Fig. 3.1 Apoptosis is activated through two major signalling pathways. The first pathway is the intrinsic or mitochondrial pathway, because the mitochondria take the key position by initiating apoptosis. Initiation by different apoptotic stimuli is still not entirely clear, but likely involves an imbalance of pro- and antiapoptotic members of the Bcl-2 protein family. This imbalance finally leads to the activation of the proapoptotic Bcl-2 family members BAX and/or BAK and the perturbance of the integrity of the outer mitochondrial membrane. This induces the release of cytochrome c and other apoptotic reg- ulators, like apoptosis-inducing factor (AIF), Smac (second mitochondria-derived activator of apoptosis)/DIABLO (direct inhibitor of apoptosis protein (IAP)-binding protein with low PI), endonuclease G or Omi/HtrA2 from the intermembrane- ous space of mitochondria. In the cytosol, cytochrome c binds to monomeric APAF-1 which then, in a dATP-dependent con- formational change, oligomerizes to assemble the apoptosome, a complex of wheel-like structure with 7-fold symmetry that triggers the activation of the initiator procaspase-9. Caspase- 9 provokes the cleavage of the executioner caspases, such as caspase-3. Furthermore, the potent endogenous inhibitors of cas- pases, the inhibitor of apoptosis proteins (IAPs), are neutralized by Smac/DIABLO or Omi/HtrA2. The second pathway is the extrinsic pathway. Extrinsic apoptosis signaling is mediated by the activation of so-called “death receptors”, which are cell sur- face receptors that transmit apoptotic signals after ligation with specific ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5. All mem- bers of the TNFR family consist of cysteine rich extracellular
subdomains which allow them to recognize their ligands with specificity, resulting in the trimerization and activation of the respective death receptor. Subsequent signalling is mediated by the cytoplasmic part of the death receptor which contains a con- served sequence termed death domain (DD). Adapter molecules like FADD or TRADD themselves possess their own DDs by which they are recruited to the DDs of the activated death receptor, thereby forming the so-called death inducing signal- ing complex (DISC). In addition to its DD, the adaptor FADD also contains a death effector domain (DED), which through homotypic DED-DED, interaction sequesters procaspase-8 to the DISC. The local concentration of several procaspase-8 molecules at the DISC leads to their autocatalytic activation and release of active caspase-8. Active caspase-8 then pro- cesses downstream effector caspases which subsequently cleave specific substrates resulting in cell death. Cells harboring the capacity to induce such direct and mainly caspase-dependent apoptosis pathways were classified to belong to the so-called type I cells. In type II cells, the signal coming from the activated receptor does not generate a caspase signalling cascade strong enough for execution of cell death on its own. In this case, the signal needs to be amplified via mitochondria-dependent apop- totic pathways. The link between the caspase signalling cascade and the mitochondria is provided by the Bcl-2 family member Bid. Bid is cleaved by caspase-8 and in its truncated form (tBID) translocates to the mitochondria where it acts in concert with the proapoptotic Bcl-2 family members BAX and BAK to induce the release of cytochrome c and other mitochondrial proapop- totic factors into the cytosol (modified from Ashkenazi, 2002)
ligand (FasL) or Apo-1 ligand, and Apo-2 ligand/TNF- related apoptosis-inducing ligand (Apo2L/TRAIL).
The FasL and Apo2L/TRAIL act on target cells through binding their specific receptors CD95 and death receptors (DR)4/DR5, respectively.
Between ligands and effector caspases, there are a number of factors that suppress apoptosis. The most proximal step to suppress a death receptor pathway is inhibition of ligand binding. This could be achieved by the lack of or mutations in death receptors or the pres- ence of antagonistic (decoy) receptors. In glioma cells, the existence of both agonistic and decoy receptors for TNF has been demonstrated: agonistic Fas receptors are present on the glioma cells (Rieger et al., 1998), but a decoy soluble receptor (DcR3) for FasL is also released by glioma cells, likely protecting them from the FasL-induced apoptosis.
