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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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Induction of programmed necrosis by TRAIL/zVAD/CHX and TNF/zVAD/CHX in human tumor cell lines. Cells were treated with 100 ng/ml of (a) TRAIL or (b) TNF in combination with 50 μM zVAD-fmk and non-toxic concentrations of CHX (U-937 0.1 μg/ml; Mz-ChA-1 2 μg/ml; BxPC-3 1 μg/ml; HT-29 5 μg/ml; Colo357 5 μg/ml; Panc89 1 μg/ml; PancTu-I 10 μg/ml; A818-4 10 μg/ml; CCRF-CEM 0.0625 μg/ml; MKN-28 5 μg/ml; SK-OV-3 1 μg/ml; KNS-62 5 μg/ml; Pt45P1 0.1 μg/ml; SK-MEL-28 10 μg/ml). After 24 h, loss of membrane integrity was measured as a marker for programmed necrosis by flow cytometric detection of PI-positive cells. Values above the respective columns represent the specific percentage of programmed necrosis (stimulus-induced minus zVAD/CHX-induced programmed necrosis), spontaneous cell death in untreated cells is shown for comparison. (c) Cells were treated with their respective LD50 concentrations of CHX alone (CCRF-CEM 60 μg/ml; MKN-28 1000 μg/ml; SK-OV-3 500 μg/ml; KNS-62 300 μg/ml; Pt45P1 150 μg/ml; SK-MEL-28 625 μg/ml) or in combination with 100 ng/ml TRAIL or TNF and 50 μM zVAD-fmk. After 24 h, viability was determined by crystal violet staining (for the adherent cell lines MKN-28, SK-OV-3, KNS-62, Pt45P1 and SK-MEL-28) or XTT assay analysis (for the suspension cell line CCRF-CEM). (d) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b in the presence of the indicated concentrations of the Smac mimetic birinapant. After 8 or 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.
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Figure 1: Induction of programmed necrosis by TRAIL/zVAD/CHX and TNF/zVAD/CHX in human tumor cell lines. Cells were treated with 100 ng/ml of (a) TRAIL or (b) TNF in combination with 50 μM zVAD-fmk and non-toxic concentrations of CHX (U-937 0.1 μg/ml; Mz-ChA-1 2 μg/ml; BxPC-3 1 μg/ml; HT-29 5 μg/ml; Colo357 5 μg/ml; Panc89 1 μg/ml; PancTu-I 10 μg/ml; A818-4 10 μg/ml; CCRF-CEM 0.0625 μg/ml; MKN-28 5 μg/ml; SK-OV-3 1 μg/ml; KNS-62 5 μg/ml; Pt45P1 0.1 μg/ml; SK-MEL-28 10 μg/ml). After 24 h, loss of membrane integrity was measured as a marker for programmed necrosis by flow cytometric detection of PI-positive cells. Values above the respective columns represent the specific percentage of programmed necrosis (stimulus-induced minus zVAD/CHX-induced programmed necrosis), spontaneous cell death in untreated cells is shown for comparison. (c) Cells were treated with their respective LD50 concentrations of CHX alone (CCRF-CEM 60 μg/ml; MKN-28 1000 μg/ml; SK-OV-3 500 μg/ml; KNS-62 300 μg/ml; Pt45P1 150 μg/ml; SK-MEL-28 625 μg/ml) or in combination with 100 ng/ml TRAIL or TNF and 50 μM zVAD-fmk. After 24 h, viability was determined by crystal violet staining (for the adherent cell lines MKN-28, SK-OV-3, KNS-62, Pt45P1 and SK-MEL-28) or XTT assay analysis (for the suspension cell line CCRF-CEM). (d) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b in the presence of the indicated concentrations of the Smac mimetic birinapant. After 8 or 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.

Mentions: 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. Since treatment with TRAIL normally activates caspase-dependent apoptosis (which would obstruct the analysis of programmed necrosis), we actively inhibited caspases/apoptosis by addition of the broad-spectrum caspase inhibitor zVAD-fmk. This treatment is not only experimentally required to suppress apoptosis, but in addition potentiates programmed necrosis by inhibiting caspase-8, which acts as a negative regulator of programmed necrosis [20] and which otherwise would prevent the induction of programmed necrosis by TRAIL. Furthermore, all cells were additionally treated with non-toxic concentrations of the protein biosynthesis inhibitor cycloheximide (CHX) that we had previously found to sensitize for programmed necrosis. As depicted in Figure 1a, treatment with TRAIL/zVAD/CHX induced programmed necrosis in eight out of 14 tested tumor cell lines. The tumor cell lines U-937, Mz-ChA-1, BxPC-3 and HT-29 exhibited the highest sensitivity, followed by Colo357, Panc89, PancTu-I and A818-4 cells. The remaining cell lines, i.e. CCRF-CEM, MKN-28, SK-OV-3, KNS-62, Pt45P1, and SK-MEL-28 displayed only a marginal or no response to treatment with TRAIL/zVAD/CHX. We obtained essentially the same results in control assays when we induced programmed necrosis with TNF/zVAD/CHX (Figure 1b). As the only exception, CCRF-CEM cells were resistant to TRAIL/zVAD/CHX- but clearly sensitive to TNF/zVAD/CHX-induced programmed necrosis.


