Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by -D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.     

Ceramide Induces Release of Pro-Apoptotic Proteins from Mitochondria by Either a Ca2+-Dependent or a Ca2+-Independent Mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide.     

Mitochondrial cytochrome c release precedes transmembrane depolarisation and caspase-3 activation during ceramide-induced apoptosis of Jurkat T cells

Whilst the role of ceramide, a second messenger of the sphingolipid family, in the initiation of receptor-mediated apoptosis is controversial, a growing body of evidence is emerging for a role of ceramide in the amplification of apoptosis via mitochondrial perturbations that culminate in the activation of execution caspases. Treatment of Jurkat T cells with the cell-permeable analog, C₂-ceramide, resulted in the rapid onset of apoptosis as evidenced by Annexin V-FITC staining of externalised phosphatidylserine residues. Cells bearing this early apoptotic marker had a reduced mitochondrial transmembrane potential (m) that was preceded by the release of cytochrome c from mitochondria. Subsequent activation of caspase-3 provides the link between these ceramide-induced mitochondrial changes and execution caspases that ultimately result in the physical destruction of the cell. Collectively these results demonstrate that ceramide signalling results in caspase-mediated apoptosis via mitochondrial cytochrome c release and are further supportive of the role of ceramide in the amplification of apoptosis.     

Identification of deficient mitochondrial signaling in apoptosis resistant leukemia cells by flow cytometric analysis of intracellular cytochrome c, caspase-3 and apoptosis

Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 processing using conformation sensitive monoclonal antibodies. This method proved to identify deficient mitochondrial apoptosis signaling in leukemia cells overexpressing Bcl-2 by a pattern of apoptosis resistance, deficient cytochrome c reduction and partial processing of caspase-3. In primary leukemia cells, reduction of cytochrome c and caspase-3 activation was induced by treatment with anticancer drugs in vitro. In leukemia cells of a patient with resistant disease, a pattern of deficient apoptosis signaling as in Bcl-2 transfected cells was observed, suggesting that deficient mitochondrial signaling contributed to the clinical phenotype of drug resistance in this patient. Flow cytometric analysis of mitochondrial apoptosis signaling may provide a useful tool for the prediction of drug resistance and treatment failure in primary leukemia.     

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 μM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 μM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 μM.     

Ion channel inhibitors block caspase activation by mechanisms other than restoring intracellular potassium concentration

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na⁺ and loss of K⁺ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA⁺) and 4-aminopyridine-sensitive K⁺ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K⁺] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K⁺]_i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K⁺ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K⁺]_i associated with apoptosis.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells

Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.     

Helix 6 of tBid is necessary but not sufficient for mitochondrial binding activity

The apoptosis effector Bid regulates cell death at the level of mitochondrial cytochrome c efflux. Bid consists of 8 -helices (designated H1 through H8, respectively) and is a soluble cytosolic protein in its native state. Proteolysis of the N-terminus (encompassing H1 and H2) of Bid yields activated tBid (truncated Bid), which translocates to the mitochondria and induces the efflux of cytochrome c. Here, we demonstrate that helix H6 of tBid is necessary, albeit not sufficient, for mitochondrial binding. In particular, a 33 amino acid long domain, which encompassed H6 and H7, behaved as the minimum domain in tBid that was sufficient for mitochondrial binding. Unexpectedly, the hydrophobic surface of these helices could be mutated without altering the binding activity of the domain, implying that the secondary structure of the helices may be the key determinant of binding. These experiments expand our mechanistic understanding of the apoptotic regulator, tBid.     

