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Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

Gillissen B, Richter A, Richter A, Overkamp T, Essmann F, Hemmati PG, Preissner R, Belka C, Daniel PT - Cell Death Dis (2013)

Bottom Line: Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes.Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype.Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany.

ABSTRACT
TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

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Downregulation or inhibition of XIAP by Mithramycin A or LBW-242, respectively, overcomes TRAIL-resistance. (a) HCT116 wt and Bax−/Bak− cells were pre-incubated with indicated concentrations of Mit A and downregulation of XIAP was observed upon immunoblotting (upper panel). Cells were then treated with TRAIL and cultured for additional 24 h. Measurement of apoptotic cells by flow cytometry revealed that Mit A sensitized Bax/Bak-deficient cells for TRAIL-induced apoptosis. Data expressed as mean values±S.D. from three experiments (lower panel). (b) To inhibit XIAP function, both cell lines were treated with 10 μM of the SMAC mimetic LBW-242 in addition to TRAIL. In HCT116 wt cells, TRAIL-induced caspase-3 processing was increased by LBW-242 (upper left). Increased caspase-3 processing was paralleled by enhanced induction of apoptosis (lower left). In HCT116 Bax−/Bak− cells, pro-caspase-3 was cleaved upon TRAIL-treatment. Proteolytic fragments, however, were detectable only upon addition of LBW-242 (upper right). Caspase-3 activation resulted in induction of apoptosis, indicating that LBW-242 can overcome TRAIL-resistance of Bax/Bak-deficient HCT116 cells (lower right). Statistical significances were determined using an unpaired Student's t-test. Levels of statistical significance are indicated with asterisks (*P<0.05; **P<0.01)
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fig5: Downregulation or inhibition of XIAP by Mithramycin A or LBW-242, respectively, overcomes TRAIL-resistance. (a) HCT116 wt and Bax−/Bak− cells were pre-incubated with indicated concentrations of Mit A and downregulation of XIAP was observed upon immunoblotting (upper panel). Cells were then treated with TRAIL and cultured for additional 24 h. Measurement of apoptotic cells by flow cytometry revealed that Mit A sensitized Bax/Bak-deficient cells for TRAIL-induced apoptosis. Data expressed as mean values±S.D. from three experiments (lower panel). (b) To inhibit XIAP function, both cell lines were treated with 10 μM of the SMAC mimetic LBW-242 in addition to TRAIL. In HCT116 wt cells, TRAIL-induced caspase-3 processing was increased by LBW-242 (upper left). Increased caspase-3 processing was paralleled by enhanced induction of apoptosis (lower left). In HCT116 Bax−/Bak− cells, pro-caspase-3 was cleaved upon TRAIL-treatment. Proteolytic fragments, however, were detectable only upon addition of LBW-242 (upper right). Caspase-3 activation resulted in induction of apoptosis, indicating that LBW-242 can overcome TRAIL-resistance of Bax/Bak-deficient HCT116 cells (lower right). Statistical significances were determined using an unpaired Student's t-test. Levels of statistical significance are indicated with asterisks (*P<0.05; **P<0.01)

Mentions: Given the therapeutic impact of our findings, we next asked if small molecules, known to downregulate or inhibit XIAP, can overcome the resistance of Bax/Bak-deficient cells. The antitumor agent Mithramycin A (Mit A) sensitizes various cancer cell lines to TRAIL-mediated apoptosis by downregulation of XIAP.19 To confirm downregulation of XIAP by Mit A, we treated HCT116 wt and HCT116 Bax−/Bak− cells with different concentrations of Mit A for 24 h. Subsequent analysis of XIAP levels confirmed reduced XIAP expression (Figure 5a). This is accompanied by an increased TRAIL sensitivity in HCT116 wt cells, mainly due to Bax/Bak-dependent, additive toxicity of Mit A. Furthermore, Mit A treatment efficiently overcame TRAIL-resistance of Bax/Bak-deficient HCT116 cells (Figure 5a, lower panel).


Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

Gillissen B, Richter A, Richter A, Overkamp T, Essmann F, Hemmati PG, Preissner R, Belka C, Daniel PT - Cell Death Dis (2013)

Downregulation or inhibition of XIAP by Mithramycin A or LBW-242, respectively, overcomes TRAIL-resistance. (a) HCT116 wt and Bax−/Bak− cells were pre-incubated with indicated concentrations of Mit A and downregulation of XIAP was observed upon immunoblotting (upper panel). Cells were then treated with TRAIL and cultured for additional 24 h. Measurement of apoptotic cells by flow cytometry revealed that Mit A sensitized Bax/Bak-deficient cells for TRAIL-induced apoptosis. Data expressed as mean values±S.D. from three experiments (lower panel). (b) To inhibit XIAP function, both cell lines were treated with 10 μM of the SMAC mimetic LBW-242 in addition to TRAIL. In HCT116 wt cells, TRAIL-induced caspase-3 processing was increased by LBW-242 (upper left). Increased caspase-3 processing was paralleled by enhanced induction of apoptosis (lower left). In HCT116 Bax−/Bak− cells, pro-caspase-3 was cleaved upon TRAIL-treatment. Proteolytic fragments, however, were detectable only upon addition of LBW-242 (upper right). Caspase-3 activation resulted in induction of apoptosis, indicating that LBW-242 can overcome TRAIL-resistance of Bax/Bak-deficient HCT116 cells (lower right). Statistical significances were determined using an unpaired Student's t-test. Levels of statistical significance are indicated with asterisks (*P<0.05; **P<0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Downregulation or inhibition of XIAP by Mithramycin A or LBW-242, respectively, overcomes TRAIL-resistance. (a) HCT116 wt and Bax−/Bak− cells were pre-incubated with indicated concentrations of Mit A and downregulation of XIAP was observed upon immunoblotting (upper panel). Cells were then treated with TRAIL and cultured for additional 24 h. Measurement of apoptotic cells by flow cytometry revealed that Mit A sensitized Bax/Bak-deficient cells for TRAIL-induced apoptosis. Data expressed as mean values±S.D. from three experiments (lower panel). (b) To inhibit XIAP function, both cell lines were treated with 10 μM of the SMAC mimetic LBW-242 in addition to TRAIL. In HCT116 wt cells, TRAIL-induced caspase-3 processing was increased by LBW-242 (upper left). Increased caspase-3 processing was paralleled by enhanced induction of apoptosis (lower left). In HCT116 Bax−/Bak− cells, pro-caspase-3 was cleaved upon TRAIL-treatment. Proteolytic fragments, however, were detectable only upon addition of LBW-242 (upper right). Caspase-3 activation resulted in induction of apoptosis, indicating that LBW-242 can overcome TRAIL-resistance of Bax/Bak-deficient HCT116 cells (lower right). Statistical significances were determined using an unpaired Student's t-test. Levels of statistical significance are indicated with asterisks (*P<0.05; **P<0.01)
Mentions: Given the therapeutic impact of our findings, we next asked if small molecules, known to downregulate or inhibit XIAP, can overcome the resistance of Bax/Bak-deficient cells. The antitumor agent Mithramycin A (Mit A) sensitizes various cancer cell lines to TRAIL-mediated apoptosis by downregulation of XIAP.19 To confirm downregulation of XIAP by Mit A, we treated HCT116 wt and HCT116 Bax−/Bak− cells with different concentrations of Mit A for 24 h. Subsequent analysis of XIAP levels confirmed reduced XIAP expression (Figure 5a). This is accompanied by an increased TRAIL sensitivity in HCT116 wt cells, mainly due to Bax/Bak-dependent, additive toxicity of Mit A. Furthermore, Mit A treatment efficiently overcame TRAIL-resistance of Bax/Bak-deficient HCT116 cells (Figure 5a, lower panel).

Bottom Line: Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes.Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype.Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Humboldt University, Berlin, Germany.

ABSTRACT
TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

Show MeSH
Related in: MedlinePlus