Adam Csordas

Resveratrol, a tumor-suppressive compound from grapes, induces apoptosis via a novel mitochondrial pathway controlled by Bcl-2

We report that resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene), a phytoalexin found in grapes and other plant food, induced a breakdown of the mitochondrial transmembrane potential (∆Ψm) in T‐acute lymphoblastic leukemia cells and swelling of isolated rat mitochondria. The breakdown of ∆Ψm was accompanied by the production of reactive oxygen species (ROS), and preceded phosphatidylserine exposure and DNA fragmentation. Breakdown of ∆Ψm was not caused by the activation of caspase‐8 or Bid, as no significant cleavage of these proteins could be detected in the induction phase of resveratrol‐induced apoptosis. Though loss of ∆Ψm was not followed by cytochrome c translocation to the cytosol, the mitochondrial changes triggered significant activation of caspase‐9, ‐2, ‐3, and ‐6. Inhibition of ∆Ψm breakdown and of ROS generation by N‐acetylcysteine, or by overexpression of Bcl‐2 protein, prevented apoptosis induction by resveratrol. The Bcl‐2 expression status of tumor cells should therefore be considered relevant for potential clinical application of resveratrol as anticancer agent.

Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), in the concentration range of 20 μM and above, induced arrest in the S-phase and apoptosis in the T cell-derived T-ALL lymphocytic leukemia cell line CEM-C7H2 which is deficient in functional p53 and p16. Expression of transgenic p16/INK4A, which causes arrest in G0/G1, markedly reduced the percentage of apoptotic cells. Antagonist antibodies to Fas or FasL, or constitutive expression of crmA did not diminish the extent of resveratrol-induced apoptosis. Furthermore, a caspase-8-negative, Fas-resistant Jurkat cell line was sensitive to resveratrol-induced apoptosis which could be strongly inhibited in the Jurkat as well as in the CEM cell line by z-VAD-fmk and z-IETD-fmk. The almost complete inhibition by z-IETD-fmk and the lack of inhibition by crmA suggested caspase-6 to be the essential initiator caspase. Western blots revealed the massive conversion of procaspase-6 to its active form, while caspase-3 and caspase-2 were proteolytically activated to a much lesser extent. Cell Death and Differentiation (2000) 7, 834–842

Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts

The histone deacetylase inhibitor and potential anti‐cancer drug sodium butyrate is a general inducer of growth arrest, differentiation, and in certain cell types, apoptosis. In human CCRF‐CEM, acute T lymphoblastic leukemia cells, butyrate, and other histone deacetylase inhibitors caused G2/M cell cycle arrest as well as apoptotic cell death. Forced G0/G1 arrest by tetracycline‐regulated expression of transgenic p16/INK4A protected the cells from butyrate‐induced cell death without affecting the extent of histone hyperacetylation, suggesting that the latter may be necessary, but not sufficient, for cell death induction. Nuclear apoptosis, but not G2/M arrest, was delayed but not prevented by the tripeptide broad‐range caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp·fluoromethylketone (zVAD) and, to a lesser extent, by the tetrapeptide ‘effector caspase’ inhibitors benzyloxycarbonyl‐Asp‐Glu‐Val‐Asp·fluoromethylketone (DEVD) and benzyloxycarbonyl‐Val‐Glu‐Ile‐Asp‐fluoromethyl‐ketone (VEID); however, the viral protein inhibitor of ‘inducer caspases’, crmA, had no effect. Bcl‐2 overexpression partially protected stably transfected CCRF‐CEM sublines from butyrate‐induced apoptosis, but showed no effect on butyrate‐induced growth inhibition, further distinguishing these two butyrate effects. c‐myc, constitutively expressed in CCRF‐CEM cells, was down‐regulated by butyrate, but this was not causative for cell death. On the contrary, tetracycline‐induced transgenic c‐myc sensitized stably transfected CCRF‐CEM derivatives to butyrate‐induced cell death.—Bernhard, D., Ausserlechner, M. J., Tonko, M., Löffler, M., Hartmann, B. L., Csordas, A., Kofler, R. Apoptosis induced by the histone deacetylase inhibitor sodium butyrate in human leukemic lymphoblasts. FASEB J. 13, 1991–2001 (1999)

