Initiation of mitochondrial-mediated apoptosis during cardiac reperfusion

Reperfusion of myocardial tissue can result in programmed cell death. Nevertheless, relatively little information exists concerning pathways initiated in vivo that ultimately commit cardiac cells to apoptosis during ischemia/reperfusion. The goal of the present study was to determine whether mitochondrial-mediated mechanisms of apoptosis are initiated during in vivo cardiac ischemia/reperfusion. We provide evidence that the content of cytochrome c in the cytosol increases exclusively during reperfusion. Over the same time interval Bax, a pro-apoptotic protein implicated in release of cytochrome c from mitochondria, was found to disappear from cytosolic extracts. This was associated with the appearance of tightly associated Bax in the mitochondrial fraction. Cytochrome c from reperfused cytosolic extracts is present as a high molecular weight oligomer consistent with formation of the apoptosome. In addition, pro-caspase-9 was found to disappear exclusively during reperfusion. Therefore, the results of the current study indicate that the mitochondrial-mediated pathway of apoptosis is initiated as a result of in vivo cardiac ischemia/reperfusion.     

Glucose induces apoptosis of cardiomyocytes via microRNA-1 and IGF-1

Glucose toxicity is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The present study investigated whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9C2 through microRNA regulated insulin-like growth factor (IGF-1) signaling pathway. Our data showed that H9C2 cells exposed to high glucose have increased miR-1 expression level, decreased mitochondrial membrane potential, increased cytochrome-c release, and increased apoptosis. Glucose induced mitochondrial dysfunction, cytochrome-c release and apoptosis was blocked by IGF-1. Using prediction algorithms, we identified 3′-untranslated regions of IGF-1 gene are the target of miR-1. miR-1 mimics, but not mutant miR-1, blocked the capacity of IGF-1 to prevent glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. In conclusion, our data demonstrate that IGF-1 inhibits glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis and IGF-1’s effect is regulated by miR-1.     

Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells

The recently developed bio-barcode (BBC) assay using polymerase chain reaction (PCR) to generate signals has been shown to be an extraordinarily sensitive method to detect protein targets. The BBC assay involves a magnetic microparticle (with antibody to capture the target of interest) and gold nanoparticle (with recognition antibody and thiolated single-stranded barcode DNAs) to form a sandwich around the target. The concentration of target is determined by the amount of barcode DNA released from the nanoparticles. Here we describe a modification using aptamers to substitute the gold nanoparticles for the BBC assay. In this study, we isolated a 76-mer monoclonal aptamer against cytochrome-c (cyto-c) and this single-stranded DNA in defined 3D structure for cyto-c was used in the BBC assay for both recognition and readout reporting. After magnetic separation, the aptamer was amplified by PCR and this aptamer-based barcode (ABC) assay was sensitive enough to detect the cyto-c in culture medium released from the apoptotic cells after drug treatment at the picomolar level. When compared to the conventional cyto-c detection by Western blot analysis, our ABC assay is sensitive, and time for the detection and quantification with ready-made probes was only 3 h.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Manganese(III)-salens induce tumor selective apoptosis in human cells

In order to explore the apoptotic and anti-tumor activities of metallo-salens, we synthesized several Mn(III)-salen derivatives (compds. 1-9) and analyzed their effects on cultured human cancer and non-cancer cells. Our results demonstrated that Mn(III)-salen derivatives affect cell viability, induce nuclear condensation and fragmentation in breast cancer cells (MCF7). Mn(III)-salen derivatives also induced caspase-3/7 activation and release of cytochrome-c from the mitochondria to cytosol suggesting that Mn(III)-salen derivatives induce apoptosis in human cells via mitochondrial pathway. Importantly, the nature of the substituent and the bridging spacer between diimino groups on the salen ligand play critical roles in determining the apoptotic activities of Mn(III)-salen derivatives. The IC₅₀ values for the active Mn(III)-salen derivatives lie within the range of 11-40 μM in MCF7 cells. Most importantly, several Mn(III)-salen derivatives showed preferential cytotoxicity (2- to 5-fold) toward malignant breast cells (MCF7) over a non-malignant breast epithelial cell line (MCF10). Notably, the level of cytotoxicity and selectivity of the Mn(III)-salen derivatives towards MCF7 and MCF10 cells are very similar to cisplatin which indicate that Mn(III)-salens are potential novel anti-tumor agent.     

