Intrinsic Mitochondrial Apoptotic Pathway Research Articles

A natural genetic variant of granzyme B confers lethality to a common viral infection.

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining ‘Asp-ase’ activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmBW mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.

A polymethoxyflavone from Laggera pterodonta induces apoptosis in imatinib-resistant K562R cells via activation of the intrinsic apoptosis pathway.

BackgroundTreatment with imatinib mesylate (IM) (a tyrosine kinase inhibitor) is the first line of standard care for patients newly diagnosed with CML. Despite the success of IM and other tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) remains largely incurable, and a number of CML patients die due to Abl mutation-related drug resistance and blast crisis. 3, 5-Dihydroxy-6, 7, 3′4′-tetramethoxyflavone (DHTMF) is a polymethoxyflavone isolated from Laggera pterodonta which is a herbal medicine used to treat cancer in the Chinese folk. In the previous study, we found DHTMF demonstrated good antiproliferative activities against a number of cancer cell lines and induced the apoptosis of CNE cells in vitro in a time- and dose-dependent manner while exhibiting low cytotoxicity in the two normal cell lines Vero and EVC304. The aim of the present study was to evaluate the proliferation inhibition and apoptosis induced by DHTMF alone and in combination with IM in the IM-resistant CML cell line K562R.MethodsCell proliferation was assayed with the cell counting kit-8 (CCK8) method. The apoptosis percentage was determined by flow cytometry (FCM). Mitochondrial transmembrane potential was detected using FCM and confocal laser-scanning microscopy. The level of proteins involved in apoptosis was detected by Western blotting.ResultsDHTMF suppressed K562R cell viability in both time- and dose-dependent manners. DHTMF combined with IM enhanced the inhibitory effects and apoptosis in K562R cells as compared with DHTMF alone. DHTMF alone and in combination with IM significantly decreased the mitochondrial membrane potential and increased the levels of cleaved caspase-9, caspase-7, caspase-3, and PARP in K562R cells.ConclusionsWe demonstrated that DHTMF could inhibit IM-resistant K562R cell proliferation and induces apoptosis via the intrinsic mitochondrial apoptotic pathway. These results suggest that DHTMF may be a potential therapeutic drug with lower side effects against IM resistance in CML cells.

FADD adaptor and PEA-15/ERK1/2 partners in major depression and schizophrenia postmortem brains: Basal contents and effects of psychotropic treatments

Enhanced brain apoptosis (neurons and glia) may be involved in major depression (MD) and schizophrenia (SZ), mainly through the activation of the intrinsic (mitochondrial) apoptotic pathway. In the extrinsic death pathway, pro-apoptotic Fas-associated death domain (FADD) adaptor and its non-apoptotic p-Ser194 FADD form have critical roles interacting with other death regulators such as phosphoprotein enriched in astrocytes of 15kDa (PEA-15) and extracellular signal-regulated kinase (ERK). The basal status of FADD (protein and messenger RNA (mRNA)) and the effects of psychotropic drugs (detected in blood/urine samples) were first assessed in postmortem prefrontal cortex of MD and SZ subjects (including a non-MD/SZ suicide group). In MD, p-FADD, but not total FADD (and mRNA), was increased (26%, n=24; all MD subjects) as well as p-FADD/FADD ratio (a pro-survival marker) in antidepressant-free MD subjects (50%, n=10). In contrast, cortical FADD (and mRNA), p-FADD, and p-FADD/FADD were not altered in SZ brains (n=21) regardless of antipsychotic medications (except enhanced mRNA in treated subjects). Similar negative results were quantified in the non-MD/SZ suicide group. In MD, the regulation of multifunctional PEA-15 (i.e., p-Ser116 PEA-15 blocks pro-apoptotic FADD and PEA-15 prevents pro-survival ERK action) and the modulation of p-ERK1/2 were also investigated. Cortical p-PEA-15 was not changed whereas PEA-15 was increased mainly in antidepressant-treated subjects (16–20%). Interestingly, cortical p-ERK1/2/ERK1/2 ratio was reduced (33%) in antidepressant-free when compared to antidepressant-treated MD subjects. The neurochemical adaptations of brain FADD (increased p-FADD and pro-survival p-FADD/FADD ratio), as well as its interaction with PEA-15, could play a major role to counteract the known activation of the mitochondrial apoptotic pathway in MD.

