Introduction

Apoptosis is an ancient Greek word which means “the falling of leaves from a tree”. John Kerr first introduced the name which refers to the morphological feature of formation of “apoptotic bodies” from a cell [1]. Apoptosis, also known as programmed cell death (PCD), plays an integral role in a variety of biological events including morphogenesis, tissue homeostasis, aging, and removal of unwanted harmful cells [2]. Changes typical for apoptosis include condensation of the nuclei, DNA fragmentation, chromatin condensation, generation of envoluted membrane segments, cellular shrinkage, and disintegration of mitochondria [3, 4]. Apoptosis can be induced by a variety of physiological and pathophysiological stimuli, such as specific receptor molecules-CD95 [5, 6], tumor necrosis factor (TNFα) [7], growth factors [8], ultraviolet light [9, 10], irradiation [11], heat shock [12], cytotoxic drugs [13, 14], oxidative stress [15], ceramide treatment [16], and bacteria [17]. Dysfunction of apoptotic pathways result in various pathological conditions including cancer, autoimmune, and neurodegenerative diseases [18–21].

A significant progress has been made toward understanding the mechanism of apoptosis and other apoptosis related dysfunction, but it needs more intensive research to better understand the whole cascade of events involved in apoptosis and also the mechanism of their regulatory pathways [22–24]. The nematode, Caenorhabditis elegans (C. elegans), has been widely used to elucidate the molecular pathways implicated in the regulation of PCD [25–27]. For elucidating the underlying genetics and biochemical pathways in PCD, three scientists, namely, S. Brenner, J. Sulston, and R. Horvitz were awarded the Noble Prize in medicine (2002).

The Bcl-2 and apoptosis

The underlying mechanism and genetics of apoptosis began a new approach of research after the landmark discovery by Horvitz, 1999 [28]. The protein expression of two C. elegans cell death genes (CED-3 and CED-4) is necessary for PCD during development, and CED-9 is the functional homolog to mammalian protein Bcl-2, which is shown to correct the phenotype of C. elegans by its anti-apoptotic function [29, 30]. CED-9 was found to act upstream of aspartate-directed cysteine protease (CED-3 and CED-4) known as caspases [31]. Previously, it was revealed that proteins that are encoded by the mutant genes discovered in C. elegans shared homology with mammalian proteins, including Bcl-2 [32].

The role of Bcl-2 (B cell lymphoma 2), a founder member of the Bcl-2 family of apoptosis regulator proteins, has been elucidated in tumor development by dysfunction in apoptotic pathways [20, 30]. In mammals, there is an extrinsic apoptotic pathway that involves death receptors and an intrinsic apoptotic pathway that involves the mitochondria. Mammalian mitochondria control apoptosis by releasing many apoptogenic mediators from intermembrane space [33] and that further leads to a cascade of reaction with the assistance of caspases, proteases that cleave key cellular proteins [34, 35]. A major difference between apoptosis in worm and mammals is the greater role of mitochondria in the mammals but not in the worm. Bcl-2 expression specifically blocks the morphologic features of apoptosis, including the plasma membrane blebbing, DNA cleavage, and nuclear condensation. Bcl-2 plays a role in cell survival and also inhibits cell death induced by various stimuli such as chemotherapeutic agents, ethanol and heat shock, indicating Bcl-2 as a negative regulator of cell death. The anti-death role is also demonstrated in vivo by generation of mice lacking the bcl-2 gene, which shows a variety of abnormalities including excessive cell death.

Members and structural classification of Bcl-2 proteins

On the basis of various structural and functional characteristics, the Bcl-2 family is divided into anti-apoptotic and pro-apoptotic proteins [36–38]. All of the members of Bcl-2 family have one or more homology domains labeled as Bcl-2 homology (BH1, -2, -3, and -4) which are important for heterodimeric interaction among members of the Bcl-2 family [27] (Fig. 1). Anti-apoptotic Bcl-2 family multidomain proteins include Bcl-2, Bcl-xL (Bcl-extra long), A1, Bcl-w, and Boo (Bcl-2 homolog of ovary) contain BH-(1-4) domains. The Bcl-xL and Bcl-2 have a carboxy-terminal hydrophobic transmembrane tail domain which helps in localization of protein in outer mitochondrial membrane (OMM) with the exception that Bcl-2 also resides in the nuclear and endoplasmic reticulum membrane and is translocated to OMM upon an apoptotic signal. Myeloid cell leukemia factor-1 (Mcl-1) is the only antiapoptotic Bcl-2 protein with three BH domains (BH-1, -2, and -3).

