Activation Of Pro-apoptotic Signaling Research Articles

16-OR: Protein Kinase C d Mediates Cytokine-Induced ß-Cell Death via Upregulation of Pro-apoptotic Signaling

In type 1 diabetes (T1D) , autoreactive immune cells secrete cytokines, such as tumor necrosis factor-α (TNF-α) , interleukin-1β (IL-1β) , and interferon-γ (IFN-γ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis; however, the exact mechanisms and role in T1D are unknown. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via nuclear translocation and activation of pro-apoptotic signaling. Islets from WT or mice with a β-cell specific knockout of PKCδ (PKCδβ-KO) were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFN-γ) , a caspase-3 inhibitor (Z-DEVD-FMK, 10µg/ml) , or an adenoviral vector for GFP-tagged PKCδ, GFP only, and a PKCδ mutant that cannot be cleaved by caspase-3 (CM-GFP-PKCδ) . Human islets were treated with 10μM PKCδ inhibitor (δV1-1) for 24hr with or without cytokines. Translocation was determined by GFP colocalization with a nuclear stain. Apoptosis was determined by fluorescent microscopy. PKCδ activity was determined with a FRET-based sensor in MIN6 cells. PKCδ’s role in activating pro-apoptotic signaling was determined via western blot. Decreasing PKCδ activity protected against cytokine-induced apoptosis in mouse (p=0.003) and human (p=0.020) islets. PKCδ translocation and activity increased in nuclei of cytokine treated islets (p<0.001, p=0.039) compared to untreated controls. Islets treated with caspase inhibitor (p<0.001) and CM-GFP-PKCδ islets were protected from cytokine-induced death. Western blot analysis demonstrates that PKCδ regulates pro-apoptotic signaling. Inhibiting PKCδ protected islets from cytokine-induced death. The role we identified for PKCδ in mediating pro-apoptotic proteins will provide novel insight to protect against cytokine-induced β-cell death. Our data suggests targeted inhibition of PKCδ has the potential to prevent cytokine-mediated β-cell death in T1D. Disclosure J.Collins: None. N.L.Farnsworth: None. R.K.Benninger: n/a. Funding American Diabetes Association (7-21-JDF-020) ; Juvenile Diabetes Research Foundation (1-FAC-2020-891-A-N)

Antiangiogenic and apoptotic effects of benzyl caffeate on human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM): In vitro and in vivo models

• Benzyl caffeate inhibits the proliferation and tube formation of endothelial cells. • Benzyl caffeate reduces the number of newly formed vessels in chick embryo chorionic membrane models. • Benzyl caffeate induces apoptosis via activation of proapoptotic signaling. • Benzyl caffeate exerts its antiangiogenic effects through induction of endothelial apoptosis. Caffeic acid and its derivatives are polyphenolic compounds, which are present in high concentration in propolis and medicinal plants. Their antioxidant activity has been reported; however, the antiangiogenic effects of benzyl caffeate remain unclear. In this study, we investigated the angiogenic and apoptotic effects of benzyl caffeate both in vitro on HUVECs and in vivo in chick embryo chorionic membrane (CAM) models. Benzyl caffeate significantly inhibited angiogenesis via HUVECs-based tube formation by inducing apoptosis and antiproliferative effects on endothelial cells. Furthermore, benzyl caffeate significantly reduced the number of newly formed vessels in CAM. Western blot analysis showed that benzyl caffeate induced apoptosis via proapototic signaling via activation of caspase-9, caspase-3 and cleavage of PARP and lamin A/C. In addition, western blot showed that benzyl caffeate treatment inhibited c-Raf/MEK/ERK signaling, and thereby suppressed angiogenesis. In conclusion, the results indicate that benzyl caffeate exerts its antiangiogenic effects via induction of endothelial apoptosis.

Therapeutic targeting of endoplasmic reticulum stress in acute graft-versus-host disease.

