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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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Impact of TRAIL/zVAD/CHX- and TNF/zVAD/CHX-induced programmed necrosis on clonogenic survival. Cells were treated with zVAD/CHX, TRAIL/zVAD/CHX or TNF/zVAD/CHX for 24 h as in Figure 1a and b. Subsequently, their ability to form colonies was analyzed by staining with crystal violet (or by soft agarose cloning for the non-adherent cell line U-937) after 7 days. Treatment with zVAD/CHX (negative control) served as reference for calculation of colony formation as well as of P values. *P < 0.05, ***P < 0.001.
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Figure 5: Impact of TRAIL/zVAD/CHX- and TNF/zVAD/CHX-induced programmed necrosis on clonogenic survival. Cells were treated with zVAD/CHX, TRAIL/zVAD/CHX or TNF/zVAD/CHX for 24 h as in Figure 1a and b. Subsequently, their ability to form colonies was analyzed by staining with crystal violet (or by soft agarose cloning for the non-adherent cell line U-937) after 7 days. Treatment with zVAD/CHX (negative control) served as reference for calculation of colony formation as well as of P values. *P < 0.05, ***P < 0.001.

Mentions: To determine whether induction of programmed necrosis is a viable strategy to block the capacity of tumor cells for unlimited proliferation, we next investigated clonogenic survival employing the tumor cell lines analyzed in Figures 3a and 4b. As shown in Figure 5, treatment with TRAIL/zVAD/CHX reduced clonogenic survival with statistical significance in four out of five sensitive cell lines (U-937 cells were only slightly above the significance threshold of 0.05 with P = 0.059), and even in the control cell line KNS-62 which had shown resistance to TRAIL/zVAD/CHX-induced programmed necrosis in cytotoxicity/viability assays (Figure 1a-c). Almost identical, a reduction of clonogenicity was detectable in five out of the six tested tumor cell lines after treatment with TNF/zVAD/CHX, with three cell lines showing a statistically significant reduction (KNS-62 cells were only slightly above the significance threshold with P = 0.057). In summary, these data confirm that induction of programmed necrosis can reduce the proliferative potential and thus the clonogenicity of tumor cells.


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)

Impact of TRAIL/zVAD/CHX- and TNF/zVAD/CHX-induced programmed necrosis on clonogenic survival. Cells were treated with zVAD/CHX, TRAIL/zVAD/CHX or TNF/zVAD/CHX for 24 h as in Figure 1a and b. Subsequently, their ability to form colonies was analyzed by staining with crystal violet (or by soft agarose cloning for the non-adherent cell line U-937) after 7 days. Treatment with zVAD/CHX (negative control) served as reference for calculation of colony formation as well as of P values. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Impact of TRAIL/zVAD/CHX- and TNF/zVAD/CHX-induced programmed necrosis on clonogenic survival. Cells were treated with zVAD/CHX, TRAIL/zVAD/CHX or TNF/zVAD/CHX for 24 h as in Figure 1a and b. Subsequently, their ability to form colonies was analyzed by staining with crystal violet (or by soft agarose cloning for the non-adherent cell line U-937) after 7 days. Treatment with zVAD/CHX (negative control) served as reference for calculation of colony formation as well as of P values. *P < 0.05, ***P < 0.001.
Mentions: To determine whether induction of programmed necrosis is a viable strategy to block the capacity of tumor cells for unlimited proliferation, we next investigated clonogenic survival employing the tumor cell lines analyzed in Figures 3a and 4b. As shown in Figure 5, treatment with TRAIL/zVAD/CHX reduced clonogenic survival with statistical significance in four out of five sensitive cell lines (U-937 cells were only slightly above the significance threshold of 0.05 with P = 0.059), and even in the control cell line KNS-62 which had shown resistance to TRAIL/zVAD/CHX-induced programmed necrosis in cytotoxicity/viability assays (Figure 1a-c). Almost identical, a reduction of clonogenicity was detectable in five out of the six tested tumor cell lines after treatment with TNF/zVAD/CHX, with three cell lines showing a statistically significant reduction (KNS-62 cells were only slightly above the significance threshold with P = 0.057). In summary, these data confirm that induction of programmed necrosis can reduce the proliferative potential and thus the clonogenicity of tumor cells.

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