Abstract
Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. One of the most important advances in cancer research in recent years is the recognition that cell death mostly by apoptosis is crucially involved in the regulation of tumor formation and also critically determines treatment response. Killing of tumor cells by most anticancer strategies currently used in clinical oncology, for example, chemotherapy, γ-irradiation, suicide gene therapy or immunotherapy, has been linked to activation of apoptosis signal transduction pathways in cancer cells such as the intrinsic and/or extrinsic pathway. Thus, failure to undergo apoptosis may result in treatment resistance. Understanding the molecular events that regulate apoptosis in response to anticancer chemotherapy, and how cancer cells evade apoptotic death, provides novel opportunities for a more rational approach to develop molecular-targeted therapies for combating cancer.
Introduction
Current cancer therapies, for example, chemotherapy, γ-irradiation, immunotherapy or suicide gene therapy, primarily exert their antitumor effect by triggering apoptosis in cancer cells (Makin and Dive, 2001; Fulda and Debatin, 2004). Apoptosis is an evolutionary conserved, intrinsic program of cell death that occurs in various physiological and pathological situations (Hengartner, 2000). Apoptosis is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, nuclear DNA fragmentation and membrane blebbing (Hengartner, 2000). The underlying mechanisms for initiation of an apoptosis response upon cytotoxic therapy may depend on the individual stimulus and have often not exactly been identified. However, damage to DNA or to other critical molecules is considered to be a common initial event which is then propagated by the cellular stress response (Rich et al., 2000). Multiple stress-inducible molecules, for example, c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK), nuclear factor kappa B (NF-κB) or ceramide, have been implied in transmitting the apoptotic signal (Davis, 2000; Karin et al., 2002). Proteolytic enzymes such as caspases are important effector molecules in apoptosis (Degterev et al., 2003). Activation of caspases in response to anticancer chemotherapy can be initiated through activation of the extrinsic (receptor) pathway or at the mitochondria by stimulating the intrinsic pathway (Fulda and Debatin, 2004). Because of the potential detrimental effects on cell survival in case of inappropriate activation of apoptosis programs, apoptosis pathways have to be tightly controlled. The antiapoptotic mechanisms regulating cell death have also been implicated in conferring drug resistance to tumor cells (Fulda and Debatin, 2004). However, the concept that apoptosis represents the major mechanism by which cancer cells are eliminated may not universally apply and caspase-independent apoptosis or other modes of cell death have also to be considered as cellular response to anticancer therapy (Brown and Wilson, 2003; Brown and Attardi, 2005). Thus, a better understanding of these diverse modes of cell death in cancer therapy will provide a molecular basis for new strategies targeting cell death pathways in resistant forms of cancer.
Apoptosis signaling pathways
In most cases, anticancer therapies eventually result in activation of caspases, a family of cysteine proteases that act as common death effector molecules in various forms of cell death (Degterev et al., 2003). Caspases are synthesized as inactive proforms and upon activation, they cleave next to aspartate residues (Degterev et al., 2003). The fact that caspases can activate each other by cleavage at identical sequences results in amplification of caspase activity through a protease cascade (Degterev et al., 2003). Caspases cleave a number of different substrates in the cytoplasm or nucleus leading to many of the morphologic features of apoptotic cell death (Degterev et al., 2003). For example, polynucleosomal DNA fragmentation is mediated by cleavage of ICAD (inhibitor of caspase-activated DNase), the inhibitor of the endonuclease CAD (caspase-activated DNase) that cleaves DNA into the characteristic oligomeric fragments (Nagata, 2000). Likewise, proteolysis of several cytoskeletal proteins such as actin or fodrin leads to loss of overall cell shape, whereas degradation of lamin results in nuclear shrinking (Degterev et al., 2003).
Activation of caspases can be initiated from different entry points, for example, at the plasma membrane upon ligation of death receptor (receptor pathway) or at the mitochondria (mitochondrial pathway) (Hengartner, 2000) (Figure 1). Stimulation of death receptors of the tumor necrosis factor (TNF) receptor superfamily such as CD95 (APO-1/Fas) or TNF-related apoptosis-inducing ligand (TRAIL) receptors results in activation of the initiator caspase-8 which can propagate the apoptosis signal by direct cleavage of downstream effector caspases such as caspase-3 (Walczak and Krammer, 2000). The mitochondrial pathway is initiated by the release of apoptogenic factors such as cytochrome c, apoptosis-inducing factor (AIF), Smac (second mitochondria-derived activator of caspase)/DIABLO (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI), Omi/HtrA2 or endonuclease G from the mitochondrial intermembrane space (Cande et al., 2002; Saelens et al., 2004). The release of cytochrome c into the cytosol triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, whereas Smac/DIABLO and Omi/HtrA2 promote caspase activation through neutralizing the inhibitory effects to the IAPs (Saelens et al., 2004).
Links between the receptor and the mitochondrial pathway exist at different levels. Upon death receptor triggering, activation of caspase-8 may result in cleavage of Bid, a Bcl-2 family protein with a BH3 domain only, which in turn translocates to mitochondria to release cytochrome c thereby initiating a mitochondrial amplification loop (Cory and Adams, 2002). In addition, cleavage of caspase-6 downstream of mitochondria may feed back to the receptor pathway by cleaving caspase-8 (Cowling and Downward, 2002).
Nonapoptotic forms of cell death
Although apoptosis pathways and signal-transducing molecules have been shown to play a crucial role for killing of tumor cells in response to cytotoxic agents used in the treatment of cancer patients, the significance of apoptosis as the main mechanism of cancer cell death in response to anticancer chemotherapy has also been challenged. So far, no clear pattern has emerged between the level of apoptosis or proteins that regulate apoptosis and treatment response in most solid tumors. In addition to apoposis, cancer cells have also been shown to be effectively eliminated after DNA damage by necrosis, mitotic catastrophe and autophagy (Brown and Wilson, 2003; Brown and Attardi, 2005). Thus, nonapoptotic modes of cell death have also been taken into consideration as response to cytotoxic therapy. Although the signaling pathways and molecules involved in these alternative forms of cell death have not yet exactly been defined, non-caspase proteases such as calpains or cathepsins, lysosomal enzymes or PARP-1 may be involved (Okada and Mak, 2004). The form of cell death may depend on the context, including the cell type, the genotype of the cell, the type of DNA damage and the dose of the agent used. Thus, the relative contribution of these different modes of cell death for chemoresponses in vitro and in vivo remains to be defined.
