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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

Increased TRAIL sensitivity by DMC is caspase-dependent. (A) Cell viability was assessed after 24h co-treatment with rhTRAIL WT (0–250 ng/ml) and 0, 25 or 50 μM DMC using a MTS assay. (B) Western blot analysis of A172 (5h) and U87 (24h) cells treated with either rhTRAIL WT (0, 10 or 100 ng/ml) and/or DMC (0, 25 or 50 μM) for caspase-8, -9, -3 and PARP. β-actin served as a loading control.
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Fig4: Increased TRAIL sensitivity by DMC is caspase-dependent. (A) Cell viability was assessed after 24h co-treatment with rhTRAIL WT (0–250 ng/ml) and 0, 25 or 50 μM DMC using a MTS assay. (B) Western blot analysis of A172 (5h) and U87 (24h) cells treated with either rhTRAIL WT (0, 10 or 100 ng/ml) and/or DMC (0, 25 or 50 μM) for caspase-8, -9, -3 and PARP. β-actin served as a loading control.

Mentions: To evaluate if the DMC effects observed in these cells were correlated with the induction of apoptosis or necrosis, A172 and U87 cells were treated with either a sub-toxic (25 μM) or a moderate cytotoxic (50 μM) dose of DMC for 24h and stained with Annexin V/PI, with Annexin V positive cells representing the apoptotic fraction. Figure 3B shows that the addition of a high concentration of DMC alone resulted in a minor increase in Annexin V positivity. In addition, after DMC treatment the level of Annexin-/PI+ cells were comparable to untreated cells. Together with the results obtained using MTS assays, these results suggest that treating cells with concentrations up to 50 μM DMC did not result in apoptosis or necrosis in these cells, contrasting with the significant reduction seen when using the MTS assay. In order to assess if DMC induced ER stress, expression levels of GRP78 and CHOP, two established markers of ER stress, were examined. Consistent with the previously described role of DMC in ER-stress induction, Western blot analysis showed a clear up-regulation of GRP78 and CHOP upon treatment with 50 μM of DMC, (Figure 3C). Next, the effect of combined exposure to rhTRAIL WT and 0, 25 or 50 μM of DMC was examined in these differentially TRAIL-responsive GBM cell lines. In this assay, A172 cells treated for 24h with 125 ng/mL rhTRAIL without DMC showed already ~75% reduction in cell viability (IC50: ~36 ng/ml), which was strongly enhanced after combination with DMC (25 μM: ~10%, IC50: ~12 ng/ml and 50 μM: ~2%, IC50: ~1 ng/ml) (Figure 4A). Notably, the cell viability of A172 cells was reduced with approximately 86% using only 31.3 ng/ml of rhTRAIL WT in combination with 25 μM of DMC, when compared to TRAIL WT in combination with 0 μM DMC, where approximately 47% reduction in cell viability could be attained. Therefore, TRAIL sensitivity can be enhanced in A172 cells by co-treatment with DMC. U87 cells remained TRAIL-resistant (0 μM: ~99%), and although the single treatment with DMC was able to lower the cell viability of U87 cells (25 μM: ~87%; 50 μM: 66%), no additive or synergistic effects could be found in combination with rhTRAIL WT (Figure 4A). Since DMC is an analogue of celecoxib, we further tested the sensitivity of these cell lines to celecoxib treatment. A172 and U87 showed similar IC50 values of approximately 88 μM for both A172 and U87 (Additional file 1: Figure S1). Next, DMC or celecoxib were tested side-by-side in combination with rhTRAIL WT. Interestingly, the combination of TRAIL/DMC (IC50: ~57 μM) was shown to be more effective than TRAIL/celecoxib (IC50: ~88 μM) (Additional file 2: Figure S2). Since DMC is a more potent ER stress inducer than celecoxib and does not target COX-2 (Pyrko et al. 2007; Chuang et al. 2008), these results suggest that ER stress activation is closely related to the reduction of cell viability, and subsequently, the enhancement of TRAIL-induced cytotoxicity in these cells.Figure 4


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)

