Apoptosis: target for novel drugs

Targeting apoptotic cell death pathways provides wide-ranging opportunities for the discovery and development of novel drugs. Some targeted therapies that selectively induce apoptosis in cancer cells are already marketed, and numerous pro-apoptotic drugs for treating cancer are currently being developed. The anti-apoptotic drugs that are most advanced in development are targeting acute disease indications such as stroke, myocardial infarction and sepsis, in which the role of apoptosis has been best defined and inhibitors of the apoptotic pathway have shown activity in various animal models. In the future, novel drugs might also result from an understanding of apoptotic pathways in chronic disorders.     

Apoptosis: live or die--hard work either way!

This review presents a brief overview of the cell's apoptotic machinery, including specific and indirect death signals. Specific death signals are transferred via death ligands, death receptors, and their intracellular signalling pathways. Indirect death signals cumulate a wide range of stimuli that potentially harm survival of cells. These include intercalating drugs, irradiation or altered intracellular signalling. Herein, a focal point is the mitochondrial control of specific death enzymes--so called caspases--by members of the pro-apoptotic Bax and BH3 subfamily or the anti-apoptotic Bcl-2 subfamily. While the initiation of cell death happens through a variety of signalling systems, the activation of caspases plays a pivotal role in the progression towards the final morphologic findings in cells undergoing apoptosis. Caspases appear to directly cleave and inactivate substrates that are clinical for the maintenance of cell structure and function but also regulate the activity of other enzymes that induce the apoptotic phenotype within the cell. The insulin-like growth factors (IGFs) are potent proliferation factors and potently inhibit apoptosis acting via the ubiquitously expressed IGF-I receptor. Within IGF-I receptor signalling, key to the inhibition of apoptosis are the RAS/RAF/mitogen-activated protein (MAP)-kinase pathway and the PI 3'-kinase pathway. To give an example of high clinical relevance of apoptosis within endocrine disorders, apoptotic death of pancreatic beta cells in type 1 diabetes disease and the involvement of IGF-II in beta cell survival and beta cell function is discussed in detail. Finally, further understanding of signalling systems that are involved in proliferation or in apoptosis might provide novel tools to treat or even heal disorders like type I diabetes.     

Regulation of apoptosis by protein S-nitrosylation

Summary. S-nitrosylation/denitrosylation of critical cysteine residues on proteins serves as a redox switch that regulates the function of a wide array of proteins. A key signaling pathway that is regulated by S-nitrosylation is apoptotic cell death. Here we will review the proteins in apoptotic pathways that are known to be S-nitrosylated by endogenous NO production. The targets and functional consequences of S-nitrosylation during apoptosis are multifaceted, allowing cells to fine tune their response to apoptotic signals.     

Mechanisms involved in tissue-specific apopotosis regulated by glucocorticoids

Physiological cell turnover is under the control of a sharp and dynamic balance of different homeostatic mechanisms such as the equilibrium between cell proliferation and cell death. These mechanisms play an important role in maintaining normal tissue function and architecture. It is well known that apoptosis is the prevalent mode of physiological cell loss in most tissues. Steroid hormones like glucocorticoids have been identified as key signals controlling cell turnover by modulating programmed cell death in a tissue- and cell-specific manner. In this sense, several reports have demonstrated that glucocorticoids are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes. In contrast, they protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, in glandular cells such as the mammary gland epithelia, endometrium, hepatocytes, ovarian follicular cells, and fibroblasts. Although several studies have provided significant information on hormone-dependent apoptosis in an specific tissue, a clearly defined pathway that mediates cell death in response to glucocorticoids in different cell types is still misunderstood. The scope of this review is held to those mechanisms by which glucocorticoids control apoptosis, emphasizing tissue-specific expression of genes that are involved in the apoptotic pathway.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

The Bcl-2 family of proteins as drug targets

Programmed cell death or apoptosis is a crucial process for normal embryonic development and homeostasis. Apoptosis is known to be coupled to multiple signalling pathways. Identification of critical points in the regulation of apoptosis is of major interest both for the understanding of control of cell fate and for the discovery of new pharmacological targets, particularly in oncology. Indeed, defects in the execution of apoptosis are known to participate in tumour initiation and progression as well as in chemoresistance. The Bcl-2 family members constitute essential intracellular players in the apoptotic machinery. Those proteins are either pro or anti-apoptotic, they interact with each other to regulate apoptosis. Inhibiting the heterodimerisation between pro- and anti-apoptotic members is sufficient to promote apoptosis in mammalian cells. Small molecules, antagonists or peptidomimetics inhibiting this heterodimerisation, represent a therapeutic prototype targeting the apoptotic cascade. They induce cell death by activating directly the mitochondrial apoptotic pathway. Considerable evidence indicate that such Bcl-2 antagonists could be useful drugs to induce apoptosis preferentially in neoplastic cells.     

