Abstract
Increasing evidence suggests that targeting ubiquitin-specific peptidase 8 (USP8) serves as an attractive anti-cancer strategy. However, the role of USP8 inhibitor, DUB-IN-1, in esophageal squamous cell carcinoma (ESCC) cells still needs to be explored. Here, immunohistochemistry was employed to examine the expression of USP8 in ESCC tissues. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation ability, and propidium iodide (PI) was selected to test the effect of DUB-IN-1 on cell cycle. AnnexinV-FITC/PI staining and the activity of caspase 3 were detedcted to evaluate apoptosis. Transmission electron microscope, microtubule-associated protein 1 light-chain 3 (LC3) expression, and acridine orange (AO) staining were selected to check if there was autophagy. Comet assay and γ-H2AX immunofluorescence was used to monitor DNA damage. Rescue experiment was used to determine the key role of of p53 in cell cycle, apoptosis, and autophagy. Results revealed that the leve of USP8 was higher in ESCC tissues than that in tissues adjacent to carcinoma. DUB-IN-1, an USP8 inhibitor, caused DNA damage, led to G2/M phase block by p53-p21 axis, and triggered apoptosis by regulating the p53 target proteins including Bax, Noxa, and Puma. Besides, DUB-IN-1 could stimulate autophagy through p53-dependent adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) activation. Taken together, this study revealed the cytotoxic effects and the mechanism of DUB-IN-1, which indicated that DUB-IN-1 may be a novel inhibitor targeting USP8 that can kill ESCC cells.
Graphical abstract
USP8 inhibitor, DUB-IN-1, treatment could inhibit esophageal squamous cell carcinoma cell growth and induce G2/M cell cycle arrest, apoptosis, and autophagy by DNA damage–induced p53 activation. DUB-IN-1 treatment led to G2/M cell cycle arrest by upregulating the protein level of p21 and triggered apoptosis by modulating the p53 target proteins including Bax, Noxa, and Puma. Meanwhile, DUB-IN-1 treatment stimulated protective autophagy through p53-dependent AMPK activation. Collectively, these findings suggested that DNA damage–triggered p53 activation, p53-Puma/Noxa/Bax, p53-p21, and p53-AMPK pathways were all involved in the effect of DUB-IN-1.
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Acknowledgements
We appreciate the Transmission Electron Microscope (TEM) Center of Henan University of Chinese Medicine for providing the TEM services and pictures.
Funding
This work was supported by the National Natural Science Foundation Grant of China (Grant No. 81672421), Program for Innovation Research Team (in Science and Technology) in University of Henan Province (Grant No. 20IRTSTHN026), and Open Project Fund of Henan Key Laboratory of Precision Clinical Pharmacy (Grant No. HKLPCP-2020–06).
National Natural Science Foundation of China,81672421,Ping Chen,Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province,20IRTSTHN026,Pei Li,Open Project Fund of Henan Key Laboratory of Precision Clinical Pharmacy,HKLPCP-2020–06,Ping Chen
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Conceptualization: Tao Hu and Ping Chen; methodology: Beibei Sha, Yaxin Sun, Shan Zhao, Miaomiao Li, Wenjing Huang, and Zheng Li; data collection and analysis: Beibei Sha and Yaxin Sun; writing—original draft preparation: Beibei Sha, Yaxin Sun and Tao Hu; writing—review and editing: Jianxiang Shi and Xuefei Han; funding acquisition: Ping Chen and Pei Li; supervision: Tao Hu and Ping Chen. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Graphical headlights
1. USP8 inhibitor, DUB-IN-1, treatment induces DNA damage and activates p53.
2. DUB-IN-1 triggers apoptosis by p53-Puma/Noxa/Bax.
3. DUB-IN-1 leads to G2/M cell cycle arrest by p53-p21.
4. DUB-IN-1 stimulates protective autophagy through p53-dependent AMPK activation.
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Sha, B., Sun, Y., Zhao, S. et al. USP8 inhibitor–induced DNA damage activates cell cycle arrest, apoptosis, and autophagy in esophageal squamous cell carcinoma. Cell Biol Toxicol 39, 2011–2032 (2023). https://doi.org/10.1007/s10565-021-09686-x
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DOI: https://doi.org/10.1007/s10565-021-09686-x