Human prostate cancer cell Human PC-3 prostate adenocarcinoma cells (PC3), Human 22RV1 prostate adenocarcinoma cells (22RV1), prostatic hyperplasia cell Human Benign Prostatic Hyperplasia Cell Line (BPH-1) and embryonic kidney cell 293T was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). All cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin antibiotics, and 0.1 mg/mL streptomycin (Gibco; Thermo Fisher Scientific, Inc.). In addition, all cells were cultured in a humidified 37 °C incubator with 5% CO${}_2$. $\beta$-ionone (I12603, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich; Merck KGaA) to make it a storage solution with a final concentration of 200 mM. PC3, 22RV1. BPH-1 cells were treated with the indicated concentrations of $\beta$-ionone for different times or the same time at gradient concentrations, and an equivalent volume of DMSO was used as a control.

PCa cells were planted into 96-well plates at 8 $\times$ 10${}^3$/well and grown to 60%–80% confluency, then cultured with a gradient concentration of $\beta$-ionone for 24 or 48 h. A medium containing 10% MTT (5 mg/mL; Sigma-Aldrich; Merck KGaA) was added to each well and cultured for 4 h. Then, the supernatant was removed and added 250 $\mu$L DMSO into each well. The 96-well microplate reader (Bio-Rad, Hercules, USA) was used to detect the absorbance at the wavelength of 490 nm.

Cell proliferation was detected using Click-tm EdU Cell Proliferation Kit (NO. C0075S, Beyotime Bio, Shanghai, China). Cells in the logarithmic phase are seeded into 6-well plates and incubated overnight in a 37 °C incubator by 4 $\times$ 10${}^5$/well. The cells were treated with $\beta$-ionone (200 mM) or DMSO for another 24 h incubation. Then the plates were added with Edu (10 $\mu$M) and returned to the incubator for 2 h, followed by immobilization of cells with 0.4% paraformaldehyde and 0.3% Triton X-100 to increase cell membrane permeability. After PBS washing, each well of the plates was supplemented with the click additive solution for 30 min and stain the nuclei with Hoechst-33,342. Finally, a fluorescence microscope was used to observe and photographed the results.

PCa cells were seeded in 6-well plates and grown in culture dishes to form a 90%–100% confluent monolayer of adherent cells. A 200 $\mu$L pipette tip was then used to score a scratch on the monolayer to create a linear wound. After washing the plates twice with pre-chilled PBS, the subsequent culture was performed in a serum-free medium containing 180 $\mu$M $\beta$-ionone. DMSO was used as the control. The wound closure was observed by an inverted microscope ($\times$ 100) at 0, 12, and 24 hours). The wound healing rate is calculated according to the following formula: wound healing rate = ($\beta$-ionone treated group gap closure rate/control group gap closure rate) $\times$ 100.

Transwell chambers (12 mm in diameter, 8 $\mu$m in pore size, Corning, Beijing, China) were used to detect the migration and invasiveness of cells after $\beta$-ionone treatment. Transwell chambers were pre-covered with 30 $\mu$L of diluted Matrigel (Sigma-Aldrich; Merck KGaA) for invasion assays. 4 $\times$ 10${}^5$ cells were dispersed in 200 $\mu$L of serum-free medium containing $\beta$-ionone or DMSO and placed in the upper chamber. The upper chamber was placed into the lower chamber filled with 800 $\mu$L of RPMI-1640 containing 10% FBS, after 24 h of incubation, he mini-cells were washed with PBS and fixed with 4% formalin at room temperature. They were then stained with crystal violet (0.1% in absolute ethanol) for 15 minutes. Five fields of view were randomly selected and photographed by a microscope to calculate the number of cells that had migrated or invaded (magnification, $\times$100).