Enhanced MTT-reducing activity under growth inhibition by resveratrol in CEM-C7H2 lymphocytic leukemia cells

Inhibition of proliferation by resveratrol of CEM-C7H2 lymphocytic leukemia cells was paradoxically associated with an enhanced cellular 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-reducing activity. This phenomenon was most pronounced at the sub-apoptotic concentration range of 5-20 microM resveratrol. The results of our study show that the MTT-reducing activity can be increased by the polyphenolic antioxidant resveratrol without a corresponding increase in the number of living cells and that this occurs at a concentration range of the antioxidant which is not sufficient to induce apoptosis but suffices to slow down cell growth. This phenomenon appears to be restricted to proliferation inhibitors with antioxidant properties and is cell type-specific. Thus, in determining the effects of flavonoids and polyphenols on proliferation, in certain cell types this might represent a pitfall in the MTT proliferation assay.

The biology behind the atherothrombotic effects of cigarette smoke

Cigarette smoke is an aerosol that contains >4,000 chemicals, including nicotine, carbon monoxide, acrolein, and oxidant compounds. Exposure to cigarette smoke induces multiple pathological effects in the endothelium, several of which are the result of oxidative stress initiated by reactive oxygen species, reactive nitrogen species, and other oxidant constituents of cigarette smoke. Cigarette-smoke exposure interferes adversely with the control of all stages of plaque formation and development and pathological thrombus formation. The reactive oxygen species in cigarette smoke contribute to oxidative stress, upregulation of inflammatory cytokines, and endothelial dysfunction, by reducing the bioavailability of nitric oxide. Plaque formation and the development of vulnerable plaques also result from exposure to cigarette smoke via the enhancement of inflammatory processes and the activation of matrix metalloproteases. Moreover, exposure to cigarette smoke results in platelet activation, stimulation of the coagulation cascade, and impairment of anticoagulative fibrinolysis. Many cigarette-smoke-mediated prothrombotic changes are quickly reversible upon smoking cessation. Public health efforts should urgently promote our understanding of current cigarette-smoke-induced cardiovascular pathology to encourage individuals to reduce their exposure to cigarette smoke and, therefore, the detrimental consequences of associated atherothrombotic disease.

Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules

Smoking is a significant risk factor for development of atherosclerosis. However, the pathophysiology of smoking‐mediated vessel wall damage is not understood. With tools ranging from analytical chemistry to cell biology, we show that cigarette smoke contains metals that catalyze the direct oxidation of cellular proteins by smoke oxidants. Oxidation of cellular proteins causes a loss of microtubule function, culminating in microtubule depolymerization and proteasome‐dependent degradation of α‐tubulin. As a consequence of the microtubule collapse, cytoskeletal structures as well as intermediate filaments break down, leading finally to a contraction of vascular endothelial cells. We observed a smoke extract‐induced, calpain‐dependent degradation of the intracellular form of platelet‐endothelial cell adhesion molecule 1/CD31, as well as a release of P‐selectin/CD62P, IL‐6, and IL‐8 from endothelial cells into the supernatant. Increased levels of soluble CD62P and IL‐6 are well known to be associated with smoking in humans. Increased permeability of the vascular endothelium is a crucial event in atherogenesis. This work highlights the compounds and mechanisms by which cigarette smoke induces leakiness of the vascular endothelium. DavidBernhard AdamCsordas BlairHenderson AndreaRossmann MichaelaKind GeorgWick Cigarette smoke metal‐catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules. FASEB J. 19, 1–12 (2005)

