Arenobufagin Inhibits Prostate Cancer Epithelial-Mesenchymal Transition and Metastasis by Down-Regulating Β-Catenin

Accepted Manuscript

Title: Arenobufagin inhibits prostate cancer epithelial-mesenchymal transition and metastasis by down-regulating ␤-catenin

Authors: Liping Chen, Weiqian Mai, Minfeng Chen, Jianyang Hu, Zhenjian Zhuo, Xueping Lei, Lijuan Deng, Junshan Liu, Nan Yao, Maohua Huang, Yinghui Peng, Wencai Ye, Dongmei Zhang

PII: S1043-6618(17)30522-4 DOI: http://dx.doi.org/doi:10.1016/j.phrs.2017.07.009 Reference: YPHRS 3640

To appear in: Pharmacological Research

Received date: 1-5-2017 Revised date: 29-6-2017 Accepted date: 7-7-2017

Please cite this article as: Chen Liping, Mai Weiqian, Chen Minfeng, Hu Jianyang, Zhuo Zhenjian, Lei Xueping, Deng Lijuan, Liu Junshan, Yao Nan, Huang Maohua, Peng Yinghui, Ye Wencai, Zhang Dongmei.Arenobufagin inhibits prostate cancer epithelial- mesenchymal transition and metastasis by down-regulating ␤-catenin.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2017.07.009

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Original article

Arenobufagin inhibits prostate cancer epithelial-mesenchymal transition and metastasis by down-regulating β-catenin

Liping Chena,b,1, Weiqian Maia,b,1, Minfeng Chena,b, Jianyang Hua,b, Zhenjian Zhuoa, Xueping Leia,b, Lijuan Denga,b, Junshan Liuc, Nan Yaoa,b, Maohua Huanga,b, Yinghui Penga,b, Wencai Ye a,b,**, Dongmei Zhanga,b,* a College of Pharmacy, Jinan University, Guangzhou 510632, P.R. China b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Jinan University, Guangzhou 510632, P.R. China c School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510632, P.R. China

*Corresponding authors. Fax: +86-20-8522-1559. **Fax: +86-20-8522-0004 E-mail address: [email protected] (Dongmei Zhang); [email protected] (Wencai Ye).

1The two authors contributed equally to this project.

Graphical abstract

ABSTRACT Epithelial-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis; thus, developing EMT inhibitors may be a feasible treatment for metastatic PCa. Here, we discovered that arenobufagin and four other bufadienolides suppressed PC3 cell EMT. These compounds modulated EMT marker expression with elevating E-cadherin and reducing ZEB1, vimentin and slug expression, and attenuated the migration and invasion of PC3 cells. Among these five compounds, arenobufagin exhibited the most potent activity. We found that the mRNA and protein expression of β-catenin and β-catenin/TCF4 target genes, which are related to tumor invasion and metastasis, were down-regulated after arenobufagin treatment. Overexpression of β-catenin in PC3 cells antagonized the EMT inhibition effect of arenobufagin, while silencing β-catenin with siRNA enhanced the inhibitory effect of arenobufagin on EMT. In addition, arenobufagin restrained xenograft tumor EMT, as demonstrated by decreased mesenchymal marker expression and increased epithelial marker expression, and reduced the tumor metastatic foci in lung. This study demonstrates a novel anticancer activity of arenobufagin, which inhibits PC3 cell EMT by down-regulating β-catenin, thereby reducing PCa metastasis. In addition, it also provides new evidence for the

development of arenobufagin as a treatment for metastatic prostate cancer.

Abbreviation : EMT, epithelial-mesenchymal transition; PCa, prostate cancer; TCF/LEF-1, T-cell factor/lymphoid-enhancer factor-1; DAPI, 4', 6-diamidino-2-phenylindole; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; FBS, fetal bovine serum; IHC, immunohistochemistry; HE, haematoxylin-eosin.

Keywords EMT, β-catenin, metastatic prostate cancer, bufadienolides, arenobufagin

