2.3.1 The extrinsic death receptor pathway

The extrinsic death receptor pathway, as its name implies, begins when death ligands bind to a death receptor. Although several death receptors have been described, the best known death receptors is the type 1 TNF receptor (TNFR1) and a related protein called Fas (CD95) and their ligands are called TNF and Fas ligand (FasL) respectively [17]. These death receptors have an intracellular death domain that recruits adapter proteins such as TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD), as well as cysteine proteases like caspase 8 [23]. Binding of the death ligand to the death receptor results in the formation of a binding site for an adaptor protein and the whole ligand-receptor-adaptor protein complex is known as the death-inducing signalling complex (DISC) [22]. DISC then initiates the assembly and activation of pro-caspase 8. The activated form of the enzyme, caspase 8 is an initiator caspase, which initiates apoptosis by cleaving other downstream or executioner caspases [24].

2.3.2 The intrinsic mitochondrial pathway

As its name implies, the intrinsic pathway is initiated within the cell. Internal stimuli such as irreparable genetic damage, hypoxia, extremely high concentrations of cytosolic Ca²⁺ and severe oxidative stress are some triggers of the initiation of the intrinsic mitochondrial pathway [24]. Regardless of the stimuli, this pathway is the result of increased mitochondrial permeability and the release of pro-apoptotic molecules such as cytochrome-c into the cytoplasm [25]. This pathway is closely regulated by a group of proteins belonging to the Bcl-2 family, named after the BCL2 gene originally observed at the chromosomal breakpoint of the translocation of chromosome 18 to 14 in follicular non-Hodgkin lymphoma [26]. There are two main groups of the Bcl-2 proteins, namely the pro-apoptotic proteins (e.g. Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim and Hrk) and the anti-apoptotic proteins (e.g. Bcl-2, Bcl-X_L, Bcl-W, Bfl-1 and Mcl-1) [27]. While the anti-apoptotic proteins regulate apoptosis by blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins act by promoting such release. It is not the absolute quantity but rather the balance between the pro- and anti-apoptotic proteins that determines whether apoptosis would be initiated [27]. Other apoptotic factors that are released from the mitochondrial intermembrane space into the cytoplasm include apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac), direct IAP Binding protein with Low pI (DIABLO) and Omi/high temperature requirement protein A (HtrA2) [28]. Cytoplasmic release of cytochrome c activates caspase 3 via the formation of a complex known as apoptosome which is made up of cytochrome c, Apaf-1 and caspase 9 [28]. On the other hand, Smac/DIABLO or Omi/HtrA2 promotes caspase activation by binding to inhibitor of apoptosis proteins (IAPs) which subsequently leads to disruption in the interaction of IAPs with caspase-3 or -9 [28, 29].

2.3.3 The common pathway

The execution phase of apoptosis involves the activation of a series of caspases. The upstream caspase for the intrinsic pathway is caspase 9 while that of the extrinsic pathway is caspase 8. The intrinsic and extrinsic pathways converge to caspase 3. Caspase 3 then cleaves the inhibitor of the caspase-activated deoxyribonuclease, which is responsible for nuclear apoptosis [30]. In addition, downstream caspases induce cleavage of protein kinases, cytoskeletal proteins, DNA repair proteins and inhibitory subunits of endonucleases family. They also have an effect on the cytoskeleton, cell cycle and signalling pathways, which together contribute to the typical morphological changes in apoptosis [30].

2.3.4 The intrinsic endoplasmic reticulum pathway

This intrinsic endoplasmic reticulum (ER) pathway is a third pathway and is less well known. It is believed to be caspase 12-dependent and mitochondria-independent [31]. When the ER is injured by cellular stresses like hypoxia, free radicals or glucose starvation, there is unfolding of proteins and reduced protein synthesis in the cell, and an adaptor protein known as TNF receptor associated factor 2 (TRAF2) dissociates from procaspase-12, resulting in the activation of the latter [22].