Cytochrome c release from rat brain mitochondria is proportional to the mitochondrial functional deficit: implications for apoptosis and neurodegenerative disease

Apoptosis may be initiated in neurons via mitochondrial release of the respiratory protein, cytochrome c. The mechanism of cytochrome c release has been studied extensively, but little is known about its dynamics. It has been claimed that release is all-or-none, however, this is not consistent with accumulating evidence of cytosolic mechanisms for 'buffering' cytochrome c. This study has attempted to model an underlying disease pathology, rather than inducing apoptosis directly. The model adopted was diminished activity of the mitochondrial respiratory chain complex I, a recognized feature of Parkinson's disease. Titration of rat brain mitochondrial respiratory function, with the specific complex I inhibitor rotenone, caused proportional release of cytochrome c from isolated synaptic and non-synaptic mitochondria. The mechanism of release was mediated, at least in part, by the mitochondrial outer membrane component Bak and voltage-dependent anion channel rather than non-specific membrane rupture. Furthermore, preliminary data were obtained demonstrating that in primary cortical neurons, titration with rotenone induced cytochrome c release that was subthreshold for the induction of apoptosis. Implications for the therapy of neurodegenerative diseases are discussed.     

Inhibition of telomerase enhances apoptosis induced by sodium butyrate via mitochondrial pathway

Telomerase activation represents an early step in carcinogenesis. Increased telomerase activity in cervical cancer suggests a potential target for the development of novel therapeutic drugs. The aim of this study is to investigate the impact of telomerase activity on the biological features of HeLa cells and the possible mechanisms of enhanced apoptosis rate induced by sodium butyrate after telomerase inhibition. We introduced vectors encoding dominate negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistance marker into HeLa cells. Thus we assessed the biological effects of telomerase activity on telomere length, cell proliferation, chemosensitivity and radiosensitivity. In order to understand the mechanisms in which DN-hTERT enhances the apoptosis induced by sodium butyrate, we detected the release status of cytochrome c and apoptosis inducing factor (AIF) from mitochondria. Ectopic expression of DN-hTERT resulted in inhibition of telomerase activity, reduction of telomere length, decreased colony formation ability, and loss of tumorigenicity in nude mice. Moreover, DN-hTERT transfected HeLa cells with shortened telomeres were more susceptible to multiple chemotherapeutic agents and radiation. WT-hTERT transfected HeLa cells with longer telomeres exhibited resistance to radiation and chemotherapeutic agents. Our data demonstrate that elevated release level of cytochrome c and AIF from mitochondria might contribute to the enhanced apoptosis in DN-hTERT transfected HeLa cells after treatment with sodium butyrate. Inhibition of telomerase might serve as a promising adjunctive therapy combined with conventional therapy in cervical cancer. Both of them contributed equally to this work.     

Induction of apoptosis by quercetin: different response of human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukemia cells

This work shows that 25 μM quercetin caused a marked inhibition of K562 cells growth together with a mild cytotoxicity, while HSB-2 cells were practically unaffected. Moreover, quercetin induced caspase-3 and cytochrome c-dependent apoptosis almost exclusively in the former cell line. Exposure of K562 cells to quercetin caused also a significant increase of cells in G₂/M phase that reached the maximum peak at 24 h (4-fold with respect to the basal value). The major sensitivity exhibited by K562 cells was only in part imputable to their higher glutathione content, as compared to HSB-2 cells, thus confirming previous reports describing the formation of intracellular quercetin–thiol toxic adducts in cells exposed to the flavonoid. In fact, after induction of intracellular glutathione increase we detected in both cell lines a significant rise of apoptotic cells, again more marked in K562 cells. By contrast, glutathione-depleted cells, failed to show a decrease of apoptosis in both cell lines, thus contradicting our previous findings and literature data. Since the yet unresolved question about the anti-oxidant or the pro-oxidant capacity of quercetin, we investigated which of these two properties worked in our experimental model. Interestingly, not only quercetin did not produce reactive oxygen species but also prevented their formation, as observed in cells exposed to the oxidizing agent ter-butylhydroperoxide, acting as an efficient oxygen radicals scavenger. This result indicates that quercetin exhibited, in these cell lines, anti-oxidant more than pro-oxidant ability.     