PGE₂ promotes apoptosis induced by cytokine deprivation through EP3 receptor and induces Bim in mouse mast cells.

Increased mast cell numbers are observed at sites of allergic inflammation and restoration of normal mast cell numbers is critical to the resolution of these responses. Early studies showed that cytokines protect mast cells from apoptosis, suggesting a simple model in which diminished cytokine levels during resolution leads to cell death. The report that prostaglandins can contribute both to recruitment and to the resolution of inflammation together with the demonstration that mast cells express all four PGE2 receptors raises the question of whether a single PGE2 receptor mediates the ability of PGE2 to regulate mast cell survival and apoptosis. We report here that PGE2 through the EP3 receptor promotes cell death of mast cells initiated by cytokine withdrawal. Furthermore, the ability of PGE2 to limit reconstitution of tissues with cultured mast cells is lost in cell lacking the EP3 receptor. Apoptosis is accompanied by higher dissipation of mitochondrial potential (ΔΨm), increased caspase-3 activation, chromatin condensation, and low molecular weight DNA cleavage. PGE2 augmented cell death is dependent on an increase in intracellular calcium release, calmodulin dependent kinase II and MAPK activation. Synergy between the EP3 pathway and the intrinsic mitochondrial apoptotic pathway results in increased Bim expression and higher sensitivity of mast cells to cytokine deprivation. This supports a model in which PGE2 can contribute to the resolution of inflammation in part by augmenting the removal of inflammatory cells in this case, mast cells.

Selective apoptotic cell death effects of oral cancer cells treated with destruxin B.

BackgroundRecent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent.MethodsDestruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses.ResultsThe results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners.ConclusionsThis is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.

The use of flow cytometry for analysis of the mitochondrial cell death

Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways - external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca2+ ions and other proteins that affect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols.

Cranberry extract initiates apoptosis in HL‐60 cell line through intrinsic apoptotic pathway (644.9)

The potential anticancer activity of cranberry was reported for the first time in 1996. Proanthocyandins (PACs), the most abundant and complex flavonoid in cranberry, have been described as the major contributors of the anticancer activity reported in the literature. One of the proposed chemopreventative mechanisms is the induction of apoptosis by cranberry PACs. However, limited studies have been done to investigate the role of cranberry in initiating apoptosis in cancer cells and its underlying mechanism of action remains undetermined. Therefore, the overall aim of this study was to investigate the apoptotic pathway that cranberry affects, intrinsic vs. extrinsic pathway. In our study, HL‐60 cells were incubated with 25 µg/ml cranberry‐(60‐65%)PAC extract for 24 hours. Cranberry treatment showed an 85 % increase in caspase 3/7 activity. The data also showed a great enhancement in the activation of caspase‐9 (379 %), but it did not exhibit any change in caspase‐8 activity. Further tests revealed that cranberry treatment caused a loss of mitochondrial transmembrane potential leading to an increase in the mitochondrial release of cytochrome C (87.5%) and Smac (41%). These findings indicate that this cranberry‐PAC extract can induce apoptosis in HL‐60 cells through triggering the intrinsic (mitochondrial) apoptotic pathway.Grant Funding Source: Funded by Agricultural Experiment Station and Ocean Spray Cranberries, Inc.

Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation

Artificial insemination (AI) with post-thawed boar spermatozoa results in low farrowing rates and reduced litter sizes mainly due to cryoinjury or damages to spermatozoa during cryopreservation. Low viability and motility of post-thawed boar spermatozoa are highly associated with apoptosis during cryopreservation. Although glycerol is widely used a cryoprotectant (CPA) for boar spermatozoa cryopreservation, the mechanism and relationship between glycerol and apoptosis-related gene expression needs to be clarified. In this study, we treated boar spermatozoa with different concentrations of glycerol in lactose egg yolk (LEY) extender to evaluate the apoptosis-related gene expression and protease activities of caspases. These results show that: (1) low concentrations of glycerol (2% and 3%) were more suitable for boar spermatozoa cryopreservation; (2) apoptosis-related genes involved in intrinsic mitochondrial and extrinsic death receptor apoptotic signaling pathways were widely expressed in different concentrations of glycerol treated boar spermatozoa; (3) there was a significant positive correlation (r=0.840, P=0.037) between the percentage of Annexin V+/PI+ staining spermatozoa and caspase-6/9 protease activity. In conclusion, 2% and 3% glycerol have the best anti-apoptotic effects, and the expression of Fas/FasL and Bcl-2/Bax have a strong correlation with spermatozoa parameters.

Mechanical stress-induced apoptosis of nucleus pulposus cells: an in vitro and in vivo rat model

BackgroundUn-physiological loads play an important role in the degenerative process of inter-vertebral discs (IVD). In this study, we used an in vitro and in vivo rat model to investigate the mechanism of nucleus pulposus (NP) cells apoptosis induced by mechanical stress. MethodsStatic compressive load to IVDs of rat tails was used as the in vivo model. For the in vitro model, NP cells were tested under the physiological and un-physiological loading. For histological examination, apoptotic index study, and apoptotic gene expression, we also selected cytokines [bone morphogenetic protein (BMP)-2/7, insulinlike growth factor (IGF)-1, platelet-derived growth factor (PDGF)] to be analyzed. ResultsUnder mechanical loading, cellular density was significantly decreased, but there was an increase of TUNEL positive cells and apoptosis index. In a dose-dependent manner; the necrosis became apparent in the unphysiologic strain. The selected cytokines (BMP-2/7, IGF-1, PDGF) can significantly reduce the percentage of apoptotic and necrotic cells. ConclusionsWe conclude that the intrinsic (mitochondrial) apoptotic pathway plays an important role in the compressive load-induced apoptosis of NP cells. Combination therapy reducing the mechanical load and selected cytokines (BMP-2/7, IGF-1 and PDGF) may have considerable promise in the treatment of spine disc degeneration.

Mechanism of the malabaricone C-induced toxicity to the MCF-7 cell line

In this study, we studied the mechanism of the cytotoxicity of malabaricone C (mal C) against human breast cancer MCF-7 cell line. Mal C dose-dependently increased the sub G1 cell population, associated with cytoplasmic oligonucleosome formation and chromatin condensation. The mal C-induced apoptosis led to mitochondrial damage as revealed by fluorescence microscopy and flow cytometry of the JC-1-stained cells as well as from the release of mitochondrion-specific nuclease proteins AIF and endo G. Mal C also released intracellular Ca2+ from the MCF-7 cells, but the Ca2+-modulators BAPTA-AM and Ru360 only partially abrogated the apoptosis. The calpain activation by mal C did not have any effect on its cytotoxicity. On the other hand, after mal C treatment significant lysosomal membrane permeabilization (LMP), along with release of cathepsin B, as well as Bid-cleavage and its translocation to mitochondria were observed much earlier than the mitochondrial damage. This suggested that cytotoxicity of mal C against human MCF-7 human breast cancer cell line may proceed through LMP as the initial event that triggered a caspase-independent, but cathepsin B and t-Bid-dependent intrinsic mitochondrial apoptotic pathway. A significant accumulation of cells in the S or G2-M phases along with upregulation of the cyclins E and A due to mal C exposure promises it to be a potential anti-cancer agent.