Fig. 1
figure 1

According to conserve domain (BH) and its anti or pro-apoptotic action, the Bcl-2 super-family can be divided into three sub-families: Anti-apoptotic or Pro-survival multi-domain, pro-apoptotic multi-domain and pro-apoptotic BH3 only. Few examples of protein from each group of the family are given in parenthesis. TM (transmembrane domain)

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Pro-apoptotic Bcl-2 family of proteins are divided into two subgroups according to the number of BH domains (e.g., Bax, Bak, and Box) or those proteins that have only the BH3 domain (e.g., Bid, Bim, and Bad) with the exception of Bcl-xS which has only BH3 and BH4 domains (Fig. 1). There are eight members of BH3 only protein, which include hara-kiri (Hrk), BH3 interacting domain death agonist (Bid), Bcl-2 interacting mediator of cell death (Bim), Bcl-2 modifying factor (Bmf), p53, promoter-upregulated modulator of apoptosis (Puma), Noxa (named for “damage”), Bcl-2 antagonist of cell death (Bad), and Bcl-2 interacting killer (Bik). Bid, Bad, and Bim have cytosolic location with respect to mitochondria. Following death signal, BH3 domain-only proteins also known as minimal death domain can neutralize or depress antiapoptotic Bcl-2 proteins allowing pro-apoptotic proteins such as Bax/Bak-like proteins to induce apoptosis [39, 40].

A restricted subset of these BH3 proteins undergoes post-translation modification such as cleavage, phosphorylation, transcriptional upregulation, and dissociation. Such modifications result in their activation and translocation to outer mitochondrial membrane (OMM) which helps it to interact with multidomain pro-apototic members, Bax/Bak-like proteins, leading to their oligomerization and formation of pore. Such pore may further lead to release of other pro-apoptotic protein on the intermembrane space (IMS) that enhances the processes of apoptosis [41]. Thus the regulation of expression and function of BH3 domain-only proteins is critical in mediating cell death and understanding their expression and function necessary for cytoprotective strategies. In general, the relative ratio of pro-survival (Bcl-2) and pro-apoptotic (Bax and BH3-only) proteins seems to determine the cell sensitivity or resistance to the apoptotic stimuli.

Mechanism of action and role of Bcl-2 family members in the regulation of mitochondrial apoptosis

Antiapoptotic or pro-survival proteins

It is now becoming evident that almost every nucleated cell requires protection by at least one of the member Bcl-2 homolog, and that regulates homeostasis in mammalian cell and tissues. Over-expression of Bcl-2 protein in hematopoietic lineages yields excess T, B, and myeloid cells and enhance the survival of these progenitor cells that are hard to avoid cytotoxic insults [29, 42]. Alternatively, inactivation of the Bcl-2 gene increases apoptosis in specific cell types, presumably because the concentrations of other Bcl-2 homolog are too low to compensate. Bcl-2 itself is required for the survival of kidney, mature lymphocytes and melanocyte stem cells [43], Bcl-w for sperm progenitors in adult mice [44], an A1 gene for neutrophils [45], Mcl-1 for zygote implantation [46], and Bcl-xL for erythroid and neuronal cells [47]. The hydrophobic carboxy-terminal domain of these antiapoptotic proteins help to anchor themselves to the cytoplasmic face of three intracellular membranes: the outer mitochondrial membrane, the nuclear envelope, and the endoplasmic reticulum (ER). Bcl-2 is an integral membrane protein, whereas Bcl-xL and Bcl-w only become tightly associated with the membrane after cytotoxic signals which induce conformational changes to protect cell. The antiapoptotic member of Bcl-2 family, including Bcl-2 itself, decreases apoptosis mainly by preventing mitochondrial outer membrane permeabilization (MOMP) either by neutralizing the activity of both pro-apoptotic members (BH3 only protein and multidomain members) [48]. If Bcl-2 or Bcl-xl cannot exert their antiapoptotic property, Bax oligomerization takes place and apoptosis is promoted.

Pro-apoptotic or anti-survival protein

It includes both multi-domain and BH3-only protein members.

Multi-domain pro-apoptotic member

Antiapoptotic members such as Bcl-2 interact with Bak and Bax and inhibit their oligomerization and/or bind to BH3 only proteins to block apoptosis [48]. Bak interacts with two mitochondrial fusion proteins (Mfn1 and Mfn2). After receiving apoptotic signal, Bak dissociates from Mfn2 to become associated with Mfn1. It has been shown that reconstitution of Bak into Bax/Bak double knockout cells restores mitochondrial fragmentation and Bak in association with Bax permeabilize the outer membrane of mitochondria rendering apoptotic cascade [49]. Bak may contribute to early mitochondrial fragmentation while Bax is probably more important for subsequent pores development and degeneration in the outer membrane [49]. A number of studies have shown that insertion of activated oligomerized Bax and/or Bak into the OMM exerts stress on the membrane, leading to supramolecular pores that include lipids (lipidic pores) in the OMM [50–52].

In healthy cells, Bak is inactive in the OMM through its association with Voltage-dependent Anion channel (VDAC-2) [48], whereas Bax is dormant in the cytosol through interactions with several proteins, including Ku-70, 14-3-3, and the humanin peptide. Many apoptotic signals can trigger BH3-only-protein-dependent translocation of Bax, followed by its insertion into the OMM and the formation of Bak or Bax homo-oligomers. Several models have been proposed for how Bcl-2 anti-apoptotic proteins antagonize the functions of Bax, Bak, and BH3-only proteins. Activation of Bax and Bak leads to a conformational change that exposes the N-terminus of the proteins, which is otherwise hidden in the inactive state [53]. After Bak and Bax form homo-oligomers, they participate in forming pores in OMM and cause permeabilization leading to the release of the contents of the mitochondrial intermembrane space (IMS) including Smac and cytochrome c into the cytosol [54, 55]. The released mitochondrial protein then activates caspases, which causes a series of cascade reactions that cleave essential proteins complement throughout the cell.

