Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

The still uncertain identity of the channel-forming unit(s) of the mitochondrial permeability transition pore

Mitochondria from different organisms can undergo a sudden process of inner membrane unselective leakiness to molecules known as the mitochondrial permeability transition (MPT). This process has been studied for nearly four decades and several proteins have been claimed to constitute, or at least regulate the usually inactive pore responsible for this transition. However, no protein candidate proposed as the actual pore-forming unit has passed rigorous gain- or loss-of-function genetic tests. Here we review evidence for -and against- putative channel-forming components of the MPT pore. We conclude that the structure of the MPT pore still remains largely undefined and suggest that future studies should follow established technical considerations to unambiguously consolidate the channel forming constituent(s) of the MPT pore.     

Differences in the Activation of the Mitochondrial Permeability Transition Among Brain Regions in the Rat Correlate with Selective Vulnerability

Mitochondria from different regions of the brain were prepared, and the activation of the mitochondrial permeability transition (MPT) by calcium was investigated by monitoring the associated mitochondrial swelling. In general, the properties of the MPT in brain mitochondria were found to be qualitatively similar to those observed in liver and heart mitochondria. Thus, swelling was inhibited by adenine nucleotides (AdNs) and low pH (<7.0), whereas thiol reagents and alkalosis facilitated swelling. Cyclosporin A and its nonimmunosuppressive analogue N-methyl-Val-4-cyclosporin A (PKF 220-384) both inhibited swelling and prevented the translocation of cyclophilin D from the matrix to the membranes of cortical mitochondria. However, the calcium sensitivity of the MPT differed in mitochondria from three brain regions (hippocampus > cortex > cerebellum) and is correlated with the susceptibility of these regions to ischemic damage. Depleting mitochondria of AdNs by treatment with pyrophosphate ions sensitized the MPT to [Ca2+] and abolished regional differences, implying regional differences in mitochondrial AdN content. This was confirmed by measurements showing significant differences in AdN content among regions (cerebellum > cortex > hippocampus). Our data add to recent evidence that the MPT may be involved in neuronal death.     

Metabolic depression is delayed and mitochondrial impairment averted during prolonged anoxia in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935)

Lepidophthalmus louisianensis burrows deeply into oxygen-limited estuarine sediments and is subjected to extended anoxia at low tides. Large specimens (> 2 g) have a lethal time for 50% mortality (LT₅₀) of 64 h under anoxia at 25 °C. Small specimens (< 1 g) have a significantly higher LT₅₀ of 113 h, which is the longest ever reported for a crustacean. Whole body lactate levels rise dramatically under anoxia and exceed 120 μmol g.f.w.^− 1 by 72 h. ATP, ADP, and AMP do not change during 48 h of anoxia, but arginine phosphate declines by over 50%. Thus arginine phosphate may help stabilize the ATP pool. Surprisingly, when compared to the aerobic resting rate, ATP production under anoxia declines only moderately during the first 12 h, and drops to only about 30% between 12 and 48 h. Finally, after 48 h of anoxia, a major metabolic depression to less than 5% occurs. Downregulation of metabolism is delayed in L. louisianensis compared to many invertebrates that exhibit facultative anaerobiosis. Bioenergetic constraints as a result of eventual metabolic depression lead to ionic disturbances like calcium overload and compromised membrane potential of mitochondria. Because these phenomena trigger apoptosis in mammalian species, we evaluated the susceptibility of ghost shrimp mitochondria to opening of the mitochondrial permeability transition pore (MPTP) and associated damage. Energized mitochondria isolated from hepatopancreas possess a pronounced capacity for calcium uptake. Exogenous calcium does not stimulate opening of the MPTP, which potentially could reduce cell death during prolonged anoxia.     

Simvastatin inducing PC3 prostate cancer cell necrosis mediated by calcineurin and mitochondrial dysfunction

In the present study we analyzed the mechanisms of simvastatin toxicity for the PC3 human prostate cancer cell line. At 10 μM, simvastatin induced principally apoptosis, which was prevented by mevalonic acid but not by cyclosporin A, the inhibitor of calcineurin and mitochondrial permeability transition (MPT). At 60 μM, simvastatin induced the necrosis of PC3 cells insensitive to mevalonic acid. Cell necrosis was preceded by a threefold increase in cytosolic free Ca²⁺ concentration and a significant decrease in both respiration rate and mitochondrial membrane potential. Both mitochondrial dysfunction and necrosis were sensitive to the compounds cyclosporin A and bongkrekic acid, as well as the calcineurin inhibitor FK506. We have concluded that simvastatin-induced PC3 cells apoptosis is dependent on 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibition and independent of MPT, whereas necrosis is dependent on mitochondrial dysfunction caused, at least in part, by calcineurin.     

