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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

A172 cells are sensitive to TRAIL-induced apoptosis in a dose-dependent manner. (A) Cell surface expression of the various TRAIL receptors was determined on A172 and U87 cells using flow cytometry analysis and expressed as the Mean Fluorescence Intensity (MFI) ratio. (B) Viability of A172 and U87 cells was assessed after treatment with various concentrations (0–1000 ng/ml) of rhTRAIL WT, 4C7 or D269H/E195R for 24h as measured by MTS assays. (C) Western blot analysis of A172 and U87 cells treated with 0, 10 or 100 ng/ml rhTRAIL WT for 5h or 24h, respectively. β-actin serves as a loading control. Error bars represent S.E.M. of three independent experiments.
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Fig1: A172 cells are sensitive to TRAIL-induced apoptosis in a dose-dependent manner. (A) Cell surface expression of the various TRAIL receptors was determined on A172 and U87 cells using flow cytometry analysis and expressed as the Mean Fluorescence Intensity (MFI) ratio. (B) Viability of A172 and U87 cells was assessed after treatment with various concentrations (0–1000 ng/ml) of rhTRAIL WT, 4C7 or D269H/E195R for 24h as measured by MTS assays. (C) Western blot analysis of A172 and U87 cells treated with 0, 10 or 100 ng/ml rhTRAIL WT for 5h or 24h, respectively. β-actin serves as a loading control. Error bars represent S.E.M. of three independent experiments.

Mentions: Analysis of receptor expression by flow cytometry revealed distinct differences in TRAIL receptor membrane expression levels of A172 and U87 cells (Figure 1A). While A172 cells express high levels of DR5, U87 cells showed significantly lower levels of surface DR5. DR4 expression was found to be low in A172 and it was undetectable in U87. Low decoy receptor expression was also detected in A172 cells and absent in U87 cells. Since both A172 and U87 cells show distinct expression profiles of DR5 on the membrane and low or absent expression of DR4, these cells were exposed to different concentrations of rhTRAIL WT, and the previously described DR4-selective variant (rhTRAIL 4C7) (Reis et al. 2010) and DR5-selective variant (rhTRAIL D269H/E195R) (van der Sloot et al. 2006). A172 cells showed a clear dose dependent sensitivity to rhTRAIL, when treated with either rhTRAIL WT or rhTRAIL D269H/E195R, but was completely resistant to rhTRAIL 4C7. The mutant D269H/E195R was more effective in reducing cell viability in A172 when compared to rhTRAIL WT. In contrast, U87 cells were highly resistant to all rhTRAIL ligands (Figure 1B). Afterwards, Western blot analysis was used to detect cleavage of caspases and PARP after treatment with 10 (low) or 100 (moderately high) ng/ml rhTRAIL WT. Since A172 cells proved to be TRAIL-sensitive these cells were exposed to rhTRAIL WT for 5h in order not to lose the cells due to massive cell death. The TRAIL-resistant U87 cells were treated for 24h. Accordingly, rhTRAIL WT exposure resulted in clear cleavage of caspase-8, -9, -3 and PARP in A172 cells, whereas in U87 cells, even upon treatment with 100 ng/ml rhTRAIL WT, no activation of these apoptotic-related proteins was observed (Figure 1C). We then tested if the differential TRAIL-sensitivities observed in A172 and U87 could be explained by the differences at the level of surface DR5 expression, with low levels of death receptor expression in U87 cells causing the insensitivity to both rhTRAIL WT and rhTRAIL D269H/E195R.Figure 1


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)

A172 cells are sensitive to TRAIL-induced apoptosis in a dose-dependent manner. (A) Cell surface expression of the various TRAIL receptors was determined on A172 and U87 cells using flow cytometry analysis and expressed as the Mean Fluorescence Intensity (MFI) ratio. (B) Viability of A172 and U87 cells was assessed after treatment with various concentrations (0–1000 ng/ml) of rhTRAIL WT, 4C7 or D269H/E195R for 24h as measured by MTS assays. (C) Western blot analysis of A172 and U87 cells treated with 0, 10 or 100 ng/ml rhTRAIL WT for 5h or 24h, respectively. β-actin serves as a loading control. Error bars represent S.E.M. of three independent experiments.
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Related In: Results  -  Collection

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Fig1: A172 cells are sensitive to TRAIL-induced apoptosis in a dose-dependent manner. (A) Cell surface expression of the various TRAIL receptors was determined on A172 and U87 cells using flow cytometry analysis and expressed as the Mean Fluorescence Intensity (MFI) ratio. (B) Viability of A172 and U87 cells was assessed after treatment with various concentrations (0–1000 ng/ml) of rhTRAIL WT, 4C7 or D269H/E195R for 24h as measured by MTS assays. (C) Western blot analysis of A172 and U87 cells treated with 0, 10 or 100 ng/ml rhTRAIL WT for 5h or 24h, respectively. β-actin serves as a loading control. Error bars represent S.E.M. of three independent experiments.
Mentions: Analysis of receptor expression by flow cytometry revealed distinct differences in TRAIL receptor membrane expression levels of A172 and U87 cells (Figure 1A). While A172 cells express high levels of DR5, U87 cells showed significantly lower levels of surface DR5. DR4 expression was found to be low in A172 and it was undetectable in U87. Low decoy receptor expression was also detected in A172 cells and absent in U87 cells. Since both A172 and U87 cells show distinct expression profiles of DR5 on the membrane and low or absent expression of DR4, these cells were exposed to different concentrations of rhTRAIL WT, and the previously described DR4-selective variant (rhTRAIL 4C7) (Reis et al. 2010) and DR5-selective variant (rhTRAIL D269H/E195R) (van der Sloot et al. 2006). A172 cells showed a clear dose dependent sensitivity to rhTRAIL, when treated with either rhTRAIL WT or rhTRAIL D269H/E195R, but was completely resistant to rhTRAIL 4C7. The mutant D269H/E195R was more effective in reducing cell viability in A172 when compared to rhTRAIL WT. In contrast, U87 cells were highly resistant to all rhTRAIL ligands (Figure 1B). Afterwards, Western blot analysis was used to detect cleavage of caspases and PARP after treatment with 10 (low) or 100 (moderately high) ng/ml rhTRAIL WT. Since A172 cells proved to be TRAIL-sensitive these cells were exposed to rhTRAIL WT for 5h in order not to lose the cells due to massive cell death. The TRAIL-resistant U87 cells were treated for 24h. Accordingly, rhTRAIL WT exposure resulted in clear cleavage of caspase-8, -9, -3 and PARP in A172 cells, whereas in U87 cells, even upon treatment with 100 ng/ml rhTRAIL WT, no activation of these apoptotic-related proteins was observed (Figure 1C). We then tested if the differential TRAIL-sensitivities observed in A172 and U87 could be explained by the differences at the level of surface DR5 expression, with low levels of death receptor expression in U87 cells causing the insensitivity to both rhTRAIL WT and rhTRAIL D269H/E195R.Figure 1

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