Formononetin, J1 and J2 have different effects on endothelial cells via EWSAT1‐TRAF6 and its downstream pathway

Abstract Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two‐way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long‐chain non‐coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1‐TRAF6 and its downstream pathways.


| INTRODUC TI ON
According to the literature, the consumption of vegetables and fruits is negatively correlated with cancer morbidity and mortality. 1 It can also be said that eating more plant-based foods has a certain effect on the incidence of cancer. Phytooestrogens are a class of non-steroidal compounds found in plants. 2 Red clover, also known as red axle, is a perennial herb of the genus Trifolium, which grows wildly in China's Xinjiang, Jilin, and Yunnan-Guizhou plateaus and Hubei's mountainous areas. 3 Research has shown that red clover contains a large number of isoflavones, which have anticancer and antitumour properties, regulate the balance of human hormones, improve osteoporosis, have antioxidant and free radical scavenging functions, etc Red clover is a natural medicine with great prospects. 4,5 Formononetin is a biologically active phytooestrogenic isoflavone extracted from red clover.
The phytooestrogens found in plant foods have non-steroidal oestrogen-like activity and are considered to be natural substitutes for oestrogen. 6 The phytooestrogens can be divided into four categories: the isoflavones, alfalfa, lignans and coumarins. 7 Among these categories, isoflavones (eg daidzein, genistein, calycosin and formononetin) dominate the research on phytooestrogens because they are the most biologically active ingredients in soya beans and they have been shown to significantly inhibit tumours. 8,9 Previous studies have shown that formononetin can be used as a pharmacological oestrogen analogue. 10,11 People have long recognized that tumour growth and metastasis require angiogenesis. 12 Currently, HUVEC is considered to be a valuable in vitro angiogenesis model because they can form a capillary structure called a tube under appropriate stimulation. 13 Studies have also shown that formononetin has antitumour effects in several types of cancer, such as breast cancer, osteosarcoma and ovarian cancer. 14,15 Our previous experimental results show that formononetin has a two-way regulatory effect on HUVEC; that is, low concentrations promote proliferation of HUVEC, while high concentrations produce inhibition, 16 but the specific mechanism remains to be understood. After conducting long-term experiments, we found that the solubility of formononetin is low and that the concentration required inhibiting cells is too high.
To compensate for these shortcomings, another laboratory that we worked with has structurally modified the drug, and in many modified species, J1 and J2 (purity > 98.0%) have been screened.
The growth of blood vessels, a process known as angiogenesis, is a complex and co-ordinated process that involves multiple cellular cytokines and signal pathways. 13 Insulin-like growth factor-1 (IGF-1) is a new target to treat cardiovascular disease, effectively inhibiting EC apoptosis and promoting cell growth and migration through binding to the IGF-1 receptor (IGF-1R), which is expressed on the surface of vascular endothelial cells (VECs). 17 Additionally, intercellular adhesion molecule 1 (ICAM-1) is an Ig-like cell adhesion molecule that is excreted by several types of cells, including leucocytes and ECs. 18 ICAM-1 is involved in polymorphonuclear leucocyte-induced angiogenesis and is an early hallmark of the angiogenesis process. It endows the adhesive interaction between VECs and the extracellular matrix, hence promoting the growth of vascular endothelial cells. 2,19 These factors stimulate the proliferation, differentiation and metastasis of vascular endothelial cells; promote the formation of new blood vessels; provide sufficient oxygen and the nutrients necessary for tumour growth; provide a good growth environment for tumours; and accelerate tumour growth and proliferation. Therefore, inhibition of the formation of blood vessels has become a key step in the treatment of cancer.
It has recently been reported that long non-coding RNAs (ln-cRNAs) are capable of regulating the proliferation, migration, invasion and chemoresistance of certain cells and are now considered to be novel regulators of gene expression. [20][21][22][23][24][25][26] In addition, lncRNA is also involved in the process of cardiovascular development and pathophysiology. 27 In this study, we initially screened several lncRNAs that were abnormally highly expressed in HUVEC but showed decreased gene expression levels after drug treatment. The difference in the expression of EWSAT1 was found to be more significant.
Next, the downstream target was predicted to be TRAF6 with EWSAT1 as the upstream target. TRAF6 is involved in the downstream pathway TAK1-C-jun/IκBα. To explore the mechanism of this pathway and the role of the drugs, we performed the following research.

