Abstract
BackgroundThe cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, its efficacy is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Programmed necrosis is an alternative, molecularly distinct mode of programmed cell death that is elicited by TRAIL under conditions when the classical apoptosis machinery fails or is actively inhibited. The potential of TRAIL-induced programmed necrosis in tumor therapy is, however, almost completely uncharacterized. We therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin.MethodsCell death/viability was measured by flow cytometry/determination of intracellular ATP levels/crystal violet staining. Cell surface expression of TRAIL receptors was detected by flow cytometry, expression of proteins by Western blot. Ceramide levels were quantified by high-performance thin layer chromatography and densitometric analysis, clonogenic survival of cells was determined by crystal violet staining or by soft agarose cloning.ResultsTRAIL-induced programmed necrosis killed eight out of 14 tumor cell lines. Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested. TRAIL synergized with chemotherapeutics in killing tumor cell lines by programmed necrosis, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. Susceptibility/resistance of the investigated tumor cell lines to programmed necrosis seems to primarily depend on expression of the pro-necrotic kinase RIPK3 rather than the related kinase RIPK1 or cell surface expression of TRAIL receptors. Furthermore, interference with production of the lipid ceramide protected all tested tumor cell lines.ConclusionsOur study provides evidence that TRAIL-induced programmed necrosis represents a feasible approach for the elimination of tumor cells, and that this treatment may represent a promising new option for the future development of combination therapies. Our data also suggest that RIPK3 expression may serve as a potential predictive marker for the sensitivity of tumor cells to programmed necrosis and extend the previously established role of ceramide as a key mediator of death receptor-induced programmed necrosis (and thus as a potential target for future therapies) also to the tumor cell lines examined here.
Highlights
The cytokine tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies
Our findings suggest that susceptibility/resistance of tumor cells to programmed necrosis is primarily determined by expression of the kinase RIPK3 and that ceramide represents a pivotal factor downstream of RIPK3 in the execution of programmed necrosis in the previously studied common laboratory cell lines, and in the clinically more relevant tumor cell systems employed here
TNF/zVAD/CHX-induced programmed necrosis We initially characterized the above human cancer cell lines with regard to their sensitivity to TRAIL-induced programmed necrosis, utilizing TNF-elicited programmed necrosis as an established control in this and subsequent experiments
Summary
The cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. Independent from apoptosis, programmed necrosis represents an alternative form of PCD that operates without detectable caspase activity [2,3]. Many tumor cells are intrinsically resistant against TRAIL-induced apoptosis and, even when combined with chemo- or radiotherapy, a resounding breakthrough in the therapy of cancer patients has not yet been achieved. We and others have previously demonstrated the ability of human and murine TRAIL receptors to induce programmed necrosis independently from their apoptotic capabilities when induction of apoptosis fails or is actively inhibited [7,10]. The induction of programmed necrosis by TRAIL may represent a novel and additional, but still largely unexplored option for the elimination of tumor cells, in addition to the well-established strategies aimed at the induction of apoptosis
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