1. Introduction Prostate cancer (PCa) is the most common malignancy in men [1]. Metastatic castration-resistant prostate cancer is progressive, and its metastasis leads to poor survival outcomes [2]. However, there are currently no effective therapeutics to treat metastatic prostate cancer. Thus, antineoplastic agents for controlling PCa metastasis are urgently needed. Epithelial-mesenchymal transition (EMT), a process of epithelial cells losing epithelial properties and acquiring mesenchymal characteristics, facilitates the invasion and metastasis of early stage tumors and contributes to cancer progression [3]. Several markers denote EMT, including vimentin, E-cadherin, N-cadherin, snail, slug and ZEB1. EMT plays an important role in PCa metastasis. Vimentin is primarily detected in poorly differentiated PCa and PCa bone metastases. Advanced prostate adenocarcinomas are characterized by the loss of E-cadherin, which leads to tumor metastasis [4]. Snail overexpression is significantly associated with increasing Gleason grade, indicating prostate cancer progression [5]. The inhibition of ZEB1 results in N-cadherin down-regulation and E-cadherin up-regulation in prostate cancer cells exhibiting a mesenchymal phenotype [4]. Therefore, inhibiting PCa cell EMT is likely to be an effective way to suppress PCa metastasis. β-Catenin, a dual-function protein, is involved in two fundamental EMT processes. It connects cadherins with the cytoskeleton to enhance cell-cell adhesion. It also acts as a transcription factor together with T-cell factor/lymphoid-enhancer factor-1 (TCF/LEF-1) to activate target genes such as c-Myc and MMP7, which are associated with tumor invasion and 3

metastasis [6, 7]. The acquisition of mesenchymal markers such as vimentin by epithelial carcinoma cells is related to nuclear overexpression of β-catenin [8]. Bufadienolides, including bufalin, arenobufagin and cinobufotalin, are the major anti-tumor active components derived from toad venom. Bufalin has been reported to induce PCa cell apoptosis by up-regulating Fas expression [9]. Previous studies of bufadienolides by our research group have shown that these compounds exhibit multiple anticancer activities[10-12], including apoptosis induction [13-15], tumor angiogenesis inhibition[11, 16] and cell cycle arrest [14, 17, 18]. However, the anticancer effects of bufadienolides and the underlying mechanisms of their inhibition of PCa EMT and metastasis remain poorly understood. Here, we verified that five bufadienolides suppressed PC3 cell EMT and determined that arenobufagin exhibited the most potent effects against EMT in PC3 cells, through a molecular mechanism involving β-catenin down-regulation. 2. Materials and methods 2.1. Materials 4',6-Diamidino-2-phenylindole (DAPI), ribonuclease A (RNase A) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640, fetal bovine serum (FBS), penicillin-streptomycin, Alexa Fluor 488 Donkey anti-Rabbit IgG, Alexa Fluor 594 Donkey anti-Goat IgG, Lipofectamine 3000, Lipofectamine LTX&PLUS reagent, RIPA lysis buffer and NE-PER nuclear and cytoplasmic extraction reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Dual-Luciferase Reporter Assay System was obtained from Promega (Madison, Wisconsin, USA). Transwell plates (6.5 mm) with 8.0 µm pore polycarbonate membrane inserts were obtained from Corning (New York, NY, USA).

The anti-Smad2/3 (#8685), anti- phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828) antibodies and the Epithelial-Mesenchymal Transition (EMT) Antibody Sample kit (#9782) were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-AREB6 (ab203829), anti-E-cadherin (ab1416), anti-ZO-1 (ab190085), anti-CD31 (ab28364), anti-VEGF Receptor 2 (ab2349), anti-PI3K (ab86714), anti-Akt (ab8805) and anti-mTOR (ab2732) antibodies were obtained from Abcam (Cambridge, MA, USA). Matrigel basement membrane matrix was supplied by BD Biosciences (Franklin Lakes, NJ). The plasmid 4

pCMV3-CTNNB1-GFPSpark and the vector pCMV3-C-GFPSpark were purchased from Sino Biological Inc. (Beijing, China). Bufadienolides (purity ≥ 98%) were obtained from Baoji Herbest Bio-Tech Co., Ltd. (Shanxi, China). Bufadienolides were dissolved in DMSO to provide a stock 500 µM solution, then stored at -20°C. Phosphatase inhibitor, protease inhibitor tablets, the Transcriptor First Strand cDNA Synthesis Kit and the TUNEL Apoptosis Assay Kit were supplied by Roche Applied Science (Mannheim, Germany). The E.Z.N.A. Total RNA Kit I was purchased from Omega Bio-Tek (Doraville, GA, USA). Recombinant Human Wnt-3A protein (CF) was obtained from R&D Systems Inc. (Minneapolis, MN, USA). LiCl was purchased from Millipore Sigma (Darmstadt, German). MG132 was supplied by Beyotime Biotechnology (Shang, China). AmpliteTM Colorimetric Lactate Dehydrogenase Assay Kit was purchased from AAT Bioquest (Sunnyvale, California, USA). Creatine Kinase (CK) Activity Colorimetric Assay Kit was obtained from BioVision (San Francisco Bay Area, California, USA). 2.2. Cell culture PC3, DU145 and LNCaP cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in RPMI-1640 supplemented with 10% FBS (v/v) and 1% penicillin-streptomycin (v/v) in a humidified atmosphere of 5% CO2 at 37°C. 2.3. Animals Four- to six-week-old male nu/nu BALB/c mice were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). All animal experiments were approved by the Experimental Animal Ethics Committee of Jinan University (Guangzhou, China), and the results of animal studies were reported in accordance with the ARRIVE guidelines. 2.4. Cell viability assay PC3 cell proliferation was measured by an MTT assay as previously described [19]. Briefly, cells (1×104/well) were seeded in 96-well plates and cultured overnight. The cells were then treated with various concentrations of bufadienolides. After 48 h of treatment, cell viability was detected by MTT assay. 2.5. Western blotting The cells were lysed in ice-cold RIPA lysis buffer containing 0.1 M PMSF, phosphatase inhibitor and protease inhibitor cocktail to obtain total cellular protein. Nuclear and 5

