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Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein.
Cancer Cell Int. 2018; 18:209.CC

Abstract

Background

It has been demonstrated that bufadienolides exert potent anti-cancer activity in various tumor types. However, the mechanisms that underlie their anti-cancer properties remain unclear. Yes-associated protein, a key effector of Hippo signaling, functions as a transcription coactivator, plays oncogenic and tumor suppressor roles under different conditions. Here, we report that arenobufagin (ABF), a representative bufadienolide, induced breast cancer MCF-7 cells to undergo apoptosis, which occurred through the JNK-mediated multisite phosphorylation of YAP.

Methods

Cytotoxicity was examined using an MTT assay. ABF-induced apoptosis was measured with a TUNEL assay and Annexin V-FITC/PI double staining assay. Western blotting, immunofluorescence, qRT-PCR and coimmunoprecipitation were employed to assess the expression levels of the indicated molecules. Lose-of-function experiments were carried out with siRNA transfection and pharmacological inhibitors. ABF-induced phosphopeptides were enriched with Ti4+-IMAC chromatography and further subjected to reverse-phase nano-LC-MS/MS analysis.

Results

ABF significantly reduced the viability of MCF-7 cells and increased the percentage of early and late apoptotic cells in a concentration- and time-dependent manner. Following ABF treatment, YAP accumulated in the nucleus and bound to p73, which enhanced the transcription of the pro-apoptotic genes Bax and p53AIP1. YAP knock-down significantly attenuated ABF-induced apoptotic cell death. Importantly, we found that the mobility shift of YAP was derived from its phosphorylation at multiple sites, including Tyr357. Moreover, mass spectrometry analysis identified 19 potential phosphorylation sites in YAP, with a distribution of 14 phosphoserine and 5 phosphothreonine residues. Furthermore, we found that the JNK inhibitor SP600125 completely diminished the mobility shift of YAP and its phosphorylation at Tyr357, the binding of YAP and p73, the transcription of Bax and p53AIP1 as well as the apoptosis induced by ABF. These data indicate that ABF induced YAP multisite phosphorylation, which was associated with p73 binding, and that apoptosis was mediated by the JNK signaling pathway.

Conclusions

Our data demonstrate that ABF suppresses MCF-7 breast cancer proliferation by triggering the pro-apoptotic activity of YAP, which is mediated by JNK signaling-induced YAP multisite phosphorylation as well as its association with p73. The present work not only provides additional information on the use of ABF as an anti-breast cancer drug, but also offers evidence that the induction of the tumor suppressor role of YAP may be a therapeutic strategy.

Authors+Show Affiliations

1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 2Formula-pattern Research Center, School of Traditional Chinese Medicine, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.4Department of Pharmacology, School of Medicine, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.5School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, 510515 People's Republic of China.Guangzhou Yucai Middle School, Fujin Road 2#, Dongshan District, Guangzhou, China.2Formula-pattern Research Center, School of Traditional Chinese Medicine, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.1Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, 510632 China. 3College of Pharmacy, Jinan University, Guangzhou, 510632 People's Republic of China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30574018

