BHX Inhibits the Wnt Signaling Pathway by Suppressing β-catenin Transcription in the Nucleus

BHX (N-(4-hydroxybenzyl)-1,3,4-triphenyl-4,5-dihydro-1H-pyrazole-5-carboxamide), a Wnt signaling pathway inhibitor, effectively inhibits tumor cell growth, but the underlying mechanism is

unclear. Thus, we aim to investigate the effects and associated mechanism of BHX action on A549 and MCF-7 cell lines. In our study,

MTT(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and xenograft model assay indicated that cell growth was inhibited by BHX at a range of concentrations in vitro and in

vivo. The expression of β-catenin and Wnt signaling pathway downstream target genes were decreased evidently under BHX treatment. Flow cytometry also revealed that BHX treatment

significantly induced G1 arrest. Further analysis showed that BHX lowered the transcriptional level of β-catenin. In conclusion, BHX inhibited the nuclear synthesis of β-catenin, thereby

suppressing the Wnt signaling pathway and further inhibiting tumor growth and proliferation.

Cancer is a condition that results from the joint involvement of multiple genes, factors, and stages. Cellular signal transduction pathways play a vital role in tumor development and

occurrence1. The Wnt signaling pathway is highly involved in cell proliferation, migration, apoptosis, and fate determination during early embryonic growth and development2,3. At present,

the Wnt/β-catenin signaling pathway is an intensively studied signal transduction pathway, which is also highly conserved throughout evolution4,5. Aberrant Wnt/β-catenin pathway activation

is strongly linked to the development of many cancers6,7. When the canonical Wnt signaling pathway is activated abnormally, the Axin/GSK-3/APC complex cannot phosphorylate β-catenin, leading

to the latter’s ubiquitination and subsequent degradation. Non-phosphorylated β-catenin accumulates in the cytoplasm, so that cytoplasmic β-catenin is stabilized and transported further

into the nucleus and interacts with TCF/LEF3,8. Subsequently, the Wnt pathway downstream target genes are involved in cell fate and proliferation regulation. These target genes include

c-myc, cyclin D1, c-Jun, c-fos, and some members of the Fra-1 and AP-1 family9,10.

Wnt signaling pathway includes a variety of proteins, abnormal transcription and expression of signaling molecules are the main reason that activate Wnt signaling pathway11,12. Therefore,

various specific targeted drugs have been developed. Inhibitors that disrupt the β-catenin and TCF interaction have been widely explored, as well as RNAi approaches, ICG-001,

PKF115–58413,14. There are also some inhibitors targeting Frizzled such as OMP-18R5 and 3289–862515. However, toxicity, side effects and other shortcomings appear. In spite of these

potential hurdles, research toward identifying potent Wnt pathway antagonists for cancer treatment is promising.

On the basis of previous studies, we generated a computer-aided design of small-molecule inhibitors and synthesized the novel pyrazoline derivative BHX

(N-(4-hydroxybenzyl)-1,3,4-triphenyl-4,5-dihydro-1H-pyrazole-5-carboxamide) to block Wnt signaling16. We discovered that BHX could inhibit the growth of cancer cell, but the mechanism was

unclear. In this study, we evaluated the anti-cancer effect of BHX on lung and breast cancers and then discovered the mechanisms of the underlying effects. Our results provide a rationale

for the further development of BHX as a prospective therapeutic agent for cancer that involves Wnt pathway activation.

To explore the tumor inhibitory effect of BHX in vitro and in vivo,We first examined the cytotoxicity of BHX in human lung cancer cell lines and normal lung epithelial cells (Beas-2b).

BHX(molecular structure were shown in Fig. 1a) displayed moderate cytotoxicity to the Beas-2b cells with an IC50 of 28.08 μM by MTT assay. However, the IC50 of the A549 cells was 15.49 μM

(Fig. 1b), which was significantly lower than that of the Beas-2b (Fig. 1c). We selected another Wnt-pathway-activated cell, i.e., the breast cancer cell line MCF-7. BHX inhibited tumor cell

proliferation in a dose-dependent manner by the MTT assay (Fig. 1d). BHX also suppressed the clonogenic activity of these two cell lines (Fig. 1e).

Antiproliferative effects of BHX on tumor and normal lung epithelial cells.

(a) Chemical structure of BHX. (b) Beas-2b and A549 cells were treated with different concentrations (0–80 μM) of BHX for 72 h. Cell viability was analyzed by MTT assays. (c,d) A549 and

MCF-7 cells were treated with different concentrations (0–80 μM) of BHX for different times. (e) Colony-formation assays of the MCF-7 and A549 cell treated with BHX at an indicated

concentration. Results are mean ± SD (n = 3). *p