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Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe.

Pérès EA, Gérault AN, Valable S, Roussel S, Toutain J, Divoux D, Guillamo JS, Sanson M, Bernaudin M, Petit E - Oncotarget (2015)

Bottom Line: Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours.In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone.Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 6301-ISTCT, CERVOxy group. GIP CYCERON, Bd Henri Becquerel, BP5229, F-14074 CAEN, France.

ABSTRACT
Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours. It has been recently reported that erythropoietin (EPO) and its receptor (EPOR) are involved in glioma growth. We now investigated whether EPOR signalling may modulate the efficacy of the GBM current treatment based on chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Using RNA interference, we showed on glioma cell lines (U87 and U251) that EPOR silencing induces a G2/M cell cycle arrest, consistent with the slowdown of glioma growth induced by EPOR knock-down. In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone. In vitro, we showed that EPOR silencing not only increases the sensitivity of glioma cells to TMZ as well as X-rays but also counteracts the hypoxia-induced chemo- and radioresistance. Silencing EPOR on glioma cells exposed to conventional treatments enhances senescence and induces a robust genomic instability that leads to caspase-dependent mitotic death by increasing the number of polyploid cells and cyclin B1 expression. Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.

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Apoptosis-dependent mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of apoptosis by cleaved caspase-3 immunostaining (red) and mitotic catastrophe by multinucleated cells revealed with Hoechst 33342 staining (blue) on U87-scrambled and U87-shEPOR cells 7 days after a single X-rays (8 Gy) or TMZ (100 μM) treatments. The arrows denote the abnormal nuclei corresponding to cells in mitotic death and scale bar=50 μm. (B) Quantification of apoptosis and mitotic catastrophe 7 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) on U87-scrambled and U87-shEPOR cells. The cells with only one nucleus and positive for cleaved caspase-3 staining were identified as apoptotic (white bar). The cells with more than two nuclei and positive for cleaved caspase-3 were considered as cells in apoptosis-dependent mitotic death (gray bar). The cells with at least two nuclei and negative for cleaved caspase-3 were described as cells in necrosis-dependent mitotic death (dark bar). Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-Scrambled untreated; # p<0.05 vs U87-shEPOR untreated and $ p<0.05 vs U87-scrambled for each treatment (Fisher's PLSD post-hoc test after a significant ANOVA).
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Figure 7: Apoptosis-dependent mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of apoptosis by cleaved caspase-3 immunostaining (red) and mitotic catastrophe by multinucleated cells revealed with Hoechst 33342 staining (blue) on U87-scrambled and U87-shEPOR cells 7 days after a single X-rays (8 Gy) or TMZ (100 μM) treatments. The arrows denote the abnormal nuclei corresponding to cells in mitotic death and scale bar=50 μm. (B) Quantification of apoptosis and mitotic catastrophe 7 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) on U87-scrambled and U87-shEPOR cells. The cells with only one nucleus and positive for cleaved caspase-3 staining were identified as apoptotic (white bar). The cells with more than two nuclei and positive for cleaved caspase-3 were considered as cells in apoptosis-dependent mitotic death (gray bar). The cells with at least two nuclei and negative for cleaved caspase-3 were described as cells in necrosis-dependent mitotic death (dark bar). Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-Scrambled untreated; # p<0.05 vs U87-shEPOR untreated and $ p<0.05 vs U87-scrambled for each treatment (Fisher's PLSD post-hoc test after a significant ANOVA).

Mentions: Apoptosis induced by EPOR silencing is further confirmed by the activation of caspase-3, as demonstrated by the appearance of its cleaved active form (Figure 7A). Morphological examination of cells also reveals the presence of some large multinucleate cells either after X-rays and TMZ treatments or when glioma cells were depleted in EPOR (Figure 7A), indicating that some cells are entering a faulty mitosis without cytokinesis, corresponding to mitotic catastrophe. We next examined whether apoptosis or necrosis were involved in the final death of these cells. To discriminate apoptotic and necrotic cell death, cells showing multiple nuclei and a co-staining for cleaved caspase-3 were scored as cells in mitotic catastrophe committed to dying by a caspase-dependent way. A quantification of the cells dying by caspase-dependent (apoptosis-dependent mitotic death) or caspase-independent way (necrosis-dependent mitotic death) was performed (Figure 7B). From these results (Figures 7A and B), and according to cell cycle data (Figure 1B), EPOR silencing increases apoptosis of glioma cells. Indeed, under basal conditions, compared to U87-scrambled cells, 37% of U87-shEPOR cells are in apoptosis (p<0.0001) and 4% of these cells are in mitotic death (Figure 7B). Ionising radiation or TMZ significantly increases apoptosis as well as in mitotic death. However, although the population of apoptotic cells remains constant, sole the population of cells entering in mitotic death is amplified when the chemo- or radiotherapy were combined with EPOR inhibition (Figure 7B). In particular, 2.5 to 3 time more of U87-shEPOR cells in apoptosis-dependent mitotic death (p<0.05) are detected compared to control cells exposed to treatment alone (U87-scrambled cells treated with X-rays and with TMZ, respectively). EPOR inhibition enhances the overall glioma death in response to radiotherapy or chemotherapy (p<0.05) relative to scrambled cells, with a total of 83% versus 50% for irradiation, and 88% versus 67% for TMZ treatment (Figure 7B).


Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe.