Citation

Deng, Li-Juan, et al. "Arenobufagin Induces MCF-7 Cell Apoptosis By Promoting JNK-mediated Multisite Phosphorylation of Yes-associated Protein." Cancer Cell International, vol. 18, 2018, p. 209.
Deng LJ, Qi M, Peng QL, et al. Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein. Cancer Cell Int. 2018;18:209.
Deng, L. J., Qi, M., Peng, Q. L., Chen, M. F., Qi, Q., Zhang, J. Y., Yao, N., Huang, M. H., Li, X. B., Peng, Y. H., Liu, J. S., Fu, D. R., Chen, J. X., Ye, W. C., & Zhang, D. M. (2018). Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein. Cancer Cell International, 18, 209. https://doi.org/10.1186/s12935-018-0706-9
Deng LJ, et al. Arenobufagin Induces MCF-7 Cell Apoptosis By Promoting JNK-mediated Multisite Phosphorylation of Yes-associated Protein. Cancer Cell Int. 2018;18:209. PubMed PMID: 30574018.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein. AU - Deng,Li-Juan, AU - Qi,Ming, AU - Peng,Qun-Long, AU - Chen,Min-Feng, AU - Qi,Qi, AU - Zhang,Jia-Yan, AU - Yao,Nan, AU - Huang,Mao-Hua, AU - Li,Xiao-Bo, AU - Peng,Yin-Hui, AU - Liu,Jun-Shan, AU - Fu,Deng-Rui, AU - Chen,Jia-Xu, AU - Ye,Wen-Cai, AU - Zhang,Dong-Mei, Y1 - 2018/12/18/ PY - 2018/10/21/received PY - 2018/12/11/accepted PY - 2018/12/22/entrez PY - 2018/12/24/pubmed PY - 2018/12/24/medline KW - Apoptosis KW - Arenobufagin KW - Breast cancer KW - Bufadienolide KW - JNK KW - YAP SP - 209 EP - 209 JF - Cancer cell international JO - Cancer Cell Int. VL - 18 N2 - Background: It has been demonstrated that bufadienolides exert potent anti-cancer activity in various tumor types. However, the mechanisms that underlie their anti-cancer properties remain unclear. Yes-associated protein, a key effector of Hippo signaling, functions as a transcription coactivator, plays oncogenic and tumor suppressor roles under different conditions. Here, we report that arenobufagin (ABF), a representative bufadienolide, induced breast cancer MCF-7 cells to undergo apoptosis, which occurred through the JNK-mediated multisite phosphorylation of YAP. Methods: Cytotoxicity was examined using an MTT assay. ABF-induced apoptosis was measured with a TUNEL assay and Annexin V-FITC/PI double staining assay. Western blotting, immunofluorescence, qRT-PCR and coimmunoprecipitation were employed to assess the expression levels of the indicated molecules. Lose-of-function experiments were carried out with siRNA transfection and pharmacological inhibitors. ABF-induced phosphopeptides were enriched with Ti4+-IMAC chromatography and further subjected to reverse-phase nano-LC-MS/MS analysis. Results: ABF significantly reduced the viability of MCF-7 cells and increased the percentage of early and late apoptotic cells in a concentration- and time-dependent manner. Following ABF treatment, YAP accumulated in the nucleus and bound to p73, which enhanced the transcription of the pro-apoptotic genes Bax and p53AIP1. YAP knock-down significantly attenuated ABF-induced apoptotic cell death. Importantly, we found that the mobility shift of YAP was derived from its phosphorylation at multiple sites, including Tyr357. Moreover, mass spectrometry analysis identified 19 potential phosphorylation sites in YAP, with a distribution of 14 phosphoserine and 5 phosphothreonine residues. Furthermore, we found that the JNK inhibitor SP600125 completely diminished the mobility shift of YAP and its phosphorylation at Tyr357, the binding of YAP and p73, the transcription of Bax and p53AIP1 as well as the apoptosis induced by ABF. These data indicate that ABF induced YAP multisite phosphorylation, which was associated with p73 binding, and that apoptosis was mediated by the JNK signaling pathway. Conclusions: Our data demonstrate that ABF suppresses MCF-7 breast cancer proliferation by triggering the pro-apoptotic activity of YAP, which is mediated by JNK signaling-induced YAP multisite phosphorylation as well as its association with p73. The present work not only provides additional information on the use of ABF as an anti-breast cancer drug, but also offers evidence that the induction of the tumor suppressor role of YAP may be a therapeutic strategy. SN - 1475-2867 UR - https://www.unboundmedicine.com/medline/citation/30574018/Arenobufagin_induces_MCF_7_cell_apoptosis_by_promoting_JNK_mediated_multisite_phosphorylation_of_Yes_associated_protein_ L2 - https://cancerci.biomedcentral.com/articles/10.1186/s12935-018-0706-9 DB - PRIME DP - Unbound Medicine ER -
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