Extrinsic pathway
Death receptors are members of the tumor necrosis factor (TNF) receptor gene superfamily that consists of more than 20 proteins with a broad range of biological functions, including regulation of cell death and survival, differentiation or immune regulation (Walczak and Krammer, 2000; Ashkenazi, 2002). Members of the TNF receptor family share similar, cysteine-rich extracellular domains. In addition, death receptors are defined by a cytoplasmic domain of about 80 amino acids called ‘death domain’, which plays a crucial role in transmitting the death signal from the cell's surface to intracellular signaling pathways. The best-characterized death receptors include CD95 (APO-1/Fas), TNF receptor 1 (TNFRI), TNF-related apoptosis-inducing ligand-receptor 1 (TRAIL-R1) and TRAIL-R2, whereas the role of DR3 (TRAMP/Apo-3/WSL-1/LARD) or DR6 has not exactly been defined (Walczak and Krammer, 2000). The corresponding ligands of the TNF superfamily comprise death receptor ligands such as CD95 ligand (CD95L), TNFα, lymphotoxin-α (the later two bind to TNFRI), TRAIL and TWEAK, a ligand for DR3 (Walczak and Krammer, 2000).
CD95
The CD95 receptor/CD95L system is a key signal pathway involved in the regulation of apoptosis in several different cell types, for example, in the immune system (Krammer, 2000). CD95, a 48 kDa type I transmembrane receptor, is expressed on activated lymphocytes, on a variety of tissues of lymphoid or non-lymphoid origin, as well as on tumor cells (Krammer, 2000). CD95 ligand is produced by activated T cells and plays a crucial role in the regulation of the immune system by triggering autocrine suicide or paracrine death in lymphocytes or other target cells (Krammer, 2000). Furthermore, CD95L expression on cancer cells has been implicated in immune escape of tumors (Krammer, 2000). By constitutive expression of death receptor ligands such as CD95L, tumors may adopt a killing mechanism from cytotoxic lymphocytes to delete the attacking antitumor T cells through induction of apoptosis via CD95/CD95L interaction. However, this model of tumor counterattack has also been challenged, since no study has so far conclusively demonstrated that tumor counterattack is a relevant immune escape mechanism in vivo (Igney and Krammer, 2002).
TNF-related apoptosis-inducing ligand and its receptors
TNF-related apoptosis-inducing ligand/Apo-2L was identified in 1995 based on its sequence homology to other members of the TNF superfamily and is constitutively expressed in a wide range of tissues (LeBlanc and Ashkenazi, 2003). The two agonistic TRAIL receptors, TRAIL-R1 and TRAIL-R2, contain a conserved cytoplasmic death domain motif, which enables them to engage the cell's apoptotic machinery upon ligand binding, whereas TRAIL-R3 to R5 are antagonistic decoy receptors, which bind TRAIL, but do not transmit a death signal (LeBlanc and Ashkenazi, 2003).
Signaling through CD95 or TRAIL receptors
Ligation of death receptors such as CD95 or the agonistic TRAIL receptors TRAIL-R1 and TRAIL-R2 by their cognate ligands or agonistic antibodies results in receptor trimerization, clustering of the receptors' death domains and recruitment of adaptor molecules such as Fas-associated death domain (FADD) through homophilic interaction mediated by the death domain (Walczak and Krammer, 2000). Fas-associated death domain in turn recruits caspase-8 to the activated CD95 receptor to form the CD95 death-inducing signaling complex (DISC) (Kischkel et al., 1995). Oligomerization of caspase-8 upon DISC formation drives its activation through self-cleavage. Caspase-8 then activates downstream effector caspases such as caspase-3. For the CD95 signaling pathway, two distinct prototypic cell types have been identified (Scaffidi et al., 1998). In type I cells, caspase-8 is activated upon death receptor ligation at the DISC in quantities sufficient to directly activate downstream effector caspases such as caspase-3 (Scaffidi et al., 1998). In type II cells, however, the amount of active caspase-8 generated at the DISC is insufficient to activate caspase-3 and a mitochondrial amplification loop is required for full activation of caspases (Scaffidi et al., 1998). Also, a similar cell type-dependent organization (type I and type II) of the TRAIL signaling pathway has been described (Fulda et al., 2002a).
Disruption of the extrinsic pathway in cancers
Cancer cells have evolved numerous strategies to resist cell death induction via the extrinsic pathway. Signaling to cell death in response to death receptor ligation can principally be inhibited by an increase in antiapoptotic molecules or by a decrease or defective function in proapoptotic proteins.
For example, surface expression of death receptors may vary between different cell types and can be downregulated or absent in resistant forms of tumors. To this end, drug-resistant leukemia or neuroblastoma cells showed strong downregulation of CD95 expression suggesting that critical levels of CD95 expression may have an impact on drug sensitivity (Friesen et al., 1997; Fulda et al., 1998b). Also, deficient transport of the agonistic TRAIL receptors TRAIL-R1 and TRAIL-R2 from intracellular stores, for example, endoplasmatic reticulum, to the cell surface was identified as a mechanism conferring TRAIL resistance in colon cancer cells (Jin et al., 2004). Mutations of the CD95 gene have been identified in a variety of hematological malignancies and solid tumors (Debatin et al., 2003). The incidence of CD95 mutations found in human tumors has been taken as evidence that the CD95 system exerts a tumor suppressor function. Moreover, decoy receptor 3 (DcR3), which act as decoy receptors by competitively binding CD95L, can interfere with CD95-triggered apoptosis and was found to be genetically amplified or overexpressed in lung carcinoma, colon carcinoma or glioblastoma (Pitti et al., 1998; Roth et al., 2001). A decoy receptor for TRAIL, TRAIL-R3, was found to be overexpressed in gastric carcinoma (Sheikh et al., 1999). Moreover, loss of expression of the agonistic TRAIL receptors TRAIL-R1 and -R2 may account for TRAIL resistance. Both receptors are located on chromosome 8p, a region of frequent loss of heterozygosity (LOH) in tumors (LeBlanc and Ashkenazi, 2003). In a small percentage of cancers, for example, non-Hodgkin's lymphoma, colorectal, breast, head and neck cancer, osteosarcoma or lung carcinoma, deletions or mutations were found, which resulted in loss of both copies of TRAIL-R1 or R2 (Pai et al., 1998; Dechant et al., 2004).
Impaired surface expression of CD95 or TRAIL receptors may also be caused by epigenetic changes such as CpG-island hypermethylation of gene promoters, for example, in neuroblastoma or colon carcinoma cells (van Noesel et al., 2002; Petak et al., 2003). Also, alterations in chromatin structure, for example, chromatin condensation because of histone deacetylation, may block transcription by preventing the access of transcription factors to the DNA (Marks et al., 2001). Interestingly, epigenetic changes in CD95 expression have been reported to determine immune escape and response to therapy (Maecker et al., 2002). In tumors with epigentically silenced CD95, restoration of CD95 expression by histone deacetylase inhibitors resulted in suppression of tumor growth and restoration of chemosensitivity in an natural killer (NK) cell-dependent manner (Maecker et al., 2002).
