Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein

Abstract

BackgroundIt 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.MethodsCytotoxicity 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.ResultsABF 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0706-9) contains supplementary material, which is available to authorized users.

Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells

“…The compound inhibited cell growth and triggered apoptosis by affecting the expression of Bax and Bcl-2 in the cells (Zhu et al 2018). Arenobufagin also reduced viability of MCF-7 cells and induced apoptosis in a time-and dose-dependent manner (Deng et al 2018).…”

“…Arenobufagin also reduced viability of MCF-7 cells and induced apoptosis in a time- and dose-dependent manner (Deng et al. 2018 ).…”

JNK and Autophagy Independently Contributed to Cytotoxicity of Arsenite combined With Tetrandrine via Modulating Cell Cycle Progression in Human Breast Cancer Cells

“…In line with previous reports showing that ERK usually served as a survival mediator implicated in cytoprotection ( Yao et al., 2012 ; Kikuchi et al., 2013 ), the combined regimen-triggered cytotoxicity was clearly augmented by PD98059, an inhibitor of ERK, suggesting that activation of ERK might compensate for the cytocidal stimuli. Although the activation of JNK has been deeply implicated in apoptosis/necrosis induction in different types of cancer cells ( Deng et al., 2018 ; Qiao et al., 2019 ), inhibition of JNK by SP600125 did not alter the induction of apoptosis/necrosis ( Figure 8 ), suggesting little involvement of JNK activation. Similarly, substantial upregulation of the expression of LC3 was not affected by SP600125 ( Figure 9 ), suggesting that JNK activation and autophagy independently contributed to the cytotoxicity of As III combined with Tetra in MDA-MB-231 cells.…”

“…ERK usually serves as a survival mediator implicated in cytoprotection (Kikuchi et al, 2013;Kawiak et al, 2019). On the other hand, JNK and p38 MAPK are generally considered to be involved in cell death induction by diverse stimuli (Hu et al, 2014b;Kikuchi et al, 2014;Deng et al, 2018;Qiao et al, 2019). Of note, recent emerging evidence has demonstrated a strong association between the activation of JNK and antitumor agent-mediated cytotoxicity such as cell cycle arrest as well as autophagic cell death in breast cancer cells (Wang et al, 2016;Xie et al, 2017;Kong et al, 2020).…”

Arenobufagin Promoted Oxidative Stress‐Associated Mitochondrial Pathway Apoptosis in A549 Non‐Small‐Cell Lung Cancer Cell Line

“…Arenobufagin (ARE, structure shown in Figure 1(a)), one of the effective constituents of toad venom, is a traditional Chinese medicine obtained from the skin and parotid venom glands of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider [11]. Anyway, a lot of studies have demonstrated its broadspectrum antitumor activities in cancers such as breast cancer, pancreatic carcinoma, and liver cancer [12][13][14]. We previously found that ARE can induce liver cancer cell apoptosis and autophagy through PI3K/Akt/mTOR signal routing [14]; induce cell cycle arrest and apoptosis in human cervical cancer HeLa cells [15]; have anticancer effect on human esophageal squamous cell carcinoma (its mechanism of exerting anticancer efficacy may be activation of cysteinecontaining aspartate proteolytic enzyme (caspase) by endogenous and exogenous pathways); promote apoptosis of esophageal cancer cells by enhancing caspase phosphorylation and activating p53 signaling [16]; promote apoptosis of human glioblastoma U-87 cells by inhibiting p38MAPK signaling pathway [17]; and inhibit epithelial-mesenchymal conversion by going down the β-catenin pathway, consequently repressing motility and invasiveness of prostate cancer PC3 cells [18].…”