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)

Induction of programmed necrosis by TRAIL/zVAD/CHX and TNF/zVAD/CHX in human tumor cell lines. Cells were treated with 100 ng/ml of (a) TRAIL or (b) TNF in combination with 50 μM zVAD-fmk and non-toxic concentrations of CHX (U-937 0.1 μg/ml; Mz-ChA-1 2 μg/ml; BxPC-3 1 μg/ml; HT-29 5 μg/ml; Colo357 5 μg/ml; Panc89 1 μg/ml; PancTu-I 10 μg/ml; A818-4 10 μg/ml; CCRF-CEM 0.0625 μg/ml; MKN-28 5 μg/ml; SK-OV-3 1 μg/ml; KNS-62 5 μg/ml; Pt45P1 0.1 μg/ml; SK-MEL-28 10 μg/ml). After 24 h, loss of membrane integrity was measured as a marker for programmed necrosis by flow cytometric detection of PI-positive cells. Values above the respective columns represent the specific percentage of programmed necrosis (stimulus-induced minus zVAD/CHX-induced programmed necrosis), spontaneous cell death in untreated cells is shown for comparison. (c) Cells were treated with their respective LD50 concentrations of CHX alone (CCRF-CEM 60 μg/ml; MKN-28 1000 μg/ml; SK-OV-3 500 μg/ml; KNS-62 300 μg/ml; Pt45P1 150 μg/ml; SK-MEL-28 625 μg/ml) or in combination with 100 ng/ml TRAIL or TNF and 50 μM zVAD-fmk. After 24 h, viability was determined by crystal violet staining (for the adherent cell lines MKN-28, SK-OV-3, KNS-62, Pt45P1 and SK-MEL-28) or XTT assay analysis (for the suspension cell line CCRF-CEM). (d) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b in the presence of the indicated concentrations of the Smac mimetic birinapant. After 8 or 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.
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Related In: Results  -  Collection

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

Figure 1: Induction of programmed necrosis by TRAIL/zVAD/CHX and TNF/zVAD/CHX in human tumor cell lines. Cells were treated with 100 ng/ml of (a) TRAIL or (b) TNF in combination with 50 μM zVAD-fmk and non-toxic concentrations of CHX (U-937 0.1 μg/ml; Mz-ChA-1 2 μg/ml; BxPC-3 1 μg/ml; HT-29 5 μg/ml; Colo357 5 μg/ml; Panc89 1 μg/ml; PancTu-I 10 μg/ml; A818-4 10 μg/ml; CCRF-CEM 0.0625 μg/ml; MKN-28 5 μg/ml; SK-OV-3 1 μg/ml; KNS-62 5 μg/ml; Pt45P1 0.1 μg/ml; SK-MEL-28 10 μg/ml). After 24 h, loss of membrane integrity was measured as a marker for programmed necrosis by flow cytometric detection of PI-positive cells. Values above the respective columns represent the specific percentage of programmed necrosis (stimulus-induced minus zVAD/CHX-induced programmed necrosis), spontaneous cell death in untreated cells is shown for comparison. (c) Cells were treated with their respective LD50 concentrations of CHX alone (CCRF-CEM 60 μg/ml; MKN-28 1000 μg/ml; SK-OV-3 500 μg/ml; KNS-62 300 μg/ml; Pt45P1 150 μg/ml; SK-MEL-28 625 μg/ml) or in combination with 100 ng/ml TRAIL or TNF and 50 μM zVAD-fmk. After 24 h, viability was determined by crystal violet staining (for the adherent cell lines MKN-28, SK-OV-3, KNS-62, Pt45P1 and SK-MEL-28) or XTT assay analysis (for the suspension cell line CCRF-CEM). (d) Cells were left untreated or stimulated with TRAIL/zVAD/CHX or TNF/zVAD/CHX as in Figure 1a and b in the presence of the indicated concentrations of the Smac mimetic birinapant. After 8 or 24 h of stimulation, programmed necrosis was analyzed by flow cytometric analysis of PI-positive cells.
Mentions: 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. Since treatment with TRAIL normally activates caspase-dependent apoptosis (which would obstruct the analysis of programmed necrosis), we actively inhibited caspases/apoptosis by addition of the broad-spectrum caspase inhibitor zVAD-fmk. This treatment is not only experimentally required to suppress apoptosis, but in addition potentiates programmed necrosis by inhibiting caspase-8, which acts as a negative regulator of programmed necrosis [20] and which otherwise would prevent the induction of programmed necrosis by TRAIL. Furthermore, all cells were additionally treated with non-toxic concentrations of the protein biosynthesis inhibitor cycloheximide (CHX) that we had previously found to sensitize for programmed necrosis. As depicted in Figure 1a, treatment with TRAIL/zVAD/CHX induced programmed necrosis in eight out of 14 tested tumor cell lines. The tumor cell lines U-937, Mz-ChA-1, BxPC-3 and HT-29 exhibited the highest sensitivity, followed by Colo357, Panc89, PancTu-I and A818-4 cells. The remaining cell lines, i.e. CCRF-CEM, MKN-28, SK-OV-3, KNS-62, Pt45P1, and SK-MEL-28 displayed only a marginal or no response to treatment with TRAIL/zVAD/CHX. We obtained essentially the same results in control assays when we induced programmed necrosis with TNF/zVAD/CHX (Figure 1b). As the only exception, CCRF-CEM cells were resistant to TRAIL/zVAD/CHX- but clearly sensitive to TNF/zVAD/CHX-induced programmed necrosis.

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