3.1.1 The Bcl-2 family of proteins

The Bcl-2 family of proteins is comprised of pro-apoptotic and anti-apoptotic proteins that play a pivotal role in the regulation of apoptosis, especially via the intrinsic pathway as they reside upstream of irreversible cellular damage and act mainly at the mitochondria level [33]. Bcl-2 was the first protein of this family to be identified more than 20 years ago and it is encoded by the BCL2 gene, which derives its name from B-cell lymphoma 2, the second member of a range of proteins found in human B-cell lymphomas with the t (14; 18) chromosomal translocation [26].

All the Bcl-2 members are located on the outer mitochondrial membrane. They are dimmers which are responsible for membrane permeability either in the form of an ion channel or through the creation of pores [34]. Based of their function and the Bcl-2 homology (BH) domains the Bcl-2 family members are further divided into three groups [35]. The first group are the anti-apoptotic proteins that contain all four BH domains and they protect the cell from apoptotic stimuli. Some examples are Bcl-2, Bcl-xL, Mcl-1, Bcl-w, A1/Bfl-1, and Bcl-B/Bcl2L10. The second group is made up of the BH-3 only proteins, so named because in comparison to the other members, they are restricted to the BH3 domain. Examples in this group include Bid, Bim, Puma, Noxa, Bad, Bmf, Hrk, and Bik. In times of cellular stresses such as DNA damage, growth factor deprivation and endoplasmic reticulum stress, the BH3-only proteins, which are initiators of apoptosis, are activated. Therefore, they are pro-apoptotic. Members of the third group contain all four BH domains and they are also pro-apoptotic. Some examples include Bax, Bak, and Bok/Mtd [35].

When there is disruption in the balance of anti-apoptotic and pro-apoptotic members of the Bcl-2 family, the result is dysregulated apoptosis in the affected cells. This can be due to an overexpression of one or more anti-apoptotic proteins or an underexpression of one or more pro-apoptotic proteins or a combination of both. For example, Raffo et al showed that the overexpression of Bcl-2 protected prostate cancer cells from apoptosis [36] while Fulda et al reported Bcl-2 overexpression led to inhibition of TRAIL-induced apoptosis in neuroblastoma, glioblastoma and breast carcinoma cells [37]. Overexpression of Bcl-xL has also been reported to confer a multi-drug resistance phenotype in tumour cells and prevent them from undergoing apoptosis [38]. In colorectal cancers with microsatellite instability, on the other hand, mutations in the bax gene are very common. Miquel et al demonstrated that impaired apoptosis resulting from bax(G)8 frameshift mutations could contribute to resistance of colorectal cancer cells to anticancer treatments [39]. In the case of chronic lymphocytic leukaemia (CLL), the malignant cells have an anti-apoptotic phenotype with high levels of anti-apoptotic Bcl-2 and low levels of pro-apoptotic proteins such as Bax in vivo. Leukaemogenesis in CLL is due to reduced apoptosis rather than increased proliferation in vivo[40]. Pepper et al reported that B-lymphocytes in CLL showed an increased Bcl-2/Bax ratio in patients with CLL and that when these cells were cultured in vitro, drug-induced apoptosis in B-CLL cells was inversely related to Bcl-2/Bax ratios [41].

3.1.2 p53

The p53 protein, also called tumour protein 53 (or TP 53), is one of the best known tumour suppressor proteins encoded by the tumour suppressor gene TP53 located at the short arm of chromosome 17 (17p13.1). It is named after its molecular weights, i.e., 53 kDa [42]. It was first identified in 1979 as a transformation-related protein and a cellular protein accumulated in the nuclei of cancer cells binding tightly to the simian virus 40 (SV40) large T antigen. Initially, it was found to be weakly-oncogenic. It was later discovered that the oncogenic property was due to a p53 mutation, or what was later called a "gain of oncogenic function" [43]. Since its discovery, many studies have looked into its function and its role in cancer. It is not only involved in the induction of apoptosis but it is also a key player in cell cycle regulation, development, differentiation, gene amplification, DNA recombination, chromosomal segregation and cellular senescence [44] and is called the "guardian of the genome" [45].

