doi: 10.3967/bes2022.058
Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells
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Abstract:
Objective To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM). Methods GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay. Results Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation. Conclusion Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair. -
Key words:
- Primary cilia /
- Glioblastoma /
- Cellular sensitivity /
- Temozolomide /
- Ionizing radiation /
- Autophagy /
- DNA damage response /
- DNA-PK
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Figure 1. GBM cells express primary cilia. (A) Representative images of the primary cilia in M059K, M059J, and GS1910 cells. Cilium was stained for ciliary membrane marker Arl13b (red) and basal body marker γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 20 µm. (B) Quantification of ciliated cell and the ciliary length counted in ten randomly selected fields from at least three independent experiments. (C) Expression levels of Ac-α-tub and HDAC6 proteins detected by Western blot in M059K, M059J, and GS1910 cells. (D) Cell growth curves. (E) Expression levels of Ac-α-tub protein measured by Western blot before and after treatment with LiCl (5 mmol/L) at 1 and 3 days. (F) Quantification of ciliated cells in three GBM cell lines treated with LiCl at 1 and 3 days. (G) Representative images of primary cilia induced by LiCl in three GBM cell lines at 1 and 3 days and stained for Arl13b (red). DNA was stained with DAPI (blue). Scale bar, 20 µm. (H) Cell proliferation assay of M059K (left) and GS1910 (right) cells in (F). (I) Representative images of primary cilia in GBM biopsies collected from 10 patients and stained for Arl13b (red). DNA was stained with DAPI (blue). Scale bar, 10 µm. Ctrl, control; white arrows indicate primary cilia; ns, not significant; *P < 0.05 and **P < 0.01. GBM: glioblastoma.
Figure 2. TMZ induces ciliogenesis in GBM cells. (A) Cell viability assay of three GBM cell lines treated with different concentrations of TMZ for 3 days. (B) Representative images of TMZ-induced primary cilium formation in M059K, M059J, and GS1910 cells. Double staining with antibodies against Arl13b (red) and γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 20 µm. Quantitative results of the frequency of ciliated cells (C) and ciliary length (D) in three GBM lines before and after treatment with 500 µmol/L TMZ for 3 days. The data are presented as the mean ± SD of three independent experiments, and more than 100 cilia were counted and measured in each individual group. (E) Western blot of Ac-α-tub expression in TMZ-induced ciliogenesis of three GBM cell lines for 1 and 3 days. (F) Representative images of TMZ-induced abnormal cilia in M059K cells treated with 500 µmol/L TMZ for 3 days and stained for Arl13b (red) and γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 10 µm. (G) Quantitative results of the multiciliated cells in M059K cells prior to and posttreatment with 500 µmol/L TMZ for 3 days. (H) Quantitative results of the length of cilium in GS1910 cells after the treatment with 500 µmol/L TMZ for 3 days. (I) Representative images of elongated cilia in GS1910 cells treated with TMZ and stained for Arl13b (red) and γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 10 µm. Ctrl, control; white arrows indicate primary cilia; ns, not significant; **P < 0.01 and ***P < 0.001. TMZ: temozolomide; GBM: glioblastoma.
Figure 3. IR triggers primary cilium formation and elongation in GBM cells. (A) Fraction of ciliated cells in M059K cells exposed to 0–10 Gy X-rays or carbon ions on day 1. (B) Average cilium length of cells in (A). (C) Frequency of ciliated cells in M059K cells treated with 10 Gy X-rays or carbon ions at 1 and 3 days after irradiation. (D) Average cilium length of cells in (C) and representative images of the elongated cilium. Double staining with antibodies against Arl13b (red) and γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 10 µm. (E) Frequency of ciliated cells in M059J cells after 0–10 Gy X-rays or carbon ion exposure at 1 day. (F) Frequency of ciliated cells in M059J cells treated with 10 Gy X-rays at 1 and 3 days after irradiation. (G) Cilium length of cells in (F). (H) Representative images of primary cilia in M059J cells during 10 Gy X-ray treatment for 3 days and double staining with antibodies against Arl13b (red) and γ-tub (green). DNA was stained with DAPI (blue). Scale bar, 10 µm. (I) Western blot of Ac-α-tub expression in M059K and M059J cells after X-ray exposure for 1 day. (J) Western blot of Ac-α-tub expression in M059K and M059J cells after 10 Gy X-ray exposure at 1 and 3 days. Ctrl, control; white arrows indicate primary cilia; *P < 0.05 and **P < 0.01 vs. Ctrl (X-ray); #P < 0.05 and ###P < 0.001 vs. Ctrl (Carbon ion). IR: ionizing radiation; GBM: glioblastoma.
Figure 4. Interference of ciliogenesis by IFT88 siRNA decreases the cellular resistance to TMZ. (A) Top, Western blot of IFT88 expression in M059K cells transfected with IFT88 siRNA (si-IFT88) or an NC siRNA at a final concentration of 50 nmol/L; bottom, representative images of primary cilia in M059K cells depleting the IFT88 expression and double staining with antibodies against Arl13b (red) and γ-tub (green); DNA was stained with DAPI (blue). Scale bar, 10 µm. (B) Quantitative results of the frequency of ciliated cells in M059K cells after the depletion of IFT88 during different periods. (C) Quantitative results of the cilium length in M059K cells treated with si-IFT88 for 3 days. (D) Western blot of IFT88 expression in GS1910 cells transfected with si-IFT88 or NC at a final concentration of 50 nmol/L. (E) Representative images of primary cilia in IFT88-deficient M059K cells treated with 500 µmol/L TMZ on day 2. Scale bar, 10 µm. Ciliary frequency (F) and length (G) of cells in (E). (H) Quantitative results of the frequency of ciliated cells in IFT88-deficient GS1910 cells treated with 500 µmol/L TMZ at 2 days. (I) Cell viability assay of three GBM cells with the depletion of IFT88 the following treatment with 500 or 1,000 µmol/L TMZ at 2 days. Ctrl, control; white arrows indicate primary cilia; *P < 0.05, **P < 0.01, and ***P < 0.001. TMZ: temozolomide; GBM: glioblastoma.