Dynamics of mitochondrial structure during apoptosis and the enigma of Opa1

“The large scale remodeling of mitochondria during apoptosis is a necessary step for the complete release of cytochrome c” has been a tenet since 2002. However, more recent findings strongly indicate that the large-scale remodeling previously described actually takes place after the release of cytochrome c and in a caspase-dependent manner, bringing into question whether mitochondria remodeling is necessary. In a more recent article, however, it was shown that a much more subtle form of remodeling is taking place which is only observable by electron tomography. In the Bcl-2 inhibitable Bax/Bak-dependent intrinsic pathway of apoptosis, the release of cytochrome c from mitochondria is a consequence of two carefully coordinated events: formation of outer membrane pores and opening of crista junctions triggered by Opa1 oligomer disassembly, and both steps are necessary for the complete release of cytochrome c. We review the recent literature pertaining to the coordinated release of cytochrome c during cell death.     

Overexpression of glutaredoxin 2 attenuates apoptosis by preventing cytochrome c release

Human mitochondrial glutaredoxin 2 (Grx2) catalyzes glutathione-dependent dithiol reaction mechanisms, reducing protein disulfides, and monothiol reactions, reducing mixed disulfides between proteins and GSH (de-/glutathionylation). Here, we have overexpressed Grx2 in HeLa cells in its mitochondrial form (mGrx2-HeLa) as well as a truncated cytosolic form, lacking the mitochondrial translocation signal (tGrx2-HeLa). The resulting clones were less susceptible to apoptosis induced by 2-deoxy-d-glucose (2-DG) or doxorubicin (Dox). Overexpression of Grx2 inhibited cytochrome c release and caspase activation induced by both agents. In addition, Grx2 prevented 2-DG- and Dox-induced loss of cardiolipin, the phospholipid anchoring cytochrome c to the inner mitochondrial membrane. Overexpression of mGrx2 provided better protection than tGrx2 overexpression, especially after treatment with 2-DG. We propose that Grx2 facilitates the maintenance of cellular redox homeostasis upon treatment with apoptotic agents, thereby preventing cardiolipin oxidation and cytochrome c release.     

A novel 21-kDa cytochrome c-releasing factor is generated upon treatment of human leukemia U937 cells with geranylgeraniol

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.     

Involvement of reactive oxygen species-independent mitochondrial pathway in gossypol-induced apoptosis

Gossypol is a component present in cottonseeds and has been demonstrated to be an effective contraceptive drug in preventing spermatogenesis in mammalian species. In the present, we reported that gossypol could induce apoptosis in human promyelocytic leukemia cells (HL-60), as characterized by DNA fragmentation, poly(ADP) ribose polymerase (PARP) cleavage. The efficacious induction of apoptosis was observed at 50 microM for 6 h. Further molecular analysis showed that gossypol induced the truncation of Bid protein, the loss of mitochondrial membrane potential (DeltaPsi m), cytochrome c release from mitochondria into cytosol, and activation of caspase-3, -8, and -9. However, gossypol did not increase the level of reactive oxygen species (ROS), and antioxidants including N-acetyl cysteine (NAC) and catalase could not block gossypol-induced apoptosis in the HL-60 cells. These data suggest that gossypol induces apoptosis in HL-60 cells through ROS-independent mitochondrial dysfunction pathway.     