Defects in the p53 tumour suppressor gene have been linked to more than 50% of human cancers [43]. Recently, Avery-Kieida et al reported that some target genes of p53 involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma cells, leading to abnormal activity of p53 and contributing to the proliferation of these cells [46]. In a mouse model with an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53, Slatter et al demonstrated that these mice had decreased survival, a different and more aggressive tumor spectrum, a marked proliferative advantage on cells, reduced apoptosis and a profound proinflammatory phenotype [47]. In addition, it has been found that when the p53 mutant was silenced, such down-regulation of mutant p53 expression resulted in reduced cellular colony growth in human cancer cells, which was found to be due to the induction of apoptosis [48].

3.1.3 Inhibitor of apoptosis proteins (IAPs)

The inhibitor of apoptosis proteins are a group of structurally and functionally similar proteins that regulate apoptosis, cytokinesis and signal transduction. They are characterised by the presence of a baculovirus IAP repeat (BIR) protein domain [29]. To date eight IAPs have been identified, namely, NAIP (BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), X-linked IAP (XIAP, BIRC4), Survivin (BIRC5), Apollon (BRUCE, BIRC6), Livin/ML-IAP (BIRC7) and IAP-like protein 2 (BIRC8) [49]. IAPs are endogenous inhibitors of caspases and they can inhibit caspase activity by binding their conserved BIR domains to the active sites of caspases, by promoting degradation of active caspases or by keeping the caspases away from their substrates [50].

Dysregulated IAP expression has been reported in many cancers. For example, Lopes et al demonstrated abnormal expression of the IAP family in pancreatic cancer cells and that this abnormal expression was also responsible for resistance to chemotherapy. Among the IAPs tested, the study concluded that drug resistance correlated most significantly with the expression of cIAP-2 in pancreatic cells [51]. On the other hand, Livin was demonstrated to be highly expressed in melanoma and lymphoma [52, 53] while Apollon, was found to be upregulated in gliomas and was responsible for cisplatin and camptothecin resistance [54]. Another IAP, Survivin, has been reported to be overexpressed in various cancers. Small et al. observed that transgenic mice that overexpressed Survivin in haematopoietic cells were at an increased risk of haematological malignancies and that haematopoietic cells engineered to overexpress Survivin were less susceptible to apoptosis [55]. Survivin, together with XIAP, was also found to be overexpressed in non-small cell lung carcinomas (NSCLCs) and the study concluded that the overexpression of Survivin in the majority of NSCLCs together with the abundant or upregulated expression of XIAP suggested that these tumours were endowed with resistance against a variety of apoptosis-inducing conditions [56].

4.1.1Agents that target the Bcl-2 family of proteins

One good example of these agents is the drug oblimersen sodium, which is a Bcl-2 antisence oblimer, the first agent targeting Bcl-2 to enter clinical trial. The drug has been reported to show chemosensitising effects in combined treatment with conventional anticancer drugs in chronic myeloid leukaemia patients and an improvement in survival in these patients [66, 67]. Other examples included in this category are the small molecule inhibitors of the Bcl-2 family of proteins. These can be further divided into: 1) those molecules that affect gene or protein expression and 2) those acting on the proteins themselves. Examples for the first group include sodium butyrate, depsipetide, fenretinide and flavipirodol while the second group includes gossypol, ABT-737, ABT-263, GX15-070 and HA14-1 (reviewed by Kang and Reynold, 2009 [68]).

Some of these small molecules belong to yet another class of drugs called BH3 mimetics, so named because they mimic the binding of the BH3-only proteins to the hydrophobic groove of anti-apoptotic proteins of the Bcl-2 family. One classical example of a BH3 mimetic is ABT-737, which inhibits anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Bcl-W. It was shown to exhibit cytotoxicity in lymphoma, small cell lung carcinoma cell line and primary patient-derived cells and caused regression of established tumours in animal models with a high percentage of cure [69]. Other BH3 mimetics such as ATF4, ATF3 and NOXA have been reported to bind to and inhibit Mcl-1 [70].