Figure 5. Interference of primary cilia by si-IFT88 decreases the cellular resistance to IR. (A) Top, Western blot of IFT88 expression in M059K cells transfected with si-IFT88 or NC at a final concentration of 50 nmol/L followed by 2 Gy X-ray exposure; bottom, representative images of primary cilia in M059K cells depleting IFT88 expression and double staining with antibodies against Arl13b (red) and γ-tub (green); DNA stained with DAPI (blue). Scale bar, 10 µm. (B) Quantitative results on the frequency of ciliated cells in M059K cells after depletion of IFT88 followed by 2 Gy X-ray exposure on day 3. (C) Cilium length of cells in (B). (D) Western blot of IFT88 expressions in GS1910 and M059J cells transfected with si-IFT88 or NC followed by 2 Gy X-rays at different times. (E) Quantitative results on the frequency of ciliated cells in GS1910 cells after IFT88 depletion followed by 2 Gy X-ray exposure on day 3. (F) Representative images of colonies of M059K cells transfected with si-IFT88 or NC following exposure to 2 Gy X-rays or carbon ions; stained with 0.5% crystal violet. At least three independent experiments were performed. (G) SF2 values of IFT88-deficient M059K cells by X-ray or carbon ion exposure. (H) Representative image of colonies of M059J and GS1910 cells transfected with si-IFT88 or NC following exposure to 2 Gy X-rays. (I) SF2 values of IFT88-deficient GS1910 cells (left), IFT88-deficient M059J cells (right), and NC cells exposed to 2 Gy X-rays. Ctrl, control; SF2, survival fraction at 2 Gy; white arrows indicate primary cilia; *P < 0.05 and ***P < 0.001.
Figure 6. Primary cilia regulate autophagy to affect the cellular sensitivity to TMZ and IR. (A) AO staining assay for the detection of autophagolysosomes levels in M059K cells transfected with si-IFT88 (50 nmol/L) or treated with CH (2 mmol/L) following the exposure to IR (10 Gy X-rays) or TMZ (500 µmol/L). Scale bar, 50 µm. (B) Bar diagram quantifies the red fluorescence signal intensity of cells in (A). At least 100 cells from each group were tested in three independent experiments. (C) Western blot of LC3B expression in M059K cells after the exposure to IR or TMZ at 1 and 3 days. (D) Western blot of LC3B and BCL2 expressions in M059K cells transfected with si-IFT88 or NC following the exposure to X-rays at 1 and 3 days. Cell viability assay of M059K (E) and GS1910 (F) cells exposed to TMZ (500 µmol/L), followed by the administration of 3-MA (10 mmol/L) or BFA (0.5 µg/mL) at 2 days. Ctrl, control; *P < 0.05 and **P < 0.01. TMZ: temozolomide; IR: ionizing radiation.
Figure 7. Primary cilia mediate cellular DNA damage repair progression. (A) Representative images of γH2AX foci formed in DNA DSB regions by immunostaining with a specific antibody against γH2AX (red) in three GBM cell lines during 10 Gy X-ray exposure at different time periods. DNA was stained with DAPI (blue). Scale bar, 10 µm. (B) Kinetic curves of γH2AX foci in three GBM cell lines. (C) Western blot of DNA-PK, pDNA-PK (Ser2056), ATM, and pATM (Ser1981) expressions in (A). (D) Representative images of γH2AX foci (red) in three GBM cell lines transfected with si-IFT88 or exposed to 2 mmol/L CH following 10 Gy X-ray exposure at 24 and 72 h and double staining with antibodies against Arl13b (green) and γH2AX (red). DNA was stained with DAPI (blue). Scale bar, 10 µm. (E) Quantity of γH2AX foci per cell in (D) at 72 h. (F) Western blot of DNA-PK, pDNA-PK, ATM, and pATM expressions in M059K and GS1910 cells transfected with si-IFT88 or NC following 10 Gy X-ray exposure at different time periods. **P < 0.01 and ***P < 0.001 vs. M059J at the same time point; Ctrl, control; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. M059J at 12 h.
Figure 8. DNA-PK contributes to ciliogenesis. (A) Representative images of subcellular localization of pDNA-PK at different cell division phases in M059K cells. Double staining with antibodies against γ-tub (green) and pDNA-PK (red). DNA was stained with DAPI (blue). Scale bar, 10 µm. (B) Representative images of subcellular localization of pDNA-PK in M059K cells during 10 Gy X-ray exposure at 24 and 72 h. Scale bar, 10 µm. (C) Western blot of DNA-PK and pDNA-PK expressions in M059K cells transfected with si-DNA-PK followed by treatment or nontreatment with 10 Gy X-rays and the quantitative results. Quantitative results of the ciliary frequency (D) and length (E) in M059K and GS1910 cells transfected with si-DNA-PK or NC. The red line indicates the mean cilium length. Quantitative results of the ciliary frequency (F) and length (G) in M059K and GS1910 cells transfected with si-DNA-PK followed by 10 Gy X-ray exposure. The red line indicates the mean cilium length. ns, not significant; *P < 0.05.
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