cytoplasmic proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents. The protein concentration was determined by the BCA protein assay. Electrophoresis and immunoblotting analysis were performed as previously described [20]. 2.6. Cell migration and invasion assays For the wound-healing assay, cells were seeded in 6-well plates and cultured until reaching confluence. The cells were then scratched in a straight line with 200-μL pipette tips and treated with the indicated concentrations of bufadienolides for 24 h and 48 h in serum-free medium containing mitomycin C (2 μg/mL) to eliminate the effect of cell proliferation [21]. Images of cells treated with bufadienolides for different lengths of time (0 h, 24 h and 48 h) were acquired with an Olympus IX70 inverted microscope (Shinjuku, Tokyo, Japan). In the Transwell migration assay, cells (4×104/well) suspended in 100 μL of RPMI medium containing 10% FBS were seeded into the chambers and cultured overnight. The cells were then treated with the indicated concentrations of bufadienolides for 48 h. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min. Cells on the upper sides of the inserts were removed with cotton swabs, and the number of cells on the bottom sides of the inserts were measured by counting 3 random fields with Image-Pro Plus 6.0 software. The Transwell invasion assay was performed using the same method as the migration assay, except that the upper sides of the inserts were pre-coated with 20 μL (2.5 mg/mL) of Matrigel. 2.7. Immunofluorescence assay The immunofluorescence assay was performed according to a previously described protocol [22]. Cells (5×104/well in 35 mm confocal dishes) were treated with 8 nM arenobufagin. After 48 h of treatment, the cells were incubated with a primary antibody overnight at 4°C followed by a fluorescent secondary antibody for 1 h at room temperature and stained with DAPI for 5 min. Images were acquired with a laser scanning confocal microscope (LSM 880, ZEISS). The fluorescence intensity of the cells was measured by Image-Pro Plus 6.0 software. 2.8. Quantitative real-time PCR Total RNA was extracted using an E.Z.N.A. Total RNA Kit I according to the manufacturer’s protocol. RNA quality was evaluated using agarose gel electrophoresis to 6

examine the 28S and 18S rRNA band intensity [23]. Reverse transcription of total RNA was performed using a Transcriptor First Strand cDNA Synthesis Kit. Primers, shown in Table 1, SYBR Green I Master Mix, and DNA templates were mixed to form the PCR system. qPCR was performed on a Roche Lightcycler 480 real-time PCR machine with the following cycling parameters: 45 cycles of 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. The mRNA expression of target genes is shown as the fold change relative to the expression of a housekeeping gene whose value was regarded as 1. Briefly, the 2-ΔΔCT method was used to calculate the relative quantity of mRNA. 2.9. Agarose gel electrophoresis Agarose gel electrophoresis was performed according to a standard protocol [24]. In brief, 2% agarose gels were prepared with a w/v percentage solution supplemented with 0.5 μg/mL ethidium bromide. The gels were electrophoresed until the loading dye had moved an appropriate distance. DNA bands were visualized after the agarose gels were exposed to UV light. 2.10. Dual-luciferase reporter gene assay Transactivation of indicated genes was assessed by pGL4.49 [luc2p/ TCF-LEF RE/Hygro] vectors, which express firefly luciferase when activated. The constitutively active pGL4.73 [hRluc/SV40] vectors, which express Renilla luciferase, were used to control for transfection efficiency. The plasmids were transfected into cells with Lipofectamine LTX&PLUS. After 6 h of transfection, the cells were cultured for another 24 h. Then, the cells were treated with arenobufagin for 48 h. The two luciferase signals were determined on a TECAN Infinite F500 platform (Männedorf, Switzerland) using the Dual-Luciferase Reporter Assay System. The results are expressed as the ratio of firefly to Renilla luciferase activity. 2.11. Transient transfection Cells were seeded into 6-well plates and grown to 80-90% confluence. The plasmid pCMV3-CTNNB1-GFPSpark and vector pCMV3-C-GFPSpark were transfected into cells using Lipofectamine LTX&PLUS reagent according to the manufacturer’s protocol. Cells were incubated with transfection reagent for 6 h. After that, the reagent was removed, and cells were harvested at 48 h. 2.12. Transfection of small interfering RNA 7