4.1.2 Silencing the anti-apoptotic proteins/genes

Rather than using drugs or therapeutic agents to inhibit the anti-apoptotic members of the Bcl-2 family, some studies have demonstrated that by silencing genes coding for the Bcl-2 family of anti-apoptotic proteins, an increase in apoptosis could be achieved. For example, the use of Bcl-2 specific siRNA had been shown to specifically inhibit the expression of target gene in vitro and in vivo with anti-proliferative and pro-apoptotic effects observed in pancreatic carcinoma cells [71]. On the other hand, Wu et al demonstrated that by silencing Bmi-1 in MCF breast cancer cells, the expression of pAkt and Bcl-2 was downregulated, rendering these cells more sensitive to doxorubicin as evidenced by an increase in apoptotic cells in vitro and in vivo[72].

4.2.1 p53-based gene therapy

The first report of p53 gene therapy in 1996 investigated the use of a wild-type p53 gene containing retroviral vector injected into tumour cells of non-small cell lung carcinoma derived from patients and showed that the use of p53-based gene therapy may be feasible [73]. As the use of the p53 gene alone was not enough to eliminate all tumour cells, later studies have investigated the use of p53 gene therapy concurrently with other anticancer strategies. For example, the introduction of wild-type p53 gene has been shown to sensitise tumour cells of head and neck, colorectal and prostate cancers and glioma to ionising radiation [74]. Although a few studies managed to go as far as phase III clinical trials, no final approval from the FDA has been granted so far [75]. Another interesting p53 gene-based strategy was the use of engineered viruses to eliminate p53-deficient cells. One such example is the use of a genetically engineered oncolytic adenovirus, ONYX-015, in which the E1B-55 kDa gene has been deleted, giving the virus the ability to selectively replicate in and lyse tumour cells deficient in p53 [76].

4.2.2 p53-based drug therapy

Several drugs have been investigated to target p53 via different mechanisms. One class of drugs are small molecules that can restore mutated p53 back to their wild-type functions. For example, Phikan083, a small molecule and carbazole derivative, has been shown to bind to and restore mutant p53 [77]. Another small molecule, CP-31398, has been found to intercalate with DNA and alter and destabilise the DNA-p53 core domain complex, resulting in the restoration of unstable p53 mutants [78]. Other drugs that have been used to target p53 include the nutlins, MI-219 and the tenovins. Nutlins are analogues of cis-imidazoline, which inhibit the MSM2-p53 interaction, stabilise p53 and selectively induce senescence in cancer cells [79] while MI-219 was reported to disrupt the MDM2-p53 interaction, resulting in inhibition of cell proliferation, selective apoptosis in tumour cells and complete tumour growth inhibition [80]. The tenovins, on the other hand, are small molecule p53 activators, which have been shown to decrease tumour growth in vivo[81].

4.2.3 p53-based immunotherapy

Several clinical trials have been carried out using p53 vaccines. In a clinical trial by Kuball et al, six patients with advanced-stage cancer were given vaccine containing a recombinant replication-defective adenoviral vector with human wild-type p53. When followed up at 3 months post immunisation, four out of the six patients had stable disease. However, only one patient had stable disease from 7 months onwards [82]. Other than viral-based vaccines, dendritic-cell based vaccines have also been attempted in clinical trials. Svane et al tested the use of p53 peptide pulsed dendritic cells in a phase I clinical trial and reported a clinical response in two out of six patients and p53-specific T cell responses in three out of six patients [83]. Other vaccines that have been used including short peptide-based and long peptide-based vaccines (reviewed by Vermeij R et al., 2011 [84]).

