Mitochondrial Membrane Perturbation Research Articles

The apoptotic pathway triggered by the Fhit protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression.

The expression of the tumour suppressor protein fragile histidine triad (Fhit) is often impaired in many human cancers and its restoration in Fhit-negative cancer cell lines suppresses tumorigenicity and induces apoptosis. Although the proapoptotic function of Fhit is well documented, little is known about its precise mechanism of action and further studies are needed in order to elucidate the putative therapeutic properties of this protein. To this end, we have engineered the lung cancer cell line NCI-H460 in order to express different molecules involved in the control of apoptotic pathways. Infection of these cells with an adenoviral vector transducing the Fhit gene (Ad-Fhit) revealed that complete protection from apoptosis was conferred by the inhibitor of caspases Cytokine response modifier A (CrmA) and by a dominant-negative form of the adapter protein Fas-associated death domain (FADD) and partial protection by a dominant-negative form of caspase-8, while cells over expressing mitochondrial mediators of the apoptotic response such as Bcl-2 or Bcl-x(L) that are resistant to treatment with cisplatin, remained highly susceptible to cell death triggered by Fhit gene transfer. In line to what was observed in H460 cells, Ad-Fhit efficacy was not affected by Bcl-2 overexpression also in two other lung cancer cell lines (A549 and Calu-1). Analysis of cytochrome c release also confirmed that in Bcl-2- or Bcl-x(L)-expressing cells apoptosis could be detected by terminal deoxynucleotidyl-transferase mediated dUTP nick-end labelling (TUNEL) assay before any evidence of mitochondrial membrane perturbation. In conclusion, our analysis indicates that the Fhit protein exerts its oncosuppressor activity through induction of an apoptotic mechanism that seems to be FADD dependent, caspase-8 mediated and independent from mitochondrial amplification.

Caspase-mediated apoptosis and cell death of rhesus macaque CD4 + T-cells due to cryopreservation of peripheral blood mononuclear cells can be rescued by cytokine treatment after thawing

Cryopreservation of peripheral blood mononuclear cells (PBMC) from animal model studies and clinical trials is utilized as a primary method for long-term storage of PBMC for future in vitro and in vivo applications. The objective of this study was to define the mechanistic pathways involved in cryopreservation-induced apoptosis of CD4 + T-cells in PBMC, and to evaluate a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved PBMC. Using cryopreserved PBMC samples isolated from naı̈ve and Simian immunodeficiency virus (SIV)-infected rhesus macaques as a model, frozen PBMC showed significantly increased levels of apoptosis-induced CD4 + T-cell death compared to fresh PBMC over a 5-day culture period as detected by Annexin V/PI and trypan blue staining. Mechanistic studies using a broad-spectrum caspase inhibitor z-VAD demonstrated a crucial involvement of caspases in cryopreservation-induced apoptosis of CD4 + T-cells. Furthermore, the ability of z-VAD to inhibit both mitochondrial membrane perturbation and apoptotic cell death implicated the involvement of caspase-mediated mitochondrial membrane damage in cryopreservation-induced apoptosis of CD4 + T-cells. Due to their known properties to promote T-cell survival and inhibit apoptosis, we evaluated the ability of IL-2, IL-4, and IL-7 combination cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4 + T-cells. The cytokine treatment resulted in a significant inhibition ( p<0.01) of apoptosis-induced cell death and rescued CD4 + T-cell survival ( p<0.01) in the cryopreserved cells. Efficient rescue of cryopreserved CD4 + T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.

Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria.

Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism, characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation. In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation through pathways that are also independent of the mitochondrial permeability transition.

Mitochondrial Damage During Ischemia Determines Post-Ischemic Contractile Dysfunction in Perfused Rat Heart

T. Iwai, K. Tanonaka, R. Inoue, S. Kasahara, N. Kamo and S. Takeo. Mitochondrial Damage During Ischemia Determines Post-Ischemic Contractile Dysfunction in Perfused Rat Heart. Journal of Molecular and Cellular Cardiology (2002) 34, 725–738. Possible mechanisms underlying sodium overload-induced ischemia/reperfusion injury in perfused rat hearts were examined. Massive accumulation of myocardial Na+ occurred during ischemia, suggesting cytosolic sodium overload in cardiac cells. Treatment of the pre-ischemic heart with 0.3μmol/l tetrodotoxin or 3μmol/l ethyl-isopropyl amiloride enhanced post-ischemic contractile recovery (72 or 82% of initialvs 24% for untreated group), which was associated with suppression of tissue Na+ accumulation (138 or 141% of initial vs 270% for untreated group), restoration of tissue high-energy phosphates, and preservation of the ability of mitochondria to produce ATP in the ischemic/reperfused heart. The release of cytochrome c from the ischemic heart was observed, which was blocked by treatment of the pre-ischemic heart with these agents. The improvement of post-ischemic contractile recovery by these agents was closely correlated with the ability of mitochondria to produce ATP during ischemia. To examine the effects of sodium overload on mitochondrial function, isolated mitochondria were incubated in the presence of various concentrations of Na+. Na+ induced mitochondrial membrane perturbations such as depolarization of the membrane potential, mitochondrial swelling, cytochrome c release from isolated mitochondria, and a reduction in oxidative phosphorylation. These events in the isolated mitochondria were not blocked by the presence of the above agents. The results suggest that cytosolic sodium overload in cardiac cells may induce deterioration of the mitochondrial function during ischemia and that this mitochondrial damage may determine post-ischemic contractile dysfunction in perfused rat hearts.

