In this study, we have investigated whether induction of TRAIL/zVAD/CHX-induced programmed necrosis represents a viable strategy for the elimination of tumor cells. Necrosis has long been regarded as an accidental, non-physiologic form of cell death, whereas caspase-dependent apoptosis was considered to be the only form of programmed and thus physiologically occurring cell death. This view has however been challenged by numerous studies which have provided evidence for the existence of programmed forms of necrosis that do not depend on caspases but nevertheless follow defined molecular steps . While caspase-dependent apoptosis is the major pathway leading to PCD, programmed necrosis can act as a backup system when the apoptotic machinery fails or is inactivated (e.g. by mutations in apoptosis-resistant cancer cells) [5, 28]. It has been shown that programmed necrosis exerts critical functions in multiple patho-physiological settings, e.g. the regulation of bone growth, ovulation, negative selection of lymphocytes , pancreatitis [22, 29], epilepsy, ischemia–reperfusion injury, Parkinson’s, Huntington’s and Alzheimer's disease, and cell destruction by Salmonella, Shigella, HIV and vaccinia virus [4, 28, 30, 31]. In contrast to apoptosis, a comprehensive picture of the signaling pathways of programmed necrosis is not yet available. In the most extensively studied model, TNF-R1 elicits programmed necrosis via activation of RIPK1 and RIPK3, a step which is stimulated by the deubiquitinase CYLD and the deacetylase SIRT2, but negatively regulated by the proteins FADD, FLIP, caspase-8 and members of the cIAP family. Downstream of RIPK3, the proteins MLKL and PGAM5 contribute to programmed necrosis by promoting mitochondrial fragmentation . We have previously demonstrated that ceramide acts as an additional key molecule in death receptor-mediated programmed necrosis [3, 6, 7]. Furthermore, enzymes of the energy metabolism, the Bcl-2-family member Bmf and production of reactive oxygen species have been implicated as additional factors in programmed necrosis .
The capacity to elicit programmed necrosis appears to be an intrinsic feature of death receptors and has been reported not only for TNF-R1 [2, 3, 6], but also for Fas/CD95  and ectodermal dysplasia receptor . Independently, we and others have demonstrated the ability to trigger programmed necrosis for human and murine TRAIL receptors [7, 10]. In contrast to programmed necrosis, the efficacy of TRAIL in the apoptotic elimination of tumor cells has been extensively demonstrated in clinical trials employing mono- or combination therapies . Consistent with the finding that TRAIL elicits apoptosis selectively in tumor but not primary cells, TRAIL was well tolerated in preclinical models at serum concentrations that were shown to be effective against cancer cells, as were agonistic TRAIL receptor antibodies applied to patients in clinical trials using mono- or combination therapies . Nevertheless, intrinsic resistance against TRAIL-induced apoptosis, even when combined with chemo- or radiotherapy, limit the therapeutic success and necessitate the search for additional, yet unexplored options for the treatment of patients.
As such a potential option, the induction of programmed necrosis by TRAIL has however been investigated only in a very limited number of studies. Our own study presented here provides strong evidence for the suitability of TRAIL/zVAD/CHX-induced programmed necrosis as a tool to eliminate tumor cells from a wide range of sources. Raising the expectation that TRAIL/zVAD/CHX-induced programmed necrosis may be even more effective under conditions that more closely resemble the in vivo situation than mere cell culture, it clearly interfered with the capacity of all tested tumor cell lines for unlimited proliferation in clonogenic survival assays (even in a tumor cell line that had shown resistance in conventional cytotoxicity/viability assays). Furthermore, our data demonstrate that cisplatin, etoposide, trichostatin A, 5-fluorouracil, irinotecan, doxorubicin, camptothecin and paclitaxel can exert cytotoxicity not only via apoptosis, but also via programmed necrosis. Providing additional encouragement for the development of future combination therapies, TRAIL/zVAD/CHX-induced programmed necrosis synergized with chemotherapeutic agents and enhanced the cytotoxic response in eight out of 10 tested tumor cell lines as well as 41 out of 80 chemotherapeutic/TRAIL/zVAD/CHX combinations. With regard to potential predictive markers, our results identify expression of RIPK3 as a primary determinant of susceptibility or resistance of tumor cells to TRAIL/zVAD/CHX-induced programmed necrosis. However, our data also show that in future screenings, it should be kept in mind that secondary factors may additionally confer resistance downstream or independent from RIPK3. Finally, our study has confirmed and extended the role of ceramide as one of the key mediators of programmed necrosis downstream of RIPK1 and RIPK3 to the clinically more relevant tumor cell systems investigated here, with the A-SMase inhibitor Arc39 additionally validating A-SMase (rather than neutral sphingomyelinase or ceramide synthase) as the main enzyme responsible for ceramide generation. Our findings are not only fully consistent with our previous data from the initially studied laboratory cell lines [3, 6, 7], but may also prove valuable for a future manipulation of intracellular ceramide levels to induce programmed necrosis in tumor therapy.
