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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 triggers FANCD2 degradation through the ubiquitin–proteasome pathway. (a) RT-PCR assays for detecting FANCD2 RNA level in A549 cells upon celastrol. (b) Effects of cycloheximide (CHX) (50 μg/mL) alone or in combination with celastrol (5 μM) on FANCD2 expression, evaluated by Western blotting in A549 cells. (c) A549 cells were pre-incubated with MG132 (10 μM) or PS341 (100 nM) for 1 h, followed by celastrol (5 μM) treatment for 6 h. Cell lysates were subjected to Western blotting using anti-FANCD2 antibody. (d) A549 cells were pretreated with PS341 (100 nM) for 1 h, followed by celastrol (5 μM) incubation for 6 h. The cells were then analyzed by immunofluorescence assay labeling with anti-FANCD2 antibody and DAPI. (e) A549 cells were pretreated with or without PS341 (100 nM) for 1 h, followed by celastrol incubation for 2 h. Cell lysates were subjected to immunoprecipitation with FANCD2 antibody, followed by Western blotting using antibodies against FANCD2 and ubiquitin. (f, g) A549 cells were treated with Biotin or Bio-Cel at 50 μM for 4 h, lysed, and the cell lysates were subjected to immunoprecipitation using streptavidin agarose and Western blotting using indicated antibodies. Mono, monoubiquitinated FANCD2.
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fig02: Celastrol triggers FANCD2 degradation through the ubiquitin–proteasome pathway. (a) RT-PCR assays for detecting FANCD2 RNA level in A549 cells upon celastrol. (b) Effects of cycloheximide (CHX) (50 μg/mL) alone or in combination with celastrol (5 μM) on FANCD2 expression, evaluated by Western blotting in A549 cells. (c) A549 cells were pre-incubated with MG132 (10 μM) or PS341 (100 nM) for 1 h, followed by celastrol (5 μM) treatment for 6 h. Cell lysates were subjected to Western blotting using anti-FANCD2 antibody. (d) A549 cells were pretreated with PS341 (100 nM) for 1 h, followed by celastrol (5 μM) incubation for 6 h. The cells were then analyzed by immunofluorescence assay labeling with anti-FANCD2 antibody and DAPI. (e) A549 cells were pretreated with or without PS341 (100 nM) for 1 h, followed by celastrol incubation for 2 h. Cell lysates were subjected to immunoprecipitation with FANCD2 antibody, followed by Western blotting using antibodies against FANCD2 and ubiquitin. (f, g) A549 cells were treated with Biotin or Bio-Cel at 50 μM for 4 h, lysed, and the cell lysates were subjected to immunoprecipitation using streptavidin agarose and Western blotting using indicated antibodies. Mono, monoubiquitinated FANCD2.

Mentions: We investigated the underlying mechanism of celastrol-induced downregulation of FANCD2. By RT-PCR assay, we showed that treatment with celastrol at 5 μM for 6 h did not perturb FANCD2 expression at mRNA level in A549 cells (Fig.2a). Protein synthesis inhibitor CHX was employed to detect the protein stability of FANCD2. We found that in A549 cells treated with CHX (50 μg/mL) alone, the expression of FANCD2 was not decreased within 6 h (Fig.2b). Interestingly, treatment with CHX in combination with celastrol dramatically decreased FANCD2 at protein level within 4 h (Fig.2b), indicating that celastrol impaired the protein stability of FANCD2 by triggering its degradation.


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 triggers FANCD2 degradation through the ubiquitin–proteasome pathway. (a) RT-PCR assays for detecting FANCD2 RNA level in A549 cells upon celastrol. (b) Effects of cycloheximide (CHX) (50 μg/mL) alone or in combination with celastrol (5 μM) on FANCD2 expression, evaluated by Western blotting in A549 cells. (c) A549 cells were pre-incubated with MG132 (10 μM) or PS341 (100 nM) for 1 h, followed by celastrol (5 μM) treatment for 6 h. Cell lysates were subjected to Western blotting using anti-FANCD2 antibody. (d) A549 cells were pretreated with PS341 (100 nM) for 1 h, followed by celastrol (5 μM) incubation for 6 h. The cells were then analyzed by immunofluorescence assay labeling with anti-FANCD2 antibody and DAPI. (e) A549 cells were pretreated with or without PS341 (100 nM) for 1 h, followed by celastrol incubation for 2 h. Cell lysates were subjected to immunoprecipitation with FANCD2 antibody, followed by Western blotting using antibodies against FANCD2 and ubiquitin. (f, g) A549 cells were treated with Biotin or Bio-Cel at 50 μM for 4 h, lysed, and the cell lysates were subjected to immunoprecipitation using streptavidin agarose and Western blotting using indicated antibodies. Mono, monoubiquitinated FANCD2.
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fig02: Celastrol triggers FANCD2 degradation through the ubiquitin–proteasome pathway. (a) RT-PCR assays for detecting FANCD2 RNA level in A549 cells upon celastrol. (b) Effects of cycloheximide (CHX) (50 μg/mL) alone or in combination with celastrol (5 μM) on FANCD2 expression, evaluated by Western blotting in A549 cells. (c) A549 cells were pre-incubated with MG132 (10 μM) or PS341 (100 nM) for 1 h, followed by celastrol (5 μM) treatment for 6 h. Cell lysates were subjected to Western blotting using anti-FANCD2 antibody. (d) A549 cells were pretreated with PS341 (100 nM) for 1 h, followed by celastrol (5 μM) incubation for 6 h. The cells were then analyzed by immunofluorescence assay labeling with anti-FANCD2 antibody and DAPI. (e) A549 cells were pretreated with or without PS341 (100 nM) for 1 h, followed by celastrol incubation for 2 h. Cell lysates were subjected to immunoprecipitation with FANCD2 antibody, followed by Western blotting using antibodies against FANCD2 and ubiquitin. (f, g) A549 cells were treated with Biotin or Bio-Cel at 50 μM for 4 h, lysed, and the cell lysates were subjected to immunoprecipitation using streptavidin agarose and Western blotting using indicated antibodies. Mono, monoubiquitinated FANCD2.
Mentions: We investigated the underlying mechanism of celastrol-induced downregulation of FANCD2. By RT-PCR assay, we showed that treatment with celastrol at 5 μM for 6 h did not perturb FANCD2 expression at mRNA level in A549 cells (Fig.2a). Protein synthesis inhibitor CHX was employed to detect the protein stability of FANCD2. We found that in A549 cells treated with CHX (50 μg/mL) alone, the expression of FANCD2 was not decreased within 6 h (Fig.2b). Interestingly, treatment with CHX in combination with celastrol dramatically decreased FANCD2 at protein level within 4 h (Fig.2b), indicating that celastrol impaired the protein stability of FANCD2 by triggering its degradation.

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