**3. Discussion**

#### *3.1. Distinct Cell Death Modalities*

Our results indicate that ricin induces distinct cell death modalities in A549 human lung epithelial cells depending upon the TNF family cytokine with which it is paired. As ricin is the common factor in each of these scenarios, it is tempting to speculate that the cytokines administered with ricin and their downstream signaling pathways are the primary factors in producing these distinct outcomes. However, when combined with CHX, another molecule that inhibits protein synthesis [54], TRAIL, TNF-<sup>α</sup>, and FasL all induced caspase-dependent apoptosis (Figures 3E and 4G–H). Therefore, there is something unique about the combination of ricin with each of these cytokines which produces different cell death responses. While ricin and CHX both inhibit protein synthesis, they do so by distinct mechanisms: ricin via depurination of rRNA [10,11,13,14] and CHX via occupation of the ribosomal E site [54]. It is possible that these fine mechanistic differences account for the distinct outcomes of cell death depending upon whether TNF family cytokines are paired with ricin or CHX. In addition, there is evidence that the protein synthesis inhibition activity of ricin does not correlate with execution of cell death in other systems [55]. The fact that the cell death caused by ricin does not correlate with protein synthesis inhibition may also provide an explanation for the difference in these cell death outcomes. While TRAIL, TNF-<sup>α</sup>, and FasL have common upstream signaling events, each pathway eventually diverges [56,57]. Thus, there is precedent for induction of distinct cell death pathways in response to TRAIL, TNF-<sup>α</sup>, and FasL. The final intersection point in these signaling pathways is likely the death-inducing signaling complex (DISC) [58,59]. We speculate that following DISC formation the signaling pathway induced by ricin/TRAIL diverges from that induced by ricin/TNF-α and ricin/FasL accounting for these distinct cell death outcomes. Future work will include a detailed study on these signaling responses to address this.

Of note is the fact that ricin alone did not appear to induce caspase-dependent apoptosis in A549 cells throughout this work. This is in contrast to in vivo findings as well as results from other human cell types [60,61]. In HeLa cervical cells, MCF7 mammary cells, and U937 monocytes, ricin alone induces caspase-dependent apoptosis [60,61]. The cell death that ricin induces in human lung epithelial cell lines is less certain. The inability of ricin alone to induce apoptosis in A549 cells does not appear to be a unique feature of this cell line as we obtained similar results in Calu3 lung epithelial cells [20]. Therefore, while ricin induces apoptosis in other human cell types, lung epithelial cells may exhibit a different cell death modality in response to this toxin.
