Reversible inhibitor of CRM1 sensitizes glioblastoma cells to radiation by blocking the NF-κB signaling pathway

Abstract

Background: Activation of nuclear factor-kappa B (NF-κΒ) through DNA damage is one of the causes of tumor cell resistance to radiotherapy. Chromosome region 1 (CRM1) regulates tumor cell proliferation, drug resistance, and radiation resistance by regulating the nuclear-cytoplasmic translocation of important tumor suppressor proteins or proto-oncoproteins. A large number of studies have reported that inhibition of CRM1 suppresses the activation of NF-κΒ. Thus, we hypothesize that the reversible CRM1 inhibitor S109 may induce radiosensitivity in glioblastoma (GBM) by regulating the NF-κΒ signaling pathway.

Methods: This study utilized the cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assay to evaluate the effect of S109 combined with radiotherapy on the proliferation and survival of GBM cells. The therapeutic efficacy of S109 combined with radiotherapy was evaluated in vivo to explore the therapeutic mechanism of S109-induced GBM radiosensitization.

Results: We found that S109 combined with radiotherapy significantly inhibited GBM cell proliferation and colony formation. By regulating the levels of multiple cell cycle- and apoptosis-related proteins, the combination therapy induced G1 cell cycle arrest in GBM cells. In vivo studies showed that S109 combined with radiotherapy significantly inhibited the growth of intracranial GBM and prolonged survival. Importantly, we found that S109 combined with radiotherapy promoted the nuclear accumulation of IκΒα, and inhibited phosphorylation of p65 and the transcriptional activation of NF-κΒ.

Conclusion: Our findings provide a new therapeutic regimen for improving GBM radiosensitivity as well as a scientific basis for further clinical trials to evaluate this combination therapy.

Keywords: CRM1; GBM; Irradiation; NF-κΒ signaling pathway; S109.

Fig. 1

S109 combined with radiotherapy significantly inhibited GBM cell proliferation. U87 a and C6 b cell lines were treated with varying doses of S109, and cell viability was assessed by CCK-8 assay at 72 h after treatment. c, e Measurement of anti-proliferation effects of S109 and/or IR by EdU incorporation assay. Represent images were showed. The EdU incorporation rate was presented as the ratio of EdU positive cells to total DAPI positive cells, scale bar: 100 μm. d, f Quantification of the percentage of EdU-positive cells. All the results were presented as the mean ± SD from 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

S109 enhances radiation sensitivity of GBM cells. a U87 cells and (C) C6 cells were cultured in 6-well plates. S109 (0.25 μM or 0.5 μM) or vehicle (DMSO) were added to culture media and the cells were irradiated the next day. Colony forming efficiency was determined 10–14 days later. b, d Killing curves were generated after normalizing for cell killing by S109. Values represent the mean ± SD of 3 independent experiments

Fig. 3

Combination of S109 and radiation results in cell cycle arrest and enhances radiation-induced DNA damage. The distribution of cell cycle after S109 and/or IR treatment and quantitative analyses were examined by flow cytometry in U87 cells a and C6 cells b. The data represent the mean ± SD from 3 independent experiments, *P < 0.05, **P < 0.01. c–f The expression levels of multiple cell-cycle- and apoptosis-associated proteins were assessed by western blot assay in time- and does-manner. g Lysates of S109-treated U87 cells were collected at 24 h after radiation (2 Gy) for Western blot analysis. The levels of γ-H2AX and H2AX were examined

Fig. 4

CRM1 inhibition synergizes with radiation in in vivo models of GBM. a Schematic diagram of mice treated with S109 combined with radiotherapy. b In vivo efficacy in U87-Luci GBM model in mice. U87-Luci-bearing mice received daily injection of S109 at a dose of 50 mg/kg, and these mice were irradiated on days 10, 12, 14, 16 and 18 with 2 Gy. Bioluminescent signal changes correlating to tumor growth were showed. c Quantification of the tumor bioluminescence signal (n = 3 mice per group). (D) Kaplan–Meier survival curve for the mice (n = 7, **P < 0.01, ***P < 0.001). e Representative images of Ki-67 immunostaining of tumors dissected from control, S109-treated mice, IR-treated mice and S109+IR groups. Scale bar: 250 μm. f Quantitative analyses of the percentages of Ki-67 positive cells. Data are presented as mean ± SD from 3 independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Combination of S109 and radiation reduces NF-κB transcriptional activation and promotes the nuclear accumulation of IκΒα. U87 and C6 cells were treated with S109 and/or IR. Lysates of cells were collected at 24 h after radiation for Western blot. The expression levels of CRM1, p-p65 and p-65 were assessed in a dose- and time-manner a–d. e The effects of S109 and/or IR treatment on the subcellular location of IκΒα. The cytoplasmic and nuclear protein extracts were used for immunoblotting with the indicated antibodies. f The transactivation ability of NF-κB were inhibited when combination of S109 and radiation. Relative NF-κB luciferase activity normalized with respect to corresponding renilla luciferase activity is shown. All the Data are presented as mean ± SD, *P < 0.05, **P < 0.01