ABSTRACT
In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , High-Throughput Screening Assays , Lung Neoplasms , Transcription Factor 4/genetics , beta Catenin/geneticsABSTRACT
Wnt/β-catenin signaling pathway plays an important role in the proliferation, growth, invasion, and metastasis of human cancers. Moreover, β-catenin/T-cell factor 4 (TCF4) interaction regulates the transcription of the key oncogenes in Wnt/β-catenin signaling pathway. Therefore, β-catenin/TCF4 interaction would be a promising therapeutic target for the development of highly selective anticancer agents. At present, most ongoing small-molecule inhibitors targeting β-catenin/TCF4 interaction, including PKF222-815, iCRT3/5/14, LF3, and sanguinarine, have been developed in preclinical studies for human cancer therapeutics. In this review, we summarized the research advances of up-to date inhibitors targeting β-catenin/TCF4 interaction, including the molecular structure and cellular functions of β-catenin in canonical Wnt signaling pathway. This review holds a hopeful avenue for the development of novel and highly selective Wnt inhibitors targeting β-catenin/TCF4 interaction for future anticancer strategy.
ABSTRACT
To develop a fluorescence polarization (FP)-based high-throughput screening (HTS) assay to identify novel small-molecule antagonists targeting β-catenin/TCF4 (T-cell factor 4) interaction, recombinant human β-catenin was expressed in Escherichia coli Rosetta (DE3) cells and purified by HisTrapTM column. The bioactivity of purified β-catenin was further analyzed by enzyme-linked immunosorbent assay (ELISA). According to FP principle, the β-catenin/TCF4 binding model was performed, and fluorescence isothiocyanate (FITC) labeled TCF4 peptide (FITC-TCF4) served as the molecular probe of adaptor for binding to β-catenin. The FITC-TCF4 and β-catenin working concentration were optimized, and the binding conditions (complex stability and dimethylsulfoxide (DMSO) tolerance) have been investigated yet for further hits screening. The results showed that recombinant human β-catenin was successfully expressed and purified β-catenin exhibited favorable bioactivity in ELISA binding assay. Subsequently, the FP-based HTS assay was performed using 20 nmol·L-1 FITC-TCF4 and 100 nmol·L-1 β-catenin. Under these optimized conditions, a high Z´factor of 0.88 was achieved in a 384-well format and this FP-based HTS assay was very stable with regard to DMSO. Through screening of a natural-based product library (NBPL) using the established FP-based HTS assay, three hits (sanguinarine, chelerythrine, and compound S720) were identified as potential β-catenin/TCF4 interaction antagonists. Taken together, we have successfully developed a simple, robust and reliable FP-based HTS assay for screening of novel antagonists targeting β-catenin/TCF4 interaction.
ABSTRACT
Objective: To investigate the effects of Wnt inhibitor IWR-1-endo (IWR-1) on the proliferation and migration of human osteosarcoma MG-63 cells, and to explore the possible mechanism. Methods: MG-63 cells were treated with different concentrations of IWR-1 (2, 4, 8 and 16 μmol/L). Then the growth inhibitory rates of MG-63 cells was determined by CCK-8 assay. The cell cycle and apoptosis rate of MG-63 cells were detected by FCM method. The migration ability of MG-63 cells was abserved by scratch wound healing assay. The expression levels of β-catenin and cyclin D1 mRNAs were detected by real-time fluorescent quantitative PCR. The expression levels of Axis inhibition protein 1 (AXIN1), β-catenin, phospho-β-catenin (p-β-catenin) and cyclin D1 proteins were detected by Western blotting. Results: The proliferation inhibitory rate of MG-63 cells after IWR-1 treatment increased in a time-and concentration-dependent manner (all P < 0.01). The proportion of MG-63 cells treated with IWR-1 in G0/G1 phase were increased (P < 0.05), and the apoptosis rate of MG-63 cells increased significantly with the increase of IWR-1 concentration (P < 0.01). IWR-1 reduced significantly the scratch healing rate of MG-63 cells (P < 0.01), decreased the mRNA (both P < 0.01) and protein (both P < 0.05) expression levels of β-catenin and cyclin D1, and increased the expression levels of AXIN 1 and p-β-catenin proteins (both P < 0.05). Conclusion: IWR-1 can inhibit the proliferation and migration of MG-63 cells by blocking Wnt/β-catenin signaling pathway.