| Cell culture
HUVEC (The Chinese Academy of Sciences) was cultured in RPMI-1640 medium (Invitrogen) containing 10% foetal bovine serum (FBS; Gibco) and cultured at 37°C, a 5% CO 2 concentration and saturated humidity. The cells were well adhered and routinely digested according to the density and fusion of the cells, and the medium was replaced with phenol red-free RPMI-1640 medium (Gibco) (containing 10% foetal bovine serum solution treated with dextran) 4 days before the experiment. Then, the cells were allowed to grow for 4 days.
In addition, the medium was changed to low-serum RPMI-1640 medium (containing 0.5% foetal bovine serum solution) for 24 hours before the cell experiments. (purity > 98.0%) were dissolved in dimethyl sulphoxide (DMSO) to prepare J1 and J2 stock solutions, which was stored at 4°C for further use. Cell proliferation after treatment with J1 and J2 was tested by the MTT assay. Human umbilical vein endothelial cell was inoculated in 96-well plates (5 × 10 3 cells per well) for 12 hours and then

| Wound healing assay
Cell migration ability was assessed with a wound healing assay. Equal numbers of cells were cultured in 6-well plates until 95% confluence was reached. Wound gaps were created by scraping the cell sheets with a sterile 10-µL pipette tip. The floating cells were removed by washing the wells with phosphate-buffered saline (PBS). Changes in the scratched area were observed daily for 4 days by an inverted microscope (Leica). The wound width was measured to calculate the cell migration ability.

| Plate colony formation experiment
Human umbilical vein endothelial cell was seeded at a low density in 6-well plates (500 cells/well in triplicate) and was cultured for 2 weeks. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes and stained with a Gram staining solution for 20 minutes.

| Microarray of lncRNAs
Total RNA was extracted from HUVEC using the miRNeasy mini kit (Qiagen) according to the manufacturer's protocol. RNA amplification and labelling were performed using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion). Labelled cDNA was subjected to hybridization using the Human lncRNA OneArray Plus microarray (Phalanx Biotech Group), followed by scanning using an Agilent scanner (Agilent Technologies). Agilent Feature Extraction software (version 11.0.1.1) was used to grid and extract the data.

| Gene function analysis
The predicted target genes of differentially expressed lncRNAs were input into the Database for Annotation, Visualization and Integrated Discovery, which utilizes Gene Ontology (GO) analysis to analyse the molecular functions represented in the lncRNA profile (XuanC Bio). Furthermore, the potential functions of these differentially expressed lncRNAs in the pathways were determined by the Kyoto Encyclopedia of Genes and Genomes database.

| Western blot analysis
Human umbilical vein endothelial cell was treated with the J1. After 48 hours, cell lysates were harvested, and the protein concentration was determined using a Bio-Rad assay kit (Bio-Rad Laboratories).

| Drug and animals
Formononetin (purity > 98.0%, Yuanye Biotechnology Co., Ltd.) was dissolved in DMSO to prepare a 160 mg/mL stock solution that was stored at 4°C for further use. Sprague Dawley rats (female, 6 weeks old, 200-220 g) were supplied by Hunan SJA Laboratory Animal Co., Ltd. Animals were bred in a specific pathogen-free room under a constant temperature of 21-25°C and 50%-60% relative humidity with a 12-hour light/12-hour dark cycle. All experimental procedures were performed in accordance with the guidelines of the Experimental Research Institute of Guilin Medical University.

| Ovariectomy
For the establishment of ovariectomized rat models, 40 female SD rats were randomly divided into 4 groups: sham operation group (SHAM group, n = 10), ovariectomized group (OVX group, n = 10), At the end of the administration, the uteri, thoracic aortas and breast tissues of the rats in the 4 groups were removed and reserved carefully for follow-up analysis.

