Mitochondrial Apoptosis-induced Channel Research Articles

Inducing breast cancer cell death: The impact of taxodone on proliferation through apoptosis

Breast cancer is the most prevalent form of cancer in women and a major contributor to cancer-related fatalities worldwide. Several factors play a role in the development of breast cancer, encompassing age, hormone levels, etc. Taxodone has shown significant anti-tumor properties in both laboratory experiments and living organisms. However, its impact on the human MCF-7 breast cancer cell line has not been researched. This investigation explores the chemo-preventive potential of taxodone in the MCF-7 breast cancer cells. The anticancer potential of taxodone against MCF-7 cells was determined by MTT assay. Further, the induction of apoptosis in MCF-7 breast cancer cells was confirmed via ELISA, which indicated the increased incidences of chromatin condensation and ssDNA breakage in the MCF-7 apoptotic cells upon 24 h of taxodone treatment. The intracellular reactive oxygen species (ROS) level was evaluated using H2DCFDA fluorescent dye to elucidate the mechanism of action triggered upon taxodone treatment. The increasing intercellular ROS level sequentially activated the caspase-mediated apoptosis pathway. Consequently, the outcomes revealed that taxodone decreased the cell viability of MCF-7 dose-dependently. Taxodone triggers apoptosis in MCF-7 cells by increasing intracellular ROS levels and activating the caspase cascade through the mitochondrial apoptosis-induced channel, an early marker of apoptosis onset. Our results indicate that taxodone exhibits anti-proliferative and apoptotic properties against human MCF-7 breast cancer cells, suggesting it to be a natural anticancer agent.

Hypoosmosis alters hepatocyte mitochondrial morphology and induces selective release of carbamoyl phosphate synthetase 1.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Optogenetic apoptosis: light-triggered cell death.

An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase-3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Identification of Inhibitors Against BAK Pore Formation using an Improved in vitro Assay System

The pro-apoptotic BCL-2 family protein BID activates BAK and/or BAX, which form oligomeric pores in the mitochondrial outer membrane. This results in the release of cytochrome c into the cytoplasm, initiating the apoptotic cascade. Here, we utilized liposomes encapsulating sulfo-rhodamine at a controlled temperature to improve upon a previously reported assay system with enhanced sensitivity and specificity for measuring membrane permeabilization by BID-dependent BAK activation. BAK activation was inhibited by BCL-XL protein but not by a mutant protein with impaired anti-apoptotic activity. With the assay system, we screened a chemical library and identified several compounds including trifluoperazine, a mitochondrial apoptosisinduced channel blocker. It inhibited BAK activation by direct binding to BAK and blocking the oligomerization of BAK.

MAC Function Triggers a Bax/Bak Dependent Bystander Effect

Collateral spread of apoptosis to nearby cells is referred to as the bystander effect - a process that is integral to tissue homeostasis and a challenge in anticancer therapies. In many systems, apoptosis relies on permeabilization of the mitochondrial outer membrane to factors like cytochrome c and Smac/DIABLO. This permeabilization occurs via formation of a mitochondrial apoptosis-induced channel, MAC, and was mimicked here by single-cell microinjection of cytochrome c in Xenopus embryos. Waves of apoptosis were observed in vivo from the injected to the neighboring cells. This finding indicates that a death signal generated downstream of cytochrome c release diffused to neighboring cells and ultimately killed the animals. The role of MAC in bystander effects was then assessed in mouse embryonic fibroblasts that did or did not express its main components Bax and/or Bak. Exogenous expression of GFP-Bax triggered permeabilization of the outer membrane and apoptosis in these cells. Time-lapse videos showed neighboring cells also underwent apoptosis, but expression of Bax and/or Bak was essential to this effect as no bystanders were observed in cells lacking both of these MAC components. In osteosarcoma cell lines, this effect relied upon gap junction intercellular communication, as bystander cell death was abrogated either by pharmacological or molecular inhibition of connexin 43. In contrast, an extracellular pathway seemed to underlie bystander effects in breast cancer cell lines. These results may impact development of novel therapeutic strategies to selectively eliminate tumors or minimize the size of tissue injury in degenerative or traumatic cell death.

Mitochondrial Apoptosis-Induced Channel (MAC) Function Triggers a Bax/Bak-Dependent Bystander Effect

Collateral spread of apoptosis to nearby cells is referred to as the bystander effect, a process that is integral to tissue homeostasis and a challenge to anticancer therapies. In many systems, apoptosis relies on permeabilization of the mitochondrial outer membrane to factors such as cytochrome c and Smac/DIABLO. This permeabilization occurs via formation of a mitochondrial apoptosis-induced channel (MAC) and was mimicked here by single-cell microinjection of cytochrome c into Xenopus laevis embryos. Waves of apoptosis were observed in vivo from the injected to the neighboring cells. This finding indicates that a death signal generated downstream of cytochrome c release diffused to neighboring cells and ultimately killed the animals. The role of MAC in bystander effects was then assessed in mouse embryonic fibroblasts that did or did not express its main components, Bax and/or Bak. Exogenous expression of green fluorescent protein-Bax triggered permeabilization of the outer membrane and apoptosis in these cells. Time-lapse videos showed that neighboring cells also underwent apoptosis, but expression of Bax and/or Bak was essential to this effect, because no bystanders were observed in cells lacking both of these MAC components. These results may guide development of novel therapeutic strategies to selectively eliminate tumors or minimize the size of tissue injury in degenerative or traumatic cell death.

