The online Oncomine database (www.oncomine.org) were used to reveal PTAFR expression patterns between normal breast tissues and invasive breast carcinomas. Involved expression data were deposited in the National Center for Biotechnology Information/Gene Expression Omnibus database entries GSE9014. Data for survival analysis of bone metastasis and PTAFR expression patterns between osteoclatogenic (OG) and non-OG cell lines were obtained from GSE2603 and GSE43811, respectively. Survival analyses were based on Kaplan-Meier method, cutoff value was determined by ROC curves.

MDA-MB-231, RAW 264.7 and 293T cells were all obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplied with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin-streptomycin (P/S; Gibco; Thermo Fisher Scientific, Inc.). RAW264.7 cells and mouse bone marrow monocytes (BMMs) were cultured in α-minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.), supplied with 10% FBS and 100 U/ml P/S. Cells were cultured in a humidified incubator (Thermo Fisher Scientific, Inc.) with 5% CO₂ at 37°C.

The viability and cytotoxicity effect of Kadsurenone was determined by the MTS method following the manual of CellTiter 96 Aqueous One Solution Cell Proliferation assay (Promega Corporation, Madison, WI, USA) with VERSA max microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) as described previously.

The transwell assay were performed with Boyden chambers (Corning Incorporated, Corning, NY, USA). MDA-MB-231 cells were collected and resuspend in blank DMEM after 12-h serum starve. 6×10⁵ cells were plated in the top chambers with or without 200 nM PAF and different concentrations (0, 0.5, 1, 2.5 and 5 µM) of Kadsurenone. The bottom chambers were filled with 600 µl mediums supplemented with 2% FBS. After 8 h incubation, migrated cells were fixed with 4% paraformaldehyde (PFA) and stained with 1% crystal violet. Images were taken using an Olympus inverted microscope (Olympus Corporation, Tokyo, Japan) and migrated cells were counted using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Dual luciferase assays were conducted in a 24-well plate format. Vectors were transfected into 70% confluent HEK293 cells, PAF or receptor activator for NF-κB ligand (RANKL) inducement and different concentrations of Kadsurenone were added. After 48-h transfection, frefly and renilla luciferase were quantified sequentially using the Dual Luciferase Assay kit (Promega Corporation) following the manufacturer's recommendations.

2×10³ MDA-MB-231 cells and 5X10³ RAW264.7 cells were pooled together and seeded into 24-well plates. Cells were cultured in α-MEM added with 10% FBS and different concentrations of Kadsurenone. After 5–7 days, cells were fixed with 4% PFA and permeabilized with 0.1% Triton-X 100 inPBS for 5 min, and subjected to tartrate-resistant acid phosphatase (Trap) staining with Leukocyte acid phosphatase kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Trap positive osteoclasts were photographed and counted thereafter.

Mouse BMMs were flushed and isolated from C57/BL6 mice as previously described. 8×10³ BMMs were seeded into 96-well plates and incubated with 10 ng/ml M-CSF, 50 ng/ml RANKL and different concentrations of Kadsurenone. After 5–7 days, cells were fixed and subjected to Trap staining and photographed. All experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of East China Normal University and Chang Zheng Hospital (Shanghai, China).

Mouse BMMs were isolation and induced for osteoclast differentiation for 5–7 days. Cells were then permeabilized with Triton X-100 and incubated with rhodamine conjugated phalloidin (Molecular Probes; Thermo Fisher Scientific, Inc.) to visualize F-actin.

Mouse BMMs were isolated and induced for osteoclast differentiation with or without indicated concentrations of Kadsurenone. Cells were collected and total RNA were extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 500 ng total RNA were reverse transcribed with PrimeScript™ RT Master Mix (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions. The complementary DNA was used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a final volume of 20 µl was adopted as follows: 10 µl 2X SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), 1 µl forward and reverse primers, 2.5 µM, 2 µl cDNA, 7 µl ddH₂O. PCR and data collection were performed on Mx3000P QPCR system (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) and data analysis was operated with the 2^−ΔΔCq method normalized to the endogenous control β-actin. Primers used in RT-qPCR are listed in Table I.

Experiments were performed with 3 or more replicates. The results were reported as the mean ± standard error of the mean. The differences between control and experimental groups were determined using an unpaired Student's t-test or one-way analysis of variance followed by Tukey's post hoc test. Survival curves were analyzed by log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Using the Oncomine database (www.oncomine.org), we compared the expression patterns of PTAFR between normal breast tissues and invasive breast carcinomas PTAFR was significantly upregulated in cancer patients with a fold change of 3.88 (P<0.001; Fig. 1A). We further examined prognostic value of PTAFR expression level in BC bone metastasis by Minn's dataset (25). Although no significance was drown (P=0.0992, P<0.1), BC with higher expression levels of PTAFR (n=19) still indicated a tendency for bone metastases (Fig. 1B). We also revealed the expression patterns of PTAFR between OG and non-OG cell lines in GSE43811. PTAFR was significantly upregulated after RANKL stimulation in OG Raw264.7 cells (Fig. 1C).

Kadsurenone is a natural product isolated from stems of Piper kadsura. Chemical structure of Kadsurenone was shown in Fig. 2A. We first tested cytotoxicity of Kadsurenone on BC cell line MDA-MB-231. No significant disturb of cell viability was observed with a highest concentration of 5 µM (Fig. 2B).

We then examined effects of Kadsurenone on PAF induced MDA-MB-231 cells migration. Transwell assay shown that Kadsurenone could dose dependently inhibit PAF induced BC cells migration (Fig. 2C and D). Luciferase assay indicated that the NF-κB activity of MDA-MB-231 cells were significantly inhibited by Kadsurenone dose dependently (Fig. 2E). Our data proved that Kadsurenone did not disturb the cell viability significantly but suppressed cell migration in MDA-MB-231 cells, which indicated that PAF/PTAFR played key role in cell migration but not cell viability.

In the bone microenvironment, BC cells could stimulate osteoclast differentiation directly through excretion of cytokines such as interleukin (IL)-1β, tumor necrosis factor-α (TNFα). BC cells could also indirectly promote osteoclast differentiation via upregulation of RANKL by osteoblast. Therefore, we intended to study whether Kadsurenone could directly inhibit BCs induced osteoclastogenesis. MDA-MB-231 cells, which could secret PAF, were co-cultured with the OG cell line RAW264.7 cells. We proposed to use this co-culture system mimic the BC bone metastases microenvironment. BC cells could induce RAW264.7 cells differentiate into Trap positive osteoclasts. However, this process could be attenuated by Kadsurenone dose dependently (Fig. 3).

It is reported that BMMs could secret PAF as an autocrine factor and promote the OG process (26). Therefore, we intend to investigate whether Kadsurenone could directly affect osteoclastogenesis. We firstly confirmed that no significant cytotoxicity of Kadsurenone on mouse BMMs under a certain concentration (Fig. 4A). However, the process of RANKL induced Trap positive osteoclasts differentiated by BMMs were significantly attenuated by Kadsurenone dose dependently (Fig. 4B-D). Meanwhile, acting ring formation were also restrained (Fig. 4E and F).

To investigate potential mechanisms of the inhibitory function of Kadsurenone on BMMs differentiation. RT-PCR (Fig. 5A) and RT-qPCT (Fig. 5B) assay were performed. Results indicated that the expression of osteoclast marker genes Ctsk, Trap, and nuclear factor of activated T cells 1 (Nfatc1) were all inhibited by Kadsurenone. Luciferase assay indicated that the transcription factors NF-κB and Nfatc1 activities were all attenuated (Fig. 5C).