Saxifragifolin B from Androsace umbellata induced apoptosis on human hepatoma cells

Bioassay-guided phytochemical study of Androsace umbellata led to the successful isolation of saxifragifolin B (SB) for the first time. The anti-tumor effect of SB was firstly reported that it was shown to have potent cytotoxicity on human hepatoma HepG2 cells with IC50 value of 11.9 microM at 24 h. Mechanistic studies were conducted, the accumulation of sub-G1 population and the externalization of phosphatidylserine suggested that SB exerted its cytotoxic effect by induction of programmed cell death, which was confirmed by activation of PARP and caspase-3. Furthermore, SB-induced apoptosis on HepG2 cells was mediated by activation of caspase-8 and -9, mitochondrial membrane potential (Deltapsim) collapse and the leakage of cytochrome c. In summary, this study provided evidence that SB isolated from A. umbellata could induce apoptosis on human hepatoma HepG2 cells and described the molecular mechanism. Our finding revealed the potential of SB as new chemotherapeutic agent for human hepatoma.     

The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis

While many studies have focused on cytochrome c release from mitochondria, little attention has been given to the specific interaction between cardiolipin (CL) and cytochrome c, the breaching of which likely represents a critical event in the initiation of mitochondrially mediated apoptosis. Mounting evidence suggests that a decrease in the level of CL affects cytochrome c binding to the inner membrane, thus leading to higher levels of soluble cytochrome c in the mitochondrial intermembrane space. Among the factors known to affect CL levels are thyroid status, plasma concentrations of free fatty acids, Ca²⁺ dysregulation, and reactive oxygen species (ROS). These factors, especially Ca²⁺ and ROS, have long been recognized as triggers of cell death and, more recently, as modulators of mitochondrially mediated apoptosis. In this review, we discuss the significance of the disruption of the CL-cytochrome c interaction for cytochrome c release and apoptosis.     

Angiostrongylus cantonensis: apoptosis of inflammatory cells induced by treatment with mebendazole or/and interleukin 12 in mice

Angiostrongylus cantonensis is the major cause of human eosinophilic meningoencephalitis. ICR mice were infected orally with 35 infective larvae and sacrificed at 4-14 days, 25 days or 32 days post infection (dpi) for pathological and immunocytochemical examinations. In the non-treated group, no apoptosis signal was found in the meninges or parenchyma of the brains (4-14 dpi). Only a few apoptotic cells were noticed at 25 dpi (3%) and 32 dpi (10%). In the groups, the animals were given a single dose of mebendazole (20 mg/kg, per os at various times) or injections of interleukin 12 (IL-12) (10 ng/daily, intraperitoneally), all the animals were sacrificed at 14 dpi; the number of apoptotic cells was increased (17-21%). In the group that received a single dose of mebendazole (4 dpi) in combination with IL-12 injections (4-13 dpi), mild meningitis was observed, and most of the infiltrated inflammatory cells were in the apoptotic program (55%). Taken together, apoptosis of the inflammatory cells (most were eosinophils) could be induced when the infected mice were treated with mebendazole or/and IL-12.     

Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells

Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.     

Disruption of Smad5 gene induces mitochondria-dependent apoptosis in cardiomyocytes

Our previous studies have shown that SMAD5, an important intracellular mediator of transforming growth factor beta (TGF-beta) family, is required for normal development of the cardiovascular system in vivo. In the current study, we reported that the lack of the Smad5 gene resulted in apoptosis of cardiac myocytes in vivo. To further investigate the mechanism of the Smad5 gene in cardiomyocyte apoptosis, the embryonic stem (ES) cell differentiation system was employed. We found that the myotubes that differentiated from the homozygous Smad5ex6/ex6 mutant ES cells underwent collapse and degeneration during the late stages of in vitro differentiation, mimicking the in vivo observation. By electron microscopy, abnormal swollen mitochondria were observed in cardiomyocytes both from Smad5-deficient embryos and from ES-differentiated cells. There was also a significant reduction in mitochondrial membrane potential (Deltapsi m) and a leakage of cytochrome c from mitochondria into the cytosol of myocytes differentiated from Smad5 mutant ES cells. The expression of p53 and p21 was found to be elevated in the differentiated Smad5 mutant myocytes, and this was accompanied by an up-regulation in caspase 3 expression. These results suggest that the Smad5-mediated TGF-beta signals may protect cardiomyocytes from apoptosis by maintaining the integrity of the mitochondria, probably through suppression of p53 mediated pathways.     

