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. 2016 Aug 5:16:604.
doi: 10.1186/s12885-016-2647-9.

A radiosensitizing effect of RAD51 inhibition in glioblastoma stem-like cells

Anaïs Balbous  1   2   3 , Ulrich Cortes  3 , Karline Guilloteau  3 , Pierre Rivet  3 , Baptiste Pinel  4 , Mathilde Duchesne  5 , Julie Godet  5 , Odile Boissonnade  4 , Michel Wager  6 , René Jean Bensadoun  4 , Jean-Claude Chomel  3 , Lucie Karayan-Tapon  7   8   9
Affiliations

A radiosensitizing effect of RAD51 inhibition in glioblastoma stem-like cells

Anaïs Balbous et al. BMC Cancer. .

Abstract

Background: Radioresistant glioblastoma stem cells (GSCs) contribute to tumor recurrence and identification of the molecular targets involved in radioresistance mechanisms is likely to enhance therapeutic efficacy. This study analyzed the DNA damage response following ionizing radiation (IR) in 10 GSC lines derived from patients.

Methods: DNA damage was quantified by Comet assay and DNA repair effectors were assessed by Low Density Array. The effect of RAD51 inhibitor, RI-1, was evaluated by comet and annexin V assays.

Results: While all GSC lines displayed efficient DNA repair machinery following ionizing radiation, our results demonstrated heterogeneous responses within two distinct groups showing different intrinsic radioresistance, up to 4Gy for group 1 and up to 8Gy for group 2. Radioresistant cell group 2 (comprising 5 out of 10 GSCs) showed significantly higher RAD51 expression after IR. In these cells, inhibition of RAD51 prevented DNA repair up to 180 min after IR and induced apoptosis. In addition, RAD51 protein expression in glioblastoma seems to be associated with poor progression-free survival.

Conclusion: These results underscore the importance of RAD51 in radioresistance of GSCs. RAD51 inhibition could be a therapeutic strategy helping to treat a significant number of glioblastoma, in combination with radiotherapy.

Keywords: Comet assay; Glioblastoma stem cells; RAD51; Radioresistance.

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Figures

Fig. 1
Fig. 1
Measurement of DNA damage and cell proliferation in GSCs following IR. a 10 GSC lines were irradiated at 4Gy and subjected to comet assay at the indicated time. Data are given as a percentage of olive tail moment (OTM) and normalized to control (***p < 0.001 versus control cells). b GSCs from group 2 were irradiated at the indicated doses and subjected to comet assay immediately thereafter. Data are given as a percentage of olive tail moment (OTM) and normalized to control (***p < 0.001 versus control cells). c GSCs from group 2 were irradiated at 12Gy and subjected to comet assay at the indicated time. Data are given as a percentage of olive tail moment (OTM) and normalized to control (***p < 0.001 versus control cells). d Cell proliferation was measured 7 days after IR (4Gy and 12Gy) using an MTS assay (T0 = IR). Each set of results was obtained from three independent experiments. Experiments were performed in sextuplicate and expressed as mean ± SD. Doubling times were extrapolated based on exponential growth equations
Fig. 2
Fig. 2
Expression of DNA repair genes in GSCs after IR. a TLDA expression levels of the most significant DNA repair genes. Relative expressions were measured 3 h following IR, data represent the mean ± SD of 10 GSCs determined by 2-ΔΔCt quantification method. Relative expressions of target genes were determined using GAPDH as endogenous control (**p < 0.01, *p < 0.05). b mRNA expression of RAD51, BRCA1, BRCA2, CHK1 and CHK2 in group 1 and group 2. The vertical scatter plot shows the log10 expression of relative quantification (RQ) values normalized to the expression before IR. Each data point represents one GSC line measured in triplicate (*p = 0.032). c Western blot analysis of RAD51 following 4Gy and 12Gy IR. Total protein were extracted after 45 min and 24 h following IR, β-actin was used as loading control. Densitometric analysis of specific signals shows relative RAD51 protein expression levels normalized with β-actin and expressed as a percentage of control in GSC-6 and GSC-11 (n = 3) (*p < 0.05) (Image J software)
Fig. 3
Fig. 3
Chemical inhibitor of RAD51, RI-1, inhibits RAD51 foci formation. a GSCs viability was measured using an MTS assay after 5 days of RI-1 treatment. IC50 values were 22.3 μM and 19.7 μM respectively for GSC-14 and GSC-1. b Western blot analysis of RAD51 was performed on GSCs treated for 24 h with 10 μM RI-1 before IR. Total protein samples were extracted after 45 min and 24 h following 4Gy and 12Gy IR. β-actin was used as a loading control. Densitometric analysis of specific signals shows relative RAD51 protein expression levels normalized with β-actin and expressed as a percentage of control in GSC-6 and GSC-11 (n = 3) (*p < 0.05) (Image J software). c Cells were treated for 24 h with 10 μM of RI-1 before 12Gy IR and harvested at the indicated times. For each time point, the number of cells with RAD51 foci > 5 was scored and expressed as a percentage of the total number of nucleus scored. (*p < 0.05, **p < 0.01, ### p < 0.001, ns = no significant). d Representative images of GSC-6 and GSC-11 treated with RI-1. RAD51 foci (green) and nucleus (blue) are shown after 24 h following 12Gy exposure. These images were captured with the Axio Imager M2 fluorescent microscope (Carl Zeiss), scale bar: 2 μm
Fig. 4
Fig. 4
Chemical inhibitior of RAD51, RI-1, selectively radiosensitizes GSCs from group 2. Comet assay was performed on GSCs from group 1 (GSC-1 and - 11) (a) and group 2 (GSC-6 and -14) (b) treated for 24 h with 10 μM RI-1 before undergoing 16Gy IR. Data are given as a percentage of olive tail moment (OTM) normalized to control (***p < 0.001, **p < 0.01 versus control cells, ns = no significant). c Apoptosis was measured in both groups 7 days after treatment with 10 μM RI-1 and IR. Annexin V/7-AAD labeling was analyzed by flow cytometry (**p < 0.01, ns = no significant)
Fig. 5
Fig. 5
RAD51 protein expression in GBM tumors is associated with shorter progression-free survival. a Representative sections of TMA stained with RAD51 were analyzed by immunohistochemistry. All images were obtained at magnification 4× (scale bar 100 μm). The left section showed no RAD51 staining and the right section showed RAD51 staining. b Kaplan-Meier curve of all glioblastoma patients plotting progression-free survival for patients with low or high expression of RAD51 protein (p = 0.065)
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