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Rotenone Inhibited Osteosarcoma Cell Growth Through USP47-Induced Decreases in FEN1 Stability and DNA Integrity
Zhen Li1, Xiang Ma2, Hengwei Ma3
1Department of Medical Oncology, The Second Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, People's Republic of China.
Background:
Osteosarcoma (OS) is the most prevalent primary malignant bone tumor in children. We previously showed that rotenone suppressed OS cell metastasis. However, its effects on OS cell growth and the underlying mechanisms remain unclear. The purpose of this study was to investigate the role of rotenone in OS and identify its direct target.
Methods:
Molecular dockingand Biacore assay were used to confirm the interaction between rotenone and USP47. Co-immunoprecipitation, immunofluorescence, and cycloheximide assays were used to verify the relationship betweenUSP47 and FEN1. Cell cycle and apoptosis were examined by flow cytometry. Comet analyses were used to determine DNA damage. Deubiquination and Ub-VME assays were carried out to assess ubiquitination status and properties. Immunohistochemistry and a xenograft mouse model were utilized to validate the effects of various proteins and rotenone in vivo.
Results:
Knockdown of FEN1 and USP47 in OS cell lines induced cell cycle arrest and apoptosis through the induction of DNA damage. FEN1 exhibited a direct interaction with USP47. Mechanistically, wild-type USP47 regulated FEN1 protein stability through deubiquitination modification, whereas mutated Cys109Ser USP47 did not. Furthermore, rotenone modulated USP47 protein expression and the combined quantity of Ub-USP47 conjugation through physical interaction. Xenograft studies further confirmed the anti-OS activity of rotenone in vivo.
Conclusion:
Rotenone is a potential therapeutic agent for OS due to its direct targeting of USP47 and resultant decrease in FEN1 stability and DNA integrity.
Insights
Rotenone targets USP47, decreasing FEN1 stability and DNA integrity, thereby inhibiting osteosarcoma (OS) growth. This study identifies rotenone as a potential therapeutic agent for OS by elucidating its anti-OS mechanisms.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Osteosarcoma (OS) is a common pediatric bone cancer.
- Previous research indicated rotenone inhibits OS metastasis.
- The impact of rotenone on OS cell growth and its mechanisms were previously unknown.
Purpose of the Study:
- To investigate rotenone's role in osteosarcoma (OS) progression.
- To identify the direct molecular target of rotenone in OS.
- To elucidate the mechanisms underlying rotenone's effects on OS cells.
Main Methods:
- Utilized molecular docking and Biacore assays to confirm rotenone-USP47 interaction.
- Employed co-immunoprecipitation, immunofluorescence, and cycloheximide assays to study USP47-FEN1 relationship.
- Assessed cell cycle, apoptosis, DNA damage, and ubiquitination status via flow cytometry, comet assays, and deubiquitination assays.
- Validated findings in vivo using immunohistochemistry and a xenograft mouse model.
Main Results:
- Knockdown of FEN1 and USP47 induced cell cycle arrest and apoptosis via DNA damage in OS cells.
- FEN1 directly interacts with USP47, which regulates FEN1 stability through deubiquitination.
- Rotenone physically interacts with USP47, modulating its expression and ubiquitination, leading to decreased FEN1 stability and DNA integrity.
- In vivo xenograft studies confirmed rotenone's anti-OS activity.
Conclusions:
- Rotenone directly targets USP47 in osteosarcoma (OS).
- Rotenone reduces FEN1 protein stability and compromises DNA integrity.
- Rotenone demonstrates potential as a therapeutic agent for OS.
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