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Radiation-induced PGE2 sustains human glioma cells growth and survival through EGF signaling.

Brocard E, Oizel K, Lalier L, Pecqueur C, Paris F, Vallette FM, Oliver L - Oncotarget (2015)

Bottom Line: We show that irradiated glioma cells produced and released PGE2 in important quantities independently of the induction of cell death.We demonstrate that the addition of PGE2 enhances cell survival and proliferation though its ability to trans-activate the Epithelial Growth Factor receptor (EGFR) and to activate β-catenin.Indeed, PGE2 can substitute for EGF to promote primary cultures survival and growth in vitro and the effect is likely to occur though the Prostaglandin E2 receptor EP2.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie Nantes Angers UMR INSERM 892, CNRS 6299, Université de Nantes, 44007 Nantes, France.

ABSTRACT
Glioblastoma Multiforme (GBM) is the most common brain cancer in adults. Radiotherapy (RT) is the most effective post-operative treatment for the patients even though GBM is one of the most radio-resistant tumors. Dead or dying cells within the tumor are thought to promote resistance to treatment through mechanisms that are very poorly understood. We have evaluated the role of Prostaglandin E2 (PGE2), a versatile bioactive lipid, in GBM radio-resistance. We used an in vitro approach using 3D primary cultures derived from representative GBM patients. We show that irradiated glioma cells produced and released PGE2 in important quantities independently of the induction of cell death. We demonstrate that the addition of PGE2 enhances cell survival and proliferation though its ability to trans-activate the Epithelial Growth Factor receptor (EGFR) and to activate β-catenin. Indeed, PGE2 can substitute for EGF to promote primary cultures survival and growth in vitro and the effect is likely to occur though the Prostaglandin E2 receptor EP2.

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Release of PGE2 from γ-irradiated cellsU251 cells were irradiated in serum-free medium at the indicated dose. PGE2 secretion was measured in supernatants after 24 h. The concentration of PGE2 (pg/ml) released from irradiated U251 cells was determined using an ELISA (see materials and methods) (A). Cell viability was determined by trypan blue exclusion using the Countess automatic cell counter (Life Technologies), 24 h after irradiation of U251 cells (B). Cell death was estimated as above at 24 h, 48 h and 72 h post-irradiation (C). U251 cells were transduced with shRNA encoding viral particles [either encoding for a non-relevant shRNA (scr) or shRNA directed against Bax mRNA]. Cells were irradiated in serum-free medium at 10 Gy; harvested at the indicated time points, fixed and labeled with active caspase 3 antibody coupled to a fluorescent secondary antibody. The percentage of labeled cells was assessed by flow cytometry (D). The corresponding PGE2 secretion was measured in supernatants during 48 h after irradiation at 5 Gy (E). Please note that in the latter experiments, the secretion of PGE2 was decreased in scr-treated U251 compared to untreated cells (compare A and E).
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Figure 1: Release of PGE2 from γ-irradiated cellsU251 cells were irradiated in serum-free medium at the indicated dose. PGE2 secretion was measured in supernatants after 24 h. The concentration of PGE2 (pg/ml) released from irradiated U251 cells was determined using an ELISA (see materials and methods) (A). Cell viability was determined by trypan blue exclusion using the Countess automatic cell counter (Life Technologies), 24 h after irradiation of U251 cells (B). Cell death was estimated as above at 24 h, 48 h and 72 h post-irradiation (C). U251 cells were transduced with shRNA encoding viral particles [either encoding for a non-relevant shRNA (scr) or shRNA directed against Bax mRNA]. Cells were irradiated in serum-free medium at 10 Gy; harvested at the indicated time points, fixed and labeled with active caspase 3 antibody coupled to a fluorescent secondary antibody. The percentage of labeled cells was assessed by flow cytometry (D). The corresponding PGE2 secretion was measured in supernatants during 48 h after irradiation at 5 Gy (E). Please note that in the latter experiments, the secretion of PGE2 was decreased in scr-treated U251 compared to untreated cells (compare A and E).

Mentions: The accumulation of PGE2 was measured 24 h after γ-irradiation of the human glioma cell line U251 at different intensities (i.e. 0, 5 and 10 Gy). As shown in Figure 1A, the amount of PGE2 found in the culture supernatant was proportional to the dose of radiation. Recent results have associated the induction of caspase activity in cancer cells to the production of PGE2 upon irradiation of cancer cells [11, 13–15]. We then assessed the viability of the cells under our conditions and found that only the high dose of irradiation (10 Gy) provoked a cell growth arrest and subsequent cell death after 48 h (Figure 1B, 1C). To determine whether the cell death was caspase 3 dependent, the number of active caspase 3 cells was quantified. As seen in Figure 1D, there was a close correlation between the percent of cell death and the percent of active caspase 3 cells. To evaluate the implication of caspase in the production of PGE2, we knocked down the expression of Bax, a central pro-apoptotic member of the BCL-2 family, in U251 cells. As illustrated in Figure 1D, we observed a significant decrease in the induction of caspase 3 activity in the absence of Bax after γ-irradiation at 10 Gy. However, quite surprisingly, the knock down of Bax appeared to promote the production of PGE2 in untreated U251 cells and the amount of PGE2 is maintained upon irradiation (Figure 1E), as previously observed in primary cultures of GBM [14]. Of note, the induction of PGE2 in control cells reached a peak 8 h after a 10 Gy irradiation to returned to normal at 16 h (Figure 1E).