Evidence for necrosis, but not apoptosis, in human hepatoma cells with knockdown of mitochondrial aquaporin-8

We previously found that mitochondrial aquaporin-8 (mtAQP8) channels facilitate mitochondrial H2O2 release in human hepatoma HepG2 cells and that their knockdown causes oxidant-induced mitochondrial dysfunction and loss of viability. Here, we studied whether apoptosis or necrosis is involved as the mode of cell death. We confirmed that siRNA-induced mtAQP8 knockdown significantly decreased HepG2 viability by MTT assay, LDH leakage, and trypan blue exclusion test. Analysis of mitochondrial proapoptotic Bax-to-antiapoptotic BclXL ratio, mitochondrial cytochrome c release and caspase-3 activation showed no alterations in mtAQP8-knockdown cells. This indicates a primary mechanism of cell death other than the intrinsic mitochondrial apoptotic pathway. Thus, nuclear staining with DAPI did not reveal any increase of apoptotic features, i.e. chromatin condensation or nuclear fragmentation. Flow cytometry studies after double cell staining with annexin V and propidium iodide confirmed lack of apoptosis and suggested necrosis as the primary mechanism of death in mtAQP8-knockdown HepG2 cells. Necrosis was further supported by the increased nuclear delocalization and extracellular release of the High Mobility Group Box 1 protein. The knockdown of mtAQP8 in another human hepatoma-derived cell line, i.e. HuH-7 cells, also induced necrotic but not apoptotic death. Our data suggest that mtAQP8 knockdown induces necrotic cell death in human neoplastic hepatic cells, a finding that might be relevant to therapeutic strategies against hepatoma cells.

Abstract C208: Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death.

Abstract The actin cytoskeleton is an ideal chemotherapeutic target due to its role in numerous biological processes essential for tumor cell growth and survival. Targeting actin however has been problematic due to unacceptable levels of toxicity associated with impacting actin containing structures essential for normal cell function. We have developed a novel class of compounds which target tropomyosin, the second core component of an actin microfilament. By targeting the cancer associated tropomyosin, Tm5NM1 we are able to discriminate between the actin filament populations in normal and transformed cells. We have demonstrated that our first in class anti-Tm compound, TR100, impacts actin filament integrity leading to tumor cell death in vitro and in vivo in neuroblastoma and melanoma models. In this study we investigate the molecular mechanisms by which disruption of the actin cytoskeleton with the next generation anti-Tm compounds leads to tumor cell death. Preliminary studies have indicated that these compounds initiate cell death via the intrinsic mitochondrial apoptotic pathway. SH-EP neuroblastoma cells treated with TR200 showed a dose dependent increase in both caspase activation and annexin V staining as measured by Western Blotting and FACS analysis respectively. Significant changes in mitochondrial membrane integrity were also observed using TMRE staining in SH-EP cells treated with 5 µM TR200 for 24h. Interestingly, mitochondrial permeabilization was independent of the presence of proapoptotic factors Bax and Bak as Bax/Bak null MEFs still displayed sensitivity to TR200. To delineate the signaling pathways leading to apoptosis, a kinexus phospho-antibody array was conducted using SH-EP cells treated with 10 µM DTR200 for 8 and 24h. DTR200 treatment for 8h reduced the phospho-activity of key intermediates of the MEK/ERK pathway, in particular a significant decrease in the phosphorylation of the RSK1/2 family of kinases. Preliminary data would suggest that the compounds at 24h are mediating their effect through the downregulation of the MEK/ERK and p38/JNK cell survival pathways. We are now validating these findings to elucidate which signaling pathways mediate the apoptotic response to the anti-Tm compounds. Understanding the molecular mechanisms involved in anti-Tm mediated cell death will allow us to determine how these compounds can be used in combination with existing therapies to lower the apoptotic threshold of a tumor cell and ultimately improve the outcome of patients with neuroblastoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C208. Citation Format: Melissa Desouza, Margaret Nguyen, Peter W. Gunning, Justine R. Stehn. Targeting the actin cytoskeleton of neuroblastoma: Elucidating the mechanism by which a novel class of anti-cancer compounds induces tumor cell death. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C208.