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF–CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIPS+L, FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8. Cell Death and Differentiation (2001) 8, 1014–1021

Cigarette smoke extract induces prolonged endoplasmic reticulum stress and autophagic cell death in human umbilical vein endothelial cells

AIMS Consumption of cigarette smoke (CS) is a well-known risk factor for early atherosclerosis; yet, the underlying mechanisms of smoking-associated atherosclerosis are poorly understood. Based on the previous results indicating that CS-induced endothelial cell death neither shows typical features of apoptosis nor of necrosis, we investigated the role of autophagy in CS extract (CSE)-induced cell death of human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS Here, we demonstrate that overexpression of the classical apoptosis inhibitor BCL-XL had no protective effect on CSE-induced cell death, whereas the autophagy inhibitor 3-methyladenin and an shRNAi-mediated knockdown of the autophagy mediator ATG5 significantly delayed cell death. Our results indicate that CSE induces an excess accumulation of misfolded proteins in the endoplasmic reticulum (ER) and consequently the onset of the unfolded protein response. We provide evidence that the ER-resident kinase PERK is a major transducer of ER stress leading to phosphorylation of eIF2α and attenuation of protein synthesis. Finally, we show that prolonged ER stress in cells subjected to CS is followed by activation of an autophagic programme. CSE-induced autophagy is characterized by an increase in LC3 II/I ratio and activation ATG12. The autophagic signalling pathway via energy depletion and consequent activation AMP-activated protein kinase could be excluded. CONCLUSION Our results confirm and extend previous findings reporting on the induction of autophagy by CSE in the lung. We show that protein damage caused by CSE activates autophagy, ultimately resulting in necrotic death of HUVECs. Via this mechanism, cigarette smoking may contribute to the deterioration of vascular endothelial function and the initiation of atherosclerosis.

Cigarette smoke is an endothelial stressor and leads to cell cycle arrest

The molecular mechanisms underlying the atherogenic activity of cigarette smoke have yet to be fully elucidated. In the present study, genome-wide microarray analysis was performed on endothelial cells exposed to an aqueous cigarette smoke extract (CSE) for 3, 7, and 24 h, to obtain a better insight into how smoking may lead to endothelial damage. Microarray analysis showed the transcriptional response to CSE was dominated by heat shock, stress responsive, and inflammatory genes, along with genes encoding for anti-oxidant and metal detoxification proteins. The heat shock response was shown to be a result of short lived reactive species of CSE, with the abrogation of the effect by the addition of old CSE, the anti-oxidant N-acetyl cysteine, or the removal of metals from CSE implying that reactive oxygen species are the main culprit. This was further supported by a strong decline in the level of intracellular protein oxidation levels seen under these conditions compared to freshly prepared CSE. Mitochondrial integrity was also found to be significantly compromised after CSE treatment, resulting in a threefold increase in depolarised mitochondria after 6 h. Finally, cell cycle analysis showed the induction of G1 cell cycle arrest. An increased stress and inflammation response indicates that endothelial damage from smoking could contribute to immune cell infiltration, while decreased growth rates reduce endothelial layer repair, promoting atherogenesis.

Structure- and configuration-dependent effects of C18 unsaturated fatty acids on the chicken and sheep erythrocyte membrane.

High concentrations of unsaturated fatty acids are known to cause hemolysis. At low concentrations, however, unsaturated cis fatty acids have been found to protect erythrocytes against hypotonic hemolysis. In the present experiments we examined the effect of oleic (18:1), linoleic (18:2), linolenic (18:3), and elaidic (18:1) acid on the osmotic fragility of chicken and sheep erythrocytes, which markedly differ in their resistance to osmotic rupture. The results are summarized as follows: (A) The phenomenon of stabilization was observed in both species alike. (B) Interaction of cells with the fatty acids under isotonic conditions led to a persistent stabilization, i.e., the cells remained more resistant against osmolysis even after several washings