The role of a membrane-bound glutathione transferase in the peroxynitrite-induced mitochondrial permeability transition pore: formation of a disulfide-linked protein complex

We have previously shown that the mitochondrial membrane-bound glutathione transferase (mtMGST1) is activated via thiol modifications and contributes to the mitochondrial permeability transition (MPT) pore. In the present study we aimed to confirm the role of mtMGST1 in the oxidant peroxynitrite (PON)-induced MPT pore opening. PON induced the swelling of mitoplasts (inner membranes including the matrix) as well as of the mitochondria. The swelling was markedly suppressed by ADP [an adenine nucleotide translocator (ANT) ligand] and partially suppressed by cyclosporin A or by GST inhibitors (tannic acid, S-hexylglutathione). Dithiothreitol (DTT), a disulfide bond-reducing reagent, prevented the swelling. Western blot analyses of mitoplast proteins after PON-induced swelling positively identified the high molecular weight protein (HMP) including mtMGST1 (monomer), ANT (48 kDa), and cyclophilin D (CypD, 30 kDa). The HMP level was decreased according to suppression of the swelling and undetectable after DTT treatment. The HMP formation and swelling were also suppressed by a Ca²⁺ chelating agent and antioxidants. These results suggest that the HMP is a disulfide-linked protein complex involving mtMGST1, ANT, CypD and function as a MPT pore in PON-induced swelling, in which the Ca²⁺ released by PON might play an important role in the complex formation.     

Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition

Opening of the permeability transition pore (PTP) has been implicated as an important mitochondrial event that occurs during apoptosis. We examined the role of the PTP in the well-characterized cell death of rat sympathetic neurons deprived of nerve growth factor (NGF) in vitro. Removal of NGF causes these neurons to undergo either a classic apoptotic cell death or, when treated with a broad-spectrum caspase inhibitor such as boc-aspartyl(OMe)-fluoromethylketone (BAF), a delayed, nonapoptotic cell death. The PTP inhibitor, cyclosporin A (CsA), blocked commitment-to-die in the presence of BAF, as defined by the ability of NGF readdition to rescue cells, but had little effect on commitment-to-die in the absence of BAF. CsA did not have trophic effects on BAF-saved cells, but did block the decrease in mitochondrial membrane potential. These data suggest that PTP opening is a critical event in caspase-independent, nonapoptotic (but not caspase-dependent, apoptotic) death of NGF-deprived rat sympathetic neurons.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Participation of the mitochondrial permeability transition pore in nitric oxide-induced plant cell death

In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)-induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine inhibited growth and caused death in suspension-cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells.     

Protective effects of EGCg or GCg, a green tea catechin epimer, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of (-)-epigallocatechin-3-gallate (EGCg) or the C-2 epimer, (-)-gallocatechin-3-gallate (GCg), afforded by their antioxidative activity among green tea catechins were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. The recovery (%) of the left ventricular developed pressure from ischemia by reperfusion was 34.4% in the control, while in the presence of EGCg (3x10(-5) M) or GCg (3x10(-6) M, a more diluted concentration than that of EGCg), it led to a maximal increase of 78.4% or 76.2%, consistent with a significant preservative effect on the tissue level of ATP at the end of ischemia or reperfusion. In the perfused preparation of mitochondria, EGCg (10(-5) M) inhibited mitochondrial Ca(2+) elevation by changes in the Ca(2+) content or the acidification of perfusate, similarly to findings with cyclosporin A, a well known inhibitor of the mitochondrial permeability transition pore. By in vitro electron paramagnetic resonance (EPR), EGCg or GCg was found to directly quench the activity of active oxygen radicals, with the strongest activity in tea catechins. EGCg or GCg decreased the caspase-3 activity induced apoptosis. Therefore, it is concluded that the beneficial effects of EGCg or GCg play an important role in ischemia-reperfusion hearts in close relation with nitric oxide (NO), active oxygen radicals and biological redox systems in mitochondria.     