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), a potentially curative treatment for leukemia. Endoplasmic reticulum (ER) stress occurs when the protein folding capacity of the ER is oversaturated. How ER stress modulates tissue homeostasis in the context of alloimmunity is not well understood. We show that ER stress contributes to intestinal tissue injury during GvHD and can be targeted pharmacologically. We observed high levels of ER stress upon GvHD onset in a murine alloHCT model and in human biopsies. These levels correlated with GvHD severity, underscoring a novel therapeutic potential. Elevated ER stress resulted in increased cell death of intestinal organoids. In a conditional knockout model, deletion of the ER stress regulator transcription factor Xbp1 in intestinal epithelial cells induced a general ER stress signaling disruption and aggravated GvHD lethality. This phenotype was mediated by changes in the production of antimicrobial peptides and the microbiome composition as well as activation of pro-apoptotic signaling. Inhibition of inositol-requiring enzyme 1α (IRE1α), the most conserved signaling branch in ER stress, reduced GvHD development in mice. IRE1α blockade by the small molecule inhibitor 4µ8c improved intestinal cell viability, without impairing hematopoietic regeneration and T-cell activity against tumor cells. Our findings in patient samples and mice indicate that excessive ER stress propagates tissue injury during GvHD. Reducing ER stress could improve the outcome of patients suffering from GvHD.

The antimalaria drug artemisinin displays strong cytotoxic effect on leukaemia lymphocytes in combination with vitamin C and pro-vitamin K3

This study investigated the anticancer effect of the anti-parasitic drug artemisinin in combination with two redox modulators: vitamin C and pro-vitamin K3 (C/K3) The experiments were conducted on leukaemia cells Jurkat. Cells were treated with either artemisinin or C/K3 alone and with all three compounds. Cell proliferation and viability were analysed using trypan blue stating and automated cell counting. The results showed that artemisinin (>10 mM) suppressed cell proliferation activity, but did not induce cell death up to 500 mM. The drug demonstrated a clear cytostatic effect at concentrations 250- 500 mM – Jurkat cells did not proliferate, but were alive. The combination C/K3 (200:2, 300:3 mM/mM) applied alone did not affect cell proliferation and viability. Vitamins C/K3 in concentration ratio 500:5 (μM/mM) decreased cell proliferation activity by ~10%. The triple combination artemisinin/C/K3 manifested synergistic anti-proliferative effects at all concentration ratios analysed. This synergistic effect increased with increasing C/K3 concentration. Based on literature data, it was assumed that the anti-proliferative effect of the triple combination was mediated by changes in the redox-homeostasis of cancer cells. The C/K3 redox system likely acted on cancer mitochondria and increased superoxide production and activation of pro-apoptotic signals, specific for cancer cells. On the other hand, artemisinin could generate hydroxyl radicals as a result of activation of Fenton reactions, depleting intracellular reducing equivalents. Both redox mechanisms lead to activation of signal pathways for induction of cancer cell death.

Pro-Oxidant Actions of Carotenoids in Triggering Apoptosis of Cancer Cells: A Review of Emerging Evidence.

Carotenoids are well known for their potent antioxidant function in the cellular system. However, in cancer cells with an innately high level of intracellular reactive oxygen species (ROS), carotenoids may act as potent pro-oxidant molecules and trigger ROS-mediated apoptosis. In recent years, the pro-oxidant function of several common dietary carotenoids, including astaxanthin, β-carotene, fucoxanthin, and lycopene, has been investigated for their effective killing effects on various cancer cell lines. Besides, when carotenoids are delivered with ROS-inducing cytotoxic drugs (e.g., anthracyclines), they can minimize the adverse effects of these drugs on normal cells by acting as antioxidants without interfering with their cytotoxic effects on cancer cells as pro-oxidants. These dynamic actions of carotenoids can optimize oxidative stress in normal cells while enhancing oxidative stress in cancer cells. This review discusses possible mechanisms of carotenoid-triggered ROS production in cancer cells, the activation of pro-apoptotic signaling by ROS, and apoptotic cell death. Moreover, synergistic actions of carotenoids with ROS-inducing anti-cancer drugs are discussed, and research gaps are suggested.