Total protein lysates were collected with lysis buffer containing protease inhibitors and phosphatase inhibitors, then the collected lysates were centrifuged at 15,000 rpm for 15 min at 4 °C. A 30 $\mu$g sample of total protein lysates were electrophoresed on an 8% or 10% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel and transferred to PVDF membranes. Polyvinylidene fluoride (PVDF) membranes were blocked with 5% Bovine serum albumin (BSA) for 1 h at room temperature, followed by incubation with the following specific primary antibodies diluted in 5% BSA (dilution 1:2000) at 4 °C overnight: Rabbit vimentin (cat. no. 5471), total $\beta$‑catenin (cat. no. 8480), phosphorylated glycogen synthase kinase (GSK)3$\beta$ (Ser9; cat. no. 5558), total GSK3$\beta$ (cat. no. 12456) and Vinculin (cat. no. 4970); which purchased from Cell Signaling Technology, Inc (Boston, MA, USA). Antibodies against epithelial (E)-cadherin (cat. no. ab15148), neural (N)-cadherin (cat. no. ab76057) and Histone H3 (cat. no. ab176842) were purchased from Abcam (San Diego, CA, USA). PVDF membranes were incubated with enzyme-conjugated secondary antibody for 1 hour at room temperature and images were obtained using the ECL system (Thermo Fisher Scientific, Rochester, NY, USA). Vinculin protein level was used as the endogenous control, and the level of the target protein was compared with vinculin in the same group, and then the expression levels of the target protein was analyzed in each group.

To overexpress $\beta$-catenin in PCa, the $\beta$-catenin cDNA was cloned into the pcDNA3.1 vector. For transfection, plasmids and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) were separately added to serum-free medium and mixed 1:1, using empty vector as a control. Twenty-four hours after transfection, the transfection efficiency was detected by qRT-PCR and western blotting, and an appropriate concentration of $\beta$-ionone was added for subsequent operations.

Cells were seeded in 6-well plates with coverslips and allowed to grow attached to the slides. After grown to a suitable density on the coverslip, the cells were treated with $\beta$-ionone for 24 h and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were washed with PBS and treated with 0.5% Triton™ X-100 solution for 15 min to increase cell permeability. The slides were washed with PBS and incubated overnight at 4 °C with an anti-$\beta$-catenin primary antibody (cat. no. 8480; dilution, 1:200; Cell Signaling Technology, Inc). The next day, the slides were washed with PBS, mixed with goat anti-rabbit IgG H&L fluorescein isothiocyanate (FITC) (cat. no. ab6717; cat. no. 1:200; Abcam), and incubated for 1 h at room temperature. The cells were stained with DAPI (1 $\mu$g/mL) for 5min again, sealed with anti-quenching resin, and the expression and distribution of $\beta$-catenin were detected by a confocal laser microscope.

Total cellular RNA was extracted with TRIzol reagent, and 1 $\mu$g of RNA was reverse transcribed with the Superscript III transcriptase (Invitrogen, Grand Island, NY) to obtain cDNA. qRT-PCR was performed to determine the mRNA expression level of $\beta$-catenin using the Bio-Rad CFX96 system. The PCR primer sequence of $\beta$-catenin was 5’-AAAGCGGCTGTTAGTCACTGG-3’ (forward) and 5’-CGAGTCATTGCATACTGTCCAT-3’ (reverse) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was 5’-GGAGCGAGATCCCTCCAAAAT-3’ (forward) and 5’-GGCTGTTGTCATACTTCTCATGG-3’ (reverse). Using GAPDH as an internal reference, relative changes in gene expression were normalized against GAPDH.

PC3 cells pretreated with $\beta$-ionone and DMSO were incubated with 50 $\mu$g/mL cycloheximide (CHX, Sigma-Aldrich) for a specified time in turnover assays. $\beta$-catenin protein level was detected by western blotting. The degradation pathway of $\beta$-catenin was further confirmed by treating PC3 cells with MG132 (proteasome inhibitor, Sigma-Aldrich) and $\beta$-ionone. 293T cells were transfected with His-Ub and Flag-$\beta$-catenin plasmids to detect the ubiquitination of $\beta$-catenin in vitro, After transfection for 24 h, 293T cells were cultured with newly replaced RPMI-1640 medium containing 180 $\mu$M $\beta$-ionone or an equal volume of DMSO for 16 h, and then 20 $\mu$g/mL of proteasome inhibitor MG132 was added for another 8 h. Then 293T cells were collected and washed with pre-cooled PBS to obtain 1 ml of cell suspension, of which 100 $\mu$L was used as the input group for routine protein extraction. The remaining 900 $\mu$L cell suspension was crushed with an ultrasonic pulverizer and then added with Ni-NTA agarose beads, and then incubated at room temperature for 3 h. The Ni-NTA agarose beads were collected by centrifugation, and the supernatant was discarded. After washed several times, the beads were added with appropriate Immunoprecipitation Buffers (IP buffer) with protein sample loading buffer, and then denatured at 95 °C for 10 minutes. The ubiquitination level of and the target protein was detected by western blotting.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and their care was in accordance with institution guidelines. Ten 4-week-old male nude mice (weight 15–20 g) were purchased from the Experimental Animal Center of Xi’an Jiaotong University, raised in a pathogen-free environment, and given a normal diet. PC3 cells (4 $\times$ 10${}^6$ cells mixed with same volume of matrigel) were injected subcutaneously into the left hind flanks of 10 mice. After the subcutaneous transplanted tumor was successfully established, the mice were randomly divided into two groups: the treatment group was treated with $\beta$-ionone (75 mg/kg) diluted with corn oil, and the control group was treated with an equal volume of corn oil, Each mouse was intraperitoneally injected once every 3 days and the tumor size were measured. The tumor volume was calculated as following formula: Tumor volume (mm${}^3$) = (length) $\times$ (width)${}^2$ $\times$ $\pi$/6. Nude mice were sacrificed after 3 weeks of treatment, and tumors were excised, weighed, and measured in volume. A small amount of tumor tissue was taken for western blotting and immunohistochemical staining to detect the expression of the target protein in the tissue. All animal experiments were conducted under the guidance of the Committee for Animal Protection and Utilization of Xi’an Jiaotong University, and executed according to standard ethical guidelines (2020-G-208).

