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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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Mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of DNA double-strand breaks identified with γH2AX immunostaining (red) to determine mitotic death on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Quantification of DNA breaks on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of γH2AX positive cells was measured relative to total cell number counted by Hoechst 33342 staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA). (C) Representative photographs of genomic instability linked to mitotic death and identified by the presence of micronuclei on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (D) Quantification of mitotic death evaluated by micronucleus assay on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of positive cells (cell having at least one micronucleus) was obtained relative to total cell number counted by Hoechst staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA).
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Figure 6: Mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of DNA double-strand breaks identified with γH2AX immunostaining (red) to determine mitotic death on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Quantification of DNA breaks on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of γH2AX positive cells was measured relative to total cell number counted by Hoechst 33342 staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA). (C) Representative photographs of genomic instability linked to mitotic death and identified by the presence of micronuclei on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (D) Quantification of mitotic death evaluated by micronucleus assay on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of positive cells (cell having at least one micronucleus) was obtained relative to total cell number counted by Hoechst staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA).

Mentions: In order to strengthen these results, we studied others hallmarks of cellular senescence including the hypermethylation of a histone H3 (trimethylK9), that is related to senescence-associated heterochromatinization [57-59] and the persistence of gamma-H2AX foci [60,61]. As illustrated on Figures 5B and 6A, both treatments lead to the appearance of these senescence markers in control glioma cells exposed to X-rays and TMZ but these phenomena are amplified by the inhibition of EPOR. The quantitative kinetic analysis of histone H3 (trimethylK9) immunostaining shows that, regardless of the time after treatment, the proportion of positive U87-shEPOR cells is significantly higher than that of U87-scrambled cells (Figures 5B and 5C). Furthermore, this enhancement of histone H3 hypermethylation persists over time. Indeed, at 168h post-radiation: 88% of U87-shEPOR cells are still positive versus 68% for U87-scrambled cells (p<0.05) and at 168h post-TMZ, 76% of U87-shEPOR cells are still positive versus 54% for the U87-scrambled cells (p<0.05).


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)

Mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of DNA double-strand breaks identified with γH2AX immunostaining (red) to determine mitotic death on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Quantification of DNA breaks on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of γH2AX positive cells was measured relative to total cell number counted by Hoechst 33342 staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA). (C) Representative photographs of genomic instability linked to mitotic death and identified by the presence of micronuclei on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (D) Quantification of mitotic death evaluated by micronucleus assay on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of positive cells (cell having at least one micronucleus) was obtained relative to total cell number counted by Hoechst staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA).
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Figure 6: Mitotic death induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of DNA double-strand breaks identified with γH2AX immunostaining (red) to determine mitotic death on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Quantification of DNA breaks on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of γH2AX positive cells was measured relative to total cell number counted by Hoechst 33342 staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA). (C) Representative photographs of genomic instability linked to mitotic death and identified by the presence of micronuclei on U87-scrambled and U87-shEPOR cells 5 days after a single exposure to X-rays (8 Gy) or TMZ (100 μM) treatments. Cell nuclei were identified with Hoechst 33342 staining (blue). Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (D) Quantification of mitotic death evaluated by micronucleus assay on U87-scrambled and U87-shEPOR cells at different times (2h, 24h, 48h, 120h and 168h) after X-rays (8 Gy) (left graph) or TMZ (100 μM) (right graph) treatments. The proportion of positive cells (cell having at least one micronucleus) was obtained relative to total cell number counted by Hoechst staining. Mean ± SD, n=9 (3 different experiments, 1 coverslip for each experiment, 3 representative fields per coverslip); * p<0.05 vs U87-scrambled or U87-shEPOR untreated for each cell type; $ p<0.05 vs U87-scrambled untreated and # p<0.05 vs U87-scrambled treated (Fisher's PLSD post-hoc test after a significant ANOVA).
Mentions: In order to strengthen these results, we studied others hallmarks of cellular senescence including the hypermethylation of a histone H3 (trimethylK9), that is related to senescence-associated heterochromatinization [57-59] and the persistence of gamma-H2AX foci [60,61]. As illustrated on Figures 5B and 6A, both treatments lead to the appearance of these senescence markers in control glioma cells exposed to X-rays and TMZ but these phenomena are amplified by the inhibition of EPOR. The quantitative kinetic analysis of histone H3 (trimethylK9) immunostaining shows that, regardless of the time after treatment, the proportion of positive U87-shEPOR cells is significantly higher than that of U87-scrambled cells (Figures 5B and 5C). Furthermore, this enhancement of histone H3 hypermethylation persists over time. Indeed, at 168h post-radiation: 88% of U87-shEPOR cells are still positive versus 68% for U87-scrambled cells (p<0.05) and at 168h post-TMZ, 76% of U87-shEPOR cells are still positive versus 54% for the U87-scrambled cells (p<0.05).

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