Two models of Bax and Bak activation, exist the direct and indirect models. According to the direct model various BH3 pro-apoptotic proteins called as activator proteins (Bid, Bim, Puma, and p53) directly interact and induce conformational changes in Bak and Bax [56]. Members of anti-apoptotic proteins bind and sequester such activator, and also bind to activated Bak and Bax proteins that might be present and thus prevent apoptosis. BH3-only proteins are further divided into activator and sensitizer categories [57]. Sensitizers bind to anti-apoptotic proteins and cause the release of activator BH3-only proteins leading to activation of Bak and Bax. However, it is likely that additional factors other than Bid and Bim can act as activators such as Puma, p53, and heat as activators of Bax and Bak [58–60], and possibly others. Gavathiotis and colleagues have demonstrated that stabilized alpha-helix of Bcl-2 domains (SAHBs) directly initiates Bax-mediated mitochondrial apoptosis [61].

According to the indirect model, anti-apoptotic proteins always bind to Bak and Bax and prevent their activation, whereas in response to death signal, BH3-only proteins bind to antiapoptotic proteins causing its release and initiating death signal through activation of Bax and Bak [62]. The sequestered forms of Bak and Bax are those fractions of the total Bak and Bax population that are already activated either spontaneously, or by other unspecified means [63]. Bax and Bak can also alternatively interact with proteins that remove the requirement for sequestration by anti-apoptotic proteins [64]. Activated population of Bak and Bax are needed to kill, and that should be sequestered by anti-apoptotic proteins to maintain survival of the cell. Activated Bak and Bax are responsible for the permeabilization of membranes. The activation is achieved, either by interacting with activator proteins or through some agents, while the anti-apoptotic proteins inhibit death by sequestering activated Bak and Bax or activator proteins.

BH3-only pro-apoptotic members

BH3-only pro-apoptotic proteins are involved in maintaining the functionality and cellular integrity of the cell in which Bim acts as sensor for cytoskeleton integrity, Bid acts as sensor for death domain receptor signaling, and Bad acts as an inhibitor for growth factors withdrawal. Individual BH3-only proteins are normally controlled by diverse mechanisms.

Cleavage of Bid

Activation of cell surface receptors such as tumor necrosis factor receptor (TNF-R) activates caspase-8, which leads to the cleavage of Bid, and then truncated Bid (active Bid or tBid) translocates from cytosol to OMM and induces cytochrome c release [65–67]. Oh and his group have demonstrated that at nanomolar concentrations of a synthetic Bid activates Bax almost as efficiently as tBid itself thus tBid engages Bax to trigger its pro-apoptotic activity [68]. It is now well established that tBid leads to oligomerization of Bax and Bak, which forms pores in OMM which in turn permits the release of IMS proteins into the cytosol [54, 69]. During apoptosis, Bid induces the mobilization of cyt c by remodeling mitochondrial cristae by interacting Bid with cardiolipin [70, 71]. Addition of tBid to permeabilized cells leads to hydrolysis of cardiolipin molecules thereby decreasing the cardiolipin levels [72, 73]. But tBid alone does not cause mitochondrial outer membrane permeabilization [74].

Phosphorylation of Bad

In the absence of various survival factors, Bad is activated by dephosphorylation [75]. The BH3 domain of Bad binds and inactivates Bcl-2 and/or Bcl-xL at the OMM, thereby promoting cell death. Conversely, in the presence of survival factors, Bad is phosphorylated, making it to dissociate from Bcl-2 and/or Bcl-xl, permitting, and survival promotion. There are several phosphatases that dephosphorylate Bad in vitro [75, 76]. However, a report by Datta and his group showed that Akt phosphorylates Bad, both in vitro and in vivo, and blocks the Bad-induced death of primary neurons [77]. Furthermore, several studies elucidate the importance of Bad phosphorylation and underlying mechanism of cell death [78–80].

Dissociation of Bim and Bmf

In response to several apoptotic stimuli, such as detachment of adherent cells from their substratum (anoikis) or ultraviolet irradiation, Bmf is released from the myosin V motor complex, translocates and binds to antiapoptotic family proteins, such as Bcl-2, Bcl-xl, and Bcl-w, but does not interact with the pro-apoptotic family protein, such as Bax, Bid, and Bad [81]. However, c-Jun N-terminal protein kinase (JNK) that causes phosphorylation of Bim is involved in ischemia-induced neuronal apoptosis through activation and translocation of Bax [82, 83]. Bim is the main regulator of hematopoietic homeostasis [84], essential for the elimination of autoreactive lymphocytes [85] and plays vital role in neuronal death [86]. Recently, it has been shown that Bim displaces Bcl-xl in the mitochondria and promotes Bax translocation during intrinsic pathways assisted by TNFα [87] or UV-induced [88] apoptosis. Histone deacetylase (HDAC) inhibitors increase ionizing radiation-induced apoptosis in several cancer cells via activation of Bmf transcription [89, 90]. In TGF beta-induced cell death, there is upregulation of Bmf and Bim, and thus, inhibition of the TGFβ provides an important therapeutic and protection of cells from apoptosis [91].