As pointed out above, only very few other studies have focused on the induction of programmed necrosis by TRAIL. One of those studies has recently reported that TRAIL induces necroptosis (i.e. a subset of programmed necrosis depending on RIPK1/RIPK3 ) in the tumor cell lines HT-29 (which was also used in this study) and Hep G2 , at first glance consistent with our results. However, unlike in our study, necroptosis was only observed under acidified (but not physiologic) conditions. Moreover, the same group had previously reported that in this very system, caspase activity is required for cell death , being inconsistent with the molecular mechanisms described for necroptosis  and thus suggesting a certain caution when interpreting the results of this study. More encouraging, Hunter and coworkers have reported that TRAIL can kill small cell lung cancer cells by inducing caspase-independent mechanisms of cell death in synergy with paclitaxel . Independently, platinum compounds in combination with TRAIL were found to be effective against breast cancer cells by inducing programmed necrosis (although to a lesser extent) in addition to apoptosis . Finally, Katz and colleagues have described that malignant pleural mesothelioma cells are killed by caspase-independent mechanisms after application of TRAIL in combination with sorafenib, and even find promising evidence of therapeutic efficacy in a xenograft mouse model , in summary corroborating our data on the synergistic action of TRAIL/zVAD/CHX and chemotherapy in the programmed necrosis of tumor cell lines.
With regard to a future therapeutic application of TRAIL/zVAD/CHX-induced programmed necrosis, further work is required. At present, it is unknown whether RIPK3-proficient tumor cells can be stimulated to undergo programmed necrosis and thus circumvent apoptosis resistance in patients. For this purpose, strategies for the induction of programmed necrosis (e.g. as used in our study) need to be adapted to the in vivo situation. As an example, the sensitizer CHX used here is cytotoxic also to healthy tissue. Therefore, therapies based on the treatment of patients with CHX are not an option. As a possible alternative (and in line with our own data presented in Figure 1d), Smac mimetics can similarly sensitize tumor cells for TRAIL- and TNF-induced programmed necrosis in cell culture models . Yet, their efficacy or toxicity under conditions of programmed necrosis has not been evaluated in vivo so far.
Since this study focuses on TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells, we explicitly want to point out that TRAIL-induced programmed necrosis in principle occurs under the condition that the normal apoptotic pathway is inhibited. It has recently become clear that caspase-8 suppresses programmed necrosis under normal conditions and that it needs to be actively inhibited (e.g. by zVAD-fmk) for programmed necrosis to be executed. Notably, even the basal activity of non-stimulated caspase-8 is already sufficient for the suppression of programmed necrosis . Therefore, the induction of programmed necrosis in apoptosis-resistant cell lines in the absence of caspase inhibitors would only be effective in tumors that carry a mutation that directly inactivates caspase-8. In all other cases (i.e. in cells that harbor apoptosis-inhibiting mutations affecting other proteins) the residual activity of caspase-8 would still be sufficient to suppress programmed necrosis. Most likely, this is the reason why the application of TRAIL alone has so far not been effective against apoptosis-resistant tumors in clinical trials. Therefore, we consider the inhibition of caspase-8 as an essential prerequisite for the successful elimination of tumor cells by TRAIL-induced programmed necrosis. In future treatment regimens this could be most conveniently achieved by combining TRAIL with a caspase inhibitor such as zVAD-fmk. With regard to its physiological and clinical relevance, zVAD-fmk has so far proven to be a non-toxic substance that has no adverse effects and which is well tolerated when administered for prolonged periods of time [3, 38, 39]. However, although TRAIL and zVAD-fmk by themselves have not shown toxicity in vivo[9, 40], it must be clarified whether their joint application (and additionally in combination with chemotherapeutic agents) is equally non-toxic in vivo.
As another topic to be addressed with regard to future therapies, cells dying by programmed necrosis can release a broad range of damage-associated molecular patterns (DAMPs) which in turn can trigger inflammatory responses. Accordingly, programmed necrosis has been associated with inflammation in several in vivo models . Therefore, it will be of high interest to clarify whether the death of tumor cells via programmed necrosis is immunogenic and may thus elicit a highly desirable anticancer immune response that would eliminate residual tumor (stem) cells . Such a beneficial inflammation elicited by tumor cells undergoing TRAIL-induced programmed necrosis could thus contribute to an even more effective treatment for cancer patients in the future.