Programmed cell death is considered a defensive mechanism to eliminate harmful and defective cells. Disturbances in the signaling pathways involved in programmed cell death may lead to uncontrolled cell proliferation and eventually cancer. Therefore, recent studies have focused on apoptosis, autophagy and necroptosis as strategic targets for novel cancer therapies (1). Apoptosis is of particular importance due to its pivotal role in controlling irregular cell proliferation through its well-defined mechanism. Apoptosis can be either initiated by ligands that bind to receptors on the cell membrane (extrinsic pathway) or initiated from intracellular signals (intrinsic or mitochondrial pathway) (2). With regard to ligand-induced apoptosis, characterized ligands and corresponding death receptors include Fas ligand/Fas receptor, tumor necrosis factor (TNF) α/TNF receptor 1, Apo-3 ligand/death receptor (DR) 3, TNF-related apoptosis-inducing ligand (TRAIL)/DR4 and TRAIL/DR5 (2).

TRAIL was first characterized in the 1990s by Wiley et al (3). Its potential use in cancer treatment was described later (4). TRAIL is characterized by its ability to selectively induce apoptosis in tumor cells but not in normal cells, qualifying as a potential drug specific for different types of cancer, including breast, bladder, lung and liver (5–9). TRAIL is a cytokine secreted by the majority of normal tissues as a part of the natural immune reaction. It has been demonstrated that breast-feeding women produce high levels of TRAIL in their milk, which may contribute to anticancer effects in infants (10). Collectively, TRAIL plays a significant role in cancer eradication and the prevention of proliferation, while being less likely to cause chemotherapeutic toxicity than established treatments (11). The growing interest in TRAIL-based interventions has led to the development of recombinant human TRAIL (rhTRAIL) as a promising therapy for different types of human cancer (12).

This review will summarize the apoptotic pathway of TRAIL monotherapy in cancer cells, and how resistance develops against it. Subsequently the outcome of studies that have used TRAIL as a part of anticancer combinatorial therapy will be summarized and a set of targets that can be subsequently targeted specifically in combination with rhTRAIL to efficiently eliminate cancer will be identified.

In addition to binding to DR4 and DR5, TRAIL can bind decoy receptor (DcR) 1, DcR2 and the soluble receptor osteoprotegerin. However, only DR4 and DR5 can produce apoptotic signals through their intracellular death domain (13). As illustrated in Fig. 1A, the apoptotic signaling pathway of TRAIL is triggered by binding of trimerized TRAIL to DR4 and/or DR5, followed by receptor clustering leading to the recruitment of Fas-associated protein with death domain (FADD). FADD adaptor protein then recruits pro-caspase 8, forming the death-inducing signaling complex (DISC) known as the primary complex. The recruitment of pro-caspase 8 causes activation of DISC and the subsequent cleavage of caspases 3, 6 and 7, resulting in membrane blebbing, DNA fragmentation and nuclear shrinkage. In certain cases, activated caspase 8 requires the engagement of a mitochondrial response in what is known as the intrinsic pathway. In the intrinsic pathway, active caspase 8 cleaves the BH3-interacting domain death agonist (Bid) to truncated Bid (tBid). tBid then binds Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (Bak), then translocates to the mitochondria. This results in a change in mitochondrial membrane polarization and the release of mitochondria-derived activator of caspase (Smac) (14). tBid also induces mitochondrial release of cytochrome c (15), which conjugates with ATP and apoptotic peptidase activating factor 1 (Apaf-1) to form a structure known as the apoptosome. This apoptosome is essential for the activation of caspase 9 and eventual activation of caspases 3, 6 and 7 (2,13,16).