Signaling by death receptors can also be negatively regulated by proteins that associate with their cytoplasmatic domains, for example, FLIP or phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15) (Hao et al., 2001; Krueger et al., 2001). Two splice variants of FLIP, a long form (FLIPL) and a short form (FLIPS), have been identified in human cells, which have sequence homology to caspase-8 and caspase-10, but lack its catalytic site (Krueger et al., 2001). Consequently, the recruitment of FLIP to the DISC instead of procaspase-8 or -10 can block caspase activation (Krueger et al., 2001). High FLIP expression has been found in many tumor cells and has been correlated with resistance to CD95- or TRAIL- and also to chemotherapy-induced apoptosis (Fulda et al., 2000; Longley et al., 2006). However, the impact of FLIP on apoptosis sensitivity towards cytotoxic drugs may vary between cell types, since overexpression of FLIP did not confer protection against cytotoxic drugs in T-cell leukemia cells (Kataoka et al., 1998), whereas FLIP antisense oligonucleotides or short interference RNA sensitized osteosarcoma or colorectal cancer cells for chemotherapeutic drugs (Kinoshita et al., 2000; Longley et al., 2006). The PEA-15 is another death effector domain (DED)-containing protein, which blocks CD95-, TRAIL- or TNFα-triggered apoptosis in a receptor-proximal manner by disrupting FADD and caspase-8 interactions (Hao et al., 2001). Recently, PEA-15 has also been implicated in mediating AKT-dependent chemoresistance in human breast cancer cells (Stassi et al., 2005).
Signaling through the extrinsic pathway in cancer therapy
The CD95 system has been implicated in chemotherapy-induced tumor cell death in a number of studies. To this end, treatment with anticancer drugs triggered an increase in CD95L expression, which stimulated the receptor pathway in an autocrine or paracrine manner by binding to its receptor CD95 (Friesen et al., 1996; Fulda et al., 1997, 1998a; Houghton et al., 1997; Muller et al., 1997). Various anticancer agents with different primary intracellular targets have been used in these studies including DNA-damaging agents such as doxorubicin, etoposide, cisplatin or bleomycin (Friesen et al., 1996; Fulda et al., 1997, 1998a; Muller et al., 1997). The CD95 receptor/ligand system has also been implicated in thymine-less death in colon carcinoma cells following treatment with 5-fluorouracil (Houghton et al., 1997). Activation of the transcription factors activator protein-1 (AP-1) and NF-κB was shown to mediate the increase in CD95L transcription and mRNA levels in response to chemotherapy and AP-1- and NF-κB-binding sites were identified in the human CD95L promotor (Kasibhatla et al., 1998). In addition to CD95L, CD95 expression on the cell's surface increased upon drug treatment, in particular in cells harboring wild-type p53 (Muller et al., 1997). p53-responsive elements have been identified in the first intron of the CD95 gene, as well as three putative p53-binding sites within the CD95 promotor, which showed limited homology with the p53 consensus-binding site (Muller et al., 1998). Moreover, antagonistic CD95L antibodies, soluble antagonistic CD95 receptors or DN-FADD were found to reduce drug-induced apoptosis under certain circumstances. In addition to upregulation of CD95L and CD95, anticancer agents have been reported to activate the CD95 pathway by modulating expression and recruitment of pro- or antiapoptotic components of the CD95 DISC to activated receptors. Upregulation of FADD and procaspase-8 was found upon treatment with doxorubicin, cisplatin or mitomycin C in colon carcinoma cells (Micheau et al., 1999). Also, increased recruitment of FADD and procaspase-8 to the CD95 receptor to form the CD95 DISC was observed in certain tumor cells upon drug treatment in a CD95L-dependent or CD95L-independent manner (Fulda et al., 2001). These findings indicate that in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may involve CD95L-initiated DISC formation and activation of downstream effector programs similar to activation-induced cell death (AICD) in T cells.
Despite the reproducibility of these findings in different model systems, other reports challenged the model that death receptor signaling is involved in drug-mediated cell death (Eischen et al., 1997; Villunger et al., 1997). To that end, antagonistic antibodies against CD95L or CD95 did not confer protection against apoptosis induced by cytotoxic drugs in other cell lines. Enforced expression of FLIP, DN-FADD or the serpin crmA did not inhibit drug-induced apoptosis, although it inhibited caspase-8 activation (Kataoka et al., 1998). Also, targeted disruption of genes involved in death receptor signaling conferred no protection against cytotoxic drug treatment, at least in nontransformed cells. The FADD−/− and caspase-8−/− fibroblasts were sensitive to cytotoxic drugs, whereas they remained resistant to death receptor stimulation (Varfolomeev et al., 1998; Yeh et al., 1998). In contrast, caspase-9−/− embryonic stem cells and Apaf-1−/− thymocytes remain sensitive to death receptor triggering, however they are resistant to cytotoxic drugs (Hakem et al., 1998; Yoshida et al., 1998)
The discrepancies in data may be explained by the relative contribution of the extrinsic versus the intrinsic pathway depending on the cytotoxic drug, dose or kinetics or on the differences between certain cell types. The discrepancies in data may also be explained by differences in the inhibitory reagents used to block CD95/CD95L interaction. The quality of CD95/CD95L-blocking agents including anti-CD95 antibody, anti-CD95L antibody or soluble decoy CD95-Fc receptor constructs may vary depending on their origin and preparation. Also, the lack of efficacy of these CD95/CD95L-neutralizing agents may be owing to inaccessibility of their proposed targets. Experiments with adenoviral delivery of a CD95L-green fluorescent protein (GFP) construct showed that CD95 and CD95L are stored intracellularly and colocalize to the same intracellular compartments, for example, the Golgi and/or endoplasmatic reticulum (Hyer et al., 2000). An anti-CD95-blocking antibody did not inhibit CD95L-induced cell death suggesting that CD95L may trigger CD95 within the same intracellular compartment and that these two molecules may already interact before surface presentation (Hyer et al., 2000). Thus, CD95/CD95L-neutralizing agents may under certain circumstances not even gain access to their targets prior to triggering of the CD95 pathway. Moreover, some studies that challenge an involvement of the CD95 system in chemotherapy of tumor cells are based on experiments performed in nontransformed cells, for example, embryonic fibroblasts, but not in cancer cells. However, the mechanisms regulating apoptosis in non-malignant cells may vary considerably from those in malignant tumor cells, which is highlighted by the differential sensitivity of these cell types to various death stimuli.