Abstract TP322: Viral Mediated Gene Delivery of TMBIM6 Protects the Neonatal Brain

Introduction: A wide variety of conditions may contribute to oxidative protein folding processes in the endoplasmic reticulum (ER) which cause ER stress and play a major role in the pathogenesis of neonatal hypoxic ischemic (HI) injury. ER stress triggers the unfolded protein response (UPR) resulting in the activation of pro-apoptotic signaling causing cells to undergo apoptosis. Here were explored the role of Bax Inhibitor-1 (BI-1) protein, an evolutionary protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, as a novel modulator of ER stress induced apoptosis after HI brain injury in a neonatal rat pup. BI-1 may modulate the UPR response via a direct physical interaction with the stress sensor receptor, IRE1α, and thus inhibiting it’s pro-apoptotic signaling. Hypothesis: Overexpression of BI-1, using viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, will protect the neonatal brain from HI by inhibiting ER stress induced apoptosis. Methods: Ten-day old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). To determine BI-1 confers protection via inhibition of IRE1α, we administered BI-1 siRNA, to silence BI-1, and an IRE1α CRISPR activation plasmid (AP), to counteract BI-1’s effects, at 48h pre-HI. Percent infarct area, immunofluorescent staining and western blot were performed at 72h post HI. Results: Overexpression of BI-1 showed to significantly reduce percent infarcted area, attenuated neuronal degeneration, reduced ER stress induced neuronal apoptosis and improved long-term neurological outcomes. Either silencing BI-1 or over activating IRE1α with a CRISPR AP significantly reversed BI-1’s protective effects. Conclusions: Overexpression of BI-1 attenuated the morphological and neurological consequences post neonatal HI via inhibition of the ER stress sensor, IRE1α. This study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and provided a basis for BI-1 as a potential therapeutic target.

Avicularin reversed multidrug-resistance in human gastric cancer through enhancing Bax and BOK expressions

5-Fluorouracil (5-Fu) and cisplatin (DDP) as important therapies in treatment of human gastric cancer have been widely determined. However, the therapeutic effects are usually hampered due to drug resistance or toxicity at high concentrations for application. Avicularin (AL, quercetin-3-α-l-arabinofuranoside), a bio-active flavonol isolated from a number of plants, has been reported to display diverse pharmacological properties. In this study, we explored the hypothesis by which AL reversed 5-Fu or DDP resistance in gastric cancer and the underlying molecular mechanism. Here, in vitro, the drug-resistant cancer cells were incubated to AL or DDP alone or the combination of AL and DDP. Then, MTT, colony formation, Hoechst 33258, flow cytometry and western blot analysis were used to investigate the effects of AL in the regulation of drug-resistance gastric cancer cells. The results indicated that AL treatment markedly re-sensitizes the drug resistant cells (SGC-7901/5-Fu and SGC-7901/DDP) to cytotoxicity of 5-Fu or DDP. Molecular mechanism analysis indicated that AL and DDP combination treatment enhanced apoptosis in SGC-7901/DDP cells, accompanied with the up-regulation of cleaved Caspase-3 and PARP, as well as the activation of pro-apoptotic signals, including Bax and BOK. Significantly, down regulation of Bax or BOK expressions using Bax siRNA or BOK siRNA decreased the inhibitory role of DDP in apoptosis of SGC-7901/DDP cells pretreated with AL, demonstrating that AL-reversed DDP resistance was associated with Bax and BOK expression. In vivo, AL and DDP combination significantly reduced gastric tumor growth. Immunohistochemical analysis indicated that co-treatment of AL and DDP significantly induced apoptosis, and reduced tumor cell proliferation in tumor tissue samples. Furthermore, we also found that the Bax, BOK, cleaved Caspase-3 and PARP expression in tumor tissues were highly induced by AL and DDP co-treatment. Together, our findings may provide a novel combination therapeutic strategy in treatment of human gastric cancer.

