Abstract
Aim:
Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells.
Methods:
A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis.
Results:
The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity.
Conclusion:
BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly.
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Acknowledgements
This work was supported in part by Shanghai Science & Technology Support Program (No 13431900401), Shanghai Science & Technology Innovation Action program (No 15140904800), the National Natural Science Foundation of China (No 81373964), and the National Science & Technology Major Project of China (No 2014ZX09301-306-03).
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Supplementary information is available at the website of Acta Pharmacologica Sinica.
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Supplemental Figure S1
BF211 induce accumulation of ubiquitinated proteins in PC-3 cells and HeLa cells. (DOC 137 kb)
Supplemental Table S1
Primers used in RT-PCR assay of mRNA levels of HSP90AA1, HSP90AB1, PSMA2, PSMB6, PSMB7, PSMB5 and GAPDH. (DOC 32 kb)
Supplemental Table S2
List of proteins differentially expressed in BF211-treated cells compared with control cells. (DOC 1000 kb)
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Sun, P., Feng, Lx., Zhang, Dm. et al. Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly. Acta Pharmacol Sin 37, 908–918 (2016). https://doi.org/10.1038/aps.2016.30
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DOI: https://doi.org/10.1038/aps.2016.30
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