Exploiting death receptor signaling pathways for tumor therapy
Death receptor as targets
The idea to specifically target the extrinsic pathway to trigger apoptosis in tumor cells, for example by ligation of death receptors, is attractive for cancer therapy since death receptors have a direct link to the cell's death machinery (Ashkenazi, 2002). In addition, apoptosis upon death receptor triggering has been reported to occur independent of the p53 tumor suppressor gene, which is deleted or inactivated in more than half of human tumors (El-Deiry, 2001). However, the clinical application of CD95Lor TNFα is hampered by severe toxic side effects (Walczak and Krammer, 2000). In contrast, TRAIL appears to be a relatively safe and promising candidate for clinical application, particularly in its non-tagged, zinc-bound homotrimeric form (LeBlanc and Ashkenazi, 2003). Studies in non-human primates such as chimpanzees and cynomolgus monkeys showed no toxicity upon intravenous infusion, even at high doses (Ashkenazi et al., 1999). In addition, no cytotoxic activity of TRAIL was reported on a variety of normal human cells of different lineages including fibroblasts, endothelial cells, smooth muscle cells, epithelial cells or astrocytes (Ashkenazi et al., 1999; Lawrence et al., 2001; Pollack et al., 2001). However, some concerns about potential toxic side effects on human hepatocytes or brain tissue have also been raised (Jo et al., 2000; Nitsch et al., 2000). The loss of tumor selectivity may be related to the TRAIL preparations used in these studies, since TRAIL preparations, which are antibody-crosslinked or not optimized for Zn content, have been reported to form multimeric aggregates thereby overpassing the threshold of sensitivity of normal cells (Lawrence et al., 2001).
Antitumor activity of TNF-related apoptosis-inducing ligand
Recombinant soluble TRAIL induced apoptosis in a broad spectrum of cancer cell lines, including colon carcinoma, breast carcinoma, lung carcinoma, pancreas carcinoma, prostate carcinoma, renal carcinoma, thyroid carcinoma, malignant brain tumors, Ewing tumor, osteasarcoma, neuroblastoma, leukemia and lymphoma and also exhibited potent tumoricidal activity in vivo in several xenograft models of colon carcinoma, breast carcinoma, malignant glioma or multiple myeloma (Chinnaiyan et al., 2000; Nagane et al., 2000; Walczak et al., 2000; Pollack et al., 2001; Rohn et al., 2001). Furthermore, monoclonal antibodies that engage the TRAIL receptors DR4 or DR5 also demonstrated potent antitumor activity against tumor cell lines and in preclinical cancer models (Chuntharapai et al., 2001; Ichikawa et al., 2001). Currently, recombinant soluble TRAIL or agonistic TRAIL receptors are being evaluated in phase I clinical trials.
Combination therapy with TNF-related apoptosis-inducing ligand
Although these studies provided ample evidence of the potential of TRAIL for cancer therapy, many tumors remain refractory towards treatment with TRAIL, which has been related to the dominance of antiapoptotic signals. Importantly, numerous studies have shown that TRAIL synergized together with cytotoxic drugs or γ-irradiation to achieve antitumor activity in various cancer cell lines including malignant glioma, melanoma, leukemia, breast, colon or prostate carcinoma and also cooperated to suppress tumor growth in different mouse models of human cancers (Gliniak and Le, 1999; Chinnaiyan et al., 2000; Keane et al., 2000; Nagane et al., 2000; Walczak et al., 2000; Belka et al., 2001; Rohn et al., 2001). Dependent on the therapeutic combinations and cell type used, different mechanisms of sensitization to TRAIL-induced apoptosis have been identified. For example, the molecular mechanisms, which account for the synergistic interaction of TRAIL and conventional chemotherapeutics, may include transcriptional upregulation of the agonistic TRAIL receptors TRAIL-R1 and -R2, which occurred in a p53-dependent or p53-independent manner (Takimoto and El-Deiry, 2000; Meng and El-Deiry, 2001). Of note, p53 has also been shown to transcriptionally activate the antagonistic TRAIL receptors TRAIL-R3 and -R4 (Meng et al., 2000). Recent evidence suggest that p53 is crucial for sensitization to TRAIL by chemotherapy through transcriptional upregulation of TRAIL-R2 in some tumors, for example, mismatch repair-deficient colorectal cancer cells harboring Bax mutations (Wang and El-Deiry, 2003). Intriguingly, pre-exposure to chemotherapy restored TRAIL sensitivity through p53-mediated increase of TRAIL-R2 expression even in resistant colorectal carcinoma cells lacking Bax expression indicating that sequential combination of anticancer agents with TRAIL may overcome some forms of resistance (Wang and El-Deiry, 2003). In addition, chemotherapy has been reported to enhance receptor assembly of CD95 or TRAIL receptors, for example, in colon carcinoma cells (Lacour et al., 2004). The synergistic interaction between cytotoxic drugs and TRAIL may also be mediated by downregulation of antiapoptotic proteins such as Bcl-2, Bcl-XL or FLIP upon drug treatment (Olsson et al., 2001). In addition, anticancer agents may sensitize tumor cells for TRAIL treatment by upregulating proapoptotic molecules incuding caspases or FADD (Micheau et al., 1999). The cooperative interaction of chemotherapeutics and TRAIL has also been explained by the simultaneous activation of the intrinsic and extrinsic pathway leading to enhanced cell death through functional complementation. As TRAIL demonstrated synergistic interaction with anticancer agents or irradiation, TRAIL may be most effective in combination with conventional cancer treatments.
Intrinsic pathway of apoptosis
Signaling through the intrinsic pathway of apoptosis
In the mitochondrial pathway of apoptosis, caspase activation is closely linked to permeabilization of the outer mitochondrial membrane by proapoptotic members of the Bcl family (Green and Kroemer, 2004). Numerous cytotoxic stimuli and proapoptotic signal-transducing molecules converge on mitochondria to induce outer mitochondrial membrane permeabilization (Decaudin et al., 1998; Green and Kroemer, 2004). This permeabilization is regulated by proteins from the Bcl-2 family, mitochondrial lipids, proteins that regulate bioenergetic metabolite flux and components of the permeability transition pore (Green and Kroemer, 2004). Upon disruption of the outer mitochondrial membrane, a set of proteins normally found in the space between the inner and outer mitochondrial membranes is released, including cytochrome c, Smac/DIABLO, Omi/HtrA2, AIF and endonuclease G (Saelens et al., 2004). Once in the cytosol, these apoptogenic proteins trigger the execution of cell death by promoting caspase activation or by acting as caspase-independent death effectors (Saelens et al., 2004).
Mitochondrial mediators of caspase-dependent apoptosis
Cytochrome c
The release of cytochrome c from mitochondria directly triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex (Cain et al., 2000). Once in the cytosol, cytochrome c binds to the C-terminal region of Apaf-1, a cytosolic protein with an N-terminal caspase-recruitment domain (CARD), a nucleotide-binding domain with homology to Caenorhabditis elegans CED-4 and a C-terminal domain containing 12–13 WD-40 repeats (Zou et al., 1997). Binding of cytochrome c to Apaf-1 facilitates the association of dATP with Apaf-1 and exposes its N-terminal CARD, which can now oligomerize and become a platform on which the initiator caspase-9 is recruited and activated through a CARD–CARD interaction (Adrain et al., 1999). Consecutively, the executioner caspase-3 is recruited to the apoptosome, where it is activated by the resident caspase-9 (Bratton et al., 2001). Caspase-3 then cleaves key substrates in the cell to produce many of the cellular and biochemical events of apoptosis.