Radiation-induced PGE2 sustains human glioma cells growth and survival through EGF signaling.

Brocard E, Oizel K, Lalier L, Pecqueur C, Paris F, Vallette FM, Oliver L - Oncotarget (2015)

Release of PGE2 from γ-irradiated cellsU251 cells were irradiated in serum-free medium at the indicated dose. PGE2 secretion was measured in supernatants after 24 h. The concentration of PGE2 (pg/ml) released from irradiated U251 cells was determined using an ELISA (see materials and methods) (A). Cell viability was determined by trypan blue exclusion using the Countess automatic cell counter (Life Technologies), 24 h after irradiation of U251 cells (B). Cell death was estimated as above at 24 h, 48 h and 72 h post-irradiation (C). U251 cells were transduced with shRNA encoding viral particles [either encoding for a non-relevant shRNA (scr) or shRNA directed against Bax mRNA]. Cells were irradiated in serum-free medium at 10 Gy; harvested at the indicated time points, fixed and labeled with active caspase 3 antibody coupled to a fluorescent secondary antibody. The percentage of labeled cells was assessed by flow cytometry (D). The corresponding PGE2 secretion was measured in supernatants during 48 h after irradiation at 5 Gy (E). Please note that in the latter experiments, the secretion of PGE2 was decreased in scr-treated U251 compared to untreated cells (compare A and E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466653&req=5

Figure 1: Release of PGE2 from γ-irradiated cellsU251 cells were irradiated in serum-free medium at the indicated dose. PGE2 secretion was measured in supernatants after 24 h. The concentration of PGE2 (pg/ml) released from irradiated U251 cells was determined using an ELISA (see materials and methods) (A). Cell viability was determined by trypan blue exclusion using the Countess automatic cell counter (Life Technologies), 24 h after irradiation of U251 cells (B). Cell death was estimated as above at 24 h, 48 h and 72 h post-irradiation (C). U251 cells were transduced with shRNA encoding viral particles [either encoding for a non-relevant shRNA (scr) or shRNA directed against Bax mRNA]. Cells were irradiated in serum-free medium at 10 Gy; harvested at the indicated time points, fixed and labeled with active caspase 3 antibody coupled to a fluorescent secondary antibody. The percentage of labeled cells was assessed by flow cytometry (D). The corresponding PGE2 secretion was measured in supernatants during 48 h after irradiation at 5 Gy (E). Please note that in the latter experiments, the secretion of PGE2 was decreased in scr-treated U251 compared to untreated cells (compare A and E).
Mentions: The accumulation of PGE2 was measured 24 h after γ-irradiation of the human glioma cell line U251 at different intensities (i.e. 0, 5 and 10 Gy). As shown in Figure 1A, the amount of PGE2 found in the culture supernatant was proportional to the dose of radiation. Recent results have associated the induction of caspase activity in cancer cells to the production of PGE2 upon irradiation of cancer cells [11, 13–15]. We then assessed the viability of the cells under our conditions and found that only the high dose of irradiation (10 Gy) provoked a cell growth arrest and subsequent cell death after 48 h (Figure 1B, 1C). To determine whether the cell death was caspase 3 dependent, the number of active caspase 3 cells was quantified. As seen in Figure 1D, there was a close correlation between the percent of cell death and the percent of active caspase 3 cells. To evaluate the implication of caspase in the production of PGE2, we knocked down the expression of Bax, a central pro-apoptotic member of the BCL-2 family, in U251 cells. As illustrated in Figure 1D, we observed a significant decrease in the induction of caspase 3 activity in the absence of Bax after γ-irradiation at 10 Gy. However, quite surprisingly, the knock down of Bax appeared to promote the production of PGE2 in untreated U251 cells and the amount of PGE2 is maintained upon irradiation (Figure 1E), as previously observed in primary cultures of GBM [14]. Of note, the induction of PGE2 in control cells reached a peak 8 h after a 10 Gy irradiation to returned to normal at 16 h (Figure 1E).

Bottom Line: We show that irradiated glioma cells produced and released PGE2 in important quantities independently of the induction of cell death.We demonstrate that the addition of PGE2 enhances cell survival and proliferation though its ability to trans-activate the Epithelial Growth Factor receptor (EGFR) and to activate β-catenin.Indeed, PGE2 can substitute for EGF to promote primary cultures survival and growth in vitro and the effect is likely to occur though the Prostaglandin E2 receptor EP2.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Cancérologie Nantes Angers UMR INSERM 892, CNRS 6299, Université de Nantes, 44007 Nantes, France.

ABSTRACT
Glioblastoma Multiforme (GBM) is the most common brain cancer in adults. Radiotherapy (RT) is the most effective post-operative treatment for the patients even though GBM is one of the most radio-resistant tumors. Dead or dying cells within the tumor are thought to promote resistance to treatment through mechanisms that are very poorly understood. We have evaluated the role of Prostaglandin E2 (PGE2), a versatile bioactive lipid, in GBM radio-resistance. We used an in vitro approach using 3D primary cultures derived from representative GBM patients. We show that irradiated glioma cells produced and released PGE2 in important quantities independently of the induction of cell death. We demonstrate that the addition of PGE2 enhances cell survival and proliferation though its ability to trans-activate the Epithelial Growth Factor receptor (EGFR) and to activate β-catenin. Indeed, PGE2 can substitute for EGF to promote primary cultures survival and growth in vitro and the effect is likely to occur though the Prostaglandin E2 receptor EP2.

Show MeSH
Related in: MedlinePlus