The development of cancer gene therapies requires agents that target pathways that maximize anti-cancer activity. EIF5A1 has been identified as a viable cancer target that can be adapted for use in gene therapy approaches since its over-expression has been demonstrated to induce apoptosis in a wide variety of cancer types [11, 13–16]. As well, suppression of hypusinated eIF5A1 using a small interfering RNA (siRNA) has been shown to inhibit activation of Nuclear Factor kappa B (NF-κB) and ERK MAPK in multiple myeloma cells  and to potentiate the pro-apoptotic activity of an eIF5AK50R expression plasmid. SNS01-T, a nanoparticle containing an eIF5AK50R expression plasmid and an eIF5A1 siRNA, is currently being evaluated in a clinical trial in patients with advanced multiple myeloma [http://www.clinicaltrials.gov; Identifier: NCT01435720].
Although the precise mechanism underlying the role of eIF5A1 in cell death is unknown, it can induce apoptosis in a p53-dependent  or independent manner [13, 14] and activate the intrinsic mitochondrial pathway of apoptosis . In this study, adenoviral-mediated over-expression of eIF5A1 or eIF5AK50A was found to induce apoptosis in A549 lung cancer cells. The similarity in cellular response to eIF5A1 and eIF5A1K50A over-expression can be attributed to the rate-limiting activity of DHS and DOHH [13, 28] available to modify the large amounts of newly translated eIF5A1 generated by the virus. Indeed, a disproportionate accumulation of unhypusinated relative to hypusinated eIF5A1 that correlated with the induction of apoptosis was observed in the present study following Ad-eIF5A1 infection of A549 cells. Another important observation is that apoptosis induced by Ad-eIF5A1 or Ad-eIF5A1K50A infection was not correlated to a reduction in hypusine-eIF5A levels, suggesting that the apoptotic response is not a result of depletion of the hypusinated form of the protein.
MAPK signaling pathways can induce either cell proliferation or cell death depending on the cell type and stimulus. Infection of A549 cells with Ad-eIF5A1 or Ad-eIF5A1K50A induced activation of ERK, p38, and JNK MAPKs. ERK can antagonize apoptosis by phosphorylating pro-apoptotic Bcl-2 proteins, e.g., Bim, and inhibiting their function [29, 30]. ERK can also promote apoptosis by binding and phosphorylating the tumor suppressor p53 on serine 15  and up-regulating pro-apoptotic Bcl-2 proteins such as Bax . The p38 and JNK MAPK pathways are activated by a variety of cell stressors, including ultraviolet light (UV), radiation, cytotoxic drugs, and cytokines such as tumor necrosis factor alpha and interleukin 1. Activation of these pathways is often correlated with stress-related apoptosis, and inhibition of p38 and JNK has been demonstrated to prevent apoptosis resulting from a wide variety of stressors, including UV , ceramide , and genotoxic stress . Inhibitors of p38 and JNK inhibited apoptosis of A549 cells in response to Ad-eIF5A1 in the present study, indicating that activation of these kinases contributes to cell death mediated by an accumulation of unmodified eIF5A1. A member of the AP-1 transcription factor family, c-Jun, has been implicated in both cell survival and apoptosis  depending on the tissue and stimulus. The transcriptional activity of c-Jun and its ability to either enhance or protect against apoptosis are largely regulated by JNK-mediated phosphorylation of its transactivation domain at serines 63 and 73 [37, 38]. P38 MAPK has also been reported to phosphorylate c-Jun at serine 63 in T lymphocytes . In accordance with an increase in JNK and p38 MAPK activity, phosphorylation of c-Jun at serine 63 was observed following Ad-eIF5A1 infection, suggesting that eIF5A1-induced apoptosis may involve the AP-1 transcription factor complex.
The p53 tumor suppressor protein is activated by a variety of cellular stressors including reactive oxygen species, DNA damage, hypoxia and oncogene stimulation, and assists in the cellular response to stress by regulating cell growth and apoptosis. Post-translational modifications, including phosphorylation, modify the activity of p53 by regulating protein stability and enhancing DNA binding and transcriptional activity. Phosphorylation of p53 at serine 15 contributes to stability of p53 by interfering with binding to the E3 ubiquitin ligase, Mdm2 , and is also critical for the transactivation activity of p53 by promoting its association with the p300 coactivator protein . Intracellular signaling resulting from DNA damage leads to phosphorylation of p53 at serines 15, 20 and 37 resulting in decreased association with Mdm2 , thereby enhancing stability and activity of the p53 protein . Phosphorylation of serine 15 is critical for p53-induced apoptosis  and has been associated with increased expression of p53-responsive pro-apoptotic genes . Oligomerization of p53, which is critical to its transcriptional activity, is regulated by phosphorylation at serine 392 . The involvement of ERK in the regulation of p53 stability and activity through direct phosphorylation has long been recognized . In the present study, eIF5A1 over-expression induced MEK-dependent accumulation and phosphorylation of the p53 tumor suppressor protein on serines 15, 37, and 392, as well as up-regulation of the p53-responsive genes, TNFR1 and p53. However, in spite of increased p53 activity in Ad-eIF5A1-infected cells, an inhibitor of p53 was not sufficient to inhibit eIF5A1-induced apoptosis. Thus, apoptosis of A549 lung cancer cells induced by eIF5A1 does not appear to be dependent on p53 activity, although increased expression/stability of p53 induced by eIF5A1 may lower the apoptotic threshold  and thereby contribute to the pro-apoptotic activity of eIF5A.
Increased expression of Bax and the BH3-only protein, Bid, was observed in response to Ad-eIF5A1 over-expression, both being pro-apoptotic proteins that are transcriptionally regulated by stress-activated p53 . Hypusine-modified eIF5A1 has been proposed to act as a tumor suppressor in Eμ-myc lymphomagenesis in mice, in part by promoting expression of Bax . However, in the present study, increased expression of both p53 and Bax was correlated with an accumulation of unmodified eIF5A, since hypusine-eIF5A1 levels were relatively unaffected by Ad-eIF5A1 infection. The pro-apoptotic BH3-only Bcl-2 family member, Bid, is cleaved by caspase 8 and then interacts with other pro-apoptotic Bcl-2 family members, specifically Bax and Bak, to connect activation of the death receptor pathway to the internal mitochondrial apoptosis pathway. In contrast to what is observed in the event of death receptor-mediated apoptosis, cleavage of Bid to tBid was not apparent during eIF5A1-induced apoptosis, although increased expression of full length Bid was observed. Although tBid is the form of Bid typically associated with the induction of apoptosis, full-length Bid has been found to associate with the mitochondrial membrane and promote apoptosis in hippocampal neurons . While tBid is typically observed in the late stages of apoptosis , full-length Bid has been reported to regulate the activation of Bax during apoptosis by facilitating its oligomerization and insertion into the mitochondrial membrane .
Malignant cells often display increased sensitivity to chemotherapy drugs and radiation. Although the molecular pathways involved in this increased sensitivity have not been completely elucidated, the sensitization of oncogenically-transformed cells to cytotoxic stresses has been attributed to the potentiation of JNK and p38 MAPK activation . In this study, WI-38 normal lung cells were found to be more resistant than transformed A549 cells to eIF5A1-induced apoptosis. Infection with adenovirus expressing eIF5A1 or eIF5A1K50A caused an induction of p38 and ERK MAPK phosphorylation in A549 cells, but had a more modest effect on p38 phosphorylation in WI-38 cells, suggesting that potentiation of p38 MAPK activation may have contributed to the increased sensitivity of A549 cells to Ad-eIF5A1 infection.