In certain cases, caspase activity instigated by cytosolic cytochrome c contributes to the drop of matrix metalloproteinase (MMP), as was shown by the use of synthetic caspase inhibitors and Apaf1 −/− cells (Waterhouse et al., 2001). In addition, caspase activity further damages the function of permeabilized mitochondria by affecting the activity of complexes I and II, inevitably leading to the loss of MMP and the generation of reactive oxygen species (Ricci et al., 2004). Thus, secondary events resulting from cytosolic changes, caused by the release of cytochrome c and other mitochondrial IMS proteins, can feed back on permeabilized mitochondria and affect their function. Importantly, this mitochondrial amplification loop of caspase activity may critically determine the response of cancer cells to cytotoxic treatments.
Moreover, there is recent evidence for the existence of a cytochrome c- and apoptosome-independent, but Apaf-1-dependent mechanism(s) for caspase activation. A targeted knock-in of a variant of cytochrome c that lacks apoptogenic properties, but has electron transfer and antioxidant activity (K72A), allowed the evaluation of the contribution of cytochrome c to apoptosis without affecting its function in oxidative phosphorylation (Hao et al., 2005). Interestingly, thymocytes from the KA/KA mice were markedly more sensitive to death stimuli including etoposide and γ-irradiation than Apaf-1(−/−) thymocytes (Hao et al., 2005). Upon treatment with γ-irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed (Hao et al., 2005) pointing to a differential requirement for cytochrome c and Apaf-1 in apoptosis.
Smac/DIABLO
Other proteins released from mitochondria, such as Smac/DIABLO and Omi/HtrA2, facilitate caspase activation through neutralizing endogenous inhibitors of caspases, the inhibitor of apoptosis proteins (IAPs). Smac and its murine homolog DIABLO are nuclear encoded mitochondrial proteins, which contain a mitochondrial localization signal, which is proteolytically removed upon mitochondrial import to yield the mature 23 kDa protein (Du et al., 2000; Verhagen et al., 2000). This maturation step exposes the IAP-binding motif (IBM) at the N-terminus of Smac/DIABLO (Du et al., 2000) (Table 1). Second mitochondria-derived activator of caspase/DIABLO was shown to bind to XIAP, cIAP1, cIAP2, survivin and Apollon in a BIR-dependent way (Vaux and Silke, 2003) (Figure 2),and acts as homodimer with the IBM present in a bivalent configuration (Chai et al., 2000). One Smac/DIABLO dimer binds one XIAP molecule by both IAP-binding motifs, one interacting with BIR2 and the other one with BIR3 (Huang et al., 2003). Intriguingly, the same BIR3 groove binds the IBM exposed at the N-terminus (Ala-Thr-Pro-Phe) of the small subunit of caspase-9 following its autocatalytic processing after Asp315, allowing Smac/DIABLO to displace caspase-9 from XIAP (Srinivasula et al., 2001) (Table 1).
The physiological mitochondrial function of Smac/DIABLO is unknown, and DIABLO −/− mice appear normal (Okada et al., 2002). Although the in vitro cleavage of procaspase-3 upon addition of cytochrome c was inhibited in lysates of Smac −/− cells, Smac −/− mice and cells responded normally to apoptotic stimuli such as UV irradiation, staurosporin, etoposide and TNF/cyclohexamide (Okada et al., 2002). These observations suggest the existence of redundant factors compensating for the loss of Smac/DIABLO, possibly HtrA2/OMI.
Omi/HtrA2 is a nuclear-encoded, 49 kDa protein with an N-terminal mitochondrial localization signal that mediates its translocation into the mitochondrial intermembrane space (Suzuki et al., 2001; Martins et al., 2002; van Loo et al., 2002). Omi/HtrA2 is processed in the intermembrane space into the 37 kDa mature form, liberating an IBM at its N-terminus (Suzuki et al., 2001; Martins et al., 2002; van Loo et al., 2002) (Table 1). Although recombinant Omi/HtrA2 can catalyse its own maturation in vitro, the protease responsible for its maturation in cells remains unknown (Martins et al., 2002). Omi/HtrA2 plays an essential role in regulating mitochondrial homeostasis requiring its proteolytic activity, although the molecular targets and interaction partners of Omi/HtrA2 in the mitochondrion have not yet been defined (Saelens et al., 2004). Once Omi/HtrA2 is released from mitochondria into the cytosol, it promotes cell death in a caspase-dependent way by antagonizing IAPs, and in a caspase-independent way as a protease (Saelens et al., 2004). Similar to Smac/DIABLO, Omi/HtrA2 blocks IAPs through its N-terminal IAP-binding motif, presented in a trimeric configuration (Li et al., 2002)
Although the release of cytochrome c into the cytosol directly triggers caspase-3 activation through formation of the cytochrome c/Apaf-1/caspase-9-containing apoptosome complex, Smac/DIABLO and Omi/HtrA2 indirectly promote caspase activation through antagonizing the inhibitory effects to IAPs (Saelens et al., 2004). Thus, a dynamic equilibrium exists between pro- and antiapoptotic effector molecules, which allows the cell to cope with limited mitochondrial damage, in which case IAPs can adequately block caspase activation initiated by a small amount of released cytochrome c. However, under circumstances where mitochondrial damage proceeds or simultaneously affects multiple mitochondria, the antiapoptotic hurdle imposed by IAPs can be overcome by the higher cytosolic concentration of their antagonists Smac/DIABLO and HtrA2/OMI, which neutralize IAPs by direct binding.
There is mounting evidence that cancer cells have an intrinsic drive to apoptosis that is held in check by IAPs. To this end, high basal levels of caspase-3 and caspase-8 activities and active caspase-3 fragments in the absence of apoptosis were detected in various tumor cell lines and cancer tissues, but not in normal cells (Yang et al., 2003a). Tumor cells, but not normal cells also expressed high levels of IAPs, suggesting that upregulated IAP expression counteracted the high basal caspase activity selectively in tumor cells (Yang et al., 2003a). Therefore, strategies targeting IAPs are considered as a promising approach to enhance the efficacy of cytotoxic therapies selectively in cancer cells. To this end, transfection-enforced expression of Smac/DIABLO lowered the threshold for TRAIL-induced killing in different tumors (Ng and Bonavida, 2002; Okano et al., 2003) and also sensitized cancer cells for chemotherapy (McNeish et al., 2003; Zhao et al., 2006). However, translocation of endogenous Smac/DIABLO into the cytosol during anticancer drug-induced apoptosis does not appear to play a major role under certain conditions, for example, in human lung carcinoma cells upon treatment with etoposide (Bartling et al., 2004). Downregulation of Smac/DIABLO by small interfering RNA (siRNA) did not influence killing by etoposide in these cells, although an IAP-binding peptide Smac-N7 enhanced etoposide-induced apoptosis (Bartling et al., 2004). These data suggest that Smac/DIABLO deficiency may be compensated for by action of redundant determinants in certain cancer cells.