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting

Background

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3’s action remain unanswered.

Results

We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants.

Conclusions

Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on mitochondria.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0152-0) contains supplementary material, which is available to authorized users.     

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established¹, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca²⁺ homeostasis², bioenergetics and redox signaling ³.mtPTP opening is triggered by matrix Ca²⁺ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids⁴. In vitro, mtPTP opening can be achieved by increasing Ca²⁺ concentration inside the mitochondrial matrix through exogenous additions of Ca²⁺ (calcium retention capacity). When Ca²⁺ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca²⁺ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca²⁺ handling (uptake and release, as measured by Ca²⁺ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca²⁺ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca²⁺, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.     

The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte/macrophage lineage

U937, THP-1, and J774 cells or human monocytes and macrophages display similar levels of sensitivity to peroxynitrite and exposure to an otherwise non-toxic concentration of the oxidant in the presence of a phospholipase A(2) inhibitor was invariably associated with the onset of mitochondrial permeability transition (MPT)-dependent toxicity. These events were prevented by exogenous arachidonic acid (AA). In general, the protective concentrations of AA were greater in those cell types releasing more AA. Thus, non-toxic concentrations of peroxynitrite commit cells belonging to the monocyte/macrophage lineage to MPT-dependent toxicity that is however prevented by endogenous AA.     

Distinctive molecular signaling in triple-negative breast cancer cell death triggered by hexadecylphosphocholine (miltefosine)

This study describes the molecular signaling involved in the different cell death modes of triple-negative breast cancer cells induced by hexadecylphosphocholine (HePC/miltefosine), a clinically relevant anticancer alkylphosphocholine. We found that the HePC treatment triggers cell-type-dependent apoptotic and non-apoptotic cell death processes. Moreover, the expression level of the apoptosis activator Fas, and Fas/Fas ligand signaling capacity are not attributing factors for the preference toward apoptosis. Using Fas siRNA and overexpression approaches we establish that Fas is not a pro-apoptotic factor but a contributor to cell protection in HePC-apoptosis-sensitive cells. The insight in the multi-modal anticancer capability of HePC in triple-negative breast cancer cells may facilitate the targeted design of therapeutic strategies against triple-negative breast cancers.     

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSC_enr-EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSC_enr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSC_enr-EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSC_enr-EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSC_enr lysate, CSC_enr-EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSC_enr-EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSC_enr-EXOs disrupted spheroids' structure. Thus, CSC_enr-EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.     

Cyclophilin D deficiency protects against the development of mitochondrial ROS and cellular inflammation in aorta

Introduction

Inflammation and oxidative stress are closely correlated in the pathology of cardiovascular disease. Mitochondrial cyclophilin D (CypD), the important modulator for mPTP opening, is increasingly recognized as a key regulator of cellular ROS generation. Besides, its association with cell inflammation is also being discovered. However, the effects of CypD in modulating vascular inflammatory response is unknown. We sought to investigate whether CypD deficiency attenutes vascular inflammation under physical conditions.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB₂) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB₂ but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.     

Safety concerns for the potential use of cyanovirin-N as a microbicidal anti-HIV agent

Based on its antiviral activity profile, cyanovirin-N (CV-N) holds great potential for anti-HIV microbicidal application. However, limited data are available on the possible side-effects of this lectin. A detailed investigation was carried out to obtain better insights in the cytotoxic, inflammatory and (anti)-proliferative properties of CV-N in comparison with several other plant-derived lectins. CV-N affected the cell morphology of PBMCs and enhanced the expression of the cellular activation markers CD25, CD69 and HLA-DR. PBMCs activated by CV-N were more susceptible for R5 HIV-1 infection. In addition, CV-N exerted a pronounced mitogenic activity and significantly enhanced in PBMCs the production of a wide variety of cytokines, as determined by the Bio-Plex human cytokine 27-plex array system. In comparison, other lectins obtained from Hippeastrum hybrid, Galanthus nivalis, and Urtica dioica induced markedly less, if any, stimulatory effects. So, the use of CV-N may be accompanied by various stimulatory effects that may compromise its application for microbicidal use.     

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.