The potential targets of $\beta$-ionone were explored and assessed utilizing public databases such as Swiss target prediction (https://www.swisstargetprediction.ch/), Similarity ensemble approach (https://sea.bkslab.org/) and SuperPred (https://prediction.charite.de/subpages/target_prediction.php). In all these databases species of target origin was limited to Homo Sapiens once the target has been predicted. The top 10 targets of the prediction results of each of the three websites were captured and intersected. Further prediction was conducted in PLIP (https://plip-tool.biotec.tu-dresden.de/) to binding residues of $\beta$-ionone with these targets. Finally the software AutoDock Vena and Pymol were applied for visualization the molecular docking of these targets and $\beta$-ionone.

All experiments were performed in three independent replicates. All statistical analyses were performed using GraphPad Prism 8.2 software (GraphPad Software Inc., San Diego, CA, USA). Student’s t-test was used for comparisons between two groups, and one-way ANOVA was used to assess mean differences between three or more groups, *p 0.05, **p 0.01, ***p 0.001 and ****p 0.0001.

To assess the effect of $\beta$-ionone on PCa and non-tumor cell viabilities, PC3, 22RV1 and BPH-1 cells were treated with gradient concentrations of $\beta$-ionone solution for 24 h or 48 h. The results showed that when the concentration of $\beta$-ionone was $\le$200 $\mu$M, there was no significant effect on the viabilities of PC3, 22RV1 and BPH-1 cells. The concentration of $\beta$-ionone $\ge$200 $\mu$M was found to affect the viabilities of PC3 and 22RV1. And $\beta$-ionone inhibited the growth of BPH-1 cells only when the concentration of $\beta$-ionone reached 400 $\mu$M (Fig. 1A). The results of EdU staining after treating cells with $\beta$-ionone (200 $\mu$M) similarly confirmed the results of MTT (Fig. 1B). Therefore, the concentration of 60/120/180 $\mu$M $\beta$-ionone was selected to treat PC3, 22RV1 and BPH-1 cells for subsequent experiments. In view of the highly invasive characteristics of prostate cancer, we investigated whether $\beta$-ionone affects cell migration and invasion of PCa. Wound healing experiments showed that $\beta$-ionone significantly delayed the rate of wound closure. However, the same concentration of $\beta$-ionone did not affect the migration of BPH-1 (Fig. 1C). The results of the transwell assays further showed that $\beta$-ionone inhibited the migration and invasion abilities of PC3 and 22RV1 cells (Fig. 1D,E).

Fig. 1.

$\beta$-Ionone inhibits migration and invasion of prostate cancer cells. (A) MTT assays were performed to determine the cell viabilities of PC3, 22RV1 and BPH-1 treated with different concentrations of $\beta$-ionone for 24 and 48 h. (B) EdU staining explore the Edu-positive rate of cells after 24 h of $\beta$-ionone treatment. (C) Wound healing assays were performed on PC3, 22RV1 and BPH-1 cells treated with DMSO or 180 $\mu$M of $\beta$-ionone. (D) Transwell migration and (E) invasion assays were used to investigate the migration and invasion abilities of PC3 and 22RV1 cells after treatment with DMSO or 180 $\mu$M $\beta$-ionone. Magnification, $\times$100. Scale bar, 20 $\mu$m. *p 0.05, **p 0.01, ***p 0.001 and ****p 0.0001.

