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Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents.

Wang GZ, Liu YQ, Cheng X, Zhou GB - Cancer Sci. (2015)

Bottom Line: In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin-proteasome pathway.We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate.Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Carcinogenesis and Targeted Therapy for Cancer, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

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Celastrol downregulates monoubiquitinated FANCD2 and attenuates DNA-damaging agent-induced FA pathway activation. (a) A549 and H1975 cells were treated with celastrol at 5 μM for indicated time points, lysed, and Western blotting was performed using anti-FANCD2 antibody. (b) FANCD2 monoubiquitination induced by different DNA-damaging agents was inhibited by celastrol. A549 cells were pre-incubated with cisplatin (CDDP; 20 μM/L), mitomycin C (MMC) (0.4 μg/mL) or hydroxyurea (HU; 1 mmol/L) for 24 h or subjected to ionizing radiation (IR) (15 Gy), then incubated with celastrol (5 μM) for additional 4 h. The cells were lysed and Western blot was performed. (c) A549 cells were pretreated with cisplatin (20 μM/L) for 24 h, followed by celastrol (5 μM) incubation for additional 4 h. The cells were subjected to immunofluorescence assay by labeling with anti-FANCD2 antibody and DAPI. (d) A549 cells were irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points; whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (e) A549 cells were transfected with siRNA against negative control (siRNA-NC) or FANCD2 (siFANCD2), irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points. The whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (f) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points, and the whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (g) A549 cells were irradiated by X-ray (15 Gy), incubated immediately with 5 μM celastrol for an additional 4 or 8 h. Cell cycle distribution was then determined. (h) A549 cells were transfected with siRNA-NC or si-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol for additional 8 h. Cell cycle distribution was then determined. (i) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), and then incubated immediately with 5 μM celastrol for an additional 8 h. Cell cycle distribution was determined.
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fig03: Celastrol downregulates monoubiquitinated FANCD2 and attenuates DNA-damaging agent-induced FA pathway activation. (a) A549 and H1975 cells were treated with celastrol at 5 μM for indicated time points, lysed, and Western blotting was performed using anti-FANCD2 antibody. (b) FANCD2 monoubiquitination induced by different DNA-damaging agents was inhibited by celastrol. A549 cells were pre-incubated with cisplatin (CDDP; 20 μM/L), mitomycin C (MMC) (0.4 μg/mL) or hydroxyurea (HU; 1 mmol/L) for 24 h or subjected to ionizing radiation (IR) (15 Gy), then incubated with celastrol (5 μM) for additional 4 h. The cells were lysed and Western blot was performed. (c) A549 cells were pretreated with cisplatin (20 μM/L) for 24 h, followed by celastrol (5 μM) incubation for additional 4 h. The cells were subjected to immunofluorescence assay by labeling with anti-FANCD2 antibody and DAPI. (d) A549 cells were irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points; whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (e) A549 cells were transfected with siRNA against negative control (siRNA-NC) or FANCD2 (siFANCD2), irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points. The whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (f) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points, and the whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (g) A549 cells were irradiated by X-ray (15 Gy), incubated immediately with 5 μM celastrol for an additional 4 or 8 h. Cell cycle distribution was then determined. (h) A549 cells were transfected with siRNA-NC or si-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol for additional 8 h. Cell cycle distribution was then determined. (i) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), and then incubated immediately with 5 μM celastrol for an additional 8 h. Cell cycle distribution was determined.

Mentions: We examined the effect of celastrol on monoubiquitinated FANCD2 in NSCLC cells, and found that compared to the unmodified FANCD2, the monoubiquitinated FANCD2 was decreased more rapidly upon celastrol treatment at 5 μM for 1–4 h (Fig.3a), suggesting that celastrol decreased monoubiquitination of FANCD2 and then reduced protein stability. Furthermore, while cisplatin, HU, MMC and IR induced monoubiquitination of FANCD2 in A549 cells, celastrol treatment (at 5 μM for 4 h) markedly attenuated this effect (Fig.3b). By immunofluorescence assay, we observed that cisplatin induced marked increase in FANCD2 in the nucleus (Fig.3c), suggesting formation of nuclear foci in A549 cells. This phenomenon was attenuated by celastrol (Fig.3c).


Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents.

