Recent advances in novel anticancer agents targeting β-catenin/TCF4 interaction for molecular cancer therapeutics / 药学学报

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.

Prediction of anti-liver fibrosis effect of Piperis Longi Fructus based on network pharmacology / 中国中药杂志

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-β1(TGF-β1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.

Development of a fluorescence polarization-based high-throughput screening assay to identify antagonists targeting β-catenin/TCF4 interaction / 药学学报

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.

Screening of small molecule inhibitors for PLK1 PBD and evaluation of antitumor activities / 药学学报

With the method of fluorescence polarization (FP), we screened small molecule inhibitors for PLK1 PBD to identify the lead compounds for antitumor drugs. FP led to the identification of a potent hit, F083-0063, whose inhibition rate was (99.7±0.4)% at 10 μg·mL-1. The IC50 was calculated to be 1.9±0.1 μmol·L-1 using Graphpad Prism 5. The effect of the compound on cells' multiplication was measured by MTT assay which showed that F083-0063 inhibited the proliferation of many tumor cell lines. Flow cytometry analysis indicated that the F083-0063 promoted cell apoptosis and induced cell G2/M arrest. Migration abilities of cells, evaluated using scratch test, increased significantly in the presence of F083-0063 with the mi-gration rate as low as (37.6±0.7)% at 20 μmol·L-1. Molecular linkage technique found F083-0063 had good affinity with PLK1 PBD. The results of Western blotting showed that the expression of cyclin-dependent proteins was increased after treatment with F083-0063. In summary, F083-0063 has an antitumor activity and is expected to be an antitumor lead compound targeting PLK1 PBD.

Reverse effect of curcumin on CDDP-induced drug-resistance via Keap1/p62-Nrf2 signaling in A549/CDDP cell

Objective To assess the effect of curcumin on CDDP-induced drug resistance and explore the underlying molecular mechanism through Nrf2 system and autophagy pathway. Methods A drug-resistant cell model was established by exposing A549/CDDP cell to 2 μg/mL CDDP. A549/CDDP cell was treated with 20 μg/mL CDDP and 10 μM curcumin. The cell viability and apoptosis level, the signals of Keap1/P62-Nrf2 and autophagy pathway were analyzed. Results CDDP induction promoted drug-resistant phenotype in A549/CDDP cell and activated autophagy as well as Nrf2 signals in A549/CDDP cell. Meanwhile, curcumin combination attenuated autophagy and Nrf2 activation induced by CDDP, and reversed the drug-resistant phenotype. Notably, curcumin combination augmented Keap1 transcription. Furthermore, Keap1 ablation with short hairpin RNAs hampered the efficacy of curcumin, suggesting Keap1 played a crucial role on reversal effect of curcumin. Conclusions The present findings demonstrate that CDDP promotes abnormal activation of Nrf2 pathway and autophagy, leading to drug resistance of A549/CDDP cell. Curcumin attenuates this process and combat drug-resistance through its potent activation on Keap1 transcription, which is essential for interplay between oxidative stress induced Nrf2 activation and autophagy/apoptosis switch.

Reverse effect of curcumin on CDDP-induced drug-resistance via Keap1/p62-Nrf2 signaling in A549/CDDP cell

OBJECTIVE@#To assess the effect of curcumin on CDDP-induced drug resistance and explore the underlying molecular mechanism through Nrf2 system and autophagy pathway.@*METHODS@#A drug-resistant cell model was established by exposing A549/CDDP cell to 2 μg/mL CDDP. A549/CDDP cell was treated with 20 μg/mL CDDP and 10 μM curcumin. The cell viability and apoptosis level, the signals of Keap1/P62-Nrf2 and autophagy pathway were analyzed.@*RESULTS@#CDDP induction promoted drug-resistant phenotype in A549/CDDP cell and activated autophagy as well as Nrf2 signals in A549/CDDP cell. Meanwhile, curcumin combination attenuated autophagy and Nrf2 activation induced by CDDP, and reversed the drug-resistant phenotype. Notably, curcumin combination augmented Keap1 transcription. Furthermore, Keap1 ablation with short hairpin RNAs hampered the efficacy of curcumin, suggesting Keap1 played a crucial role on reversal effect of curcumin.@*CONCLUSIONS@#The present findings demonstrate that CDDP promotes abnormal activation of Nrf2 pathway and autophagy, leading to drug resistance of A549/CDDP cell. Curcumin attenuates this process and combat drug-resistance through its potent activation on Keap1 transcription, which is essential for interplay between oxidative stress induced Nrf2 activation and autophagy/apoptosis switch.