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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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Senescence induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of senescence detected by β-galactosidase staining performed 5 days following a single dose of treatment (X-rays = 8 Gy or TMZ = 100 μM) on U87-scrambled and U87-shEPOR cells. The positive cells for senescence assay showed a blue coloration. Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Representative photographs of senescence detected by histone H3 (triMethylK9) immunostaining (red) on U87-scrambled and U87-shEPOR cells 5 days after 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. (C) Quantification of senescence 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 histone H3 (TriMethylK9) positive cells was expressed 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).
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Figure 5: Senescence induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of senescence detected by β-galactosidase staining performed 5 days following a single dose of treatment (X-rays = 8 Gy or TMZ = 100 μM) on U87-scrambled and U87-shEPOR cells. The positive cells for senescence assay showed a blue coloration. Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Representative photographs of senescence detected by histone H3 (triMethylK9) immunostaining (red) on U87-scrambled and U87-shEPOR cells 5 days after 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. (C) Quantification of senescence 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 histone H3 (TriMethylK9) positive cells was expressed 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).

Mentions: All of these data indicate that cell cycle perturbations observed after treatment of shEPOR-glioma cells with radiation or TMZ are mediated by an abrogation of G2/M checkpoint leading mainly to polyploid cells. It suggests that the inhibition of EPOR expression might increase the cell death induced by radio- or chemotherapy in comparison to control cells. We next investigated the nature of the sensitisation to chemo- and radiotherapy induced by EPOR silencing by focusing on cell death especially cellular senescence. As depicted on Figure 5 and according to our previous cell cycle data (Figures 1 and 4), in control conditions, inhibition of EPOR expression increases the proportion of senescent cells compared to U87-scrambled cells (Figure 5A). We confirm that the X-rays exposure causes the senescence of control cells, but this phenomenon is amplified by the inhibition of EPOR since, proportionally, the number of blue cells is more important in U87-shEPOR group compared to U87-scrambled group. As described for the radiotherapy, chemotherapy-induced senescence is potentiated by EPOR down-regulation (Figure 5A).


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)

Senescence induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of senescence detected by β-galactosidase staining performed 5 days following a single dose of treatment (X-rays = 8 Gy or TMZ = 100 μM) on U87-scrambled and U87-shEPOR cells. The positive cells for senescence assay showed a blue coloration. Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Representative photographs of senescence detected by histone H3 (triMethylK9) immunostaining (red) on U87-scrambled and U87-shEPOR cells 5 days after 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. (C) Quantification of senescence 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 histone H3 (TriMethylK9) positive cells was expressed 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Senescence induced by X-rays treatment or temozolomide exposure is potentiated by EPOR inhibition on glioma cells(A) Representative photographs of senescence detected by β-galactosidase staining performed 5 days following a single dose of treatment (X-rays = 8 Gy or TMZ = 100 μM) on U87-scrambled and U87-shEPOR cells. The positive cells for senescence assay showed a blue coloration. Scale bar=100 μm for low magnification and scale bar=25 μm for high magnification. (B) Representative photographs of senescence detected by histone H3 (triMethylK9) immunostaining (red) on U87-scrambled and U87-shEPOR cells 5 days after 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. (C) Quantification of senescence 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 histone H3 (TriMethylK9) positive cells was expressed 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).
Mentions: All of these data indicate that cell cycle perturbations observed after treatment of shEPOR-glioma cells with radiation or TMZ are mediated by an abrogation of G2/M checkpoint leading mainly to polyploid cells. It suggests that the inhibition of EPOR expression might increase the cell death induced by radio- or chemotherapy in comparison to control cells. We next investigated the nature of the sensitisation to chemo- and radiotherapy induced by EPOR silencing by focusing on cell death especially cellular senescence. As depicted on Figure 5 and according to our previous cell cycle data (Figures 1 and 4), in control conditions, inhibition of EPOR expression increases the proportion of senescent cells compared to U87-scrambled cells (Figure 5A). We confirm that the X-rays exposure causes the senescence of control cells, but this phenomenon is amplified by the inhibition of EPOR since, proportionally, the number of blue cells is more important in U87-shEPOR group compared to U87-scrambled group. As described for the radiotherapy, chemotherapy-induced senescence is potentiated by EPOR down-regulation (Figure 5A).

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