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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

DMC reduces proliferation in GBM cells. Acridine orange staining was used to visualize apoptosis in A172 (A) and U87 (B). Cells were treated with 0, 10 or 100 ng/ml rhTRAIL WT and 0, 25 or 50 μM DMC. After 24h, cells were stained using acridine orange dye.
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Fig5: DMC reduces proliferation in GBM cells. Acridine orange staining was used to visualize apoptosis in A172 (A) and U87 (B). Cells were treated with 0, 10 or 100 ng/ml rhTRAIL WT and 0, 25 or 50 μM DMC. After 24h, cells were stained using acridine orange dye.

Mentions: To confirm that the combinatorial effects described above were correlated with enhanced apoptosis activity, the effect of DMC on TRAIL-induced caspase cleavage was further analysed. Figure 4B shows that after 5h treatment, A172 cells display a significant activation of the caspases-8, -9, -3 and PARP cleavage when exposed to rhTRAIL WT alone, and enhanced cleavage when combined with DMC. Treatment with 100 ng/ml of rhTRAIL WT and 50 μM DMC resulted in massive cell death in A172 cells. Notably, DMC alone did not induce any activation of downstream apoptosis-related proteins, as indicated by a lack of caspase activation or PARP cleavage, and in agreement with our earlier observations using Annexin V staining assays (Figure 3B). Furthermore, acridine orange staining of DMC-treated cells showed no evidence of nuclear condensation and nuclear/cellular fragmentation, as shown after rhTRAIL WT treatment in A172 cells, but it does appear that DMC reduces cell proliferation, especially in A172 cells (Figure 5). Notably, in U87 cells the combined treatment of rhTRAIL WT with 50 μM DMC led to activation of the initiator pro-caspase-8 but no subsequent pro-caspase-9, -3 or PARP cleavage. Treatment of U87 cells with both TRAIL WT and DMC resulted in the down-regulation of pro-caspase-3 in this cell line (Figure 4B). Finally, the effect of combined exposure to rhTRAIL WT and DMC was also examined in U87-control and U87-DR5 cells, with no significant effect of DMC on TRAIL-sensitivity being observed for these cell lines (Additional file 3: Figure S3).Figure 5


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)

DMC reduces proliferation in GBM cells. Acridine orange staining was used to visualize apoptosis in A172 (A) and U87 (B). Cells were treated with 0, 10 or 100 ng/ml rhTRAIL WT and 0, 25 or 50 μM DMC. After 24h, cells were stained using acridine orange dye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554544&req=5

Fig5: DMC reduces proliferation in GBM cells. Acridine orange staining was used to visualize apoptosis in A172 (A) and U87 (B). Cells were treated with 0, 10 or 100 ng/ml rhTRAIL WT and 0, 25 or 50 μM DMC. After 24h, cells were stained using acridine orange dye.
Mentions: To confirm that the combinatorial effects described above were correlated with enhanced apoptosis activity, the effect of DMC on TRAIL-induced caspase cleavage was further analysed. Figure 4B shows that after 5h treatment, A172 cells display a significant activation of the caspases-8, -9, -3 and PARP cleavage when exposed to rhTRAIL WT alone, and enhanced cleavage when combined with DMC. Treatment with 100 ng/ml of rhTRAIL WT and 50 μM DMC resulted in massive cell death in A172 cells. Notably, DMC alone did not induce any activation of downstream apoptosis-related proteins, as indicated by a lack of caspase activation or PARP cleavage, and in agreement with our earlier observations using Annexin V staining assays (Figure 3B). Furthermore, acridine orange staining of DMC-treated cells showed no evidence of nuclear condensation and nuclear/cellular fragmentation, as shown after rhTRAIL WT treatment in A172 cells, but it does appear that DMC reduces cell proliferation, especially in A172 cells (Figure 5). Notably, in U87 cells the combined treatment of rhTRAIL WT with 50 μM DMC led to activation of the initiator pro-caspase-8 but no subsequent pro-caspase-9, -3 or PARP cleavage. Treatment of U87 cells with both TRAIL WT and DMC resulted in the down-regulation of pro-caspase-3 in this cell line (Figure 4B). Finally, the effect of combined exposure to rhTRAIL WT and DMC was also examined in U87-control and U87-DR5 cells, with no significant effect of DMC on TRAIL-sensitivity being observed for these cell lines (Additional file 3: Figure S3).Figure 5

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