Cells were transfected with the indicated siRNA duplexes or a negative control siRNA using Lipofectamine 3000. After 6 h of transfection, cells were cultured for another 24 h before arenobufagin treatment. β-Catenin was knocked down with the following siRNA duplex: 5’-GUCAACGUCUUGUUCAGAATT-3’ and 5’-UUCUGAACAAGACGUUGACTT-3’. The negative siRNA duplex that does not target any gene product was used as a negative control, and its sequences were 5’-UUCUCCGAACGUGUCACGUTT-3’ and 5’-ACGUGACACGUUCGGAGAATT-3’. 2.13. Tumor xenografts in nude mice

PC3 cells (1×107) were subcutaneously inoculated into the armpits of nude mice. When the tumor volume developed to approximately 100 mm3, the mice were randomly assigned into two groups (10 mice per group). Pre-experiment showed that the mice tolerated administration of 1 mg/kg arenobufagin (dissolved in normal saline containing 5% HS15) and showed no obvious abnormalities. Therefore, we selected the dose of 1 mg/kg in the in vivo studies. Thus, the mice were then intravenously injected with saline or 1 mg/kg arenobufagin every day. Tumor size was measured every two days using a digital caliper, and tumor volume was calculated using the following formula: 0.5×a×b2, where a refers to the longest diameter and b is the shortest diameter. In addition, the mice were weighed every two days. At the end of the experiment, the mice were euthanized. Tumors and viscera were obtained and fixed in 4% paraformaldehyde for histological and immunohistochemical examination.

2.14. Development of lung metastasis models

PC3 cells (2×106) were suspended in 250 μL of serum-free RPMI-1640 and injected into the tail veins of the mice. The mice were randomly divided into two groups (10 mice per group) and intravenously injected with saline or arenobufagin (1 mg/kg) every day for 1 month. Finally, the mice were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium, and the lungs were fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) staining.

2.15. Histology and immunohistochemistry (IHC)

The visceral tissues and tumors were fixed in 4% paraformaldehyde for 24 h, dehydrated in

a graded alcohol series (75%, 85%, 95% and 100%, v/v) successively and embedded in paraffin. The samples were sectioned at a 5-μm thickness. For histological examination, the sections were stained with HE to distinguish tumorous metastatic foci and normal tissues. For IHC, the sections were stained with anti-ZEB1, anti-N-cadherin, anti-β-catenin, anti-vimentin, anti-slug/snail, anti-ZO-1, and anti-E-cadherin antibodies and then incubated with the appropriate secondary antibody. Protein expression was then detected with a DAB kit. Images were obtained using an Olympus IX70 inverted microscope.

2.16. Assay for lactate dehydrogenase (LDH) and creatine kinase (CK) level

After the last treatment, animals were anesthetized with pentobarbital sodium. Blood was collected and centrifuged at 3000 rpm for 10 min for two times at 4 °C to obtain serum. Enzyme activities of LDH and CK in serum were detected using AmpliteTM Colorimetric Lactate Dehydrogenase Assay Kit and Creatine Kinase (CK) Activity Colorimetric Assay Kit according to the manufacturer’s protocol.

2.17. Statistical analysis

Data are displayed as mean ± SEM. The statistical analysis was performed by GraphPad Prism 5.0 software. An unpaired Student’s t-test and one-way ANOVA were used to analyze the differences between two variables and multiple variables, respectively. P<0.05 was considered statistically significant.

3. Results 3.1. Bufadienolides inhibit EMT and attenuate the migration and invasion of PC3 cells The chemical structure and functional groups of bufadienolides are displayed in Fig. 1A. The non-toxic concentrations of these bufadienolides were determined using MTT assays. Cinobufotalin, bufarenogin, arenobufagin, 19-oxocinobufotalin and 19-hydroxybufalin inhibited 10% of PC3 cell growth at the doses of 20 nM, 3 μM, 8 nM, 50 nM and 12 nM, respectively (Fig. 1B). During EMT, epithelial cells exhibit morphological changes and signs of cytoskeleton reconstruction, such as the formation of stress fiber bundles, partly due to the decrease in E-cadherin [25-27]. Thus, we characterized the morphology and status of F-actin

