Abstract
The antiproliferative effect of cardamonin on mTORC1 is related with downregulation of Raptor. We investigated the mechanism that cardamonin decreases Raptor expression through caspase-mediated protein degradation. SKOV3 cells and HeLa cells were pretreated with caspase inhibitor z-VAD-fmk for 30 min and then exposed to different doses of cardamonin and cisplatin, respectively. We analyzed the gene expression of caspases based on TCGA and GTEx gene expression data in serous cystadenocarcinoma and normal tissues, monitored caspase activity by caspase colorimetric assay kit, detected expression of mTORC1-associated proteins and apoptosis-associated proteins by western blotting, and finally detected cell viability by methyl thiazolyl tetrazolium (MTT) assay. A different expression of caspases except caspase-1 was found between serous cystadenocarcinoma and normal tissues. Raptor was cleaved when caspases were activated by cisplatin and caspase-6/caspase-8 was activated by cardamonin in SKOV3 cells. We further used a monoclonal antibody recognizing the N-terminal part of Raptor to find that Raptor was cleaved into a smaller fragment of about 70 kDa by cardamonin and was rescued by z-VAD-fmk treatment. As a result of Raptor cleavage, mTORC1 activity was decreased and cell viability was inhibited, while cell apoptosis was induced in SKOV3 cells. Notably, similar results are only observed in HeLa cells with a high dose of cardamonin. We concluded that caspase-mediated cleavage of Raptor might be an important mechanism in that cardamonin regulated Raptor and mTORC1 activity.
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This study was supported by the Natural Science Foundation of Fujian Province of China (2019 J01508), Fujian Provincial Health Technology Project (2017-1-18, 2017-1-19), and Foundations of Fujian Maternity and Child Health Hospital (YCXZ 18-07, YCXQ 18-02, YCXM 19-05), China.
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DS and YZ conceived and designed research. YZ and JZ conducted experiments. HC and PN analyzed data. YZ wrote the manuscript. All authors read and approved the manuscript. All data were generated in-house, and we did not use a paper mill.
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Zhu, Y., Zhou, J., Niu, P. et al. Cardamonin inhibits cell proliferation by caspase-mediated cleavage of Raptor. Naunyn-Schmiedeberg's Arch Pharmacol 394, 809–817 (2021). https://doi.org/10.1007/s00210-020-01986-z
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DOI: https://doi.org/10.1007/s00210-020-01986-z