Epigallocatechin-3-Gallate Inhibits Hepatoblastoma Growth by Reactivating the Wnt Inhibitor SFRP1

Activation of Wnt signaling plays a central role in the formation of hepatoblastoma (HB), the most common pediatric liver cancer. Blocking this pathway with specific inhibitors is currently the target of various research endeavours. This study provides evidence that the naturally occurring flavonoid epigallocatechin-3-gallate (EGCG) is highly effective against HB growth through inhibition of Wnt signaling. We demonstrate that EGCG has a strong cytotoxic effect on HB cells in a time- and dose-dependent manner by impinging on cell viability, while leaving normal fibroblasts unaffected. Apoptotic features, including morphological changes, caspase 3 activity, and proteolytic cleavage of poly(ADP-ribose) polymerase, were frequently found in EGCG-treated HB cells, thereby suggesting involvement of the mitochondrial intrinsic apoptotic pathway. We furthermore show that EGCG effectively inhibits Wnt signaling, as evidenced by down-regulation of Wnt-responsive reporter gene activity and expression of the Wnt target genes MYC and CCND1. Interestingly, EGCG induced reexpression of the tumor suppressor gene SFRP1, which is transcriptionally silenced in HB cells and known to down-regulate Wnt signaling. Considering the lack of toxic effects on normal cells, EGCG should be preclinically validated as an adjuvant therapy in vivo with the ultimate goal of determining its efficacy in human trials.

5-Lipoxygenase Antagonist therapy: a new approach towards targeted cancer chemotherapy

Leukotrienes are the bioactive group of fatty acids and major constituents of arachidonic acid metabolism molded by the catalytic activity of 5-lipoxygenase (5-LOX). Evidence is accumulating in support of the direct involvement of 5-LOX in the progression of different types of cancer including prostate, lung, colon, and colorectal cancers. Several independent studies now support the correlation between the 5-LOX expression and cancer cell viability, proliferation, cell migration, invasion through extracellular matrix destruction, metastasis, and activation of anti-apoptotic signaling cascades. The involvement of epidermal growth factor receptor and 5-oxo-ETE receptor (OXER1) is the major talking point in the downstream of the 5-LOX pathway, which relates the cancer cells to the proliferative pathways. Antisense technology approaches and use of different kinds of blocker targeted to 5-LOX, FLAP (5-LOX-activating protein), and OXER1 have shown a greater efficiency in combating different cancer cell types. Lastly, suppression of 5-LOX activity that reduces the cell proliferation activity also induces intrinsic mitochondrial apoptotic pathway in either p53-dependent or independent manner. Pharmacological agents that specifically inhibit the LOX-mediated signaling pathways have been used during last few years to treat inflammatory diseases such as asthma and arthritis. Studies of these well-characterized agents are therefore warranted for their use as possible candidates for chemotherapeutic studies against the killer disease cancer.

Induction of apoptosis by collinin from Zanthoxylum schinifolium is mediated via mitochondrial pathway in human Jurkat T cells

Collinin, which was isolated from the leaves of Zanthoxylum schinifolium, could exert cytotoxic effect on various human tumor cells with IC50 values in the range of 38.1–111.6μM, whereas the IC50 value for human normal mammary epithelial MCF-10A cells was 124.4μM. To examine the contribution of apoptosis to the cytotoxicity of collinin toward tumor cells, collinin-induced apoptotic events of Jurkat T cells transfected with vector (JT/Neo) were compared with those of Jurkat T cells transfected with Bcl-2 gene (JT/Bcl-2). Treatment of JT/Neo cells with collinin (30–60μM) resulted in induction of sub-G1 peak representing apoptotic cells along with activation of Bak and Bax, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -3, -8, and -7, degradation of PARP, and DNA fragmentation dose-dependently, but these apoptotic events were abrogated by overexpression of Bcl-2, which could prevent the induced activation of Bak and Bax, and subsequent mitochondrial damage. Under these conditions, necrosis was not accompanied. Pretreatment of JT/Neo cells with the pan-caspase inhibitor z-VAD-fmk completely blocked collinin-induced apoptotic sub-G1 cells and caspase cascade activation, whereas it failed to suppress Bak activation and Δψm loss. Neither FADD-deficiency nor caspase-8-deficiency affected the susceptibility of Jurkat T cells to collinin-induced cytotoxicity and apoptotic cell death. These results demonstrate that the apoptogenic activity of collinin was mediated by the intrinsic mitochondrial apoptotic pathway which was preceded by activation of pro-apoptotic multidomain Bcl-2 family members Bak and Bax, mitochondrial damage, and resultant activation of caspase cascade, leading to PARP degradation, which could be regulated by Bcl-2.