Role of mitochondrial ion channels in cell death

Ion channels located in the outer and inner mitochondrial membranes are key regulators of cellular signaling for life and death. Permeabilization of mitochondrial membranes is one of the most critical steps in the progression of several cell death pathways. The mitochondrial apoptosis-induced channel (MAC) and the mitochondrial permeability transition pore (mPTP) play major roles in these processes. Here, the most recent progress and current perspectives about the roles of MAC and mPTP in mitochondrial membrane permeabilization during cell death are presented. The crosstalk signaling of MAC and mPTP formation/activation mediated by cytosolic Ca(2+) signaling, Bcl-2 family proteins, and other mitochondrial ion channels is also discussed. Understanding the mechanisms that regulate opening and closing of MAC and mPTP has revealed new therapeutic targets that potentially could control cell death in pathologies such as cancer, ischemia/reperfusion injuries, and neurodegenerative diseases.

The mitochondrial apoptosis-induced channel, MAC, and Bcl-2 family proteins are co-conspirators in a deadly plot

The mitochondrial apoptosis-induced channel, MAC, and Bcl-2 family proteins are co-conspirators in a deadly plot

MAC inhibitors suppress mitochondrial apoptosis

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis. Compounds that blocked the release of fluorescein from liposomes by recombinant Bax were tested for their ability to directly close MAC and suppress apoptosis in FL5.12 cells. Low doses of these compounds (IC50 values ranged from 19 to 966 nM) irreversibly closed MAC. These compounds also blocked cytochrome c release and halted the onset of apoptotic markers normally induced by IL-3 (interleukin-3) deprivation or staurosporine. Our results reveal the tight link among MAC activity, cytochrome c release and apoptotic death, and indicate this mitochondrial channel is a promising therapeutic target.

Mitochondrial apoptosis is amplified through gap junctions

The death of one cell can precipitate the death of nearby cells in a process referred to as the bystander effect. We investigated whether mitochondrial apoptosis generated a bystander effect and, if so, by which pathway. Microinjection with cytochrome c mimicked function of the mitochondrial apoptosis-induced channel MAC and caused apoptosis of both target and nearby osteoblasts. This effect was suppressed by inhibiting gap junction intercellular communication. A bystander effect was also observed after exogenous expression of tBid, which facilitates MAC formation and cytochrome c release. Interestingly, in connexin-43 deficient osteoblasts, microinjection of cytochrome c induced apoptosis only in the target cell. These findings indicate that a death signal was generated downstream of MAC function and was transmitted through gap junctions to amplify apoptosis in neighboring cells. This concept may have implications in development of new therapeutic approaches.

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane alpha-helices in Bax and Bak, mature MAC pores would typically contain approximately 9 monomers and have diameters of 5.5-6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.

Apoptosis in FL5.12 cells is suppressed by inhibitors of the Mitochondrial Apoptosis-induced Channel MAC

The Mitochondrial Apoptosis-induced Channel (MAC) forms early in apoptosis and orchestrates cell death by releasing cytochrome c from the intermembrane space of mitochondria. MAC is a potential therapeutic target, as modulation of its opening could induce or prevent cell death. Compounds previously found to block Bax-induced release of fluorescein from liposomes were tested for their ability to directly inhibit the channel activity of MAC. Patch clamp techniques were applied to proteoliposomes containing mitochondrial outer membranes of apoptotic FL5.12 cells to monitor MAC activity. Several antagonists irreversibly blocked MAC with the IC50's ranging from 25 to 900 nM. These Inhibitors of MAC, or iMACs, were also effective in preventing cytochrome c release and progression of apoptosis induced by IL3 deprivation or staurosporine treatment. A matrix-targeted GFP facilitated visualization of the collapse of the mitochondrial network during staurosporine-induced apoptosis; this collapse was also prevented by iMACs. The action of these inhibitors demonstrates the tight links between MAC activity, cytochrome c release and apoptosis. Future studies will evaluate the impact of MAC on mitochondrial dynamics.