Increased TRAIL sensitivity by DMC is caspase-dependent. (A) Cell viability was assessed after 24h co-treatment with rhTRAIL WT (0–250 ng/ml) and 0, 25 or 50 μM DMC using a MTS assay. (B) Western blot analysis of A172 (5h) and U87 (24h) cells treated with either rhTRAIL WT (0, 10 or 100 ng/ml) and/or DMC (0, 25 or 50 μM) for caspase-8, -9, -3 and PARP. β-actin served as a loading control.
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Related In: Results  -  Collection

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Fig4: Increased TRAIL sensitivity by DMC is caspase-dependent. (A) Cell viability was assessed after 24h co-treatment with rhTRAIL WT (0–250 ng/ml) and 0, 25 or 50 μM DMC using a MTS assay. (B) Western blot analysis of A172 (5h) and U87 (24h) cells treated with either rhTRAIL WT (0, 10 or 100 ng/ml) and/or DMC (0, 25 or 50 μM) for caspase-8, -9, -3 and PARP. β-actin served as a loading control.
Mentions: To evaluate if the DMC effects observed in these cells were correlated with the induction of apoptosis or necrosis, A172 and U87 cells were treated with either a sub-toxic (25 μM) or a moderate cytotoxic (50 μM) dose of DMC for 24h and stained with Annexin V/PI, with Annexin V positive cells representing the apoptotic fraction. Figure 3B shows that the addition of a high concentration of DMC alone resulted in a minor increase in Annexin V positivity. In addition, after DMC treatment the level of Annexin-/PI+ cells were comparable to untreated cells. Together with the results obtained using MTS assays, these results suggest that treating cells with concentrations up to 50 μM DMC did not result in apoptosis or necrosis in these cells, contrasting with the significant reduction seen when using the MTS assay. In order to assess if DMC induced ER stress, expression levels of GRP78 and CHOP, two established markers of ER stress, were examined. Consistent with the previously described role of DMC in ER-stress induction, Western blot analysis showed a clear up-regulation of GRP78 and CHOP upon treatment with 50 μM of DMC, (Figure 3C). Next, the effect of combined exposure to rhTRAIL WT and 0, 25 or 50 μM of DMC was examined in these differentially TRAIL-responsive GBM cell lines. In this assay, A172 cells treated for 24h with 125 ng/mL rhTRAIL without DMC showed already ~75% reduction in cell viability (IC50: ~36 ng/ml), which was strongly enhanced after combination with DMC (25 μM: ~10%, IC50: ~12 ng/ml and 50 μM: ~2%, IC50: ~1 ng/ml) (Figure 4A). Notably, the cell viability of A172 cells was reduced with approximately 86% using only 31.3 ng/ml of rhTRAIL WT in combination with 25 μM of DMC, when compared to TRAIL WT in combination with 0 μM DMC, where approximately 47% reduction in cell viability could be attained. Therefore, TRAIL sensitivity can be enhanced in A172 cells by co-treatment with DMC. U87 cells remained TRAIL-resistant (0 μM: ~99%), and although the single treatment with DMC was able to lower the cell viability of U87 cells (25 μM: ~87%; 50 μM: 66%), no additive or synergistic effects could be found in combination with rhTRAIL WT (Figure 4A). Since DMC is an analogue of celecoxib, we further tested the sensitivity of these cell lines to celecoxib treatment. A172 and U87 showed similar IC50 values of approximately 88 μM for both A172 and U87 (Additional file 1: Figure S1). Next, DMC or celecoxib were tested side-by-side in combination with rhTRAIL WT. Interestingly, the combination of TRAIL/DMC (IC50: ~57 μM) was shown to be more effective than TRAIL/celecoxib (IC50: ~88 μM) (Additional file 2: Figure S2). Since DMC is a more potent ER stress inducer than celecoxib and does not target COX-2 (Pyrko et al. 2007; Chuang et al. 2008), these results suggest that ER stress activation is closely related to the reduction of cell viability, and subsequently, the enhancement of TRAIL-induced cytotoxicity in these cells.Figure 4

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