Mitochondrial mediators of caspase-independent apoptosis
Upon its release from the mitochondrial intermembrane space, HtrA2/OMI contributes to cell death also in a caspase-independent way as a protease in addition to promoting apoptosis in a caspase-dependent way by antagonizing IAPs (Suzuki et al., 2001; Martins et al., 2002; van Loo et al., 2002). In vitro data demonstrated the degradation of XIAP, cIAP1, cIAP2 and Apollon by the protease activity of HtrA2/OMI (Suzuki et al., 2004). In addition, Trencia et al. (2004) demonstrated the interaction of antiapoptotic PED/PEA-15 with, and its degradation by, cytosolic HtrA2/OMI. Decreasing the levels of HtrA2/OMI in cells by antisense or RNA interference lowers the sensitivity of different cancer cell lines to cell death induced by staurosporin, Fas, UV or cisplatin (Martins et al., 2002).
Furthermore, AIF and endonuclease G are released from mitochondria upon outer mitochondrial membrane permeabilization and translocate into the nucleus to contribute to nuclear chromatin condensation and large-scale DNA fragmentation (Cande et al., 2004; Saelens et al., 2004). Whether these two proteins are released before, together or after cytochrome c has been controversially discussed (Arnoult et al., 2002). Also, it is still not exactly clear how AIF contributes to nuclear DNA fragmentation as it lacks intrinsic DNase activity. In mammalian cells, cyclophilin A, a peptidyl-prolyl cis–trans isomerase, cooperates with AIF to induce breakdown of DNA (Cande et al., 2004).
Disruption of the intrinsic pathway in cancer
Mutations in genes involved in the regulation of the mitochondrial pathway are very common in cancer cells. As the majority of anticancer therapies induce apoptosis in cancer cells by triggering the intrinsic pathway, such mutations are usually associated with treatment resistance. For example, overexpression of Bcl-2, as a result of chromosomal translocation of the bcl-2 oncogene into the immunglobulin heavy chain gene locus, is associated with approximately 85% of human follicular lymphoma (Tsujimoto et al., 1984). Experiments with transgenic mice have demonstrated that Bcl-2 overexpression can promote neoplastic transformation of B and T lymphocytes and myeloid cells (McDonnell and Korsmeyer, 1991; Traver et al., 1998).
Given the fact that overexpression of antiapoptotic Bcl-2 family members promotes oncogenesis, it follows that multi BH domain proapoptotic members of this family act as tumor suppressors. However, since Bax and Bak have largely overlapping roles in apoptosis, it has been difficult to determine whether this hypothesis holds true and indeed bax−/− mice are not markedly predisposed to neoplasia (Knudson et al., 2001). However, somatic mutations that inactivate the bax gene have been found in certain solid tumors and hematological malignancies. To this end, single nucleotide substitution or frameshift mutations, which inactivate the Bax gene in mismatch repair-deficient (MMR) colon cancer or hematopoetic malignancies, have been described (Rampino et al., 1997; Kitada et al., 2002).
Moreover, there is mounting evidence that BH3-only proteins may contribute to the suppression of malignant transformation, indicating that they can function as bona fide tumor suppressors. For example, loss of a single allele of Bim was shown to accelerate B-cell lymphomagenesis induced by expression of a c-myc transgene (Egle et al., 2004), in line with the key role of Bim as a regulator of lymphoid homeostasis (Strasser, 2005). Interestingly, homozygous deletions in the chromosomal region harboring the bim gene have recently been identified in patients with mantle cell lymphoma (Tagawa et al., 2005). Mice lacking bid spontaneously develop a myeloproliferative disorder that can progress to a malignancy resembling chronic myelomonocytic leukemia (CMML) (Zinkel et al., 2003). Moreover, RNAi-mediated suppression of Puma was reported to accelerate Myc-induced lymphomagenesis (Hemann et al., 2004).
In addition to genetic alterations, aberrant expression of Bcl-2 family proteins is mostly regulated at the transcriptional or post-transcriptional level. For example, expression of several antiapoptotic Bcl-2 family proteins, for example, Bcl-2, Bcl-XL, Mcl-1 or Bfl-1, is transcriptionally regulated by NF-κB (Cory and Adams, 2002).
Besides Bcl-2 family proteins, decreased or absent activity of Apaf-1 was found in ovarian cancer, melanoma and leukemia. Furthermore, mutations in the tumor suppressor gene p53, the most common genetic defect in human cancers, impinge on the intrinsic pathway. For example, activation of p53 upregulates a number of genes possessing p53-responsive elements present in their promoters, such as the proapoptotic BH3-only proteins Puma, Noxa and Bid (Oda et al., 2000; Yu et al., 2001; Sax et al., 2002). As a result, cells in which p53 is stabilized are sensitized for activation of the mitochondrial cell death pathway. In addition, p53 may directly affect mitochondrial integrity without the need for gene activation. Indeed, it has been reported that p53 can bind to Bcl-2 and Bcl-XL at the mitochondria, thereby promoting mitochondrial destabilization (Mihara et al., 2003).
Signaling through the intrinsic pathway in cancer therapy
Most conventional chemotherapeutic agents, for example, etoposide, doxorubicin, cisplatin or paclitaxel, elicit mitochondrial permeabilization in an indirect fashion by triggering perturbations of intermediate metabolism or by increasing the concentration of proapoptotic second messengers, for example, by inducing p53 expression, by inducing the ceramide/GD3 pathway, by inducing the CD95/CD95L ligand system, affecting Bcl-2-like proteins and/or by compromising the redox or energy balance.
There is mounting evidence for the existence of a nucleo-mitochondrial cross-talk following DNA damage. For example, apoptotic signals arising from DNA damage can be transmitted via the tumor suppressor p53 to mitochondria, which in turn release apoptogenic factors into the cytoplasm that activate downstream destruction programs (Moll et al., 2005). p53 can indirectly engage the mitochondrial pathway by transcriptionally activating the expression of proapoptotic Bcl-2 proteins, for example, Bid, Puma or Noxa (Oda et al., 2000; Yu et al., 2001; Sax et al., 2002). Additionally, p53 can directly trigger permeabilization of the outer mitochondrial membrane in a transcription-independent fashion through direct activation of proapoptotic Bcl-2 proteins Bax or Bak or through binding and inactivating of antiapoptotic Bcl-2 proteins such as Bcl-2 or Bcl-XL (Mihara et al., 2003; Chipuk et al., 2004; Moll et al., 2005). Importantly, mitochondrially targeted p53 has recently been shown to possess tumor suppressor activities also in vivo (Talos et al., 2005).