Intracellular localization of the BCL-2 family member BOK and functional implications.

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

Phytotoxic hazards of NiO-nanoparticles in tomato: A study on mechanism of cell death

Nickel oxide nanoparticles (NiO-NPs) in the concentration range of 0.025–2.0mg/ml were examined for the induction of oxidative stress, mitochondrial dysfunction, apoptosis/necrosis in tomato seedling roots, as an in vivo model for nanotoxicity assessment in plants. Compared to the control, catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and lipid peroxidation (LPO) in 2.0mg/ml NiO-NPs treatments exhibited 6.8, 3.7, 1.7 and 2.6-fold higher activities of antioxidative enzymes. At 2.0mg/ml, 122% and 125.4% increase in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) of seedling roots confirmed the oxidative stress and mitochondrial dysfunction. Comet assay exhibited a significant increase in the number of apoptotic (21.8%) and necrotic (24.0%) cells in 2.0mg/ml treatment groups vis-á-vis in control 7% apoptotic and 9.6% of necrotic cells were observed. Flow cytometric analysis revealed 65.7% of apoptotic/necrotic cell populations and 2.14-fold higher caspase-3 like protease activity were recorded in 2.0mg/ml treatment groups. Ultrastructure analysis revealed NiO-NPs translocation, nuclear condensation, abundance in peroxisomes and degenerated mitochondrial cristae. The dissolution of Ni ions from NiO-NPs signifies its potential to induce cell death presumably by Ni ions, triggering the mitochondrial dependent intrinsic apoptotic pathway.

BCL-2 Proapoptotic Proteins Distribution in U-87 MG Glioma Cells before and after Hypericin Photodynamic Action

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards the intrinsic mitochondrial apoptotic pathway, where Bcl-2 family of proteins plays the major role. We were particularly interested in two pro-apoptotic players Bak and Bax. Here we investigated their role in apoptosis triggered by photodynamic action. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action caused Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.View Large Image | View Hi-Res Image | Download PowerPoint Slide

In vitro investigation of integrin-receptor antagonist-induced vascular toxicity in the mouse

An αVβ3 receptor antagonist (SB-273005) induced unique vascular lesions in the aorta of mice, but not other pharmacologically responsive species. Vascular smooth muscle cell (VSMC) necrosis was observed ∼6h postdose followed by VSMC loss with no evidence of hemorrhage/thrombosis, inflammation or damage to endothelium. Since direct drug-induced vascular toxicity is uncommon, involvement of VSMC–endothelial cell (EC) interactions was hypothesized. In vitro model systems of murine aortic VSMC and EC monocultures and cocultures were established and used to investigate the mechanism of toxicity. Incubation of cultures with SB-273005 within a dose range and timeframe comparable to in vivo studies, showed a concentration-dependent decrease in viability with increases in cytotoxicity for monocultures and VSMC/EC cocultures; however, VSMC monocultures responded at lower doses (were most sensitive) suggesting a direct effect on VSMC which is not mediated or enhanced through EC/VSMC interactions. Further studies revealed increased caspase-9 and caspase-3/7 activation in VSMC beginning as early as 0.5 and 1h following treatment, respectively. These findings suggest SB-273005 causes direct chemical vascular toxicity in murine VSMC which involves apoptosis mediated through the intrinsic (mitochondrial) apoptotic pathway. To our knowledge, this is the first report to provide a link between VSMC apoptosis and treatment with an αVβ3 receptor antagonist.