Preconditioning prevents loss in mitochondrial function and release of cytochrome c during prolonged cardiac ischemia/reperfusion

Loss in mitochondrial function and induction of mitochondrial-mediated apoptosis occur as a result of cardiac ischemia/reperfusion. Brief and repeated cycles of ischemia/reperfusion, termed ischemic preconditioning, prevent or minimize contractile dysfunction and apoptosis associated with prolonged episodes of cardiac ischemia and reperfusion. The effects of preconditioning on various indices of ischemia/reperfusion-induced alterations in mitochondrial function and structure were therefore explored. Utilizing an in vivo rat model data is provided indicating that preconditioning completely prevents cardiac ischemia/reperfusion-induced: (1) loss in the activity of the redox sensitive Krebs cycle enzyme alpha-ketoglutarate dehydrogenase; (2) declines in NADH-linked ADP-dependent mitochondrial respiration; (3) insertion of the pro-apoptotic Bcl-2 protein Bax into the mitochondrial membrane; and (4) release of cytochrome c into the cytosol. The results of the current study indicate that preconditioning prevents specific alterations in mitochondrial structure and function that are known to impact cellular viability and provide insight into the collective benefits of preconditioning.     

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨ_m) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨ_m, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

Augmentation of drug-induced cell death by ER protein BRI3BP

To determine the contribution of the endoplasmic reticulum (ER) to cell fate decision, we focused on BRI3-binding protein (BRI3BP) residing in this organelle. BRI3BP, when overexpressed, augmented the apoptosis of human embryonic kidney 293T cells challenged with drugs including the anti-cancer agent etoposide. In contrast, the knockdown of BRI3BP reduced the drug-triggered apoptosis. BRI3BP overexpression enhanced both mitochondrial cytochrome c release and caspase-3 activity in etoposide-treated cells. In response to etoposide, the ER reorganized into irregularly shaped lamellae in mock-transfected cells, whereas in BRI3BP-overexpressing cells, such reorganization was not observed. These observations suggest that BRI3BP is involved in the structural dynamics of the ER and affects mitochondrial viability. Taken together, BRI3BP, widely expressed in animal cell types, seems to possess a pro-apoptotic property and can potentiate drug-induced apoptosis.     

Heat shock proteins,cellular chaperones that modulate mitochondrial cell death pathways

Stress or heat shock proteins (HSPs) are ubiquitous and highly conserved proteins whose expression is induced in response to a wide variety of physiological and environmental insults. They allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. These proteins play an essential role in intracellular "house-keeping" by assisting the correct folding of nascent and stress-accumulated misfolded proteins and preventing their aggregation. Several HSPs have also demonstrated to directly interact with various components of the tightly regulated programmed cell death machinery, upstream, and downstream of the mitochondrial events. Finally, HSPs could play a role in the proteasome-mediated degradation of selected proteins under stress conditions. Altogether, these properties could make HSPs appropriate targets for modulating cell death pathways.     

Regulation by sphingosine 1-phosphate of Bax and Bad activities during apoptosis in a MEK-dependent manner

Herein we report that the prosurvival sphingolipid sphingosine 1-phosphate regulates the activities of both Bad and Bax during apoptosis of Jurkat cells. First, sphingosine 1-phosphate treatment results in Bad inactivation via the ERK/Rsk-1 pathway. Second, sphingosine 1-phosphate blocks the translocation of Bax to the mitochondria induced by Fas ligation. MEK inhibition by PD98059 or U0126 not only abrogates sphingosine 1-phosphate-induced Bad phosphorylation, but also its cytoprotective effect. Furthermore, inhibition of both mitochondrial cytochrome c efflux and Bax translocation to the mitochondria by sphingosine 1-phosphate could be overcome by PD98059 or U0126. Hence, the MEK/ERK pathway seems to be crucial for the survival effects initiated by sphingosine 1-phosphate.