Transcriptional regulation of BH3-only proteins

BH3-only proteins are also transcriptionally regulated as in DNA damage-induced apoptosis which requires synthesis of new protein. Transcription of Noxa and Puma, the BH3-only group members, is induced by p53 [92–94]. Apoptosis in fibroblast in response to DNA damage is decreased in mice knocked out in Noxa/puma whereas mutations of the BH3 domain suppress the pro-apoptotic activity of Noxa [94], and Noxa-induced apoptosis is inhibited by Bcl-xl and Bcl-2 [95]. Recently, it has been reported that Puma expressed independent of p53 regulation [96, 97] and initiates apoptosis by dissociating Bcl-xl and Bax, promoting Bax multimerization and mitochondrial translocation [9, 96]. Puma rapidly induces apoptosis in cells lacking not only the BH3-only proteins but in the absence of Bid and Bim [98]. Puma induced apoptosis is associated by regeneration of superoxide and H2O2 which is Bax-dependent and that can be confirmed by the presence of antioxidants that prevent Puma-dependent apoptosis [99]. Whereas Bax inactivation confers a resistance to Puma-dependent apoptosis [100].

Apoptotic pathways

Apoptosis or PCD has conserved genetic and biochemical pathways [27, 101]. In vertebrates, caspase-dependent apoptosis occurs through two main interconnected pathways which are intrinsic and extrinsic pathways [102]. The intrinsic pathway or intracellular path is mediated by Bcl-2 family, whereas the death receptor or extrinsic pathway is activated by signal from other cells [7, 103, 104].

Intrinsic pathway

The intrinsic pathways also known as mitochondrial pathways or stress pathways are activated by a diverse array of death stress, genomic stress, metabolic stress, presence of unfolded proteins, and other stimuli that lead to permeabilization of OMM and release of apoptotic proteins into the cytosol (Fig. 2). Several of these proteins including cytochrome c (cyt c) initiate or regulate caspase activation. The cyt c plays the main role in this pathway which is activated after its interaction with apoptotic protease-activating factor (Apaf1) and deoxyadenosine triphosphate (dATP) to form apoptosome [102, 105]. The apoptosome creates a platform to bring together molecules of the initiator caspase of the intrinsic pathway. Progression through the pathway usually leads to activation of caspase-9, enabling their auto-activation. Caspase-8 also has a major role to play by activating the Bid leading to the formation of activated truncated Bid (tBid), which translocates to mitochondria and releases cyt c. The activated caspase-8 then cleaves procaspase-3, giving activated caspase-3, which acts as an executioner, by cleaving multiple of other substrate within the cells [34, 106].

Fig. 2
figure 2

The underlying mechanisms in the execution of apoptotic pathways. The Apoptotic stimulus received are communicated to the cell by extrinsic or intrinsic pathways. The extrinsic pathway involves the engagement of various transmembrane death receptors such as FAS and TNF-α and their corresponding extracellular ligands (FASL, TNF-αL). Fas recruits, Fas-associated death domain protein (FADD) whereas TNF-R recruits TNF-R1 associated death domain protein (TRADD) which again recruits FADD. Such association leads to formation of Death-inducing signaling complex (DISC) which acts as platform for many co-factors and regulator for cleavage and activation of procaspase-8 (or 10), an initiator caspase of the extrinsic pathway. The activated caspase-8 (or 10) leads to their own autoactivation which further activate the caspase-3 and caspase-7, an effectors caspase. The intrinsic pathway involves the mitochondria and after receiving the stimulus, the pro-apototic BH3-only members (Bid, bad, bim, etc.) bind and neutralize the antiapoptotic proteins (Bcl-2, Bcl-xL, etc.). This leads to oligomerization of multi-domain pro-apoptotic member (Bax/Bak) present on mitochondrial membrane surface whose activation leads to permeabilization and formation of pores or VDAC in outer mitochondrial membrane (OMM), releasing various apoptotic mediators (HtrA2/Omi, Smac/DIABLO, Cyt c, endo G and AIF). The release of Cyt c in cytosol causes the association of Apaf-1 and ATP/dATP to form apoptosome, the initiator caspase of the intrinsic pathway, caspase-9, enabling their autoactivation. This in turn activates the series of downstream of cascade to activate the caspase-3 and caspase-9, an effectors caspase involved in cell death. The role of IAP is to act a guardian inside a cell to defend against the mediator of apoptosis (HtrA2/Omi, Smac/DIABLO) by binding to Caspase 3/7/9 where as Endo G and AIF acts in a caspase independent manner by causing chromatin condensation and fragmentation. The pro-apototic BH3-only members, Bid, is also activated by caspase-8, making it an important converging point of both pathways. The activated truncated (t)Bid further activates downstream targets, such as BAX and BAK