Pérès EA, Gérault AN, Valable S, Roussel S, Toutain J, Divoux D, Guillamo JS, Sanson M, Bernaudin M, Petit E - Oncotarget (2015)

Apoptosis-dependent mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of apoptosis by cleaved caspase-3 immunostaining (red) and mitotic catastrophe by multinucleated cells revealed with Hoechst 33342 staining (blue) on U87-scrambled and U87-shEPOR cells 7 days after a single X-rays (8 Gy) or TMZ (100 μM) treatments. The arrows denote the abnormal nuclei corresponding to cells in mitotic death and scale bar=50 μm. (B) Quantification of apoptosis and mitotic catastrophe 7 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) on U87-scrambled and U87-shEPOR cells. The cells with only one nucleus and positive for cleaved caspase-3 staining were identified as apoptotic (white bar). The cells with more than two nuclei and positive for cleaved caspase-3 were considered as cells in apoptosis-dependent mitotic death (gray bar). The cells with at least two nuclei and negative for cleaved caspase-3 were described as cells in necrosis-dependent mitotic death (dark bar). Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-Scrambled untreated; # p<0.05 vs U87-shEPOR untreated and $ p<0.05 vs U87-scrambled for each treatment (Fisher's PLSD post-hoc test after a significant ANOVA).
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Figure 7: Apoptosis-dependent mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of apoptosis by cleaved caspase-3 immunostaining (red) and mitotic catastrophe by multinucleated cells revealed with Hoechst 33342 staining (blue) on U87-scrambled and U87-shEPOR cells 7 days after a single X-rays (8 Gy) or TMZ (100 μM) treatments. The arrows denote the abnormal nuclei corresponding to cells in mitotic death and scale bar=50 μm. (B) Quantification of apoptosis and mitotic catastrophe 7 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) on U87-scrambled and U87-shEPOR cells. The cells with only one nucleus and positive for cleaved caspase-3 staining were identified as apoptotic (white bar). The cells with more than two nuclei and positive for cleaved caspase-3 were considered as cells in apoptosis-dependent mitotic death (gray bar). The cells with at least two nuclei and negative for cleaved caspase-3 were described as cells in necrosis-dependent mitotic death (dark bar). Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-Scrambled untreated; # p<0.05 vs U87-shEPOR untreated and $ p<0.05 vs U87-scrambled for each treatment (Fisher's PLSD post-hoc test after a significant ANOVA).
Mentions: Apoptosis induced by EPOR silencing is further confirmed by the activation of caspase-3, as demonstrated by the appearance of its cleaved active form (Figure 7A). Morphological examination of cells also reveals the presence of some large multinucleate cells either after X-rays and TMZ treatments or when glioma cells were depleted in EPOR (Figure 7A), indicating that some cells are entering a faulty mitosis without cytokinesis, corresponding to mitotic catastrophe. We next examined whether apoptosis or necrosis were involved in the final death of these cells. To discriminate apoptotic and necrotic cell death, cells showing multiple nuclei and a co-staining for cleaved caspase-3 were scored as cells in mitotic catastrophe committed to dying by a caspase-dependent way. A quantification of the cells dying by caspase-dependent (apoptosis-dependent mitotic death) or caspase-independent way (necrosis-dependent mitotic death) was performed (Figure 7B). From these results (Figures 7A and B), and according to cell cycle data (Figure 1B), EPOR silencing increases apoptosis of glioma cells. Indeed, under basal conditions, compared to U87-scrambled cells, 37% of U87-shEPOR cells are in apoptosis (p<0.0001) and 4% of these cells are in mitotic death (Figure 7B). Ionising radiation or TMZ significantly increases apoptosis as well as in mitotic death. However, although the population of apoptotic cells remains constant, sole the population of cells entering in mitotic death is amplified when the chemo- or radiotherapy were combined with EPOR inhibition (Figure 7B). In particular, 2.5 to 3 time more of U87-shEPOR cells in apoptosis-dependent mitotic death (p<0.05) are detected compared to control cells exposed to treatment alone (U87-scrambled cells treated with X-rays and with TMZ, respectively). EPOR inhibition enhances the overall glioma death in response to radiotherapy or chemotherapy (p<0.05) relative to scrambled cells, with a total of 83% versus 50% for irradiation, and 88% versus 67% for TMZ treatment (Figure 7B).

Bottom Line: Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours.In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone.Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR 6301-ISTCT, CERVOxy group. GIP CYCERON, Bd Henri Becquerel, BP5229, F-14074 CAEN, France.

ABSTRACT
Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours. It has been recently reported that erythropoietin (EPO) and its receptor (EPOR) are involved in glioma growth. We now investigated whether EPOR signalling may modulate the efficacy of the GBM current treatment based on chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Using RNA interference, we showed on glioma cell lines (U87 and U251) that EPOR silencing induces a G2/M cell cycle arrest, consistent with the slowdown of glioma growth induced by EPOR knock-down. In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone. In vitro, we showed that EPOR silencing not only increases the sensitivity of glioma cells to TMZ as well as X-rays but also counteracts the hypoxia-induced chemo- and radioresistance. Silencing EPOR on glioma cells exposed to conventional treatments enhances senescence and induces a robust genomic instability that leads to caspase-dependent mitotic death by increasing the number of polyploid cells and cyclin B1 expression. Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.

Show MeSH
Related in: MedlinePlus