Design Of An ELISA Protocol For A Rapid Quantification Of Activated/oligomerized Bax During Apoptosis

The release of cytochrome c from mitochondria into the cytosol is considered to be the commitment step of early apoptosis. During this step, the activation/oligomerization of the pro-apoptotic protein Bax leads to the formation of the Mitochondrial Apoptosis-Induced Channel (MAC) through which cytochrome can be released. Remarkably, Bax activation/oligomerization leads to the exposure of the previously inaccessible N-terminal domain of the pro-apoptotic protein Bax. This property was extensively used to detect activated/oligomerized Bax by immunocytochemistry and immunoprecipitation but these assays are long and tedious; and they are only qualitative or semi-quantitative at best.During this study, we developed an ELISA protocol based on the utilization of monoclonal (6A7) and polyclonal (N-20) antibodies which specifically recognize Bax N-terminus (i.e. the activated/oligomerized form of Bax). Monomeric human recombinant Bax (hBax) was used in all the experiments. As previously described, activation/oligomerization of hBax was artificially triggered by an exposure of the recombinant protein to the detergent Triton X-100. We first confirmed that the N-20 and 6A7 antibodies were able to fully immunoprecipitate the activated/oligomerized form but not the monomeric form of hBax. We then develop a sandwich ELISA using the 6A7 and the N-20 respectively as capture and detection antibodies. Reproducible standard curves were obtained when using increasing amounts of activated/oligomerized hBax. Monomeric hBax was never detected during these assays, even when previously mixed with activated/oligomerized hBax. Taken together, these results show that this ELISA allows a specific and quantitative detection of activated/oligomerized recombinant Bax. Finally, experiments of quantification of activated/oligomerized Bax with this technique in protein extracts from control and apoptotic cells are currently underway.Supported by NYU College of Dentistry Research Faculty Funds to LD.

Bcl-2 Does Not Inhibit Bax Insertion During Intrinsic Apoptosis

Mitochondrial outer membrane (MOM) permeabilization and cytochrome c release from mitochondria into the cytosol are considered to be the commitment steps of the intrinsic apoptotic pathway. Cytochrome c release is regulated by the Bcl-2 family proteins, which contains both pro-apoptotic (e.g. BAX) and anti-apoptotic (e.g. Bcl-2) members. It is now well established that after a death signal, cytosolic BAX is translocated to the mitochondrial outer membrane, inserted then in the double leaflet and activated through a conformational change. Activated BAX oligomerizes and might be associated to other mitochondrial proteins, leading to the formation of the Mitochondrial Apoptosis-induced Channel (MAC) into the MOM. This channel allows the release of cytochrome c into the cytosol. Bcl-2 inhibits MAC formation and therefore, cytochrome c release. However, the molecular mechanisms through which Bcl-2 affects earlier steps of BAX-mediated apoptosis are not fully understood.In this study we investigated the effects of Bcl-2 over-expression on BAX-mediated apoptosis. We were able to confirm that Bcl-2 over-expression inhibits BAX translocation to the MOM and activation/oligomerization, as previously reported. Bax translocation is generally considered as the primary target of Bcl-2. Surprisingly, Bcl-2 over-expression did not alter the insertion status of BAX into the MOM. These data point out the further step, BAX activation/oligomerization, as the primary target of Bcl-2. Since Bcl-2 does not inhibit BAX insertion, we hypothesize that an event occurring after cytochrome c release triggers somehow a positive feedback on Bax expression and translocation. Thus, the inhibition of cytochrome c release by Bcl-2 could explain the further blocking on BAX translocation.Supported by NYU Research Challenge Funds to LD.

Construction and analysis of a modular model of caspase activation in apoptosis

BackgroundA key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins.ResultsA model of caspase activation is constructed and analyzed. The apoptosis signaling network is simplified through modularization methodologies and equilibrium abstractions for three functional modules. The mathematical model is composed of a system of ordinary differential equations which is numerically solved. Multiple linear regression analysis investigates the role of each module and reduced models are constructed to identify key contributions of the extrinsic and intrinsic pathways in triggering apoptosis for different cell lines.ConclusionThrough linear regression techniques, we identified the feedbacks, dissociation of complexes, and negative regulators as the key components in apoptosis. The analysis and reduced models for our model formulation reveal that the chosen cell lines predominately exhibit strong extrinsic caspase, typical of type I cell, behavior. Furthermore, under the simplified model framework, the selected cells lines exhibit different modes by which caspase activation may occur. Finally the proposed modularized model of apoptosis may generalize behavior for additional cells and tissues, specifically identifying and predicting components responsible for the transition from type I to type II cell behavior.

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

A tale of two mitochondrial channels, MAC and PTP, in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.

Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins

Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis-induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl-2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl-2 family proteins and pharmacological agents. Protocols include the use of time-lapse video-microscopy to monitor the onset of apoptosis in living cells and patch-clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.

Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins

Programmed cell death or apoptosis is central to many physiological processes and pathological conditions such as organogenesis, tissue homeostasis, cancer, and neurodegenerative diseases. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other pro-apoptotic factors. Control of the formation of the mitochondrial apoptosis-induced channel, or MAC, is central to the regulation of apoptosis by Bcl-2 family proteins. MAC is detected early in apoptosis by patch clamping the mitochondrial outer membrane. The focus of this review is on the regulation of MAC activity by Bcl-2 family proteins. The role of MAC as the putative cytochrome c release channel during early apoptosis and insights concerning its molecular composition are also discussed.