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Extrinsic pathway

The extrinsic pathway involves the association of receptor-mediated transmembrane death receptor (FAS and TNF-α) and its extracellular ligand (FAS-L and TNF-αL) [107–109]. For association the receptor trimerizes and death adapter molecules are recruited on the cytoplasmic side of the mitochondrial membrane. Fas recruits Fas-associated death domain protein (FADD) whereas TNF-R recruits TNF-R1-associated death domain protein (TRADD) which again recruits FADD [7]. Such association leads to formation of death-inducing signaling complex (DISC) consisting of a complex of receptor, its ligand, the initiator procaspase-8 (or procaspase-10), and some other regulators and co-factor [110]. The complex helps in recruiting more of procaspase-8 (or procaspase-10) and enables their autoactivation (Fig. 2).

Apoptotic mediators

Permeabilization of the outer mitochondrial membrane allows the leakage of at least five apoptotic mediators (apoptogenic proteins) from the mitochondrial intermembrane space, such as cyt c, second mitochondrial activator of caspases/Direct IAP protein with low pI (Smac/DIABLO), HtrA2/Omi, apoptosis-inducing factors (AIF), and endonuclease G [32, 37]. These proteins induce apoptosis in different ways. Smac/DIABLO and HtrA2/Omi suppress the ability of IAPs (inhibitors of apoptosis proteins) to inhibit caspases. Endonuclease G and AIF are involved in DNA fragmentation, and AIF is also involved in chromatin condensation (Fig. 2). The release of these apoptotic mediators from mitochondria is known to be regulated by Bcl-2 family of proteins [55, 111]. However, caspase-independent mitochondrial cell death results from loss of respiration and not from the release of cytotoxic apoptotic mediators [112].

Release of cytochrome c

Cyt c is a water soluble 13 kDa protein encoded by nuclear gene that is translated in cytosol to be finally imported into mitochondria. It normally resides in the spaces within cristae of the inner mitochondrial membrane (IMM) and at narrow cristae junctions [113]. The role of cyt c in the intrinsic pathways in mammalian cells is well known. Addition of dATP to cytosolic extract induces caspase activity [114] and depletion of cyt c in cell extract inhibits its apoptotic potential and also microinjection of cyt c in various cell types enhances the apoptotic pathways [115, 116]. Thus, cyt c is the main mediator of apoptosis [117, 118], and the release of cyt c occurs due to DNA damage [119, 120]. Over-expression of Bcl-2 blocks the release of cyt c from mitochondria and inhibits the initiation of apoptosis [121].

Release of endonuclease G (EndoG)

EndoG is a 30 kDa nuclease protein located in mitochondrial intermembrane space [122, 123]. The release of EndoG after apoptotic signal leads to DNA fragmentation as found in inhibitor of caspase-activated DNase (ICAD)-deficient cells after induction of apoptosis by TNF treatment and UV-irradiation [123, 124]. Once released, EndoG participates in DNA fragmentation but without assistance of caspases [125–127].

Release of apoptosis-inducing factors (AIF)

Apoptosis-inducing factor (AIF), which resembles bacterial oxidoreductase, is a 57 kDa flavoprotein present in the mitochondrial intermembrane space [128]. Upon induction of apoptosis, AIF translocates from the mitochondria to the nucleus and causes DNA fragmentation and chromatin condensation [128]. These effects are independent of its oxidoreductase and caspases activity [129, 130]. Disruption of AIF in mice prevents normal apoptosis necessary for the activation of embryoid bodies in the embryo [131]. In addition, AIF is required for specific cell death pathways including lethal responses to excitotoxins such as glutamate and N-methyl-D-aspartate (NMDA), DNA-alkylating agents, hypoxia–ischemia, or growth factor deprivation [132]. Recently, Schulthess and coworkers have shown a protective role of AIF on β-cell turnover and the loss of AIF increases β-cell apoptosis. AIF is essential for maintaining β-cell mass and oxidative stress response [133]. The mechanism of AIF-induced large-scale chromatin condensation and DNA fragmentation is still not clear [128, 131, 134–136]. More recently, the role of AIF in 12/15-lipoxygenase (LOX)-dependent organelle damage pathway has been reported showing that AIF and 12/15-LOX are important mediators in a common cell death pathway in stroke-induced brain damage [137].

Release of Smac/DIABLO

SMAC (Second Mitochondrial Activator of Caspases) or DIABLO (Direct IAP Binding Protein with Low pI) are a 25 kDa, pro-apoptotic protein released from the intermembrane space that neutralizes the inhibitory activity of IAP leading to activation of caspases and apoptosis [138–140]. During reovirus-induced apoptosis, Smac/DIABLO are released that decreases the level of IAP’s and thus activates apoptosis [139]. Sphingosine 1-phosphate inhibits the release of Smac/DIABLO from mitochondria and antagonizes apoptosis of human leukemia cells. Smac/DIABLO is involved in many cancer manifestation and progression [141] such as cervical cancer [142], colon cancer [143], and hepatocellular carcinoma [144].