Osmotic stress resistance imparts acquired anti-apoptotic mechanisms in lymphocytes

Apoptosis is a stochastic, physiological form of cell death that is characterized by unique morphological and biochemical properties. A defining feature of apoptosis in all cells is the apoptotic volume decrease or AVD, which has been considered a passive component of the cell death process. Most cells have inherent volume regulatory increase (RVI) mechanisms to contest an imposed loss in cell size, however T-cells are unique in that they do not have a RVI response. We utilized this property to explore potential regulatory roles of a RVI response in apoptosis. Exposure of immature T-cells to hyperosmotic stress resulted in a rapid, synchronous, and caspase-dependent apoptosis. Multiple rounds of osmotic stress followed by recovery of cells in normal media resulted in the development of a population of cells that were resistant to osmotic stress induced apoptosis. These cells were also resistant to other apoptotic stimuli that activate via the intrinsic cell death pathway, while remaining sensitive to extrinsic apoptotic stimuli. Interestingly, these osmotic stress resistant cells showed no increase in anti-apoptotic proteins, and released cytochrome c from their mitochondria following exposure to intrinsic apoptotic stimuli. The osmotic stress resistant cells developed a RVI response, and inhibition of the RVI restored sensitivity to apoptotic agents. Analysis of apoptotic signaling pathways showed a sustained increase in phospho-AKT, whose inhibition also prevented an RVI response resulting in apoptosis. These results define a critical role of volume regulation mechanisms in apoptotic resistance.     

PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.     

Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψm) evaluated by three-parameter flow cytometric analysis

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ_m) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ_m did not always externalize PS, while some late apoptotic cells had polarized Δψ_m. The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS–PI and Δψ_m assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.     

A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes.     

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip_L expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.     

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.     

Inhibition of Drug-Induced Apoptosis by Survival Factors in PC12 Cells

Abstract: Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2 . We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum-deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl-2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl-2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.     

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.     

Cell nucleus and DNA fragmentation are not required for apoptosis

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.     

Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species

Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c-Jun N-terminal kinases and p38-MAP kinase, which are responsible for inducing apoptotic cell death. This pathway plays a pivotal role in transduction of signals from different apoptotic stimuli. In the present review, we summarized the recent evidence concerning MAP kinase-dependent apoptotic pathway and its regulation in the mammalian cells and organism in vivo. We have shown that the key messengers of regulation of this pathway are the reactive oxygen and nitrogen species. The role of protein oxidation and S-nitrosation in induction of apoptotic cell death via ASK1 is discussed. Also we have outlined other recently discovered signal transduction processes involved in the regulation of ASK1 activity and downstream pathway.     

Oxidative stress induces caspase-independent retinal apoptosis in vitro

Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase-independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity.     

25-Hydroxycholesterol activates a cytochrome c release-mediated caspase cascade

We have previously shown that 25-hydroxycholesterol (25-OHC) treated CHO-K1 cells could be used as a model to investigate the signaling pathway of apoptosis induced by oxidized LDL in vascular cells. In the present study, we examine the execution phase of the apoptotic pathway in CHO-K1 cell death induced by 25-OHC. Oxysterol-induced apoptosis in CHO-K1 was accompanied by caspase activation and was preceded by mitochondrial cytochrome c release. The addition of a competitive caspase-3 inhibitor, Ac-DEVD-CHO, prevented 25-OHC-induced apoptotic cell death. Furthermore, immunoblot analysis showed that 25-OHC treatment induced the degradation of poly(ADP-ribose) polymerase (PARP)-a substrate for caspase 3 and a key enzyme involved in genome surveillance and DNA repair. Thus, we could demonstrate in CHO-K1 cells that 25-OHC activates the apoptotic machinery through induction of the release of cytochrome c from mitochodria into the cytosol and activation of a typical caspase cascade.