The efficacy of TNF-induced apoptosis is enhanced by protein synthesis inhibition, which points to the role of expression of factors with specific antiapoptotic function. The TNF-to-TNFR ligation provokes the for- mation of the DISC, initiating the apoptotic cascade (Fig.3.1). However, such activation corresponds to a concurrent and parallel activation of the transcription factor nuclear factor (NF)-κB. The nuclear factor-κB is a dimeric transcription factor controlling the expres- sion of several regulators of immune, inflammatory, and acute phase responses (Conti et al.,2007). A role of NF-κB in the genesis and progression of cancer has also been demonstrated; in particular a constitu- tive NF-κB activation has been described in a variety of epithelial and lymphoid cancers. As seen in other cell systems, TNF-induced NF-κB activation in astro- cytoma cells may be mediated by the TNFR-associated factor (TRAF) family, which consists of a group of six adapter proteins (TRAF1–TRAF6) that participate in the intracellular signalling activity of several members of the TNFR superfamily. Through appropriate ligand stimulation of TNFRs found on the surface of these cells, TRAF proteins can induce activation of NF-κB, resulting in both cytokine secretion and resistance to apoptosis (Conti et al.,2005).
The signal transduction mechanism emanating from the TNFR is thought to be mediated by TRAF2, a signalling intermediate that has been shown to be recruited to the cytoplasmic tail of TNFR through a TNFR/TRADD/TRAF2 interaction. On the basis of this hypothesis, TNF can either induce apoptosis through FADD (FAS associating protein with death domain) and caspase recruitment or promote survival
through TRAF2 recruitment and NF-κB induction. As the cytosolic NF-κB concentration rises, the expression of several antiapoptotic genes is amplified. Candidate antiapoptotic genes for NF-κB induction include, c- IAP (inhibitor of apoptosis) 1 and 2, Bcl-2, Bcl-XL, XIAP, and survivin. Characteristics and the role of these antiapoptotic factors will discussed in specific sections in this chapter.
There are other possible anti-apoptotic mechanisms related to the death ligands and receptors. The U373- MG cell line appears to be resistant to death receptor mediated apoptosis due to lack of crucial signalling components. Expression of caspase-8 sensitizes this cell line to FasL and TRAIL mediated apoptosis.
Furthermore, recent studies have reported methylation of the caspase-8 gene as a mechanism for decreased levels of protein expression in neuroblastomas, render- ing cells resistant to apoptosis (Teitz et al.,2000).
Recent progress in the understanding of the varying susceptibility of glioma cell lines to Apo2L/TRAIL- induced apoptosis has revealed that resistant cell lines expressed 2-fold higher levels of the apop- tosis inhibitor phosphoprotein enriched in dia- betes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). This phosphoprotein protects astro- cytes from TNF-α-induced apoptosis through interrup- tion of FADD-Caspase-8 binding (Condorelli et al., 1999).
Preclinical studies have established the potential for using TNF factors as a therapy in gliomas. In the field of death ligands and receptors, inducing cancer cell apoptosis via local or systemic applica- tion of Apo2L/TRAIL is one of the most promising strategies. Local injection of TRAIL exerted strong antitumor activity on intracranial human malignant glioma xenografts in athymic mice without neurotoxi- city (Roth et al.,1999). However, a significant number of glioma cell lines remain resistant to TRAIL when it is used as monotherapy. In combination with conven- tional DNA-damaging chemotherapy, TRAIL showed synergistic cytotoxicity for human gliomas in vivo and in vitro. Of particular concern is the fact that TRAIL and FasL administration have been shown to have profound toxicity toward normal human hepatocytes, resulting in a massive and rapid induction of cell death.
The local application of adenoviral vectors express- ing FasL may be a strategy to circumvent systemic side effects in gliomas. Transferring the gene encoding FADD into glioma cells also inhibits glioma growth in vitro and in vivo. Finally, even more down-stream
effectors of death receptor-mediated apoptosis, the caspases, have been successfully employed to promote glioma cell death.