Wang GZ, Liu YQ, Cheng X, Zhou GB - Cancer Sci. (2015)

Celastrol downregulates monoubiquitinated FANCD2 and attenuates DNA-damaging agent-induced FA pathway activation. (a) A549 and H1975 cells were treated with celastrol at 5 μM for indicated time points, lysed, and Western blotting was performed using anti-FANCD2 antibody. (b) FANCD2 monoubiquitination induced by different DNA-damaging agents was inhibited by celastrol. A549 cells were pre-incubated with cisplatin (CDDP; 20 μM/L), mitomycin C (MMC) (0.4 μg/mL) or hydroxyurea (HU; 1 mmol/L) for 24 h or subjected to ionizing radiation (IR) (15 Gy), then incubated with celastrol (5 μM) for additional 4 h. The cells were lysed and Western blot was performed. (c) A549 cells were pretreated with cisplatin (20 μM/L) for 24 h, followed by celastrol (5 μM) incubation for additional 4 h. The cells were subjected to immunofluorescence assay by labeling with anti-FANCD2 antibody and DAPI. (d) A549 cells were irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points; whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (e) A549 cells were transfected with siRNA against negative control (siRNA-NC) or FANCD2 (siFANCD2), irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points. The whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (f) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points, and the whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (g) A549 cells were irradiated by X-ray (15 Gy), incubated immediately with 5 μM celastrol for an additional 4 or 8 h. Cell cycle distribution was then determined. (h) A549 cells were transfected with siRNA-NC or si-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol for additional 8 h. Cell cycle distribution was then determined. (i) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), and then incubated immediately with 5 μM celastrol for an additional 8 h. Cell cycle distribution was determined.
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fig03: Celastrol downregulates monoubiquitinated FANCD2 and attenuates DNA-damaging agent-induced FA pathway activation. (a) A549 and H1975 cells were treated with celastrol at 5 μM for indicated time points, lysed, and Western blotting was performed using anti-FANCD2 antibody. (b) FANCD2 monoubiquitination induced by different DNA-damaging agents was inhibited by celastrol. A549 cells were pre-incubated with cisplatin (CDDP; 20 μM/L), mitomycin C (MMC) (0.4 μg/mL) or hydroxyurea (HU; 1 mmol/L) for 24 h or subjected to ionizing radiation (IR) (15 Gy), then incubated with celastrol (5 μM) for additional 4 h. The cells were lysed and Western blot was performed. (c) A549 cells were pretreated with cisplatin (20 μM/L) for 24 h, followed by celastrol (5 μM) incubation for additional 4 h. The cells were subjected to immunofluorescence assay by labeling with anti-FANCD2 antibody and DAPI. (d) A549 cells were irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points; whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (e) A549 cells were transfected with siRNA against negative control (siRNA-NC) or FANCD2 (siFANCD2), irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points. The whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (f) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol at indicated time points, and the whole-cells extracts were used for detection of FANCD2 and γ-H2AX. (g) A549 cells were irradiated by X-ray (15 Gy), incubated immediately with 5 μM celastrol for an additional 4 or 8 h. Cell cycle distribution was then determined. (h) A549 cells were transfected with siRNA-NC or si-FANCD2, irradiated by X-ray (15 Gy), then incubated immediately with 5 μM celastrol for additional 8 h. Cell cycle distribution was then determined. (i) A549 cells were transfected with pDONR201-vector or pDONR201-FANCD2, irradiated by X-ray (15 Gy), and then incubated immediately with 5 μM celastrol for an additional 8 h. Cell cycle distribution was determined.
Mentions: We examined the effect of celastrol on monoubiquitinated FANCD2 in NSCLC cells, and found that compared to the unmodified FANCD2, the monoubiquitinated FANCD2 was decreased more rapidly upon celastrol treatment at 5 μM for 1–4 h (Fig.3a), suggesting that celastrol decreased monoubiquitination of FANCD2 and then reduced protein stability. Furthermore, while cisplatin, HU, MMC and IR induced monoubiquitination of FANCD2 in A549 cells, celastrol treatment (at 5 μM for 4 h) markedly attenuated this effect (Fig.3b). By immunofluorescence assay, we observed that cisplatin induced marked increase in FANCD2 in the nucleus (Fig.3c), suggesting formation of nuclear foci in A549 cells. This phenomenon was attenuated by celastrol (Fig.3c).

Bottom Line: In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin-proteasome pathway.We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate.Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Carcinogenesis and Targeted Therapy for Cancer, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

Show MeSH
Related in: MedlinePlus