*3.2. Cathepsin-Dependent Cell Death*

We originally hypothesized that the combination of ricin with TRAIL, TNF-<sup>α</sup>, or FasL would produce caspase-dependent apoptosis. However, this was only the case when ricin was combined with TRAIL (Figures 2A and 3). The fact that the pan-caspase inhibitor, zVAD-fmk, prevented cell death by ricin/TNF-α and ricin/FasL suggested that, like TRAIL, these cytokines also provoke caspase-dependent apoptosis when administered with ricin (Figure 1C,D). However, it is clear that there is no role for caspases in cell death by ricin/TNF-α or ricin/FasL (Figure 4A–F and Figure S5). Moreover, these signaling molecules remain inactivated in response to ricin/TNF-α and ricin/FasL (Figure 2B,C and Figure S2). In addition, we confirmed that alternative caspases (caspases-1 and -2) and downstream apoptosis effectors (Bax) do not play a role in cell death by ricin/TNF-α or ricin/FasL (Figure 5).

Cathepsins are proteases originally discovered to be resident to lysosomes but are present in the cytoplasm and nucleus as well [62]. These molecules may have roles in apoptosis, necroptosis, and pyroptosis [51,62,63]. The results presented in Figure 6 clearly indicate that ricin/TNF-α and

ricin/FasL each induce cathepsin-dependent death of A549 human lung epithelial cells. However, we believe that this is a distinct cathepsin-driven cell death pathway and not a component of another cell death pathway (e.g., apoptosis, necroptosis, etc.). Indeed, we ruled out contributions from apoptosis (Figures 2B,C and 4), necroptosis (Figure S2), and pyroptosis (Figure 5A,B) to cell death induced by ricin/TNFα and ricin/FasL. Cathepsin-driven cell death is often accompanied by ROS activity [51]. Therefore, our findings that N-acetylcysteine (NAC) prevents cell death by ricin/TNFα or ricin/FasL (Figure S4) are consistent with known mechanisms of cathepsin-dependent cell death, particularly since we have ruled out contributions from apoptosis, necroptosis, and pyroptosis (Figures 4 and 5A,B and Figure S8). It is possible that the cathepsin-dependent cell death induced by ricin/TNFα and ricin/FasL is a form of lysosome-dependent cell death, which requires the release of cathepsins from lysosomes via permeabilization [51,63]. However, cathepsins are known to be resident to the cytoplasm as well [62], making lysosomal involvement in these pathways unclear. If lysosomal permeabilization is the basis for cathepsin involvement in these pathways, it is likely to be ROS-mediated, particularly in light of the results from Figure S12. The other major stimulus for lysosomal permeabilization is Bak/Bax [51], whose involvement we have ruled out (Figure 5E,F).

Interestingly, in Figure S11 NAC did not prevent cell death induced by ricin alone. This is in contrast to other reports that ROS are involved in cell death induced by ricin [64,65]. Data on the involvement of ROS in ricin-induced death of human lung epithelial cells are lacking, however. As noted earlier, ricin alone clearly induces caspase-dependent apoptosis in human monocytes, mammary cells, and cervical cells [60,61]. However, in this work as well as our prior work we have shown that ricin alone does not induce caspase-dependent apoptosis of human lung epithelial cells lines A549 (Figures 2–4) and Calu3 [20]. We hypothesize that NAC did not a ffect cell death by ricin alone in A549 cells as these cells undergo a cell death response to ricin which di ffers from the caspase-dependent apoptosis that ricin induces in other human cell types. This also agrees with the accepted concept that ROS have a key role in apoptosis [66].
