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TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells.

Voigt S, Philipp S, Davarnia P, Winoto-Morbach S, Röder C, Arenz C, Trauzold A, Kabelitz D, Schütze S, Kalthoff H, Adam D - BMC Cancer (2014)

Bottom Line: 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.Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Immunologie, Christian-Albrechts-Universität, Michaelisstrasse 5, 24105 Kiel, Germany. dadam@email.uni-kiel.de.

ABSTRACT

Background: The 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.

Methods: Cell 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.

Results: TRAIL-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.

Conclusions: Our 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.

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Flow cytometric analysis of TRAIL-R1 and TRAIL-R2 cell surface expression. Cell lines clearly expressing both TRAIL-R1 and TRAIL-R2 (a) or primarily TRAIL-R2 with low or absent expression of TRAIL-R1 (b) are shown. Cells were stained with specific monoclonal antibodies for either TRAIL-R1 or TRAIL-R2 as indicated in the figure (blue) or with isotype matched control antibodies (red), with each curve representing 10,000 counted cells.
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Figure 2: Flow cytometric analysis of TRAIL-R1 and TRAIL-R2 cell surface expression. Cell lines clearly expressing both TRAIL-R1 and TRAIL-R2 (a) or primarily TRAIL-R2 with low or absent expression of TRAIL-R1 (b) are shown. Cells were stained with specific monoclonal antibodies for either TRAIL-R1 or TRAIL-R2 as indicated in the figure (blue) or with isotype matched control antibodies (red), with each curve representing 10,000 counted cells.

Mentions: The susceptibility/resistance of cells to TRAIL/zVAD/CHX- or TNF/zVAD/CHX-induced programmed necrosis is initially dependent on the expression of the corresponding receptors on the cell surface. We limited our analyses to cell surface expression of TRAIL-R1 and TRAIL-R2 on the tumor cell lines used in this study since TNF-R1 is known to be expressed on the surface of every cell type in the human body except red blood cells [23]. Whereas TRAIL-R1 differentially showed pronounced (Figure 2a) or low to no cell surface expression (Figure 2b), TRAIL-R2 was highly expressed on the surface of all analyzed tumor cell lines (Figures 2a and b). Since Guo and coworkers have shown that TRAIL-R2 can mediate programmed necrosis by itself [10], this indicates that susceptibility or resistance of the investigated tumor cell lines to programmed necrosis is not determined at the level of receptor expression.


TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells.

Voigt S, Philipp S, Davarnia P, Winoto-Morbach S, Röder C, Arenz C, Trauzold A, Kabelitz D, Schütze S, Kalthoff H, Adam D - BMC Cancer (2014)

Flow cytometric analysis of TRAIL-R1 and TRAIL-R2 cell surface expression. Cell lines clearly expressing both TRAIL-R1 and TRAIL-R2 (a) or primarily TRAIL-R2 with low or absent expression of TRAIL-R1 (b) are shown. Cells were stained with specific monoclonal antibodies for either TRAIL-R1 or TRAIL-R2 as indicated in the figure (blue) or with isotype matched control antibodies (red), with each curve representing 10,000 counted cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927850&req=5

Figure 2: Flow cytometric analysis of TRAIL-R1 and TRAIL-R2 cell surface expression. Cell lines clearly expressing both TRAIL-R1 and TRAIL-R2 (a) or primarily TRAIL-R2 with low or absent expression of TRAIL-R1 (b) are shown. Cells were stained with specific monoclonal antibodies for either TRAIL-R1 or TRAIL-R2 as indicated in the figure (blue) or with isotype matched control antibodies (red), with each curve representing 10,000 counted cells.
Mentions: The susceptibility/resistance of cells to TRAIL/zVAD/CHX- or TNF/zVAD/CHX-induced programmed necrosis is initially dependent on the expression of the corresponding receptors on the cell surface. We limited our analyses to cell surface expression of TRAIL-R1 and TRAIL-R2 on the tumor cell lines used in this study since TNF-R1 is known to be expressed on the surface of every cell type in the human body except red blood cells [23]. Whereas TRAIL-R1 differentially showed pronounced (Figure 2a) or low to no cell surface expression (Figure 2b), TRAIL-R2 was highly expressed on the surface of all analyzed tumor cell lines (Figures 2a and b). Since Guo and coworkers have shown that TRAIL-R2 can mediate programmed necrosis by itself [10], this indicates that susceptibility or resistance of the investigated tumor cell lines to programmed necrosis is not determined at the level of receptor expression.

Bottom Line: 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.Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Immunologie, Christian-Albrechts-Universität, Michaelisstrasse 5, 24105 Kiel, Germany. dadam@email.uni-kiel.de.

ABSTRACT

Background: The 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.

Methods: Cell 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.

Results: TRAIL-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.

Conclusions: Our 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.

Show MeSH
Related in: MedlinePlus