| Quantitative real-time PCR (qRT-PCR) assay in vivo
For the analysis of IGF-1R and ICAM-1 mRNA transcription, the uteri from different groups were collected, and the total RNAs

| Western blot assay in vivo
For the analysis of IGF-1R and ICAM-1 protein expression, the uteri from different groups were collected, and the total protein was extracted by RIPA reagent (Solarbio) by adding 1% PMSF protease inhibitors (Solarbio) according to the standard protocol. Then, the mixtures were sonicated on ice with a Sonifier cell disruptor (75%, 5 minutes). After incubation for 30 min on ice, the mixtures were centrifuged at 16 099.2 g for 10 min at 4°C. The concentration of the supernatant was determined with a BCA protein assay kit. Ten micrograms of protein was separated by 10% or 8% SDSpolyacrylamide gel, and then, the protein in the gel was transferred to the activated PVDF membrane. After sealing with 5% skim milk, the PVDF membranes were incubated with the correspond-

| Immunohistochemistry
The uteri, thoracic aortas and breast tissues from the different groups were collected and fixed in 4% paraformaldehyde over-

| Statistical analysis
All data are presented as the mean ± standard deviation (SD).
Statistical significance was tested by two-tailed Student's t test or one-way ANOVA using SPSS 19.0 software. Statistical significance was set at P < .05, and extreme significance was set at P < .01. All statistical diagrams were performed using GraphPad Prism 5.0.

| Inhibitory effects of different concentrations of formononetin and J1 and J2 on endothelial cells
Due to the solubility of formononetin, the concentration of the drug required to inhibit cells was too high, so modification of the formononetin structure was carried out to obtain two modified drugs, J1 and J2. Figure 1A shows the molecular structure of each drug. To elucidate the effects of these two plus formononetin on endothelial cells, MTT experiments were performed to examine the effects of these drugs on cells and to determine which J1 and J2 have better effects. As shown in Figure 1B, with increasing concentrations of formononetin and J1, an inhibitory effect was observed on HUVEC.
However, the inhibition of J1 is stronger than that of J2. It can also be seen in Figure 1C,D that high concentrations of J1 and J2 have an inhibitory effect on HUVEC, and similarly, the inhibitory effect of J1 is significantly stronger than J2.

| Effect of formononetin on ovariectomized rats
The bodyweight of the rats slightly increased during the administration ( Figure 3A). The uterine coefficient represents the relative weight of the uterus to the bodyweight, and the results showed that after 16 mg/kg/d administration, the uterine coefficient significantly increased compared with the OVX model group (Figure 3B Here, we also used the trend effects of J1 and J2 on rats for in vivo experiments. The trending effects of J1 and J2 in the in vivo experiments were not statistically significant.

| EWSAT1-TRAF6 participates in the influence of the HUVEC mechanism
To explore the underlying mechanisms of formononetin, J1 and J2 regulation of endothelial cells, lncRNA levels were measured with microarrays after treatment with formononetin, J1 and J2 (Figure 4). J1 significantly down-regulated the expression level of EWSAT1.
Next, the downstream target of EWSAT1 was analysed, and it was determined that TRAF6 is a downstream target of EWSAT1.

| Effect of J1 on the expression of EWSAT1 and its related downstream pathway factors
In this study ( Figure 5A), after treating HUVEC with J1, the expression level of EWSAT1 in HUVEC was found to gradually decrease with an increasing concentration of J1. Moreover, the expression level of TRAF6 was also found to decrease accordingly. Simultaneously, the expression of TRAF6 and IGF-1R and the phosphorylation of the related pathway proteins were also detected. As shown in Figure 5B-F, the expression levels of TRAF6 and IGF-1R gradually decreased with increasing J1 concentration, and the phosphorylation level of the related pathway protein factor TAK1 and its downstream pathway-related protein also appeared to change. This indicates that J1 reduces the expression levels of TRAF6 and IGF-1R, thereby affecting the phosphorylation of TAK1 and IGF-1R and the downstream branches.