Abstract 2328: Race specific hyper-activation of CCR9-mediated survival signals and its impact on efficacy of docetaxel in prostate cancer

Abstract Current approaches to treat and manage prostate cancer (PCa) have failed to reduce racial disparity primarily due to undefined molecular mechanism. We have shown higher expression of chemokine receptor-9 in PCa cells and CCR9 expression was higher in PCa cells (MDA-PCa-2b) derived from African American (AA) patient compared to PCa cells derived form European American (EA) patients (LNCaP and PC3). Using antibody microarray we observed hyper activation of survival molecules and down-regulation of apoptotic molecules in AA PCa cells compared to EA PCa cell lines, following CCL25 stimulation. Comparative heat map analysis showed significant increase (~2-3 fold) in phosphorylation of pro-survival proteins in AA cells compared to EA cells treated with CCL25, which was further confirmed by western blot analysis. Furthermore, CCL25 treated PCa cells showed decreased expression of pro-apoptotic proteins (Bim, Bid, Bak), which was significantly abrogated by CCR9 shRNA. This effect was more pronounced in AA cells compared to EA cells. Efficacy of docetaxel (DTX) was higher after CCR9 blockade in presence of CCL25 compared to cells treated with CCL25 without blocking CCR9. Improvement in efficacy of DTX was due to inhibition of cell survival and activation of pro-apoptotic signals following CCR9 blockade. These observations suggest potential involvement of CCR9 mediated molecular pathways in disparity associated with outcome of PCa. Citation Format: Neeraj Kapur, Hina Mir, Guru Sonpavde, Shailesh Singh. Race specific hyper-activation of CCR9-mediated survival signals and its impact on efficacy of docetaxel in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2017-2328

Ceramide generation during curcumin-induced apoptosis is controlled by crosstalk among Bcl-2, Bcl-xL, caspases and glutathione

Curcumin exhibits anti-cancer properties manifested by activation of pro-apoptotic signaling. We have demonstrated earlier that apoptosis of HL-60 human leukemia cells induced by curcumin is controlled by ceramide generated by neutral sphingomyelinase (nSMase) which contributes to sphingomyelin synthase (SMS) inhibition favoring accumulation of ceramide in cells. Here we report that the activity of nSMase, ceramide accumulation and death of HL-60 cells are inhibited by overexpression of Bcl-xL or Bcl-2 proteins, while down-regulation of nSMase interferes with degradation of Bcl-2 but not Bcl-xL. Activation of nSMase in curcumin-treated cells requires the activity of apoptosis initiator caspase-8 and executioner caspase-3, whereas nSMase depletion prevents activation of caspase-3, but not caspase-8. These data place nSMase activation downstream of caspase-8 and Bcl-xL and indicate a mutual regulation between nSMase and caspase-3 activity on one hand, and Bcl-2 level on the other hand in curcumin-treated cells. The activation of nSMase and ceramide accumulation also depended on the depletion of glutathione. The depletion of glutathione required the activity of caspase-8 and caspase-3 as well as the down-regulation of Bcl-2 and Bcl-xL. Together, the data indicate a crosstalk among Bcl-2, Bc-xL, caspases and glutathione during curcumin-induced apoptosis and point to the superior role of caspase-8 activity, Bcl-xL down-regulation and glutathione depletion in the pro-apoptotic cascade leading to nSMase activation and generation of ceramide.

Neuroprotective and Neurotherapeutic Effects of Bee Venom on Neurodegenerative Diseases

