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By Alex Almasan, PhD
The success or failure of clinical cancer treatments depends in great part on their ability to induce programmed cell death (apoptosis). Because apoptosis is deregulated in most cancers, apoptotic-modulating therapies offer an attractive opportunity for many tumors. For most cancers, ionizing radiation is a major modality of treatment. The radiation response of mammalian cells includes apoptosis, a process which requires activation of multiple genes; however, their role in radiation-induced apoptosis is poorly understood. One important pathway to apoptosis is mediated by surface-receptor signaling, which depends on ligand-receptor interactions for cell death effector activity. These cell surface receptors are part of the tumor necrosis factor (TNF)-receptor family and include Fas (also known as CD95) and death receptor (DR) 4 and DR5 molecules.1 Engagement of these receptors by Fas ligand (FasL), or the apoptosis-inducing ligand Apo2L (also known as TRAIL) can lead to cell death.2,3
Apo2L as an Antitumor Agent
Apo2L has emerged as a powerful antitumor agent, as evidenced by increasing interest in its potential use in cancer therapy. The DR4 and DR5 receptors for Apo2L contain a cytoplasmic "death domain" capable of engaging the cell suicide apparatus through the adaptor molecule, Fas-associated death domain protein (FADD).4 Most importantly, it was reported that while many human tumor cell lines are sensitive to cell-surface or soluble Apo2L, the majority of normal cells are not. This apparent protection of normal cells from the cytotoxic effect of Apo2L is believed to be based on a unique set of decoy receptors (DcR); these cells either lack the DcR1 or have a truncated DcR2 and are unable to signal, but compete instead for receptor binding to Apo2L. An alternative view is that levels of an intracellular inhibitor, FLICE-inhibitory protein (FLIP), may provide resistance in normal cells.5 The mechanism of activation of this receptor-mediated cell death pathway during cancer therapy is still not well understood.
A recent study has shown Apo2L mRNA induction following g-irradiation of a variety of human T lineage-derived normal and tumor cells.6 Increased Apo2L protein levels also were found in these cells. Radiation also activated the Apo2L death receptor DR5, but only in those cells which harbored a wild-type p53. Similar to radiation treatment, exogenously added Apo2L induced the typical cellular and molecular events known to be associated with apoptosis, such as phosphatidylserine exposure on cell membranes and activation of caspases.
These events constitute a signaling cascade consisting of regulator caspases, such as caspase 8 and 9, and effector caspases, such as caspase 3, 7, and 6.7 It has been clearly demonstrated using caspase cleavage site substrates and inhibitors, and by monitoring the cleavage
of caspase cellular substrates, such as poly(ADP-ribose)polymerase (PARP) protein in vivo, that at least four known caspases are activated following exposure to radiation.8 Caspase 8 is an apical caspase known to be the first caspase activated in receptor-mediated apoptosis. This recent study showed a substantial decrease in the levels of pro-caspase 8 protein, as well as an increase in caspase 8 enzymatic cleavage activity, indicating that the caspase cascade is activated by Apo2L through
Proteolytic activation following cleavage of Bid, a Bcl-2 family pro-apoptotic protein also known to contribute to receptor-mediated apoptosis by inserting into mitochondrial membranes, indicates the requirement for mitochondria in this process. Mitochondria serve as stores for apoptogenic molecules, such as cytochrome c, which once released into the cytosol further contribute to caspase activation and apoptosis.9 Bid levels were significantly reduced upon Apo2L treatment, indicating processing of the full length Bid to its active proteolytic fragment. Activation of caspase 8 and caspase 3 indicate a caspase-dependent apoptosis, with Bid most likely contributing to the amplification of the caspase cascade. The functional significance of Apo2L for radiation-induced apoptosis was demonstrated by a significantly higher cell survival of those cells expressing the dominant negative Apo2L receptor DR5, which blocks Apo2L signaling.8 This finding is consistent with a recent suggestion that radiation kills lymphocytes by a Fas-independent mechanism.10 Apo2L induction in T cells could be most important because radiation could selectively kill tumor cells, which express only the DR5 and/or DR4 receptors, but not normal cells, which also express decoy receptors or FLIP.