formation in PC3 cells. As shown in Fig. 1C, the morphology of PC3 cells treated with the five bufadienolides for 48 h transformed from a mesenchymal-like shape (spindle) to an epithelial-like shape (spherical). The cell cytoskeletons also showed similar changes (Fig. 1D), indicating that F-actin formation in PC3 cells was strongly suppressed by the bufadienolides. Western blotting results showed that the mesenchymal markers vimentin, ZEB1 and slug decreased and the epithelial marker E-cadherin increased (Fig. 1E). EMT enhances cell migration and invasion by disrupting intercellular junctions [28]. We further analyzed the migratory capacity of PC3 cells treated with the five bufadienolides using wound-healing and Transwell assays. The bufadienolides significantly inhibited wound closure in a time-dependent manner (Fig. 2A and 2D) and cellular migration across the membrane (Fig. 2B and 2E). To further investigate whether bufadienolides inhibit PC3 cell invasion, we performed a Transwell invasion assay using Matrigel-coated membranes. As shown in Fig. 2C and 2F, the invasive capability of PC3 cells was weakened after bufadienolides treatment for 48 h. In particular, arenobufagin caused the most significant attenuation of cell mobility and invasiveness among the five bufadienolides, thus we chose arenobufagin for further investigation.

3.2. Arenobufagin modulates the expression of EMT markers in PC3 cells

EMT is characterized by the up-regulation of mesenchymal markers such as vimentin, snail, slug, ZEB1, N-cadherin and Twist1, accompanied by the down-regulation of epithelial markers like E-cadherin, ZO-1 and Claudin1 [29]. Thus, Western blotting and immunofluorescence assays were performed to determine whether arenobufagin alters the expression of EMT markers in PC3 cells. Cells were treated with 8 nM arenobufagin for 0 h, 24 h, 36 h and 48 h. Western blotting results showed that the epithelial markers ZO-1, E-cadherin, and Claudin1 were elevated and that the mesenchymal markers ZEB1, N-cadherin, vimentin, snail, slug and Twist1 were reduced (Fig. 3A and 3B). Immunofluorescence assays further confirmed a significant increase in the expression of E-cadherin and ZO-1 and a decrease in the expression of ZEB1 and vimentin in PC3 cells treated with arenobufagin (Fig. 3C and 3D).

3.3. Arenobufagin down-regulates β-catenin mRNA and protein in PC3 cells

Some studies have shown that the up-regulation of β-catenin promotes EMT [30-32]. Thus, we assessed the effect of arenobufagin on β-catenin. As shown in Fig. 4A and 4B, arenobufagin down-regulated the mRNA and protein expression of β-catenin in a time-dependent manner. β-Catenin acts a transcription factor together with TCF/LEF-1 to activate target genes such as c-Myc and MMP7. Immunofluorescence assays and Western blotting showed that the nuclear translocation of β-catenin was reduced after arenobufagin treatment (Fig. 4C, 4D, 4E and 4F). A dual-luciferase reporter gene assay showed that TCF/LEF transcriptional activity was weakened by arenobufagin in a dose-dependent manner (Fig. 4G). β-Catenin/TCF/LEF target genes, such as Met, LEF, TCF, c-Myc and cyclin D1, were down-regulated at the mRNA level after exposure to 8 nM arenobufagin for 48 h. (Fig. 4H).

3.4. β-Catenin is involved in the EMT inhibitory effect of arenobufagin in PC3 cells

To confirm that arenobufagin suppresses EMT of PC3 cells via the down-regulation of β-catenin, we construct β-catenin-overexpressing PC3 cells. As shown in Fig. 5A, β-catenin overexpression antagonized the inhibitory effect of arenobufagin on EMT, activating mesenchymal markers and down-regulating epithelial markers. The enhanced migratory and invasive abilities of PC3 cells induced by β-catenin overexpression were weakened by arenobufagin (Fig. 5B), while β-catenin knockdown in combination with arenobufagin significantly down-regulated mesenchymal markers and up-regulated epithelial markers (Fig. 5C). In addition, β-catenin knockdown attenuated the migration and invasion of PC3 cells, and these effects were augmented by arenobufagin treatment (Fig. 5D). These results suggest that the inhibition of EMT by arenobufagin in PC3 cells is dependent on β-catenin.