EMT plays a crucial role in tumor metastasis and progression. To investigate whether the inhibition of migration and invasion of PCa cells by $\beta$-ionone is related to EMT, we detected the expression of EMT markers using western blotting. The results showed that the $\beta$-ionone treatment increased the expression level of E-cadherin while downregulating the expression of N-cadherin and Vimentin in a concentration and time-dependent manner (Fig. 2A,B). To further demonstrate the effect of $\beta$-ionone on EMT, we treated cells with EMT-induced TGF-$\beta$1. The results indicated that $\beta$-ionone could reverse TGF-$\beta$1-induced EMT in PCa cells (Fig. 2C). In addition, the results of migration and invasion assays demonstrated that cell migration and invasion enhanced by TGF-$\beta$1 could be inhibited by $\beta$-ionone (Fig. 2D,E). These results confirmed that $\beta$-ionone could inhibit PCa cell migration and invasion by suppressing EMT.

Fig. 2.

$\beta$-ionone inhibits EMT in prostate cancer cells. The expressions of E-cadherin, N-cadherin and vimentin were detected by Western blotting in prostate cancer cells PC3 and 22RV1 were treated with gradient concentrations of $\beta$-ionone for 24 h (A) or 180 $\mu$M of $\beta$-ionone for 0, 12, 24, and 36 h (B) by Western blotting. (C) After treating PC3 and 22RV1 with 180 $\mu$M $\beta$-ionone or (and) 5 ng/mL TGF-$\beta$1 for 24 h, the variation trend in EMT markers were detected by western blotting. (D) Transwell migration and (E) invasion assays were used to determine the migration and invasion abilities of PC3 and 22RV1 cells after treatment with 180 $\mu$M $\beta$-ionone or (and) 5 ng/mL TGF-$\beta$1. Magnification, $\times$100. Scale bar, 20 $\mu$m. *p 0.05, **p 0.01, ***p 0.001 and ****p 0.0001.

The Wnt/$\beta$-catenin signaling pathway has been found to act as an upstream pathway of EMT and promote PCa progression. Therefore, we further determined whether $\beta$-ionone inhibition of EMT and migration was related to the Wnt/$\beta$-catenin pathway. The results demonstrated the expression levels of $\beta$-catenin and p-GSK-3$\beta$ were down-regulated in PCa cells treated with $\beta$-ionone (Fig. 3A). After overexpression of $\beta$-catenin in PC3, $\beta$-ionone still downregulated the expression level of $\beta$-catenin protein (Fig. 3B). At the same time, $\beta$-ionone also decreased the expression of $\beta$-catenin in the cytoplasm and nucleus (Fig. 3C). Immunofluorescence analysis further confirmed the decreased expression of intracellular $\beta$-catenin (Fig. 3D), and a negative control group was used to exclude nonspecific binding (Supplementary Fig. 1). Hence, $\beta$-ionone can inhibit EMT by negatively regulating the Wnt/$\beta$-catenin signaling pathway in prostate cancer cells.

Fig. 3.

$\beta$-ionone inhibits the Wnt/$\beta$-catenin pathway in prostate cancer cells. (A) The expression of $\beta$-catenin, GSK-3$\beta$ and p-GSK-3$\beta$ were detected in prostate cancer cells PC3 and 22RV1 treated with gradient concentrations of $\beta$-ionone for 24 h. (B) Intracellular $\beta$-catenin was overexpressed and cells were treated with 180 $\mu$M $\beta$-ionone or DMSO, and relevant protein expression levels of the EMT were assayed by Western blot. (C) Nucleoplasmic protein separation and (D) Cell immunofluorescence detection of the effect of $\beta$-ionone treatment on the expression levels of $\beta$-catenin in the cytoplasm and nucleus. Magnification, $\times$100. Scale bar, 20 $\mu$m. *p 0.05, **p 0.01, ***p 0.001 and ****p 0.0001.