4.3.1 Targeting XIAP

When designing novel drugs for cancers, the IAPs are attractive molecular targets. So far, XIAP has been reported to be the most potent inhibitor of apoptosis among all the IAPs. It effectively inhibits the intrinsic as well as extrinsic pathways of apoptosis and it does so by binding and inhibiting upstream caspase-9 and the downstream caspases-3 and -7 [85]. Some novel therapy targeting XIAP include antisense strategies and short interfering RNA (siRNA) molecules. Using the antisense approach, inhibition of XIAP has been reported to result in an improved in vivo tumour control by radiotherapy [86]. When used together with anticancer drugs XIAP antisense oligonucleotides have been demonstrated to exhibit enhanced chemotherapeutic activity in lung cancer cells in vitro and in vivo[87]. On the other hand, Ohnishi et al reported that siRNA targeting of XIAP increased radiation sensitivity of human cancer cells independent of TP53 status [88] while Yamaguchi et al reported that targeting XIAP or Survivin by siRNAs sensitise hepatoma cells to death receptor- and chemotherapeutic agent-induced cell death [89].

4.3.2 Targeting Survivin

Many studies have investigated various approaches targeting Survivin for cancer intervention. One example is the use of antisense oligonucleotides. Grossman et al was among the first to demonstrate the use of the antisense approach in human melanoma cells. It was shown that transfection of anti-sense Survivin into YUSAC-2 and LOX malignant melanoma cells resulted in spontaneous apoptosis in these cells [90]. The anti-sense approach has also been applied in head and neck squamous cell carcinoma and reported to induce apoptosis and sensitise these cells to chemotherapy [91] and in medullary thyroid carcinoma cells, and was found to inhibit growth and proliferation of these cells [92]. Another approach in targeting Survivin is the use of siRNAs, which have been shown to downregulate Survivin and diminish radioresistance in pancreatic cancer cells [93], to inhibit proliferation and induce apoptosis in SPCA1 and SH77 human lung adenocarcinoma cells [94], to suppress Survivin expression, inhibit cell proliferation and enhance apoptosis in SKOV3/DDP ovarian cancer cells [95] as well as to enhance the radiosensitivity of human non-small cell lung cancer cells [96]. Besides, small molecules antagonists of Survivin such as cyclin-dependent kinase inhibitors and Hsp90 inhibitors and gene therapy have also been attempted in targeting Survivin in cancer therapy (reviewed by Pennati et al., 2007 [97]).

4.3.3 Other IAP antagonists

Other IAP antagonists include peptidic and non-peptidic small molecules, which act as IAP inhibitors. Two cyclopeptidic Smac mimetics, 2 and 3, which were found to bind to XIAP and cIAP-1/2 and restore the activities of caspases- 9 and 3/-7 inhibited by XIAP were amongst the many examples [98]. On the other hand, SM-164, a non-peptidic IAP inhibitor was reported to strongly enhance TRAIL activity by concurrently targeting XIAP and cIAP1 [99].

4.4.1 Caspase-based drug therapy

Several drugs have been designed to synthetically activate caspases. For example, Apoptin is a caspase-inducing agent which was initially derived from chicken anaemia virus and had the ability to selectively induce apoptosis in malignant but not normal cells [100]. Another class of drugs which are activators of caspases are the small molecules caspase activators. These are peptides which contain the arginin-glycine-aspartate motif. They are pro-apoptotic and have the ability to induce auto-activation of procaspase 3 directly. They have also been shown to lower the activation threshold of caspase or activate caspase, contributing to an increase in drug sensitivity of cancer cells [101].

4.4.2 Caspase-based gene therapy

In addition to caspase-based drug therapy, caspase-based gene therapy has been attempted in several studies. For instance, human caspase-3 gene therapy was used in addition to etoposide treatment in an AH130 liver tumour model and was found to induce extensive apoptosis and reduce tumour volume [102] while gene transfer of constitutively active caspse-3 into HuH7 human hepatoma cells selectively induced apoptosis in these cells [103]. Also, a recombinant adenovirus carrying immunocaspase 3 has been shown to exert anti-cancer effects in hepatocellular carcinoma in vitro and in vivo[104].