Event Abstract Back to Event Neuroprotective and Neurotherapeutic Effects of Bee Venom on Neurodegenerative Diseases Miran K. Rakha1* 1 Suez Canal University, Biotechnology Research Center, Egypt Acute and chronic neurodegenerative diseases are illnesses associated with high morbidity and mortality, and few or no effective options are available for their treatment. A characteristic of many neurodegenerative diseases — which include stroke, brain trauma, spinal cord injury, amyotrophic lateral sclerosis, Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease — is neuronal cell death. Given that central nervous system tissue has very limited, if any, regenerative capacity, it is of utmost importance to limit the damage caused by neuronal death. Bee venom, which is also known as apitoxin, consists of several biologically active peptides, including melittin, adolapin, mast cell degranulating peptide and phospholipase A2. Moreover, bee venom contains a variety of bioamines, such as apamin, histamine, procamine, serotonin, and norepinephrine, which facilitate nerve transmission and healing in a variety of nerve disorders. This gives bee venom the ability to travel along the neural pathways from the spine to various trigger points and injured areas to help repair nerve damage and restore mobility. This review overviews; (1) causes and mechanisms of neurodegenerative diseases which pertains to neuronal cell death, (2) evidence linking composition comprising bee venom to its substantial potential for preventing and treating of neurodegenerative diseases associated with neuronal cell death, and (3) how improving our knowledge of the mechanisms mediating neuroprotective and neurotherapeutic activities of bee venom against neuronal cell death may led to novel therapeutic strategies for the treatment of neurodegenerative diseases. Future challenges remaining will be to elucidate signaling responses activated by bee venom in neurons. In other words, bee venom inhibits neuronal cell death and activation of proapoptotic signaling in neurons. These findings emphasize the clinical importance of bee venom for treatment of neurodegenerative diseases. Further investigation is necessary to elaborate the mechanisms involved and to permit full exploitation of neuroprotective and neurotherapeutic potentials of bee venom. References Doo AR et al. (2012): Bee venom protects SH-SY5Y human neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptotic cell death. Brain Res, 1429: 106-115. Lee SM et al. (2012): Effects of Bee Venom on Glutamate-Induced Toxicity in Neuronal and Glial Cells. Evidence-Based Complementary and Alternative Medicine, 2012: 368196, doi:10.1155/2012/368196. Rakha MK (2011): Impact of Beehive Products on the Cardiovascular Neurophysiology Expands Novel Horizons in Apitherapy. Conference Abstract: 10th Meeting of the Société des Neurosciences, May 24-27, 2011, Marseille, France. Yang EJ et al. (2011): Melittin restores proteasome function in an animal model of ALS. J Neuroinflammation, 8: 69, doi: 10.1186/1742-2094-8-69. Yang EJ et al. (2010): Bee venom attenuates neuroinflammatory events and extends survival in amyotrophic lateral sclerosis models. J Neuroinflammation, 7: 69, doi: 10.1186/1742-2094-7-69. Keywords: Bee Venom, Neuroprotective Activity, Neurotherapeutic Potential, Neuronal Cells, Neurodegenerative Diseases. Conference: 4th Conference of the Mediterrarnean Neuroscience Society, Istanbul, Turkey, 30 Sep - 3 Oct, 2012. Presentation Type: Poster Presentation Topic: Abstracts Citation: Rakha MK (2013). Neuroprotective and Neurotherapeutic Effects of Bee Venom on Neurodegenerative Diseases. Conference Abstract: 4th Conference of the Mediterrarnean Neuroscience Society. doi: 10.3389/conf.fnhum.2013.210.00057 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jan 2013; Published Online: 11 Apr 2013. * Correspondence: Dr. Miran K Rakha, Suez Canal University, Biotechnology Research Center, Ismailia, Egypt, mirankhalil@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Miran K Rakha Google Miran K Rakha Google Scholar Miran K Rakha PubMed Miran K Rakha Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Role of Glutathione S-Transferase Class Mu During VCD-Induced Ovotoxicity in Neonatal Rat Ovaries.