Previous studies have demonstrated that ubiquitin-proteasomes maintain an inactive state of the Wnt/$\beta$-catenin pathway by degrading $\beta$-catenin. The mRNA level of $\beta$-catenin did not change significantly after the $\beta$-ionone treatment of cells (Fig. 4A), so we sought to consider its ubiquitination and degradation.

As expected, $\beta$-ionone accelerated the degradation of the $\beta$-catenin protein (Fig. 4B,C), which was slowed by proteasome inhibition with MG132 (Fig. 4D). The ubiquitination assays further confirmed that $\beta$-ionone significantly increased the ubiquitination level of $\beta$-catenin (Fig. 4E). In summary, $\beta$-ionone promotes the ubiquitination and degradation of $\beta$-catenin, thereby inhibiting the Wnt/$\beta$-catenin pathway.

Fig. 4.

$\beta$-ionone inhibitis the Wnt/$\beta$-catenin signaling pathway by regulating its ubiquitination and degradation. (A) Prostate cancer cells PC3 and 22RV1 were treated with gradient concentrations of $\beta$-ionone for 24 h (A) to detect the mRNA levels of $\beta$-catenin. ns p $>$ 0.05. (B,C) Protein synthesis was inhibited using CHX and the effect of 180 $\mu$M $\beta$-ionone on the rate of $\beta$-catenin degradation in cells was determined. (D) Effects of $\beta$-ionone or DMSO on $\beta$-catenin protein levels after treatment with MG132. (E) It was investigated the effect of 180 $\mu$M $\beta$-ionone treatment on the ubiquitination level of $\beta$-catenin in 293T cells by Immunoprecipitation. ***p 0.001.

To verify the results in vitro , we used human prostate cancer PC3 cells to construct subcutaneous xenograft tumors in nude mice as an in vivo model. According to the previous studies, the dose of $\beta$-ionone was set at 75 mg/kg, and there were no abnormal changes in diet and body weight of the two groups of mice during the treatment. In the subcutaneous xenografts of nude mice, $\beta$-ionone showed an inhibitory effect on the proliferation of xenografts (Fig. 5A–C). We speculate that it may be related to its anti-inflammatory effect in vivo, which needs to be further explored. Western blot showed that the expression levels of $\beta$-catenin and Vimentin were down-regulated in tumor tissues in the $\beta$-ionone treatment group, while promoting the expression of E-cadherin (Fig. 5D). Immunohistochemical results showed that protein expression the positive rate of $\beta$-catenin, Vimentin and Ki67 protein in the treatment group was lower than in the control group, indicating that $\beta$-ionone may inhibit tumor proliferation in vivo. The related proteins of Wnt/$\beta$-catenin and EMT are consistent with the western blot analysis (Fig. 5E). In conclusion, these results are consistent with the results in vitro, indicating that $\beta$-ionone inhibits PCa EMT in vivo by down-regulating the Wnt/$\beta$-catenin pathway.

Fig. 5.

$\beta$-ionone inhibits the Wnt/$\beta$-catenin pathway and EMT in vivo. (A) PC3 cells were injected subcutaneously into male nude mice, and tumors were dissected after treatment with $\beta$-ionone or corn oil for 22 days. (B) Tumor volume was measured every three days in corn oil and $\beta$-ionone treated mice. (C) Mice were sacrificed in 22 days and tumor weights were weighed. (D) The expression levels of $\beta$-catenin, E-cadhrin, and Vimentin in subcutaneous xenografts of mice were detected by Western blot and Immunohistochemical analysis (E). Immunohistochemical analysis of the expression levels of $\beta$-catenin, E-cadhrin, Vimentin and Ki67 in subcutaneous xenografts. (F) Pattern diagram was drawn to depict $\beta$-ionone inhibiting EMT process in prostate cancer cells. *p 0.05, **p 0.01, ***p 0.001 and ****p 0.0001.

To explore the possible targets of $\beta$-ionone, we used the website Swiss target prediction, Similarity ensemble approach and SuperPred for predictions. The intersection of the top ten prediction results of each website was obtained, and finally the proteins encoded by the three genes of RXRA, RXRB, and RXRG were most likely to be used as targets for $\beta$-ionone. Further analysis using PLIP revealed that $\beta$-ionone could bind to 243A, 244A, 249A, and 316A of RXRA-encoded proteins, 504A, 505A, and 508A of RXRB-encoded proteins, and 274B, 275B, and 278B residues of RXRG-encoded proteins through hydrophobic interaction (Supplementary Fig. 2).