Release of Omi/HtrA2

The mammalian serine protease Omi/HtrA2 (high-temperature requirement) is a 49 kD protein, homologous to the bacterial endoprotease also known as DegP [145]. Unlike Smac/DIABLO, the pro-apoptotic activity of Omi/HtrA2 involves both IAP binding and serine protease activity. Omi/HtrA2 has a dual function, when residing inside the mitochondria it promotes cell survival, but when released into the cytoplasm it participates in both caspase-dependent and -independent cell death. It prevents the IAP’s action via amino-terminal reaper-related motif which induces caspase activity [99, 146–148]. It also mediates caspase-independent cell death through its own protease activity, by the fact that simultaneous deletion of the other IAP binding protein, Smac/DIABLO, does not alter the phenotype of Omi/HtrA2 knockout mice or cells derived from them [149]. The caspase-independent role of Omi/HtrA2 in apoptosis is evident in human cardiac-specific inhibitor of cell cycle protein, Thanatos-Associated Protein 5 (THAP5) that are cleaved by pro-apoptotic Omi/HtrA2 during cardiomyocytic cell death [150]. The role of Omi/HtrA2 in colon cancer [151] and oxidative stress of pigment epithelial cell [152] further supports its application of mediating apoptosis in cells. The role of Omi/HtrA2 in promoting cell death by binding and degrading ped/pea-15, an anti-apoptotic protein, establishes the pro-apoptotic effect of Omi/HtrA2 [153] and its role in the apoptosis of prostate cancer cell, PC-3 [154]. However, a contradictory role of mitochondrial Omi/HtrA2 has been reported. In response to some extracellular inducers of mitochondrial stress, Omi/HtrA2 stabilizes mitochondrial membrane potential and inhibits mitochondrial superoxide generation and hence controls apoptosis [155].

Regulation of apoptosis by IAPs

Inhibitor of apoptosis (IAPs) is a family of antiapoptotic proteins that associate with caspases in response to diverse stimuli. IAP has been discovered both in invertebrate and vertebrates. So far, eight human IAP homologs have been identified which includes X-chromosome-linked IAP (XIAP, also known as hILP, MIHA, or BIRC4), survivin (also known as TIAP or BIRC5), cellular IAP1 (c-IAP1, also known as HIAP2, MIHB or BIRC2), c-IAP2 (also known as HIAP1, MIHC, or BIRC3), neuronal apoptosis inhibitory protein (also known as BIRC1), IAP-like protein 2 (also known as BIRC8, or Ts-IAP), apollon (also known as Bruce or BIRC6), melanoma IAP (ML-IAP, also known as KIAP, livin, or BIRC7) [156, 157]. The best characterized IAPs such as XIAP, c-IAP1, and c-IAP2, bind caspase-3, caspase-7, and caspase-9, thereby inhibiting their activation and preventing apoptosis (Table 1) [156, 158–160]. Also, cIAP1 and cIAP2 have been shown to bind caspases, although how they inhibit apoptosis at the molecular level is not completely understood [161]. Direct inhibition of caspase activity by c-IAPs is an important means of regulation in order to protect cell death. Activity of XIAP is blocked by binding to Omi/Htr2A and Smac/DIABLO proteins released from mitochondria after pro-apoptotic stimuli. Thus, Smac/DIABLO is a negative regulator of IAPs [140, 162]. Similarly, Omi/HtrA2 also inhibits the XIAP through a reaper-like motif [148] and has a prognostic significance in hepatocellular [144] and renal cell carcinoma [163]. Inhibition of apoptosis increases the survival rate of cancer cells and facilitates their escape from cytotoxic therapies and immune surveillance [144, 164, 165].

Table 1 Subfamily of caspase showing their functional role, functional domain and corresponding specificity to member of Inhibitor of Apoptosis (IAP’s)
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Caspases and underlying mechanism in controlling cellular apoptosis

Caspases are not only involved in the process of apoptosis but also needed for the development and maturation of cytokines leading to cell growth and differentiation [166]. Apoptotic cell death is dependent on a family of aspartate-specific cysteine proteases (caspases) that cleave certain vital structural proteins (e.g., lamins, gelsolin) and proteolytically activate latent enzymes (e.g., nucleases) that contribute to cell death. These enzymes exist in most cells as inactive precursors (procaspases) that are converted into their active forms by proteolytic cleavage at internal aspartic acid residues, which separates the caspase into small and large subunits [167] and then they become activated by autoproteolysis.

Through genetic analysis of cell-death defective (CED) mutants, it was found that the product of the aspartate-directed cysteine protease (CED-3) is required for all developmental-related programmed cell deaths in the worm [168]. In this multicellular model, various genes that encode for proteins essential for the regulation and execution of apoptosis were identified and their mammalian homologs were described [33, 76]. For example, ced-9 and Bcl-2 encode proteins that inhibit apoptosis, Apaf-1 and ced-4 encode an adaptor protein that permits the interaction between initiator proteins ced-3 and caspase-9. Ced-3 encodes a protein homologous to caspase-9 which is responsible for the apoptotic initiation process. Fifteen mammalian caspases have been identified, with caspase-11 and 12 identified only in mouse as shown in Table 1.

All caspases are synthesized as zymogens sharing a common domain structure consisting of a large (p10) and a small (p20) catalytic subunit. Among two fundamentally different groups, the first group, termed as initiator caspases, is characterized by a long prodomain which provides a protein–protein interaction platform [167, 169]. Prodomains allow the recruitment of procaspases into an activating protein complex. Long prodomain caspases are caspase-1, -2, -4, -5, -9, -11, and -12 with an N-terminal caspase-activating recruitment domain (CARD), and caspase-8 and -10 with an N-terminal death effector domain (DED) (Table 1). In contrast to the initiator caspases, the second group consisting of executioner caspases-3, -6, and -7 lack the large N-terminal non-enzymatic domain and they are responsible for the majority of cellular destruction during apoptosis [170]. When procaspase-8 or -10 recruited to ligate death receptors by Fas-Associated Death Domain (FADD), they undergo autocatalysis, releasing the p10 and p20 subunits that form the active (tetrameric) enzyme. Caspase-9 is activated in the presence of ATP and cyt c by an allosteric change on a heptameric scaffold of apoptotic protease-activating factor 1 (Apaf1) proteins termed as apoptosome. Caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. In a heat shock-induced death, Caspase-2 induces apoptosis via cleavage of Bid [171].

Interdependence of caspases and Bcl-2 in the regulation of apoptosis

Numerous studies suggest that many apoptotic signaling pathways converge at the mitochondria, where signals are processed through a series of molecular events culminating in the release of potent death factors that trigger either through the extrinsic or the intrinsic pathway. Basically, the release of the pro-apoptotic proteins from the intermembrane space triggers apoptosis, in a caspase-dependent (through cyt c, Omi/HtrA2, and SMAC) as well as in a caspase-independent form (through AIF and Endo G).

In mammalian cells, caspases-9, -8, and -2 rely on the formation of apoptosome (Apaf-1), death-inducing signaling complex (DISC), and PIDDosome, respectively, for activation of apoptotic signals. Apoptosome is composed of seven molecules (heptamer) of Apaf-1 bound to cyt c in the presence of ATP/dATP, Fig. 2. DISC is assembled following binding of death ligand to its receptor and contains FADD and caspase-8 (or –10) whereas PIDDosome contains at least three components, PIDD, RAIDD, and caspase-2.

Apoptosome complex

Apoptosome is a multimeric protein complex that mediates activation of an initiator caspase at the onset of apoptosis. Biochemical and structural investigations revealed insights into the assembly and function of the various apoptosomes from fruit fly (Drosophilia melanogaster), worm (C. elgans) and mammals [172, 173]. The assembly of the mammalian apoptosome which is responsible for the activation of caspase-9 requires the binding of Apaf-1, cyt c, and ATP/dATP. The apoptosome is an oligomeric signaling platform that has a core of seven apoptotic Apaf1. Each Apaf-1 monomer contains an N-terminal caspase recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD), and a string of WD40 (tryptophan-aspartic acid) repeats at the C terminus (Fig. 2). The WD40 repeats are thought to be the site of cyt c binding [102, 174].

Death-inducing signaling complex (DISC)

DISC consist of complex of the death receptor (FAS), the adaptor FAS-associated death domain protein (FADD), the initiator caspase procaspase-8 (or procaspase-10), and possibly other co-factors and regulators [175]. Kischkel and coworkers reported the formation of a protein complex in the dying cell and named it as DISC. Upon receiving the death signal the activated death ligands homo-trimerizes, which in turn, induces oligomerization of the Fas death receptors (Fig. 2) [176, 177]. Dimerization of caspase-8 is a crucial factor for activation and suggests that DISC may facilitate the activation of caspase-8 through dimerization [178]. The DISC creates a platform that brings together molecules of the initiator caspase-8 (or caspase-10) and other co-factors and regulators for execution of extrinsic apoptotic pathway which finally leads to their auto-activation [179, 180].

p53-Inducible death domain containing protein complex (PIDDosome)

The PIDDosome complex under physiological conditions contains PIDD and associate with the activation of another initiator caspase, caspase-2 [181]. Although, the role of the PIDDosome in apoptosis remains controversial, its expression is inducible upon DNA damage [173, 181, 182]. However, PIDD-deficient mice undergo apoptosis not only in response to DNA damage, but also in response to various p53-independent stress signals and to death receptor engagement. In the absence of PIDD, both caspase-2 processing and activation occur in response to DNA damage indicating that PIDD does not play an essential role for all p53-mediated or p53-independent apoptotic pathways [182]. The role of caspase-2 in the mitochondrial pathway is now widely accepted [183–185]. Thus, the initial stage of DNA damage facilitated by p53-mediated apoptosis occurs by a PIDD and caspase 2-dependent mechanism. For events that are downstream of cyt c release, p53’s full transcriptional regulatory functions are required [186].

Apoptosis and the caspase-independent pathway

There also exists a caspase-independent apoptotic pathway that is associated to AIF [131, 134], endonuclease G [187], as well as Omi/HtrA2 [150–152]. As already discussed above in this review, cells after receiving the apoptotic signals release the nuclear AIF molecule and endonuclease G protein which are translocated to the nucleus causing a large-scale chromatin condensation and DNA fragmentation independently of caspase activation. Similarly Omi/HtrA2 mediates caspase-independent cell death through its own protease activity as can be observed during apoptosis. A specific Omi/HtrA2 inhibitor can stop degradation of THAP5 protein (THAP family of proteins), which leads to reduced cell death [150]. A few studies have focused the role of Heat Shock Proteins (HSPs) that are either constitutively expressed or expressed under variety of stresses stimuli [188], have shown their role in apoptosis via caspase-independent pathway [189]. Members of HSP protein such as HSP27 and HSP70 participate in oncogenesis, probably by interfering apoptotic pathways. First, they act as chaperones and play a role in proteasome-mediated degradation of apoptosis-regulatory proteins. Second, they inhibit key effectors of the apoptotic machinery including the apoptosome and apoptosis-inducing factor [190]. However recent work on caspase-independent apoptotic pathway has lead to the discovery of various other molecules which has a major role in the pathway. For example, the work by Yuan and his group have shown that Ste20-like protein kinase 3 respond to apoptosis of HeLa cells to trigger the caspase-independent apoptotic pathway [191]. Similarly activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY) is being used for the therapy for hematological malignancies and autoimmune disorders through caspase-independent T-cell apoptosis [192].

Therapeutic implication in the control of apoptosis

Lack of the phenomena of apoptosis results in excessive increase of cell number and has implications in autoimmunity and tumorigenesis. On the other hand, excessive apoptosis decreases the cell population, which is linked to many neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy. Alzheimer’s disease is a complex neurological disorder in which the beta-amyloid peptides are formed in the brain. In this, the Bcl-2 is down-regulated and Bax is up-regulated [193, 194]. With respect to the role of Bcl-2 proteins and its role in Parkinson’s disease, it has been anticipated that pro-apoptotic family members participate in neuronal death in a variety of Parkinson’s disease models [195]. Numerous studies show that activation of apoptosis has also been found to be involved in the pathogenesis of other human diseases such as chronic heart failure [196], diabetes [197, 198], and atherosclerosis [199]. The increased expression of Bcl-2 in the vascular endothelium inhibits the diabetes-induced degeneration of retinal capillaries and superoxide generation [198, 200].

An imbalance among the Bcl-2 family of proteins, in favor of the anti-apoptotic members, is a phenomenon that naturally, and frequently occurs in cancer cells [201–203]. Over-expression of anti-apoptotic Bcl-2 or Bcl-xl probably occurs in more than half of all cancers. Moreover, loss of expression of Bax is also found in some colorectal cancers and in hematopoietic malignancies, whereas the expression of a highly apoptogenic variant of Bax (Baxψ) is correlated with an increased survival of patients with glioblastoma multiforme, an aggressive form of brain tumors [204]. The defects may arise from the fact that neoplastic cells are under strong selective pressure to stabilize their mitochondrial permeability, even if they harbor alterations in the p53 tumor suppressor pathway. c-Myc, for instance, can induce mitochondrial damage independently from the transcriptional activity of p53. Since p53 also activates the mitochondrial death pathway, the mitochondrion appears to integrate the diverse pro-apoptotic mechanisms induced by oncogenes. Studies in transgenic mice have revealed that Bcl-2 (and/or Bcl-xl) over-expression and p53 mutations (or ARF loss) are selected independently during Myc-induced lymphomagenesis [205–207].

The emerging knowledge about proteins that are involved in apoptosis, including their 3-D structures and biochemical mechanisms, has provided therapeutic avenues by discovering molecules or targets which may modulate apoptosis. A number of therapeutic approaches are undergoing using an antisense RNA [208], various small molecules [209–218, 222, 223], and peptidic compounds [219–221, 224, 225] classified as potential therapeutics to target the pathway of apoptosis, as briefly summarized in Table 2. Most of these therapeutics involve the targeting of structurally defined multidomain of the member of Bcl-2 family of protein. Some of these molecules/agents are in clinical trials.

Table 2 Compounds on high clinical development against the Bcl-2 and IAP’s family of proteins involved in the mitochondria-mediated apoptosis pathway
Full size table

The combination of IAP antagonists with drugs that target ErbB receptors promotes apoptosis thereby reduces the cell turnover of breast cancer cells [226]. The apoptotic response to most chemotherapeutic drugs in mammalian cells involves the induction of mitochondrial pathway in which mitochondrial membrane permeabilization controlled by the Bcl-2 protein family, is induced. Other strategies include the IAP proteins as therapeutic targets that are expressed in the majority of human tumor through the inhibition of cellular death and participation in signaling pathways associated with malignancies [157, 227]. Further more, Table 2 has summarized the list of antisense [228], small molecules [229–235], and peptidic compounds [234, 236, 237] that are under investigation to develop potential therapeutics against IAPs protein which are involved in cancer.