Moreover, caspase-2 possesses the ability to engage the mitochondrial apoptotic pathway in response to DNA damage by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane. Hence, cells stably transfected with procaspase-2 antisense, or transiently expressing siRNA, were refractory to cytochrome c release and various downstream events, such as caspase activation and DNA fragmentation, induced by DNA damage (Lassus et al., 2002; Robertson et al., 2002). Caspase-2 can act indirectly on the mitochondria, for example, by cleaving the proapoptotic protein Bid, followed by its translocation to the mitochondria to induce cytochrome c release (Guo et al., 2002). Additionally, caspase-2 can directly permeabilize the outer mitochondria membrane and stimulate the release of cytochrome c and Smac/DIABLO, possibly as a result of direct interaction of processed caspase-2 with putative proteins and/or phospholipids located in the outer mitochondrial membrane, or at contact sites between the outer and inner membranes (Robertson et al., 2004). Permeabilization of the outer mitochondria membrane requires processing of the caspase-2 zymogen but not the associated proteolytic activity and occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2 (Robertson et al., 2004). Further, caspase-2 was also shown to have the surprising ability to disrupt the association between cytochrome c and anionic phospholipids, notably cardiolipin, thereby making additional cytochrome c available for release into the cytosol (Enoksson et al., 2004). Upon DNA damage, caspase-2 has recently been reported to become activated in the so-called PIDDosome, a complex of the p53-inducible, death domain-containing protein PIDD, caspase-2 and the adaptor protein RAIDD (Tinel and Tschopp, 2004) pointing to the existence of a nucleo-mitochondrial apoptotic pathway.
Additionally, histone H1.2 has been reported to play an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks (Konishi et al., 2003). Nuclear histone H1.2 is released into the cytoplasm through a p53-dependent mechanism after DNA double-strand breaks and induced cytochrome c release from isolated mitochondria in a Bak-dependent manner (Konishi et al., 2003). Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide (Konishi et al., 2003).
Moreover, the orphan nuclear receptor Nur77 (also known as TR3) has recently been coupled with the Bcl-2 apoptotic machinery at the mitochondria (Lin et al., 2004). Nur77 binding to the Bcl-2 N-terminal loop region, located between its BH4 and BH3 domains, induces a Bcl-2 conformational change that exposes its BH3 domain, resulting in conversion of Bcl-2 from a protector to a killer (Lin et al., 2004). Interestingly, elevated levels of an Nur77-family member have been associated with favorable responses to chemotherapeutic agents in patients (Shipp et al., 2002).
Furthermore, upon cellular stress including chemotherapeutic drugs, specific proapoptotic Bcl-2 family members are activated, derepressed or induced, and thereby act as sensors. The activity of BH3-only proteins is kept in check by several mechanisms, keeping these proteins away from the multidomain Bcl-2 counterparts under normal circumstances, yet allowing their rapid activation under stress conditions (Bouillet and Strasser, 2002). As mentioned above, Bcl-2 family protein such as Bid, Puma or Noxa are under the transcriptional control of the tumor suppressor p53 and hence, upregulated in response to DNA-damaging agents (Oda et al., 2000; Yu et al., 2001; Sax et al., 2002). The BH3-only protein Bim, which is associated with the cytoskeleton by binding to microtubules, is set free and activates the intrinsic pathway following treatment with taxol, which acts on microtubule assembly (Sunters et al., 2003). Recently, paclitaxel has been demonstrated to induce Bim accumulation and Bim-dependent apoptosis in epithelial tumors in vitro and also in vivo (Tan et al., 2005). Active BH3-only proteins bind and counteract antiapoptotic and in some cases activate multidomain proapoptotic Bcl-2 family members, leading to the loss of mitochondrial membrane permeability (Bouillet and Strasser, 2002). How Bcl-2 proteins induce disturbance of the mitochondrial outer membrane is still a matter of debate and may involve the pore-forming and self-oligomerizing capacity of some Bcl-2 family proteins, modulation of the mitochondrial permeability transition pore by Bcl-2 family proteins and/or lipid changes and lipid protein interactions within the mitochondrial membranes. Moreover, chemotherapeutic agents such as paclitaxel cause hyperphosphorylation and inactivation of Bcl-2, and, simultaneously, favor opening of the permeability transition (PT) pore (Ruvolo et al., 2001).
Chemotherapeutic drugs may also induce or facilitate permeabilization of the outer mitochondrial membrane through changes in cellular redox potentials owing to an enhanced generation of reactive oxygen species (or a decrease in their detoxification), depletion of reduced glutathione or depletion of NADPH, as the mitochondrial megachannel possesses several redox-sensitive sites (Debatin et al., 2002). In addition, changes in energy metabolism, for example depletion in ADP and ATP, might facilitate permeability transition pore complex (PTPC) opening, since ADP and ATP are the physiologic ligands of the adenine nucleotide translocator, which function as endogenous inhibitors of the PTPC (Costantini et al., 2000). Also, uncoupling or inhibition of the respiratory chain or matrix alkalinization may favor mitochondrial membrane permeabilization. In addition, lipid messengers such as ceramide, which is generated in cells exposed to several apoptosis-inducing stimuli, including cytotoxic drugs, can contribute to mitochondrial membrane permeabilization (Susin et al., 1997). At high concentrations. some chemotherapeutic drugs, for example, etoposide or paclitaxel, can also induce mitochondrial outer membrane permeabilization in isolated mitochondria (Robertson et al., 2000; Kidd et al., 2002).
In addition, an increasing number of experimental anticancer drugs, including arsenite, lonidamide, the synthetic retinoid CD437 or the natural product betulinic acid, have been shown to act directly on mitochondria (Debatin et al., 2002). For example, betulinic acid has been reported to trigger apoptosis by directly inducing loss of mitochondrial membrane potential in isolated mitochondria in a way that is not affected by the caspase inhibitor Z-VAD-fmk and yet is inhibited by BA, an inhibitor of the PTPC, or by Bcl-2 and Bcl-XL (Fulda et al., 1998b).
Strategies targeting the intrinsic pathway
Bcl-2 family proteins
Since antiapoptotic Bcl-2 proteins, which potently block the intrinsic apoptosis pathway, are found at elevated levels in human cancers of both hematological and nonhematological origin (Cotter, 2004), they represent promising targets for therapeutic interventions. Consequently, several strategies have been developed to target Bcl-2 proteins, for example, antisense techniques, BH3-domain peptides or synthetic small molecule drugs interfering with Bcl-2-like protein function. To downregulate Bcl-2 expression, Bcl-2 antisense oligonucleotidex (genasense) were developed by Genta Incorporated (Berkeley Heights, NJ, USA). Genasense is a synthetic, 18-base, single-stranded phosphorothioate oligonucleotide that selectively targets the first six codons (i.e., 18 bases) of the mRNA open reading frame encoding the Bcl-2 protein (Cotter, 2004). Treatment with genasense markedly enhanced the antitumor activity of many chemotherapeutic drugs, for example, taxanes, anthracyclines, alkylators or platinum-containing agents (Cotter, 2004). In a preclinical model of melanoma, pretreatment with genasense increased the chemosensitivity of human melanoma (Jansen et al., 1998). Also in a clinical trial, genasense was reported to act as chemosensitizer for dacarbazine in patients with malignant melanoma (Jansen et al., 2000). To optimize antisense-based therapy, bispecific antisense oligonucleotides directed against a sequence, which is highly homologous in Bcl-2 and Bcl-xL, but missing in Bcl-xS mRNA, were subsequently designed (Zangemeister-Wittke et al., 2000). Simultaneous downregulation of both Bcl-2 and Bcl-xL induced apoptosis and enhanced chemosensitivity in various cancer cells (Gautschi et al., 2001; Tortora et al., 2003; Milella et al., 2004; Yamanaka et al., 2005).
