The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.     

Radiation induces apoptosis primarily through the intrinsic pathway in mammalian cells

Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in “initiator” caspases, “effector” caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both “initiator” and “effector” caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.     

Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells

A family of cystein proteases, the caspases, plays a central role in mediating cell death. In this study, we measured the activation of the initiator and effector caspase in real time, and studied the relationship between caspase activity and mitochondrial membrane potential in living cells by means of bioimaging. We also designed and developed a fluorescence resonance energy transfer (FRET)-based genetically encoded fluorescent indicator, which consisted of yellow fluorescent protein (YFP), a peptide sequence which can be cleaved by specific caspases, and cyan fluorescent protein (CFP). Two peptide sequences which could be cleaved by initiator caspases and effector caspases, respectively, were used. Simultaneous real-time measurements of the caspase activity and mitochondrial membrane potential in the cells treated with TNF-alpha and staurosporine revealed that dying cells showed caspase activation and mitochondrial depolarization, and that these events, however, were not firmly linked. Although it takes anywhere from 1 to over 10 h after the addition of the cell death inducer for the caspases to begin to be activated, initiator caspases and effector caspases are activated within a short period of time at the last stage in the entire process leading to cell death.     

Caspase-8-mediated intracellular acidification precedes mitochondrial dysfunction in somatostatin-induced apoptosis

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pH(i). (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pH(i) is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.     

Novel piperazine induces apoptosis in U937 cells

The effect of 1,4-bis-(4-(1H-benzo[d]imidazol-2-yl-phenyl)) piperazine (BIPP), a newly synthesized piperazine derivative, on U937 leukemia cell viability was investigated. We show that BIPP induces dose-responsive apoptotic cell death in U937 cells by intrinsic mechanisms of apoptosis. Maximum apoptotic effect of BIPP on U937 cells was observed at 12.8μM. BIPP-induced apoptosis was evident by characteristics such as altered annexin-V binding, caspase activation, loss of mitochondrial membrane potential (MMP) and cytochrome c release. BIPP also differentially activates initiator and effector caspases combined with the loss of MMP strongly suggesting that BIPP causes an intrinsic apoptosis in U937 leukemia cells. Due to our observations that BIPP induces leukemia cell death without significantly affecting normal cells, our data suggests that it may be a potential therapeutic agent for human myeloid leukemia.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.     

Quantitative analysis of pathways controlling extrinsic apoptosis in single cells

Apoptosis in response to TRAIL or TNF requires the activation of initiator caspases, which then activate the effector caspases that dismantle cells and cause death. However, little is known about the dynamics and regulatory logic linking initiators and effectors. Using a combination of live-cell reporters, flow cytometry, and immunoblotting, we find that initiator caspases are active during the long and variable delay that precedes mitochondrial outer membrane permeabilization (MOMP) and effector caspase activation. When combined with a mathematical model of core apoptosis pathways, experimental perturbation of regulatory links between initiator and effector caspases reveals that XIAP and proteasome-dependent degradation of effector caspases are important in restraining activity during the pre-MOMP delay. We identify conditions in which restraint is impaired, creating a physiologically indeterminate state of partial cell death with the potential to generate genomic instability. Together, these findings provide a quantitative picture of caspase regulatory networks and their failure modes.     

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.     

Apoptosis: checkpoint at the mitochondrial frontier.

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Caspase- and p53-dependent apoptosis in breast carcinoma cells induced by a synthetic selenadiazole derivative

Selenadiazole derivative is one kind of synthetic organoselenium compounds with potent and broad-spectrum antitumor activity. In this study, we showed that anthrax [1,2-c] [1,2,5] selenadiazolo-6,11-dione (ASDO), an novel selenadiazole derivative, induced time- and dose-dependent apoptotic cell death in MCF-7 human breast carcinoma cells, as indicated by accumulation of sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and PARP cleavage. ASDO-induced apoptosis was significantly inhibited by a general caspase inhibitor z-VAD-fmk, demonstrating the important role of caspases in ASDO-induced apoptotic pathway. Treatment of MCF-7 cells with ASDO resulted in a rapid depletion of mitochondrial membrane potential and release of cytochrome c and Smac/Diablo through up-regulation of Bax, Bad and PUMA expression and down-regulation of Bcl-xl expression. Moreover, ASDO treatment up-regulated the expression levels of total p53 and its target gene p21Waf1. Silencing of p53 activation with RNA interference effectively blocked the ASDO-induced cell PARP cleavage, DNA fragmentation and caspase activation. Furthermore, ASDO-induced apoptosis was interestingly found to be independent of reactive oxygen species production. Taken together, we conclude that ASDO induces MCF-7 cell apoptosis through a p53-dependent and mitochondria-mediated pathway.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.     

