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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 cell viability and induces ER stress in GBM cell lines. (A) Cell viability was assessed in a panel of GBM cell lines after 24h exposure to 0–100 μM of DMC using a MTS assay. Error bars represent S.E.M. of three independent experiments. (B) A172 and U87 cells were treated with 0, 25 or 50 μM DMC for 24h after which apoptosis induction was determined using Annexin V/PI using flow cytometry. (C) Treatment of A172 and U87 cells with 0, 25 or 50 μM DMC for 24h resulted in a dose-dependent up-regulation of CHOP and GRP78 proteins levels as showed by Western blotting. β-actin serves as a loading control. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.
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Fig3: DMC reduces cell viability and induces ER stress in GBM cell lines. (A) Cell viability was assessed in a panel of GBM cell lines after 24h exposure to 0–100 μM of DMC using a MTS assay. Error bars represent S.E.M. of three independent experiments. (B) A172 and U87 cells were treated with 0, 25 or 50 μM DMC for 24h after which apoptosis induction was determined using Annexin V/PI using flow cytometry. (C) Treatment of A172 and U87 cells with 0, 25 or 50 μM DMC for 24h resulted in a dose-dependent up-regulation of CHOP and GRP78 proteins levels as showed by Western blotting. β-actin serves as a loading control. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.

Mentions: Next, we examined the potential of the ER stress inducer DMC alone and in combination with TRAIL across several GBM cell lines. We started by determining the effect of DMC on the cell viability of GBM cells. As shown in Figure 3A, DMC was able to efficiently promote reduction in cell viability as measured by MTS in all the GBM cell lines studied. SNB75 cells showed the highest sensitivity to DMC, whereas U251, A172 and U87 cells were almost equally sensitive to DMC (Figure 3A).Figure 3


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 cell viability and induces ER stress in GBM cell lines. (A) Cell viability was assessed in a panel of GBM cell lines after 24h exposure to 0–100 μM of DMC using a MTS assay. Error bars represent S.E.M. of three independent experiments. (B) A172 and U87 cells were treated with 0, 25 or 50 μM DMC for 24h after which apoptosis induction was determined using Annexin V/PI using flow cytometry. (C) Treatment of A172 and U87 cells with 0, 25 or 50 μM DMC for 24h resulted in a dose-dependent up-regulation of CHOP and GRP78 proteins levels as showed by Western blotting. β-actin serves as a loading control. 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

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

Fig3: DMC reduces cell viability and induces ER stress in GBM cell lines. (A) Cell viability was assessed in a panel of GBM cell lines after 24h exposure to 0–100 μM of DMC using a MTS assay. Error bars represent S.E.M. of three independent experiments. (B) A172 and U87 cells were treated with 0, 25 or 50 μM DMC for 24h after which apoptosis induction was determined using Annexin V/PI using flow cytometry. (C) Treatment of A172 and U87 cells with 0, 25 or 50 μM DMC for 24h resulted in a dose-dependent up-regulation of CHOP and GRP78 proteins levels as showed by Western blotting. β-actin serves as a loading control. Presented data are representative for at least three independent experiments and mean cell viability levels ± S.E.M. are shown.
Mentions: Next, we examined the potential of the ER stress inducer DMC alone and in combination with TRAIL across several GBM cell lines. We started by determining the effect of DMC on the cell viability of GBM cells. As shown in Figure 3A, DMC was able to efficiently promote reduction in cell viability as measured by MTS in all the GBM cell lines studied. SNB75 cells showed the highest sensitivity to DMC, whereas U251, A172 and U87 cells were almost equally sensitive to DMC (Figure 3A).Figure 3

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