Furthermore, BH3-domain peptides or synthetic small molecule inhibitors were developed to target anti-apoptotic Bcl-2-like proteins. The BH3 domain comprises a nine-amino-acid amphipathic α-helix that binds to a hydrophobic pocket of Bcl-2-like proteins (Cory and Adams, 2002). Similarly, BH3-domain peptides aim at disrupting this complex, thereby sensitizing cancer cells to apoptosi (Letai et al., 2002). Additionally, substitution of the Bid BH3 domain with non-natural amino acids on the surface opposite to the interacting region by hydrocarbon stapling resulted in stabilized BH3 peptides termed SAHBs (stabilized α-helix of Bcl-2 domains), with improved pharmacological properties (Walensky et al., 2004). These stabilized BH3 peptides triggered apoptosis in a variety of leukemic cell lines and also inhibited the growth of leukemia xenografts in mice without adverse side effects (Walensky et al., 2004).
Also, several small molecule compounds interfering with Bcl-2/Bcl-xL function have been identified. Screening of a chemical library for compounds able to bind to the BH3 pocket of Bcl-2 proteins resulted in the identification of HA14-1, a compound that competes with Bak for the binding to Bcl-2 (Wang et al., 2000). By screening a library of 16 320 preselected compounds for the ability to displace a fluorescent Bak BH3 peptide from Bcl-xL in a fluorescent polarization assay, Degterev et al. (2003) identified two classes of agents called BH3 inhibitors (BH3Is), which also disrupt the Bcl-xL complex with Bax and Bad in intact cells.
Using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, a small-molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, ABT-737, was recently discovered. ABT-737 was effective as a single agent against certain lymphomas and solid tumors and displayed synergistic cytotoxicity with chemotherapeutics and radiation (Oltersdorf et al., 2005).
Smac/DIABLO agonists
For the design of potentially therapeutic small molecules to target XIAP, the binding groove of the BIR3 domain of XIAP, to which Smac/DIABLO binds to after its release from mitochondria, has attracted most attention. Structural analysis has provided a clear rationale for the synthesis of small compounds that can mimic the caspase-9 displacing activity of Smac/DIABLO from XIAP BIR3 (Chai et al., 2000; Wu et al., 2000). To enhance intracellular delivery, Smac peptides were linked to a carrier, for example, the protein transduction motif of the HIV Tat protein (Fulda et al., 2002), the Drosophila antennapaedia penetratin sequence (Arnt et al., 2002) or a polyarginine stretch (Yang et al., 2003b). A heptapeptide representing the N-terminus of mature Smac/DIABLO, which is essential for binding to XIAP, was reported to promote caspase activation and to sensitize various tumor cell lines and also primary patients' derived tumor cells for apoptosis induced by death-receptor ligation or cytotoxic drugs (Fulda et al., 2002). Importantly, Smac peptides even enhanced the antitumor activity of TRAIL in vivo in an intracranial malignant glioma xenograft model (Fulda et al., 2002b). Likewise, an 8-mer peptide (AVPIAQKS) fused to the protein transduction domain of Drosophila antennapaedia penetratin was able to enter breast cancer cells, bind XIAP and cIAP1, and potentiate caspase activity induced by a number of anticancer drugs, including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38) and doxorubicin (Arnt et al., 2002). Moreover, on the basis of the three-dimensional structure of Smac/DIABLO in complex with XIAP BIR3, Smac peptidomimetics were designed, which cooperated with TRAIL, TNFα, cisplatin or etoposide to trigger apoptosis in tumor cells (Li et al., 2004; Sun et al., 2004a, 2004b, 2005).
Subsequently, non-peptidic small molecule antagonists of XIAP, which were derived by screening of a phage library or polyphenylurea library, were developed for targeting IAPs (Schimmer et al., 2004; Wang et al., 2004). In addition, the natural product embelin from the Japanese Ardisia herb was recently discovered as a cell-permeable, non-peptidic, small-molecular weight inhibitor of XIAP through structure-based computational screening of a traditional herbal medicine three-dimensional structure database (Nikolovska-Coleska et al., 2004). Embelin was shown to effectively overcome the protective effect of XIAP in prostate cancer cells with high endogenous levels of XIAP or in Jurkat cells transfected with XIAP through binding to the XIAP BIR3 domain (Nikolovska-Coleska et al., 2004). Thus, Smac agonists or low molecular weight XIAP antagonists may be promising candidates for cancer therapy by potentiating the efficacy of cytotoxic therapies selectively in cancer cells.
Conclusions
Continued efforts over many years to understand the mechanisms controlling tumor cell proliferation and cell death have delineated key molecular pathways involved in cancer formation, progression and treatment resistance. One of the key discoveries in cancer research has been the recognition that anticancer chemotherapy kills cancer cells by activating the intrinsic and/or extrinsic apoptosis pathway. Although a considerable amount of data support a role of the CD95 system in anticancer drug-induced apoptosis, at least under certain circumstances, most cytotoxic drugs are considered to primarily initiate cell death by triggering a cytochrome c/Apaf-1/caspase-9-dependent pathway linked to mitochondria. Collectively, these data point to a key role of the mitochondrial pathway in chemotherapy-induced apoptosis, whereas the CD95 system may amplify killing by cytotoxic drugs under certain conditions. Importantly, this amplification of the chemoresponse may have important clinical implications, since it may critically affect the time required for execution of the death program. Thus, defects in the intrinsic or extrinsic pathway may promote tumor progression and therapy resistance. Accordingly, targeting defective apoptosis programs may restore sensitivity in resistant forms of cancer. However, apoptosis does not represent the sole killing mechanism by which cancers are eradicated, and other modes of cell death, for example, necrosis, autophagy, mitotic catastrophe or some forms of cell death that cannot be easily classified at present, have also to be considered. Further insight into the complex signaling network activated in response to anticancer therapy using cancer cell lines, primary tumor cells and animal models are necessary to see to what extent the current knowledge can be exploited for the design of apoptosis-based cancer therapies. Also, further studies on the role of apoptosis signaling molecules in clinical samples using DNA or proteomic arrays are warranted to assess the impact of these molecular parameters on clinical outcome. Hopefully, these studies may eventually allow the identification of novel therapeutic targets thereby providing the basis for tailored, individual tumor therapy.
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