3.5. Arenobufagin blocks EMT in a xenograft model and metastasis in a tail vein injection model

To examine the inhibitory effect of arenobufagin on EMT and metastasis in vivo, we determined the expression levels of EMT marker proteins in xenograft tumors using IHC. As shown in Fig. 6A and 6B, the epithelial marker proteins ZO-1 and E-cadherin were up-regulated, and the mesenchymal marker proteins ZEB1, β-catenin, vimentin, snail and slug were down-regulated after arenobufagin treatment. In addition, arenobufagin suppressed

tumor growth (Fig. 6C and 6D). Arenobufagin restrained xenograft tumor EMT, and thus, we developed a pulmonary metastases model to assess the relative effects of arenobufagin on the development of cancer metastasis. Tail vein injection of PC3 cells into nu/nu BALB/c mice was used to establish the lung metastases model. The circulating tumor cells establish themselves in the lungs of mice and gradually grow into metastatic foci. As demonstrated in Fig. 6E and 6F, arenobufagin reduced the number and size of tumor metastatic foci in lung tissues. To evaluate the toxicity of arenobufagin in vivo, we examined pathological lesions in the heart, liver, spleen, lung and kidney by HE staining. As shown in Fig. 7A, no abnormal pathological changes were observed in the visceral organs of arenobufagin-treated nude mice. Clinically, CK and LDH are considered to be important markers of heart damage [33, 34]. The activities of LDH and CK in serum were detected for further confirming the cardiotoxicity caused by arenobufagin. As shown in Fig. 7B, treatment with arenobufagin did not significantly change the levels of CK and LDH, suggesting that arenobufagin at a dose of 1 mg/kg/day shows negligible cardiotoxicity. Besides, the mice exhibited normal weight gain throughout the arenobufagin treatment and did not show any abnormalities in food intake or behavior (Fig. 7C).

Discussion In the present study, we identified five bufadienolides, cinobufotalin, bufarenogin, arenobufagin, 19-oxocinobufotalin and 19-hydroxybufalin, that were capable of suppressing EMT and weakening the migratory and invasive potential of PC3 cells. Arenobufagin which exhibited the strongest EMT inhibitory activity among the five compounds, was chosen for further research. Our results suggested that the EMT-suppressing effect of arenobufagin depends on the down-regulation of β-catenin. Moreover, we demonstrated that arenobufagin inhibited xenograft tumor EMT and decreased PC3 cell metastases in lung tissues without significant toxicity towards several important organs, which supports its potential for further clinical investigation for PCa treatment. To our knowledge, this is the first report of the inhibition of PCa EMT by arenobufagin. A structure-activity analysis was performed to highlight the most important substituents associated with the EMT inhibitory activity of bufadienolides in PCa. The difference between arenobufagin and bufarenogin is that arenobufagin has an 11β-hydroxyl group and a 12

12-carbonyl group, whereas bufarenogin has an 11-carbonyl group and a 12α-hydroxyl group. The 10% growth inhibitory activity of arenobufagin (8 nM) is 375 times higher than that of bufarenogin (3 μM). Arenobufagin significantly inhibited PC3 cell migration and invasion (P<0.001), while bufarenogin only slightly suppressed the mobility and infiltrating capacity of PC3 cells (P<0.05). Based on these results, we reasonably speculated that the 11β-hydroxyl and 12-carbonyl groups are associated with better activity than the 11-carbonyl and 12α-hydroxyl groups. Interestingly, we found that arenobufgin suppresses EMT of DU145 cells (Supplementary Fig. 2), but not that of LNCaP cells (data are not shown), indicating that arenobufagin has the EMT inhibitory effect on androgen-independent prostate cell line (PC3 and DU145), but not on androgen-dependent prostate cell lines (LNCaP). It has been reported that arenobufagin blocks VEGF-mediated angiogenesis and induces apoptosis via (PI3K)/Akt/ (mTOR) inhibition pathway for several other solid tumor cell lines [10, 11], our data demonstrated the similar effect of arenobufagin on PCa xenografts (Supplementary Fig. 3). In the future, we will develop luciferase expressing PCa model to track the effect of arenobufagin on the tumor growth and metastasis in vivo. β-Catenin is closely associated with EMT markers. ZEB1, a direct target of β-catenin/TCF4, is a key inducer of EMT and inhibits the epithelial phenotype [35]. Vimentin, a mesenchymal marker, has been demonstrated to be a target of the β-catenin-TCF/LEF-1 complex [36]. The zinc-finger transcription factor snail is activated when β-catenin translocates to the nucleus and binds with TCF/LEF-1 to form a functional complex, which results in E-cadherin down-regulation and enhanced tumor invasion [37]. Slug, a member of the snail family [38], is activated by the nuclear translocation of β-catenin and leads to reduced E-cadherin expression in colorectal carcinoma [39]. As some mesenchymal markers are directly or indirectly regulated by β-catenin, our results indicate that arenobufagin suppresses EMT via down-regulating β-catenin mRNA and protein expression. In addition, β-catenin overexpression antagonizes the EMT inhibitory effect of arenobufagin, while its knockdown enhanced this effect, suggesting that β-catenin plays a crucial role in EMT inhibition by arenobufagin in PC3 cells. In the Wnt/β-catenin signaling pathway, β-catenin is phosphorylated by GSK3β, subsequently ubiquitinated by β-Trcp and eventually degraded by the proteasome [40-42]. 13