Apo2L as an Adjuvant Therapy
While tumor-specific, Apo2L kills only about 60% of tumors. For the remaining tumors, a second therapeutic agent could be used in combination with Apo2L for an effective response. A recent study used clonogenic assays to show that low levels of purified, recombinant soluble Apo2L enhanced the lethality of therapeutic doses (1-2 Gy) of g-irradiation.6 This indicates that production of Apo2L may cooperate synergistically with the cytotoxic effect of radiation, and that combinations of Apo2L and radiation may become a powerful tool in clinical therapy. The observation that radiation can induce Apo2L, and that low-dose radiation can cooperate synergistically with Apo2L in enhancing cell death, may have implications for clinical therapy.
These findings are reminiscent of the previously reported synergistic or additive cell killing between TNF and radiation.11 Gene therapy approaches have been proposed based on radiation-responsive promoters driving TNF expression,12 and TNF expression has been shown to sensitize certain radiation-resistant tumors. Indeed, some tumor cells also may be sensitive to the death effects of Fas or TNF (applied alone or in combination with radiation). However, the therapeutic use of TNF or FasL has been hampered by severe side effects, since systemic administration of TNF causes a septic shock-like response, likely mediated by the inflammatory response triggered by activated nuclear factor-kB (NFkB), and Fas causes hepatotoxicity. Compared to TNFa and Fas, Apo2L is promising to be a safer agent, because most normal cells seem to be resistant to it and it activates NFkB only weakly, if at all.
Moreover, unlike TNFa and Fas, systemic administration of Apo2L has no toxicity in mouse and non-human primates.13,14 In contrast to TNF and FasL, Apo2L is present in most human tissues, indicating that it is not cytotoxic in these tissues. However, it is not expressed in brain and liver, which may be one reason why some sources of Apo2L can induce apoptosis in primary hepatocytes.15 It is important to note that other sources of Apo2L, which in fact are the ones soon to be tested in humans, are not cytotoxic to these cells, indicating that cells may respond differently to different sources of Apo2L. This has become a very controversial issue that may delay the use of Apo2L for clinical therapy until it is resolved satisfactorily. However, if this issue is addressed, understanding the regulation of Apo2L gene expression in brain, liver, and cancer cells may provide the basis for clinical application of Apo2L as an antitumor agent.
In addition to radiation, which induces Apo2L mRNA and protein in lymphoma and leukemia, interferons (IFN) also induce Apo2L in multiple myeloma cells (unpublished results). IFNs are a family of pleiotropic cytokines that play an essential role in the antiviral and antitumor host defense and have been widely used clinically to treat certain types of cancers, including myeloma. Cell death induced by radiation, IFNs, and Apo2L can be prevented by blocking the Apo2L receptor DR5, demonstrating the functional significance of Apo2L in mediating the cytotoxic effects of these therapeutic agents. This Apo2L induction is transcriptional and is mediated through 5’ flanking DNA sequences likely to be responsible for radiation or induction of Apo2L by IFN.16
The observed synergy between Apo2L and therapeutic doses of radiation can form a basis for developing strategies for pharmacological intervention, with a potential for clinical application. In particular, the specificity of Apo2L cytotoxicity for tumor cells and its systemic distribution reaching metastases,17 and the surgical precision with which IR now can be delivered could constitute a very attractive combination for enhancing the clinical response of tumors resistant to radiation therapy. Importantly, radiation therapy usually requires the function of the p53 tumor-suppressor gene for antitumor activity; however, more than one-half of human tumors acquire inactivating p53 mutations, thereby becoming resistant to therapy. Apo2L induces apoptosis independently of p53, and thus, may offer a complementary approach to conventional cancer therapy. Further studies are needed to elucidate the mechanism by which Apo2L can induce apoptosis in combination with radiation or chemotherapy. (Dr. Almasan is Assistant Staff, Department of Cancer Biology, Lerner Research Institute, and Department of Radiation Oncology, The Cleveland Clinic Foundation, Cleveland, OH.)
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