry (FCM) was performed to detect the cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Caspase-3 activity was detected using a commercial kit. The protein expression of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 was detected using western blot. A pan-caspase inhibitor, z-VAD-fmk, was applied to block the apoptotic pathway and the apoptosis status were re-tested. We found that oleandrin significantly induced the increased apoptosis of U2OS cells. Meanwhile, the intracellular ROS was elevated, but the MMP decreased. The cytochrome c in mitochondria was notably decreased but increased in cytoplasm. The caspase-3 activity was also enhanced with the increase of drug concentration and treatment time. Oleandrin also down-regulated the level of bcl-2, but remarkably up-regulated the expression of bax, cleaved caspase-9, Fas, FasL, cleaved caspase-8 and cleaved caspase-3. Furthermore, the pre-treatment with z-VAD-fmk almost completely reverted the oleandrin-induced apoptosis. The results suggested that oleandrin induces the apoptosis of osteosarcoma cells via mitochondrial- and death receptor-dependent pathways.

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Regulation of Caspase 9 through Phosphorylation by Protein Kinase C Zeta in Response to Hyperosmotic Stress

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10543-10555.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10543-10555 ◽

Cited By ~ 44

Author(s):

Suzanne C. Brady ◽

Lindsey A. Allan ◽

Paul R. Clarke

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Signaling Pathways ◽

Caspase 3 ◽

Hyperosmotic Stress ◽

Intrinsic Apoptotic Pathway ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Kinase C ◽

In Cells

ABSTRACT Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKCζ). Prevention of Ser144 phosphorylation by inhibition of PKCζ or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKCζ and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKCζ restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.

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Role of the Mitochondrial Signaling Pathway in Murine Coronavirus-Induced Oligodendrocyte Apoptosis

Journal of Virology ◽

10.1128/jvi.80.1.395-403.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 395-403 ◽

Cited By ~ 21

Author(s):

Yin Liu ◽

Yinghui Pu ◽

Xuming Zhang

Keyword(s):

Hepatitis Virus ◽

Mitochondrial Pathway ◽

Caspase 8 ◽

Caspase 9 ◽

Apoptotic Pathway ◽

Antiapoptotic Proteins ◽

Mitochondrial Signaling ◽

Drastic Increase ◽

Infected Cells

ABSTRACT A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.

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