| D ISCUSS I ON
The proliferation of endothelial cells plays a key role in the process of angiogenesis, which is necessary for tumour growth and metastasis. 28 Therefore, the endothelium is positioned as a barrier between the blood and blood vessels, and anticancer agents are damaging to the endothelial layer. 29 Angiogenesis is thought to be a hallmark of cancer progression in solid tumours larger than 1-2 mm. 3,30 Cancer cells grow rapidly due to the increased vascular supply, which maintains the delivery of oxygen and nutrients as well as the elimination of the waste generated by cells. 31 Hypoxic and nutrient-starved tumours secrete pro-angiogenic factors that target specific receptors on the surface of vascular cells to promote neovascularization. 6 Therefore, controlling the formation of blood vessels has become one of the primary tasks involved in treating tumours. Human umbilical vein endothelial cell is considered to be valuable in vitro angiogenesis model because they can form a capillary structure called a tube under appropriate stimulation. 13 This study also selected the most experimental model objects of HUVEC.
Our results indicate that formononetin has a two-way regulatory effect on endothelial cells, that is, a low concentration (>16 μmol/L) promotes the proliferation of HUVEC, and a high concentration (<16 μmol/L) inhibits HUVEC, but the specific mechanism is still unclear. 16 In addition, long-term experimental studies have found some shortcomings of formononetin, that is, the solubility of the drug is poor and the concentration required for the drug effect is large.
Considering these problems, the drug was modified to obtain formononetin modifier, J1 and J2, which were selected from numerous modifier. However, the trend of J1 and J2 in vivo is not statistically significant.
Next, six samples of HUVEC were prepared, which were formononetin (0, 10 μmol/L), J1 (0, 10 μmol/L) and J2 (0, 10 μmol/L), and subjected to gene screening. Several lncRNAs were found to be highly expressed in HUVEC, and the fold change of EWSAT1 after treatment with J1 was significantly higher than that of the other two F I G U R E 4 lncRNAs expression profile differences between the formononetin-, J1-and J2-treated groups compared to the control group. (A) The heat map, (B) the volcano plots, (C) the downstream pathway analysis drugs after treatment with drugs. At the same time, based on the above results, J1 had better effects on HUVEC than formononetin and J2. Therefore, mechanism experiment was carried out using J1.
After EWSAT1 was screened, its downstream target was analysed and predicted, and TRAF6 was found to be the downstream target of EWSAT1.
TRAF6 is a member of the TNF receptor-associated factor (TRAF) family. 32 Recent studies have shown that inhibition of the TRAF6/NF-κB/MMP2 pathway can affect endothelial cell invasion and migration. 33 Studies have also shown that inhibition of the AT1R-NFκB-TRAF6-mitogen-activated protein kinase (MAPK) pathway affects endothelial cell proliferation and vascular regeneration. 34 These results may indicate that TRAF6 also plays an important role in endothelial cell proliferation, invasion and migration.
Upon stimulation, TRAF6 is recruited into the receptor complex and activated by the IL-1 receptor-associated kinase 1 (IRAK1), which binds to the TRAF domain of TRAF6. 35 The IRAK1/TRAF6 complex is then excised from the receptor and binds to the membrane portion of TAK1 and the TAK1-associated binding proteins TAB1 and TAB2. 35  Vascular growth, a process called angiogenesis, is a complex and co-ordinated process involving multiple cytokines and signalling pathways. 41,42 Insulin-like growth factor-1 (IGF-1) is a novel target that inhibits endothelial cell apoptosis by binding to the IGF-1 receptor (IGF-1R) and promoting cell growth and migration. Recently, studies have shown that IGF-1R can inhibit the proliferation of osteosarcoma cells through its downstream pathways, JNK and C-jun N-terminal kinase. 43 In this study, after treatment of HUVEC with different concentrations of the formononetin derivative J1 for 48 hours, the expression levels of TRAF6, IGF-1R and C-jun and the phosphorylation levels of TAK1, IκBα and C-jun were detected. The levels of phosphorylation change, and it was found that with increasing J1 concentration, the expression level of the TRAF6 protein gradually decreased, the phosphorylation level of the corresponding pathway proteins (including TAK1) changed accordingly, and In summary, the above experimental results show that formononetin and J1 and J2 have the ability to inhibit endothelial cell proliferation, invasion and migration. It was found that J1 is superior to formononetin and J2. In addition, the selected lncRNA (EWSAT1) and its downstream targets were analysed, and the corresponding downstream pathways were found. This study confirmed that the formononetin derivative J1 regulates the expression of TRAF6 and its downstream TAK1-and IκBα/C-jun-related factors by mediating the expression of EWSAT1, and the IGF-1R-C-jun pathway also has an effect on ECs. These advances in the understanding of the drug action of formononetin and formononetin modifier and their inhibition of the angiogenic processes necessary for tumour growth and metastasis may provide opportunities for their clinical application in tumour therapy.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
Zhaoquan Huang and Jian Chen conceived and designed the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.