IWR-1 reportedly inhibits EMT of colorectal cancer cells through the Wnt/β-catenin pathway, which involves in promoting the phosphorylation of β-catenin by destroying β-catenin destruction complex [43]. Bufalin inhibits hepatocellular carcinoma cell invasion and metastasis by suppressing the phosphorylation of GSK-3β Ser9 site, which indirectly modulates the expression of downstream factors in the Wnt/β-catenin signaling pathway [44]. However, in this study, when we activated Wnt/β-catenin signaling using wnt3a, blocked the activity of GSK3 with LiCl and inhibited the ubiquitination of β-catenin using MG132, the down-regulation of β-catenin modulated by arenobufagin was abolished (Supplementary Fig. 1A, 1B and 1C), indicating that no crosstalk exists between the molecular mechanism of β-catenin down-regulation by arenobufagin and Wnt/β-catenin signaling. In addition, arenobufagin reduces β-catenin mRNA expression, further suggesting that the mechanism by which arenobufagin inhibits EMT in PC3 cells does not involve the Wnt/β-catenin pathway. TGF-β signaling pathway plays a crucial role in EMT [25]. However, we found that arenobufagin had no effect on phospho-Smad2/3 in PC3 cells (Supplementary Fig. 1D), suggesting that the classical TGF-β/Smad signaling pathway is not involved in the inhibitory effect of arenobufagin on EMT. In fact, the mechanism underlying arenobufagin-induced down-regulation of β-catenin remains undetermined and has yet to be further investigated. In conclusion, our study verified that five bufadienolides could inhibit EMT as well as migration and invasion in PC3 cells. In addition, we firstly report the novel activity of arenobufagin, which inhibits EMT of PC3 cells by down-regulating β-catenin. Arenobufagin exhibits promising results and may be developed as a potential agent for treating metastatic prostate cancer. Conflicts of interest The authors declare that there are no conflicts of interest.

Chemical compounds studied in this article Cinobufotalin (PubChem CID: 259776); Bufarenogin (PubChem CID: 167607); Arenobufagin (PubChem CID: 12305198); 19-oxocinobufotalin (PubChem CID: 102093790).

Acknowledgments This work was supported by the Science and Technology Program of China 14

(2017ZX09101003-008-008), the National Science Foundation of China (81573455), Guangdong Province (S2013050014183 and 2013CXZDA006), the Program for New Century Excellent Talents in University and Guangdong Pear River Scholar Funded Scheme (D.M. Zhang).

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Figure Legends Fig. 1 Bufadienolides inhibit EMT of PC3 cells. (A) Chemical structure of bufadienolides isolated from toad venom. (B) The viability of PC3 cells treated with bufadienolides at the indicated concentrations for 48 h was determined by MTT assays. (C) After 48 h treatment with 20 nM cinobufotalin, 3 μM bufarenogin, 8 nM arenobufagin, 50 nM 19-oxocinobufotalin or 12 nM 19-hydroxybufalin, the morphology of PC3 cells was observed using a microscope (200× magnification) (scale bar = 100 μm). (D) PC3 cells were stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Images were obtained using a laser scanning confocal microscope (63× oil lens magnification) (scale bar = 20 μm). (E) PC3 cells were exposed to 20 nM cinobufotalin, 3 μM bufarenogin, 8 nM arenobufagin, 50 nM 19-oxocinobufotalin or 12 nM 19-hydroxybufalin for 48 h, and the expression of E-cadherin, vimentin, ZEB1 and slug was then detected by Western blotting. β-Actin was used as a loading control. Representative figures (left) and the densitometric analysis (right) of the expression of EMT markers are shown. Data are presented as mean ± SEM, n=3. **P<0.01, ***P<0.001 versus the control group. Cin, Buf, Are, 19-Oxo and 19-Hy represent cinobufotalin, bufarenogin, arenobufagin, 19-oxocinobufotalin and 19-hydroxybufalin, respectively. Fig. 2 Bufadienolides attenuate the migration and invasion of PC3 cells. The migratory and invasive properties of PC3 cells were analyzed by wound-healing assays (A) and Transwell migration (B) and invasion assays (C). 100× magnification in A and 200× magnification in B and C. The relative wound width (E) and the relative amount of migrated (F) and invaded cells (G) were analyzed using GraphPad Prism 5.0. Data are exhibited as mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 versus the control group.