Regulation of ceramide channel formation and disassembly: Insights on the initiation of apoptosis

Sphingolipid research has surged in the past two decades and has produced a wide variety of evidence supporting the role of this class of molecules in mediating cellular growth, differentiation, senescence, and apoptosis. Ceramides are a subgroup of sphingolipids (SLs) that are directly involved in the process of initiation of apoptosis. We, and others, have recently shown that ceramides are capable of the formation of protein-permeable channels in mitochondrial outer membranes under physiological conditions. These pores are indeed good candidates for the pathway of release of pro-apoptotic proteins from the mitochondrial intermembrane space (IMS) into the cytosol to initiate intrinsic apoptosis. Here, we review recent findings on the regulation of ceramide channel formation and disassembly, highlighting possible implications on the initiation of the intrinsic apoptotic pathway.     

Respiration and Mitochondrial Membrane Potential Are not Required for Apoptosis and Anti-apoptotic Action of Bcl-2 in HeLa Cells

The release of cytochrome c from intermembrane space of mitochondria into cytosol is one of the critical events in apoptotic cell death. The important anti-apoptotic oncoprotein Bcl-2 inhibits this process. In the present study it was shown that apoptosis and release of cytochrome c induced by staurosporine or by tumor necrosis factor- in HeLa cells were not affected by inhibitors of respiration (rotenone, myxothiazol, antimycin A) or by uncouplers (CCCP, DNP) that decrease the membrane potential at the inner mitochondrial membrane. The inhibitors of respiration and the uncouplers did not affect also the anti-apoptotic activity of Bcl-2.     

Mechanisms of N-acetylcysteine-driven enhancement of MK886-induced apoptosis

N-Acetylcysteine (NAC), besides being a precursor of glutathione, has an array of other effects including an ability to scavenge free radicals, modulate gene expression and signal transduction pathways, and regulate cell survival and apoptosis. At concentrations lower than 20 mmol/L, NAC is nontoxic to cultured cells and can protect against apoptosis induced by a number of agents. A few recent reports, however, have indicated that NAC can also increase apoptosis. MK886, a 5-lipoxygenase activating protein (FLAP) inhibitor, induces apoptosis in many cell lines by an unknown mechanism that is independent of FLAP and lipoxygenase activity but is possibly related to effects on kinases such as Akt. In Jurkat T lymphocytes, NAC pretreatment (10 mmol/L) enhanced MK886-induced apoptosis by 2.4-fold. Following NAC-MK886 treatment, there was a significant increase in caspase-3 activity, and a decrease in mitochondrial transmembrane potential compared to MK886 alone. However, the extent of cytochrome c release was comparable between MK886 alone and MK886-NAC treatments. The enhancement of MK886-induced apoptosis by 10 mmol/L NAC appears to be partly related to a decrease in pH caused by this concentration of NAC, because an acidic environment favors activation of effector caspases and triggering of mitochondrial apoptosis. However, because neutralized NAC also enhanced apoptosis (1.6-fold), a direct role for NAC in augmenting the apoptotic pathways initiated by MK886 is suggested.     

Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m², but not at 150 J/m², induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m² UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m² UVB, but not at 150 J/m². However, with both 150 and 500 J/m² UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m² UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

生物大分子从头合成和设计的研究进展

生物大分子指生物体内存在的DNA、蛋白质、多糖等物质,其对生物体正常生命活动至关重要.从头合成和设计技术在生物大分子的合成和结构设计上具有自由度高、前体简单等特点,能够按照特定研究目的对生物大分子进行全新设计和高效合成.近年来,从头合成与设计技术在人造基因组合成、新型蛋白质类药物设计、糖缀合物合成等领域已开始受到重视.基于生物大分子从头合成和设计技术,可以定向制备全新设计的DNA或全新的基因表达产物,以及具有识别功能的糖链或糖缀合物,将大大推进诸如细胞因子模拟物、基因治疗递送载体等生物活性物质的开发,为人工生物系统的构建、罕见疾病的治疗等提供新的解决方法.本文就DNA、蛋白质和多糖的从头合成和设计进行了综述,阐述了相关方法及应用,最后概括分析了三者之间的关系.