Hyperthermia engages the intrinsic apoptotic pathway by enhancing upstream caspase activation to overcome apoptotic resistance in MCF-7 breast adenocarcinoma cells

Febrile hyperthermia enhanced TNF-stimulated apoptosis of MCF-7 cells and overcame resistance in a TNF-resistant, MCF-7 variant (3E9), increasing their TNF-sensitivity by 10- and 100-fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF-7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase-8 activation, as the specific inhibitor, Z-IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase-8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF-dependent loss of mitochondrial membrane potential and activation of capspase-9 that was initiated and dependent on upstream caspases. MCF-7 and 3E9 cells were equally sensitive to exogenous C(6)-ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro-palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF-7 cells and overcomes TNF-resistance by sustained activation of caspase-8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase-dependent apoptosis by febrile hyperthermia.     

Apoptosis-like cell death of human breast cancer cell line MCF-7 induced by buprenorphine hydrochloride

The analgesic buprenorphine hydrochloride (Bph) induced apoptosis-like cell death in the caspase-3-deficient human breast cancer cell line, MCF-7. This apoptosis-like cell death activated key molecules in the mitochondrial apoptotic pathway: cytochrome c, caspase-9, caspase-7, and caspase-6. Bph caused the release of fluorescent protein from the mitochondria of MCF-7 cells transfected with the pDsRed2-Mito-vector in a time-dependent manner, suggesting disruption of the mitochondrial membrane. Zn(2+) as high as 2 mM did not inhibit the DNase that took part in this apoptosis. Thus, this unidentified DNase might resemble other DNases involved in apoptosis-like cell death whose activity is not inhibited by zinc ion.     

Apoptotic cell death induced by hydrogen peroxide in NT2 parental and mitochondrial DNA depleted cells

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.     

The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function.

ARC is an apoptotic regulatory protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it has been shown to block the activation of some initiator caspases. Because ARC also blocks caspase-independent events associated with apoptosis, such as hypoxia-induced cytochrome c release, we examined its role in cell death triggered by exposure to hydrogen peroxide (H(2)O(2)) in the myogenic cell line, H9c2. Cell death in this model was caspase-independent and characterized by dose-dependent reduction in ARC expression accompanied by disruption of the mitochondrial membrane potential (Delta psi(m)) and loss of plasma membrane integrity, typical of necrotic cell death. Ectopic expression of ARC prevented both H(2)O(2)-induced mitochondrial dysfunction and cell death without affecting the stress kinase response, suggesting that ARCs protective effects were downstream of early signaling events and not due to quenching of H(2)O(2). ARC was also effective in blocking H(2)O(2)-induced loss of membrane integrity and/or disruption of Delta psi(m) in two human cell lines in which it is not normally expressed. These results demonstrate that, in addition to its ability to block caspase-dependent and -independent events in apoptosis, ARC also prevents necrosis-like cell death via the preservation of mitochondrial function.     

Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers

Kinetic analysis of dexamethasone-induced apoptosis in the human lymphoblastoid cell line CCRF CEM C7A has revealed a point when cells, morphologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicity. Since all cells that fail to clone eventually died through apoptosis, measurements of clonogenicity in this system provide an accurate measure of commitment to apoptotic death. Inhibition of caspases by peptide inhibitors blocked proteolysis of endogenous substrates and reduced nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contrast to the results with caspase inhibitors, expression of BCL-2 in CCRF CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitochondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological features of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent execution phase characterised as apoptosis.