4-vinylcyclohexene diepoxide (VCD) is an occupational chemical that selectively destroys ovarian small pre-antral follicles in both rats and mice via apoptosis. Glutathione S-transferase (GST) enzymes catalyze glutathione (GSH) conjugation to xenobiotics. In cultured neonatal mouse ovaries exposed to VCD, mRNA encoding the GST isoform mu (GSTm) increased (d4) prior to the onset of follicle loss (d6). GST protein can also regulate cell signaling, and in extra-ovarian tissues GSTM prevents the action of apoptosis signal-regulating kinase 1 (ASK1) through formation of a protein complex. Upon xenobiotic exposure, this protein complex dissociates resulting in activation of pro-apoptotic signaling by ASK1. This study was designed to investigate an involvement of GSTm in VCD-induced ovotoxicity in rats. Post-natal day 4 (PND4) rat ovaries were incubated in control medium ± VCD (30 μM) for 2, 4, 6 or 8 d. Following culture, total RNA or protein were isolated (n=3; 10-20 ovaries per pool). GSTm mRNA and protein levels were determined using quantitative RT-PCR and Western blotting, respectively. To assess the presence of an GSTM:ASK1 protein complex, immunoprecipitation using an anti-ASK1 primary antibody was followed by Western blotting to measure GSTM protein level. There was no effect of VCD exposure on Gstm mRNA expression at any time point. Despite lack of a transcriptional effect GSTM protein was increased (P < 0.05) by VCD exposure after 6 and 8 d, relative to control. A GSTM:ASK1 protein complex was detected in the rat ovary. Relative to control, there was no effect of VCD on the GSTM:ASK1 protein complex after 4 d of exposure. GSTM-bound to ASK1 increased (P < 0.05) after 6 d of VCD exposure, when follicle loss is underway. Because there was no increase in the amount of GSTM protein unbound to ASK1, this indicates that the increase in total GSTM reflected an increase in the ASK1-bound form. These findings support that GSTM protein is involved in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. (Supported by ES016818 to AFK, ES09246 to PBH).

Impact of acute kidney injury on lung injury

classic animal models of critical illness have focused on injury of a single end-organ, with the goal of better understanding and attenuating that injury, and ultimately developing novel disease-focused therapies. For example, acute lung injury (ALI) can be induced in animal models by administration

Receptors that mediate cellular dependence

Cells depend for their survival on stimulation by trophic factors and other prosurvival signals, the withdrawal of which induces apoptosis, both via the loss of antiapoptotic signaling and the activation of proapoptotic signaling via specific receptors. These receptors, dubbed dependence receptors, activate apoptotic pathways following the withdrawal of trophic factors and other supportive stimuli. Such receptors may feature in developmental cell death, carcinogenesis (including metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Mechanistic studies of dependence receptors suggest that these receptors form ligand-dependent complexes that include specific caspases. Complex formation in the absence of ligand leads to caspase activation by a mechanism that is typically dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Cellular dependence receptors, considered in the aggregate, may thus form a system of molecular integration, analogous to the electrical integration system provided by dendritic arbors in the nervous system.

Membrane cholesterol but not putative receptors mediates anandamide-induced hepatocyte apoptosis

The endogenous cannabinoid anandamide, a lipid mediator, induces various physiologic events such as vascular relaxation, inhibition of gap-junctions formation, tumor proliferation, neurologic analgesia, and apoptosis. Although increased concentration of anandamide in plasma has been implicated in pathophysiologic states including endotoxin-induced hypotension, the effects of anandamide on hepatocytes still remain unclear. In this study, we present evidence that plasma anandamide concentration is highly increased in severe hepatitis and cirrhosis patients. In addition, concentrations of anandamide within the pathophysiologic range potently induced apoptosis of hepatoma cell line (Hep G2) and primary hepatocytes, suggesting a possible link between increased anandamide level and hepatocyte damage. Anandamide-induced cell death was preceded by G0/G1 cell-cycle arrest, activation of proapoptotic signaling (i.e., p38 MAPK and JNK), and inhibition of antiapoptotic signaling (i.e., PKB/Akt) pathways. Moreover, anandamide increased susceptibility to oxidative stress-induced hepatocyte damage. In this context, methyl-beta-cyclodextrin (MCD), a membrane cholesterol depletor, or mevastatin, an HMG-CoA reductase inhibitor, or N-acetyl cysteine, an antioxidant, potently inhibited the anandamide-induced proapoptotic events and cell death, whereas putative cannabinoid receptor antagonists did not exhibit an inhibitory effect on anandamide-induced cell death. Furthermore, binding assay using polymyxin beads revealed that anandamide could interact with cholesterol. In conclusion, our data suggest that cholesterol present in the cell membrane determines the fate of hepatocytes exposed to anandamide, possibly functioning as an anandamide receptor.

Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals.

Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4-12 h with peak changes occurring at 3-7 days. Maximal apoptosis was also evident at distant limb muscles at 3-7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting.