APOPTOSIS SIGNALING; A DRUG DESIGN IN CANCER THERAPY: A REVIEW

APOPTOSIS SIGNALING; A DRUG DESIGN IN CANCER THERAPY: A REVIEW

Md. Ataur Rahman*

Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University, Chuncheon, Gangwon-do, 200-702, South Korea

ABSTRACT

Apoptosis plays an important role in development, growth, differentiation, altered gravity conditions, tissue homeostasis, immune defense, and cancer to control cell number in tissues and to eliminate individual cells that threaten the animal’s survival. The purposes of this review are to describe the signaling pathways and the cellular changes that occur with apoptosis. Currently, two pathways for activating apoptosis have been studied in detail. One starts with ligation of a death ligand to its transmembrane death receptor, followed by recruitment and activation of caspases in the death-inducing signalling complex. The second pathway involves the participation of mitochondria, which release caspase-activating proteins into the cytosol, thereby forming the apoptosome where caspases will bind and become activated. The novel agents include those targeting for the design of more effective and selective therapeutic strategies. This review providing an overview of the recent understanding of apoptotic signaling pathways, the main mechanisms by which cancer cells resist apoptotic insults, and discuss some recent attempts to target for restoring efficient cell death signaling in cancer cells to develop a potential anticancer drug.

Drug

INTRODUCTION:Apoptosis plays a central role both in development and in homeostasis of metazoans ¹. Cells die by apoptosis in the developing embryo during morphogenesis or synaptogenesis and in the adult animal during tissue turnover or at the end of an immune response. Because the physiological role of apoptosis is crucial, aberration of this process can be detrimental.

Thus, unscheduled apoptosis of certain brain neurons contributes to neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, whereas the failure of dividing cells to initiate apoptosis after sustaining severe DNA damage contributes to cancer ². Extensive research in the cancer field has explored numerous pathways in cancer progression to elucidate effective measures to target these cancer cells. Though cancer therapeutics favor a multiple approach where individual treatment varies between surgery, radiation, chemicals, antibodies and/or cells of the immune system, the effectiveness of the treatment differs largely between individuals and the cancer background ³. In this regard, improving therapeutic efficacy and selectivity and overcoming drug resistance are the major goals in developing anti cancer agent’s today ⁴.

To fulfill these goals, a thorough understanding of apoptotic signaling pathways and how tumor cells become apoptosis is imperative, because they provide directions to unravel novel therapies and key targets to surpass or supplement current cancer treatments. Therefore, in this review I would like to summarize the apoptotic signaling pathways and how cancer cells resist apoptotic insults to cancer therapy.

Apoptotic Pathway: Apoptosis is characterized by cell shrinkage, blebbing of plasma membrane, maintenance of organelle integrity, condensation and fragmentation of DNA, followed by ordered removal of phagocytes ⁵. It works like a “suicide” program and it causes minimal damage to surrounding tissues. Apoptosis has been subclassified into two types of death pathways, namely, the extrinsic pathway and the intrinsic mitochondria-mediated pathway. These two processes however, are not exclusive and evidence suggests that they can be linked and that molecules in one pathway can influence the other ⁶. Moreover, recent evidences support non-apoptotic roles for many effectors of the apoptotic signaling pathways. For instance, caspase-2, the most conserved member of the caspase family, also plays a role in cell cycle regulation, DNA repair, and tumor suppression⁷.

1. Extrinsic pathway: The extrinsic or receptor mediated death pathway requires effective engagement between the death receptors found on the surface of the cell membranes and their respective ligands ⁸ (Fig. 1). The receptor-mediated pathway involves death receptors from the tumor necrosis factor (TNF) superfamily such as TNF, CD95 (Fas) and TNF-related apoptosis inducing ligand (TRAIL) receptors. These receptors have an extracellular domain to engage the ligands and an intracellular cytoplasmic domain that is also referred to as the death domain. This death domain is responsible for transmitting the death signal from the surface to the intracellular signaling pathways ⁹.

Activation of CD95 or TNF receptors often leads to receptor clustering and intracellular recruitment of proteins into a death-inducing signaling complex (DISC), which then activates an initiator caspase, procaspase-8. Activated caspase-8 then triggers the execution phase of apoptosis via the activation of the downstream effector caspase, caspase-3 ¹⁰. The activated caspases can also induce mitochondrial damage and reinforce the death signal by facilitating the egress of death amplifying proteins from the mitochondrial inter-membranous space^11-14.

2. Intrinsic pathway: The intrinsic or mitochondria pathway of cell death can be activated by a variety of receptor-independent stimuli such as radiation, free radicals, viral infections and serum/growth factor withdrawal (Fig. 1). Initially, it was demonstrated that these triggers invariably result in changes in the inner mitochondrial membrane permeability due to the opening of the mitochondrial permeability transition (MPT) pore. The major consequences of this change of permeability are the loss of the mitochondrial transmembrane potential (ΔΨ_m), the release of pro-apoptotic proteins and the arrest of the bioenergetic function of the organelle.

The proteins that are released can be broadly classified into two categories. The first category comprises of proteins that can activate the caspase-dependent pathway. This group includes proteins such as cytochrome c (cyt c) and Smac/ DIABLO (second mitochondria-derived activator of caspases).

The holocytochrome c induces Apaf-1 oligo merization, leading to the activation of caspase-9. This active cytc/Apaf-1/caspase-9 complex forms the apoptosome and activates the executioner caspases-3 and -7 resulting in the dismantling of the cell through nuclear fragmentation ^{10, 15, 16}. Smac/DIABLO binds to IAPs (inhibitor of apoptosis proteins) and deactivates them, thus preventing the IAPs from arresting the apoptotic process, permitting apoptotic progression.

The second group includes other pro-apoptotic proteins such as apoptosis inducing factor (AIF) and endonuclease G (Endo G). In some models, the release of these proteins is a late event in apoptosis, which occurs once the cells are committed to die. Following the release of AIF, it translocates to the nucleus where it promotes DNA fragmentation. Both AIF and Endo G act in a caspase-independent manner to execute cell death ¹⁷. For example, in an ovarian stem cell model, Endo G was shown to mediate caspase-independent cell death in response to chemotherapeutic agents ¹⁸. Alternatively, AIF has also been recently proposed to participate in a different form of cell death, namely programmed necrosis ¹⁹.

FIGURE 1: THE EXTRINSIC AND INTRINSIC PATHWAYS OF APOPTOSIS: Death receptor pathway (left) is initiated by the ligation of the ligands to their respective surface receptors. Ligation of death receptors is followed by the formation of the death-inducible signalling complex (DISC), which results in the activation of pro-caspase-8 which cleaves caspase-8 and activates pro-caspase-3, which cleaves target proteins, leading to apoptosis. The intrinsic pathway (right) is activated by death signals function directly or indirectly on the mitochondria, resulting in the formation of the apoptosome complex. This cell death pathway is controlled by Bcl-2 family proteins (regulation of cytochrome c release), inhibitor of apoptosis proteins (IAPs) (inhibition of caspases), and second mitochondrial activator of caspases (Smac) and Omi (negative regulation of IAPs). The apoptosome function is also regulated by the oncoprotein pro-thymosin- (Pro-T) and the tumor suppressor putative HLA-DR-associated protein (PHAP). The intrinsic pathway might also operate through caspase-independent mechanisms, which involve the release from mitochondria and translocation to the nucleus of at least two proteins, apoptosis inducing factor (AIF) and endonuclease G (EndoG). The nuclear location of AIF is linked to chromatin condensation and the appearance of high-molecular-mass chromatin fragments, whereas the role of EndoG is still unclear.

Molecular mechanism of regulation of Apoptosis Pathway:

1.  Members of the Bcl-2 family proteins: The Bcl-2 superfamily of apoptosis regulatory gene products includes proteins commonly ascribed as death antagonists (Bcl-2, Bcl-Xl, Bcl-W, Bfl-1, and Mcl-1), or death agonists (Bax, Bak, Bcl-Xs, Bad, Bid, Bik, Bim, and Hrk) and these proteins act mainly at the level of the external mitochondrial membrane²⁰. These proteins also contain a hydrophobic carboxy terminal domain that enables them to dock onto the outer mitochondria membrane (OM), the nucleus and ER ²¹.

It has been shown that Bcl-2 and Bcl-X_L can protect the cells by interacting with mitochondrial proteins such as the adenine nucleotide translocase (ANT) or the voltage dependent anion channel (VDAC), thus preventing them from forming mitochondrial pores, protecting membrane integrity, and inhibiting the release of apoptogenic factors such as cytochrome c ²².

More recent evidence has highlighted novel functional biology of the anti-apoptotic protein Bcl-2 ²³. Overexpression of Bcl-2 was associated with a slight increase in steady state intracellular superoxide (O₂^-) production, which was linked to increases in mitochondrial oxygen consumption and the activity of the rate limiting enzyme in the electron transport chain, cytochrome c oxidase (COX). Interestingly, pharmacological or molecular inhibition of the NADPH oxidase restored apoptosis sensitivity of Bcl-2 overexpressing cells, thus implicating the pro-oxidant activity of Bcl-2 in its anti-apoptotic function.

The altered ratios favoring the pro-survival proteins such as Bcl-X_L and Bcl-2 over the pro-apoptotic proteins such as Bak and Bax have been shown to lead to chemoresistance in cancer cells. The need to drive the ratio toward death in cancer cells has led to the development of anti-cancer compounds that can either act to suppress the pro-survival proteins such as Bcl-2 and Bcl-X_L, or mimic the pro-death BH3 only proteins such as Bid, Bim, Bad, Bmf, Noxa and PUMA to promote cell death. Another attractive drug target is the naturally occurring Bcl-2 inhibitor gossypol, which is derived from cottonseeds ²⁴.

Gossypol, like HA14-1 was also shown to bind to the BH3 domain of Bcl-2. The anti-apoptotic effects of Gossypol have been demonstrated in head and neck cancer, colon, prostate, pancreatic and leukemia cell lines ^25-27. The second category of Bcl-2 family of proteins contains BH domains 1, 2 and 3. These proteins would include Bax and Bak. Bax is a pro-apoptotic protein that resides in the cytosol under physiological conditions.

An apoptotic trigger however, can lead to its translocation to the mitochondria and its subsequent insertion into the OMM. At the mitochondria, Bax can homodimerize or heterodimerize with other pro-apoptotic members such as Bak or truncated Bid, thus disrupting the integrity of the OMM by forming mitochondrial pores and increasing its permeability. These pores can then lead to the release of apoptogenic factors such as cyt c ²⁸.

Some reports have also suggested that Bax engages in a close molecular cooperation with proteins from the PTPC, such as ANT and/or VDAC, to induce mitochondria membrane permeabilization (MMP) ²⁹.

Bcl-2 and Bcl-X_L have been shown to antagonize the apoptotic cascade by a direct interaction and sequestration of these pro-apoptotic proteins³⁰. Similarly Bak, which is normally inhibited by its interaction with VDAC, can also homodimerize and result in pore formation at the mitochondria when freed. The third group of Bcl-2 family of proteins is the BH3-only proteins. The BH3-only family members include Bim, Bad, Bmf, Noxa and Puma. They act by neutralizing the anti-apoptotic proteins ³¹.

For instance, Bim, Puma, Bad and Bmf heterodimerize with Bcl-2 and Bcl-X_L and sequester them, thereby blocking their anti-apoptotic action at the mitochondria. While drugs that could suppress anti-apoptotic proteins have yielded some promising results, attempts to increase sensitivity by mimicking the structures of pro-apoptotic proteins has also led to the discovery of some novel BH3 mimetics. One such well understood BH3 mimetic is ABT-737. ABT-737 mimics the BH3-only protein Bad and has been found to bind and inhibit Bcl-2, Bcl-w and Bcl-X_L.

However, it displays a weak affinity to MCL-1³². Thus cancer cells that overexpress MCL-1, are resistant to this small molecule compound. To that end, in a small cell lung cancer model, ABT-737 was shown to sensitize cells to apoptosis induced by chemotherapeutic agents ³³. Of note, ABT-737 is highly effective against cancers with elevated expression of Bcl-2 and is an efficient apoptosis inducer in the presence of Bax and Bak ³⁴. Another group of BH3 peptides synthesized by Shangary et al. to mimic Bax and Bad BH3 domains demonstrated effective engagement of the intrinsic pathway with the release of cyt c, even in Bcl-2 and Bcl-X_L overexpressing cells ³⁵.

BH3 peptides have also been shown to trigger oligomerization of Bax and Bak, resulting in MOMP and cyt c release ³⁶. Though the use of BH3 mimetics holds much promise, the use of BH3 peptides in cancer therapeutics has been limited due their poor cell permeability, bioavailability, solubility and metabolic stability in vivo. To address these issues, tagged proteins ³⁷ or proteins with chemical modifications ³⁸ have been employed.

2. Apoptotic Inhibitor proteins:The inhibitors of apoptosis (IAP) are a family of antiapoptotic proteins that directly bind to caspases, inhibiting their functional activity. Cell fate is tightly regulated by the interactions between pro and anti-apoptotic proteins which act to tweak the balance between survival and cell death ³¹. In cancers, the crippling of pro-apoptotic pathways or enhancement of the anti-apoptotic pathways via the modulation of the regulatory proteins largely confers survival advantages onto the cells in the face of death triggers.

The inhibitors of apoptosis (IAPs) family were first identified in baculovirus and to date, eight mammalian IAPs have been described ³⁹. This would include neuronal apoptosis inhibitory protein (NAIP), cellular IAP1 and IAP2 (cIAP1 and cIAP2), X-linked inhibitor of apoptosis (XIAP), Survivin, Testis-specific IAP (Ts-IAP), BIR-containing ubiquitin conjugating enzyme (BRUCE) and Livin ⁴⁰.

IAPs are characterized by the presence of 70–80 amino acid baculoviral IAP repeat (BIR) domain(s), which are important for the binding and inhibition of caspases. They play a critical role in blocking cell death by regulating the caspase cascade, and then, may influence both the intrinsic and extrinsic pathway in the cells. Of the IAPs, XIAP has been best described, possibly due to the extensive studies on its anti-apoptotic role and the plausible therapeutic benefits in targeting it. XIAPs have been shown to antagonize the apoptotic cascade via the direct inhibition of caspases and via proteasome-dependant degradation of caspases.

In addition to these caspase inhibitory roles, it has also been found to activate nuclear factor kappa B (NF-κB) by promoting the nuclear localization of NF-κB. IAPs have been shown to be regulated by IAP binding proteins such as second mitochondrial activator of caspases (Smac/DIABLO) ⁴¹. Smac /DIABLO normally resides in the mitochondria and upon receiving an apoptotic stimulus via the intrinsic pathway is proteolytically cleaved and released into the cytosol through the Bax/Bak channels or via Bid-induced permeabilization of the outer mitochondrial membrane.

Subsequently, Smac/DIABLO associates with IAPs and prevents them from inhibiting caspases, thus promoting apoptosis.

3.  Apoptotic pro-survival proteins:Upregulation of the anti-apoptotic family of proteins has been a frequent explanation for the resistance observed in cancer cells. Increased expression of the anti-apoptotic family members such as Bcl-2, Bcl-X_Land Mcl-1 has been often observed in cancer cells⁴², where they serve to antagonize mitochondria-mediated cell death pathway.

Specifically, Bcl-2/Bcl-X_L upregulation is clearly associated with poor prognosis in cancer ⁴³. The ability of these proteins to antagonize the pro-apoptotic family of proteins such as Bax and Bak has been the key mechanism by which these cells acquire resistance to apoptosis. As such, measures to target Bcl-2 or Bcl-X_L via anti-sense oligo nucleotides have been employed in clinical studies to sensitize cancer cells to apoptotic triggers ^{44, 45}.

However, IAPs are a class of anti-apoptotic proteins upregulated in a variety of human cancers. IAPs inhibit the activity of caspases and hence protect cells from the deleterious effects of active caspases. Smac/DIABLO, a natural antagonist of IAPs, has been shown to sensitize cells to drug- and receptor-induced apoptosis by binding to XIAP and releasing caspase-9 in vitro. Increased resistance to drugs that activate the extrinsic death pathway has also been observed with upregulation of proteins such as c-Flip and decoy receptors.

c-Flip inhibits the autoproteolytic cleavage of pro-caspase-8, which is involved in DISC formation, and hence, the downstream transduction of the death signals via the extrinsic death pathway ⁴⁶. Decoy receptors can compete with death receptors for the ligands such as TRAIL and CD95, thus inhibiting/abolishing death signal transduction ⁴⁷.

4. Repression of pro-apoptotic proteins: In addition to the upregulation of pro-survival factors, suppression of pro-apoptotic proteins also contributes to resistance against apoptosis-inducing therapeutic regimens. Thus, Bax is one key pro-apoptotic member that is frequently suppressed or mutated in cancers.

The mutations include frameshifts and/or mutations at the BH domains, which lead to a loss of function. Indeed tumors with reduced Bax expression have been found to have a poorer prognosis ^{48, 49}. In addition to Bax deficiencies, it has also been reported that Bak deficiencies can also lead to substantial inhibition of mitochondria-mediated apoptotic cell death. The activation of Bax and Bak is mediated via caspase-8-induced cleavage of the BH3 only protein Bid to tBid.

Activation and oligomerization of BAX or BAK have been proposed to result in the formation of a VDAC-containing pore and permeabilization of mitochondrial membranes. This leads to the release of cyt c and the subsequent engagement of the Apaf-1-caspase-9 apoptosome complex, which activates downstream effector caspases. In this regard, suppression of components which act downstream of the mitochondria such as cyt c, Apaf-1 and caspases can protect the cells from apoptotic insults. Accordingly, the expression levels of several pro-apoptotic members of Bcl-2 family, such as Bim and Puma have been shown to correlate with colon carcinoma susceptibility to chemotherapy ⁵⁰.

Apoptosis and Cancer Therapy: Most drugs currently used in anti-cancer therapy kill target cells by induction of apoptosis, both by receptor-mediated and mitochondrial-mediated pathways. Disruption of the mitochondrial membrane potential, cytochrome c release and activation of different caspases have been described following treatment of cells with diverse chemotherapeutic agents ^{51, 52}.

For example, chemotherapy-induced increase in the transcription of the p53 response gene Bax leads to cytochrome c release and caspase activity. Activation of the Fas system has been observed in different systems, e.g. induction of FasL and upregulation of Fas following treatment of different tumour cell lines with doxorubicin, cisplatin, methotrexate, cytarabine and etoposide ^53-57. In addition, treatment of CML with the death receptor ligand interferon α brings about the upregulation of Fas on CML progenitors ⁵⁸. Patients with Fas-positive AML were shown to have a better therapeutic response in comparison with Fas-negative AML patients ⁵⁹. The improved understanding of the mechanisms of apoptosis and resistance to apoptosis has provided new insights for the development of new anti-cancer agents. TRAIL, a member of the TNF family of ligands, binds to the cell surface death receptors DR4 and DR5 and is expressed by most cells; most normal cells appear to be resistant to TRAIL-induced apoptosis due to expression of decoy receptors (DcR1 and DcR2), while transformed cells are sensitive^60-62. The dysregulation of different members of the Bcl-2 family in many cancer types led to the search for small inhibitors of this protein family.

Small molecules with high affinity for the BH3-domain on the surface of Bcl-2 induce apoptosis ^63-66. Gossypol, a natural product found in cottonseeds, interacts with the BH3-binding pocket of four anti-apoptotic proteins ^{67, 68}.  The suppression of NF-κB is another strategy for developing new cancer therapies. This transcription factor induces expression of several anti-apoptotic genes (Bcl-2, Bcl-Xl, c-IAP-2), and NF-κB-overexpression has been found in different cancers ^{69, 70}. Recently, the Smac/Diablo protein was discovered. This protein binds to and inhibits the IAP-family proteins and promotes apoptosis ⁷¹.

CONCLUSION: For all these therapeutic options, the basic idea of selective activation of apoptosis in transformed cells remains the key issue and may result in the development of new therapeutic agents, more active and/or less toxic than the ones used currently. In the future, patient-specific profiles of apoptosis-related genetic alterations and genetic comparisons between chemotherapy-sensitive and chemotherapy-resistant cells will open the way for patient-specific apoptosis-based therapy with hopefully fewer adverse effects ^{72, 73}.

Indeed, this pathway is frequently impaired in cancer cells and contributes to the development of resistance to conventional chemotherapy. Several small molecules, targeted anti/pro-oxidants and antisense oligonucleotides have been designed to activate pro-apoptotic proteins as well as to block anti-apoptotic proteins and are currently under clinical evaluation. Results of clinical trials will determine whether the promise that these strategies hold will be realized for a significant improvement in the clinical management of cancers that are refractory to conventional interventions.

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    How to cite this article:

    Rahman Md. A: Apoptosis Signaling; A Drug Design in Cancer Therapy: A Review. Int J Pharm Sci Res, 2012; Vol. 3(7): 1946-1954

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