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. 2018 Oct 18;9(11):1063.
doi: 10.1038/s41419-018-0957-3.

Autophagy Suppresses Self-Renewal Ability and Tumorigenicity of Glioma-Initiating Cells and Promotes Notch1 Degradation

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Free PMC article

Autophagy Suppresses Self-Renewal Ability and Tumorigenicity of Glioma-Initiating Cells and Promotes Notch1 Degradation

Zhennan Tao et al. Cell Death Dis. .
Free PMC article

Abstract

Autophagy is a vital process that involves degradation of long-lived proteins and dysfunctional organelles and contributes to cellular metabolism. Glioma-initiating cells (GICs) have the ability to self-renew, differentiate into heterogeneous types of tumor cells, and sustain tumorigenicity; thus, GICs lead to tumor recurrence. Accumulating evidence indicates that autophagy can induce stem cell differentiation and increase the lethality of temozolomide against GICs. However, the mechanism underlying the regulation of GIC self-renewal by autophagy remains uncharacterized. In the present study, autophagy induced by AZD8055 and rapamycin treatment suppressed GIC self-renewal in vitro. We found that autophagy inhibited Notch1 pathway activation. Moreover, autophagy activated Notch1 degradation, which is associated with maintenance of the self-renewal ability of GICs. Furthermore, autophagy abolished the tumorigenicity of CD133 + U87-MG neurosphere cells in an intracranial model. These findings suggest that autophagy regulating GICs self-renewal and tumorigenicity is probably bound up with Notch1 degradation. The results of this study could aid in the design of autophagy-based clinical trials for glioma treatments, which may be of great value.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. AZD8055 and rapamycin inhibited mTOR signaling pathway followed by inducing autophagy.
a, b U87 and U251 GICs were treated with AZD8055 and rapamycin for 24, 48, 72, and cell viability was detected using CCK-8 assay. ce U87 and U251 GICs were treated with various concentrations of AZD8055 for 48 h and rapamycin for 24 h. The cells were harvested, and the effects of AZD8055 and rapamycin on the protein expression of the mTOR pathway and autophagy-related were detected by western blot. Data are shown as means ± s.d., n = 3, #P = NS, *P < 0.05, **P < 0.01, Student’s t-test. f Immunofluorescence staining of U87 and U251 GICs, which treated by DMSO, AZD8055 (0.3 μM) and rapamycin (3 μM). The nuclei were stained with DAPI and the antibody against LC3B and p62. Images were captured by laser confocal microscope ( × 400), scale bar = 100 μm
Fig. 2
Fig. 2. GICs treated with AZD8055 and rapamycin showing that autophagy suppressed GICs proliferation and self-renewal.
a The representative images of GICs neurospheres showed that neurosphere formation ability of GICs was significantly inhibited by AZD8055 and rapamycin treatment. n = 5, Scale bar = 200 µm. b The quantification of numbers and diameter of the GICs neurospheres showing that neurosphere formation ability of GICs were obviously inhibited after AZD8055 and rapamycin treatment. c The ability of GICs proliferation were showed by cell viability assay. d The ability of GICs self-renewal was detected by in vitro limiting dilution assay. Data are shown as means ± s.d, n = 5, #P = NS, *P < 0.05, **P < 0.01, likelihood ratio test. Data in b, c are shown as means ± s.d., n = 5, *P  < 0.05, **P  < 0.01, Student’s t-test
Fig. 3
Fig. 3. 3-MA counteracted the suppression of AZD8055 on GICs self-renewal and proliferation.
a The representative images of GICs neurospheres showed that self-renewal ability of GICs was significantly inhibited by DMSO, AZD8055, and AZD8055 combined with 3-MA treatment. n = 5, Scale bar = 200 µm. b The quantification of numbers and diameter of the GICs neurospheres showed that neurosphere formation ability of GICs was rescued after 3-MA treatment. c The ability of GICs proliferation was detected by cell viability assay. d The ability of GICs self-renewal were detected by in vitro limiting dilution assay. Data are shown as means ± s.d, n = 5, *P < 0.05, **P < 0.01, likelihood ratio test. Data in b, c are shown as means ± s.d., n = 5, *P  < 0.05, **P < 0.01, Student’s t-test
Fig. 4
Fig. 4. Autophagy effectively downregulated the expression of CD133, Nestin, and GFAP.
ad U87 and U251 cells were treated the same as in (Picture 2). Protein expressions of CD133, Nestin, and GFAP were detect by western blot. Data are shown as means ± s.d., n = 3, *P < 0.05, **P < 0.01, Student’s t-test. e Immunofluorescence staining of U87 and U251 GICs, which treated by DMSO, AZD8055 (0.3 μM), and rapamycin (3 μM). The nuclei were stained with DAPI and the antibody against CD133 and nestin. Images were captured by laser confocal microscope ( × 400), scale bar = 100 μm
Fig. 5
Fig. 5. Autophagy reduced the protein expression of Notch pathway.
ad U87 and U251 cells were treated the same as in (Picture 2). Protein expressions of Notch1 pathway were detect by western blot. Data are shown as means ± s.d., n = 3, #P = NS, *P < 0.05, **P < 0.01, Student’s t-test. e Immunofluorescence staining of U87 and U251 GICs, which treated by DMSO, AZD8055 (0.3 μM) and rapamycin (3 μM). The nuclei were stained with DAPI and the antibody against Notch1 and NICD. Images were captured by laser confocal microscope ( × 400), scale bar = 100 μm
Fig. 6
Fig. 6. Autophagy regulated Notch1 degradation.
a U87 and U251 GICs were treated with AZD8055 (0.3 µM) for 48 h and rapamycin (3 µM) for 24 h. Cells were immunostained for Notch1 (green) and LC3 (red) and stained with DAPI (blue). The white box indicates the location of the enlargement. The white triangle shows LC3B and Notch1 colocalization with some yellow points. Scale bar = 5 µm. b Cells were immunostained for NICD (green) and LC3B (red) and stained with DAPI (blue) after the same treatment as a. Scale bar = 5 µm. c The expression of Notch1, DLL1 on the membrane of U87 and U251 GICs were detected by western blot. Drugs treatment were the same as in a. d Bar chart were used to compare the expression of Notch1 in three groups treatment. Data are shown as means ± S.D. n = 3, *P < 0.05, **P < 0.01, Student’s t-test. e Mechanism diagram described the line of Notch pathway activation and the progress of Autophagy regulating Notch1 degradation
Fig. 7
Fig. 7. AZD8055 and rapamycin inhibited the tumorigenicity of CD133 + U87-MG neurosphere cells in vivo.
The mice were treated with intraperitoneal injection with DMSO, AZD855 (10 mg/kg/day), or rapamycin (1.5 mg/kg/day) for 5 days a week. The treatment started from the 7th day after implantation and lasted for ~21 days. a Representative images of bioluminescence of mice on days 7, 14, and 28 after implantation. b Quantitative analysis of these bioluminescence images for the DMSO, AZD8055, and rapamycin treatment groups. c The overall survival of mice in the DMSO, AZD8055, and rapamycin treatment groups. Data are shown as the mean ± S.D. n = 5, **P < 0.01 compared to the control, ANOVA test. d Representative images of the HE staining in tumor sections ( × 400 magnification)

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