Current TRAIL-induced apoptosis strategies are hampered by the scarcity of death receptors expressed on the cell surface, and thus the inefficient targeting of these cells by TRAIL/agonistic monoclonal antibody (mAb). In addition, development of resistance to rhTRAIL/agonistic mAb has unfavorable negative implications for such therapies (16). Although DISC is considered a critical step in the initiation of apoptotic signaling through the activation of pro-caspase 8, cellular FLICE inhibitory protein (c-FLIP), which shares sequence homology with caspase 8, may inhibit caspase activation by competing for FADD binding, as illustrated in Fig. 1B. In the presence of c-FLIP, FADD and pro-caspase 8, together with receptor-interacting protein (RIP), TNF receptor-associated factor 2 (TRAF2), IκB kinase and TNFR1-associated death domain (TRADD), form a secondary complex responsible for the activation of non-apoptotic signals initiated through the phosphoinositide 3-kinase (PI3K)/Akt, nuclear factor κB and mitogen-activated protein kinase (MAPK) pathways. However, a previous report refers to c-FLIP as a pro-apoptotic protein and therefore the survival process may require further clarification (17).

Another group of molecules involved in the resistance mechanism is the inhibitor of apoptosis (IAP) family, which includes X-linked IAP, cellular IAP (c-IAP) 1, c-IAP2 and survivin. This group of molecules can inhibit the activity of caspases 3, 7 and/or 9. Nevertheless, this effect can be antagonized by Smac/direct inhibitor of apoptosis binding protein with low pi (DIABLO), which is released from mitochondria during apoptosis (18).

Facing acquired resistance to TRAIL-targeted cell death, an alternative approach has been utilized through which TRAIL is combined with other drugs that can be more effective than a single therapy. The major objective of combinatorial TRAIL is to either synergize the activity of TRAIL or to sensitize TRAIL-resistant cells. Previous studies by the authors demonstrated that several natural compounds, including curcumin, cinobufotalin and berberine may be used solely or in combination to treat various disorders, including cancer (19–22). To that end, natural compounds are involved in the majority of combinatorial strategies directed towards synergizing TRAIL and/or sensitizing resistant cancers to TRAIL.

Combinatorial strategies mainly initiate their action through endoplasmic reticulum (ER) stress, resulting in the upregulation of DR5 and/or DR4 followed by increased TRAIL-induced apoptosis (23–25) (Fig. 2). ER stress primarily causes the release of reactive oxygen species (ROS) (26,27), which is considered a central checkpoint from which several signaling pathways can be triggered. Another downstream checkpoint is the activation of CCAAT-enhancer-binding protein homologous protein (CHOP) via p38/extracellular-signal-regulated kinase (ERK) MAPKs, which in turn increase the transcription of DR5 (28,29), enhance pro-apoptotic proteins (such as Bim) (30) or downregulate the Bcl-2 and Mcl-1 survival proteins (29,31). The third member of the MAPK family, the c-Jun N-terminal kinases (JNKs), can also upregulate DR5 (via an Sp1-mediated mechanism) and downregulate Bcl-2 and Mcl-1 (32). ROS may also cause DNA damage and p53 activation, leading to direct DR5 upregulation (the extrinsic apoptotic pathway) (33,34) or activation of p53 upregulated modulator of apoptosis (PUMA), phorbol-12-myristate-13-acetate-induced protein 1 (Noxa) and Bax pro-apoptotic proteins (the intrinsic apoptotic pathway) (35,36).

In addition to ER stress, TRAIL combinations can act by downregulating NFκB, PI3K/Akt or Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. Previous studies have also revealed that the downregulation of c-FLIP appears to be an important mechanism for improved apoptotic response (37).

The remainder of this review focuses on candidates that can be targeted in combination with TRAIL as a part of emerging treatments for unresponsive cancer.

This review has summarized the outcome of various studies carried out during the past fifteen years and the role of TRAIL combinations in enhancing apoptotic signaling pathways. It has highlighted the pathways activated or downregulated by those combinations which enhance apoptotic cell death and eliminate resistance to single TRAIL therapy. Future therapeutic strategies should capitalize on selective modulators that regulate those pathways as a part of a combined TRAIL therapy. In addition, the study has outlined several promising targets for direct intervention together with rhTRAIL therapy. It remains to be verified whether these new combinations are effective therapies.