Neuroprotection by a Bile Acid in an Acute Stroke Model in the Rat

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is a strong modulator of apoptosis in both hepatic and nonhepatic cells, and appears to function by inhibiting mitochondrial membrane perturbation. Excitotoxicity, metabolic compromise, and oxidative stress are major determinants of cell death after brain ischemia-reperfusion injury. However, some neurons undergo delayed cell death that is characteristic of apoptosis. Therefore, the authors examined whether TUDCA could reduce the injury associated with acute stroke in a well-characterized model of transient focal cerebral ischemia. Their model of middle cerebral artery occlusion resulted in marked cell death with prominent terminal deoxynucleotidyl transferase-mediated 2;-deoxyuridine 5;-triphosphate-biotin nick end labeling (TUNEL) within the ischemic penumbra, mitochondrial swelling, and caspase activation. Tauroursodeoxycholic acid administered 1 hour after ischemia resulted in significantly increased bile acid levels in the brain, improved neurologic function, and an approximately 50% reduction in infarct size 2 and 7 days after reperfusion. In addition, TUDCA significantly reduced the number of TUNEL-positive brain cells, mitochondrial swelling, and partially inhibited caspase-3 processing and substrate cleavage. These findings suggest that the mechanism for in vivo neuroprotection by TUDCA is, in part, mediated by inhibition of mitochondrial perturbation and subsequent caspase activation leading to apoptotic cell death. Thus, TUDCA, a clinically safe molecule, may be useful in the treatment of stroke and possibly other apoptosis-associated acute and chronic injuries to the brain.

Amyloid β-Peptide Disrupts Mitochondrial Membrane Lipid and Protein Structure: Protective Role of Tauroursodeoxycholate

Mitochondria have been implicated in the cytotoxicity of amyloid β-peptide (Aβ), which accumulates as senile plaques in the brain of Alzheimer's disease patients. Tauroursodeoxycholate (TUDC) modulates cell death, in part, by preventing mitochondrial membrane perturbation. Using electron paramagnetic resonance spectroscopy analysis of isolated mitochondria, we tested the hypothesis that Aβ acts locally in mitochondrial membranes to induce oxidative injury, leading to increased membrane permeability and subsequent release of caspase-activating factors. Further, we intended to determine the role of TUDC at preventing Aβ-induced mitochondrial membrane dysfunction. The results demonstrate oxidative injury of mitochondrial membranes during exposure to Aβ and reveal profound structural changes, including modified membrane lipid polarity and disrupted protein mobility. Cytochrome c is released from the intermembrane space of mitochondria as a consequence of increased membrane permeability. TUDC, but not cyclosporine A, almost completely abrogated Aβ-induced perturbation of mitochondrial membrane structure. We conclude that Aβ directly induces cytochrome c release from mitochondria through a mechanism that is accompanied by profound effects on mitochondrial membrane redox status, lipid polarity, and protein order. TUDC can directly suppress Aβ-induced disruption of the mitochondrial membrane structure, suggesting a neuroprotective role for this bile salt.

A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.

Allylamine cardiovascular toxicity: VI. subcellular distribution in rat aortas

The cardiovascular toxin allylamine (3-aminopropene) has been shown to concentrate in elastic and muscular tissues. In this study the 14C-moiety of [ 14C] allylamine was traced in aortas of adult Sprague-Dawley rats after intravenously injecting 30 μ;Ci of [ 14C] allylamine (spec. act. = 0.4 mCi/mM). At 5, 10, 15 and 20 min after injection 33.3–29.8% of the 14C-moiety was sequestered in aortas; at 30 min 16.8% was still present. Subcellular fractionation of the postnuclear supernatant by isopycinic centrifugation in sucrose demonstratated that 5 min after administration of [ 14C] allylamine, the 14C-moiety displayed a modal density peak of 1.20 g/ml. Similar activities were observed up to 30 min exposure. This modal density was similar to the distribution pattern of mitochondria based on analysis of malate dehydrogenase activities. As early as 20 min post-exposure, mitochondrial malate dehydrogenase activities of aortic mitochondria decreased, while cytosolic malate dehydrogenase activities increased, suggesting mitochondrial membrane perturbation. We suggest that the subcellular site for allylamine injury to the aorta is the mitochondrion.