Fig. 3 Arenobufagin modulates the expression of EMT markers in PC3 cells. (A) PC3 cells were exposed to 8 nM arenobufagin for the indicated time. The mesenchymal markers ZEB1, N-cadherin, vimentin, slug, snail, and Twist1 and the epithelial markers ZO-1, E-cadherin and Claudin-1 were detected by Western blotting. (B) Data represent the relative protein expression, shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 versus the control group. (C) PC3 cells were exposed to 8 nM arenobufagin for 48 h, and the expression levels of E-cadherin, ZO-1, vimentin and ZEB1 were confirmed by immunofluorescence staining. 20

Images were obtained at 400× magnification (scale bar = 50 μm). (D) The relative fluorescence intensities are expressed as mean ± SEM, n=3. *P<0.05, **P<0.01 compared with the control group.

Fig. 4 Arenobufagin down-regulates β-catenin mRNA and protein expression in PC3 cells. (A) PC3 cells were exposed to 8 nM arenobufagin for the indicated time, and the mRNA level of β-catenin was analyzed by RT-PCR. (B) The protein level of β-catenin was detected by Western blotting. (C) Immunofluorescence assays were performed to detect the expression of β-catenin in the nucleus and cytoplasm, and images were obtained using a laser scanning confocal microscope (400× magnification) (scale bar = 20 μm). The expression of β-catenin in the membrane, cytoplasm and nucleus was visualized by the green fluorescent signal. (D) RFI, namely relative fluorescence intensities, are expressed as mean ± SEM. ***P<0.001 compared with the control group. (E) The expression of β-catenin in the nucleus and cytoplasm was detected by Western blotting. GAPDH and histone3 were used as the internal controls for the cytosol and nucleus, respectively. (F) Densitometric analysis of the expression of β-catenin in the nucleus and cytoplasm. *P<0.05, **P<0.01 compared with the control group. (G) HEK293T cells transiently transfected with pGL4.49[luc2p/TCF-LEF RE/Hygro] and pGL4.73[hRluc/SV40] vectors were incubated with the indicated concentrations of arenobufagin for 48 h. TCF-LEF reporter activity is reported as relative light units normalized to the Renilla luciferase activity. (H) PC3 cells were exposed to 8 nM arenobufagin for 48 h, and the relative mRNA expression of target genes of the β-catenin/TCF4 complex, including Met, LEF, TCF, c-Myc, cyclin D1 and MMP7, was detected with qPCR. Data represent mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 versus the control group.

Fig. 5 β-Catenin is involved in the EMT inhibitory effect of arenobufagin in PC3 cells. (A and B) PC3 cells were transiently transfected with the plasmid pCMV3-CTNNB1-GFPSpark and the pCMV3-C-GFPSpark vector for 24 h and subsequently treated with 8 nM arenobufagin for 48 h. β-Catenin, ZO-1, E-cadherin, ZEB1, and vimentin were analyzed using Western blotting (A). The migratory and invasive abilities of PC3 cells were examined using Transwell migration and invasion assays (B). (C and D) PC3 cells were transfected with β-catenin siRNA for 24 h and then treated with arenobufagin for 48 h. β-Catenin, slug, ZEB1,

N-cadherin and E-cadherin were detected with Western blotting (C). Cell migration and invasion were analyzed by Transwell migration and invasion assays (D). Data represent mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 versus the vector-transfected or NC group. #P<0.05, ##P<0.01 versus the β-catenin-transfected or β-catenin siRNA group.

Fig. 6 Arenobufagin blocks EMT in a xenograft model and metastasis in a tail vein injection model. PC3 cells-xenografted nude mice (n = 10 per group) were injected with normal saline (control group) or arenobufagin (1 mg/kg body weight) for 35 days. (A and B) The expression of ZEB1, vimentin, snail, slug, β-catenin, ZO-1 and E-cadherin was detected using immunohistochemical staining in xenograft tumor sections (scale bar = 100 μm). (C) Tumor volume and (D) tumor weight are presented. (E and F) Lung tissue sections were obtained from control and arenobufagin-treated mice after 1 month of tail vein injections and stained with HE. Images were obtained under a microscope (100× magnification) (scale bar = 400 μm). Data are presented as mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 versus the control group.

Fig. 7 Arenobufagin shows no obvious toxicity to nude mice. (A, B and C) PC3 tumor-bearing mice were treated intravenously with normal saline or arenobufagin, and the mouse body weight was measured every 2 days (C). The heart, liver, spleen, lung and kidney were stained with HE for histological analysis of the PCa xenograft mice. Images were obtained using a microscope (100× magnification for the spleen, lung and kidney; 200× magnification for the heart and liver) (A). The serum levels of LDH and CK in PCa xenograft mice treated with or without arenobufagin (B). Data are exhibited as mean ± SEM, n = 3.