Direct targeting of HSP90 with daurisoline destabilizes β-catenin to suppress lung cancer tumorigenesis
Introduction
Aberrant Wnt/β-catenin signaling is involved in the pathological processes of multiple human diseases including cancer and maybe a potential therapeutic target for cancer treatment [1]. As a key component in Wnt/β-catenin signaling, β-catenin is phosphorylated by casein kinase I (CK1) and the complex consisting of glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC) and axin in the absence of Wnt, after which it undergoes ubiquitin-mediated proteasomal degradation [2,3]. In the presence of Wnt ligand, Wnt binds to Frizzled receptors and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) on the cell surface to form a trimer, which transmits signals and activates cytoplasmic dishevelled proteins to attenuate the stability of the APC/axin/GSK-3β complex and prevent β-catenin phosphorylation and degradation [4,5]. Stabilized β-catenin transfers to the nucleus to interact with T-cell factor/lymphoid enhancing factor (TCF/LEF), and then activates transcription of downstream target genes including c-myc and cyclin D1, thereby leading to cancer development and progression [6,7]. Emerging evidence suggests that the stability and transcriptional activity of β-catenin can be affected by a large number of partners that bind to β-catenin and control its activity [8,9]. Therefore, the identification of novel therapeutic strategies that target β-catenin is urgently needed.
Lung cancer is the leading cause of cancer death in the world. Although recently there have been further advances in the understanding of the mechanism of lung tumorigenesis and in the development of anticancer drugs, the treatment outcomes in clinic are still unsatisfactory due to limited efficacy and significant side effects. In this study, we aim to screen for novel anticancer agents with good treatment efficacy and safety in a small molecule library consisting of 429 natural products. Daurisoline, a bis-benzylisoquinoline alkaloid, isolated from the Chinese herbal medicine Rhizoma Menispermi [10], was found to exert the strongest inhibitory effect on lung cancer proliferation. Daurisoline has displayed the potential for treating some diseases, such as focal ischemia-reperfusion injury, arrhythmia and platelet aggregation [11,12], but up until now, the bioactivity of daurisoline in the treatment of cancer and its underlying mechanism are unknown.
Quantitative proteomics coupled with bioinformatic analysis is an excellent strategy for exploring the molecular mechanism of biological processes [13]. With the recent rapid advances in proteomics and chemical biology, Drug Affinity Responsive Target Stability (DARTS), a straightforward method, could be used to identify the target proteins that small molecules directly bind to, without the use of any probe [14,15]. It relies on the principle that the target protein in the cell lysate is resistant to protease digestion when it binds to the ligand molecule, after which mass spectrometry can be used to identify the specific proteins [16]. In the present study, the data from quantitative proteomics and DARTS suggested that daurisoline exerted its anticancer effect through the regulation of β-catenin signaling and may directly target heat shock protein 90 (HSP90), which has been reported to regulate the degradation and expression of β-catenin as an interaction partner [17]. By performing quantitative proteomics, co-immunoprecipitation, and a series of functional assays in vitro and in vivo, we investigated whether daurisoline inhibits the β-catenin pathway and transcription of its downstream target genes to suppress lung tumorigenesis by directly targeting HSP90.
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Cell lines and culture
Human lung cancer cell lines A549, Hop62 and H1299 (ATCC, Rockville, MD, USA) were cultured in DMEM medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies) at 37 °C in 5% CO2.
Cell viability assay
Cell viability was measured by a WST-1 assay (Beyotime, Shanghai, China) as described previously [18]. Lung cancer cells were seeded in 96-well plates and treated with daurisoline at various concentrations for different time points. WST-1 was added and incubated
Screening of a food-source small molecule library identifies daurisoline as a novel anticancer compound
To search for novel cancer therapeutic agents, a small molecule library consisting of 429 natural products was initially screened by a literature study, and 54 compounds that have been rarely documented for anticancer bioactivity, were selected for functional experiments. Three lung cancer cell lines A549, Hop62 and H1299, were treated with the 54 compounds (10 μM) individually for 48 h, and the inhibitory effect of each compound was determined by WST-1 assays. As shown in Fig. 1A–C,
Discussion
HSP90, a member of the heat shock protein family, is abundant in all eukaryotes and prokaryotes, and plays an important role in multiple cellular functions [34], including the regulation of stability of key transcription factors and kinases required for normal physiological processes [35]. HSP90 is commonly overexpressedin cancer cells and is involved in the biological processes of various malignant phenotypes, thus making it a potent therapeutic target for cancer treatment [36,37]. HSP90
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2017YFA0505100), and the National Natural Science Foundation of China (31770888, 81773085, 81803551, 81973339, 81672953).
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2022, Pharmacological ResearchDaurisoline alleviated experimental colitis in vivo and in vitro: Involvement of NF-κB and Wnt/β-Catenin pathway
2022, International ImmunopharmacologyCitation Excerpt :The efficacy of TCM for UC has been proven to have indispensable natural advantages [32]. DS is the main active component of Menispermum Dauricum DC, which has anti-inflammatory, anti-arrhythmic, anti-Alzheimer's disease, and anti-cancer activities [21,33]. Although some studies have indicated the effect of total alkaloids in Menispermum on UC, there are no studies on the effect and mechanism of DS on colitis.
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These authors contributed equally to this work.