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The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

van Roosmalen IA, Reis CR, Setroikromo R, Yuvaraj S, Joseph JV, Tepper PG, Kruyt FA, Quax WJ - Springerplus (2014)

Bottom Line: We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells.Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells.Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen, 9713 AV The Netherlands ; Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen, 9713 GZ The Netherlands.

ABSTRACT
Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

No MeSH data available.


Related in: MedlinePlus

Sensitivity of A172 to TRAIL-induced apoptosis is slightly enhanced by down-regulation of survivin. (A) Western blot analysis of survivin siRNA transfected A172 cells treated with or without 10 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R for 5h. Lysates were tested for survivin down-regulation and apoptosis induction by PARP cleavage. β-actin served as a loading control. (B) A172 cells transfected with survivin siRNA were treated with 0–100 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R. Cell viability was assessed after 24h using MTS assay. The graphs are corrected for control or survivin knockdown induced cytotoxicity. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.
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Fig7: Sensitivity of A172 to TRAIL-induced apoptosis is slightly enhanced by down-regulation of survivin. (A) Western blot analysis of survivin siRNA transfected A172 cells treated with or without 10 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R for 5h. Lysates were tested for survivin down-regulation and apoptosis induction by PARP cleavage. β-actin served as a loading control. (B) A172 cells transfected with survivin siRNA were treated with 0–100 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R. Cell viability was assessed after 24h using MTS assay. The graphs are corrected for control or survivin knockdown induced cytotoxicity. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.

Mentions: In order to determine if down-regulation of survivin by DMC could explain the increase in TRAIL sensitivity observed in A172 cells, its expression was selectively silenced using a siRNA approach. Figure 7A shows efficient down-regulation of survivin upon siRNA transfection, which resulted in apoptosis activation as indicated by PARP cleavage. Interestingly, the addition of only 10 ng/ml rhTRAIL WT enhanced PARP cleavage in survivin knockdown cells (Figure 7A), whereas the addition of D269H/E195R resulted in massive cell death, such that an insufficient amount of lysate could be prepared. Finally, analysis of cell viability upon TRAIL treatment in non-transfected, control-siRNA and survivin-siRNA treated cells indicate that the down-regulation of survivin slightly increased rhTRAIL WT- and rhTRAIL D269H/E195R-mediated apoptosis in A172 at low concentrations, when compared to both untransfected and control-siRNA treated cells (Figure 7B).Figure 7


The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma.

van Roosmalen IA, Reis CR, Setroikromo R, Yuvaraj S, Joseph JV, Tepper PG, Kruyt FA, Quax WJ - Springerplus (2014)

Sensitivity of A172 to TRAIL-induced apoptosis is slightly enhanced by down-regulation of survivin. (A) Western blot analysis of survivin siRNA transfected A172 cells treated with or without 10 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R for 5h. Lysates were tested for survivin down-regulation and apoptosis induction by PARP cleavage. β-actin served as a loading control. (B) A172 cells transfected with survivin siRNA were treated with 0–100 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R. Cell viability was assessed after 24h using MTS assay. The graphs are corrected for control or survivin knockdown induced cytotoxicity. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig7: Sensitivity of A172 to TRAIL-induced apoptosis is slightly enhanced by down-regulation of survivin. (A) Western blot analysis of survivin siRNA transfected A172 cells treated with or without 10 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R for 5h. Lysates were tested for survivin down-regulation and apoptosis induction by PARP cleavage. β-actin served as a loading control. (B) A172 cells transfected with survivin siRNA were treated with 0–100 ng/ml rhTRAIL WT or rhTRAIL D269H/E195R. Cell viability was assessed after 24h using MTS assay. The graphs are corrected for control or survivin knockdown induced cytotoxicity. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.
Mentions: In order to determine if down-regulation of survivin by DMC could explain the increase in TRAIL sensitivity observed in A172 cells, its expression was selectively silenced using a siRNA approach. Figure 7A shows efficient down-regulation of survivin upon siRNA transfection, which resulted in apoptosis activation as indicated by PARP cleavage. Interestingly, the addition of only 10 ng/ml rhTRAIL WT enhanced PARP cleavage in survivin knockdown cells (Figure 7A), whereas the addition of D269H/E195R resulted in massive cell death, such that an insufficient amount of lysate could be prepared. Finally, analysis of cell viability upon TRAIL treatment in non-transfected, control-siRNA and survivin-siRNA treated cells indicate that the down-regulation of survivin slightly increased rhTRAIL WT- and rhTRAIL D269H/E195R-mediated apoptosis in A172 at low concentrations, when compared to both untransfected and control-siRNA treated cells (Figure 7B).Figure 7

Bottom Line: We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells.Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells.Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen, 9713 AV The Netherlands ; Department of Medical Oncology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, Groningen, 9713 GZ The Netherlands.

ABSTRACT
Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

No MeSH data available.


Related in: MedlinePlus