DADS was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The following antibodies were obtained: Rabbit anti-uPA primary antibody (cat. no. ABO10669; Abgent, Inc., San Diego, CA, USA); Rabbit anti-MMP-9 primary antibody (cat. no. ab73734; Abcam, Cambridge, UK); Mouse anti-TTP and mouse anti-β-actin primary antibodies (cat. no. SAB4200565 & A1978; Sigma-Aldrich; Merck KgaA, Darmstadt, germany.); horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG secondary antibodies (cat. nos. 70-GAR0072 & 70-GAM007; Wuhan Boster Biological Technology, Ltd., Wuhan, China). The bicinchoninic acid (BCA) protein assay and other reagents were obtained from Kangwei Biotechnology (CWBiotech, Beijing, China). Transwell^® inserts were purchased from Corning Incorporated (Corning, NY, USA). MCF-7 and MDA-MB-231 human breast cancer cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco by Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified atmosphere containing 5% CO₂.

A total of 18 4-week-old female nude mice (weighing 16–18 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All mice were kept in a barrier facility under high-efficiency particulate air filtration and maintained in a specific pathogen-free environment with shavings as bedding. The cages and bedding were sterilized at 121°C for 20 min. All mice were allowed free access to food and water, the feed was purchased from Ke'aoxieli Feed Co. Ltd (Peking, China), drinking water was sterilized by ⁶⁰Co irradiation. Approximately 1×10⁷ MDA-MB-231 or MCF-7 cells were subcutaneously injected into the subcutis of the right axilla of each mouse. Tumor volume (mm³) was examined every 4 days and calculated using a standard formula [width² × (length/2)]. When the tumor volume reached 80 mm³, the mice were divided into two groups. The control group used saline as vehicle treatment while the treatment group received 50 mg/kg DADS via intraperitoneal injection every 2 days. After 28 days, the xenografts were removed. The tumor weights were measured in both groups. All experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Ethics Committee of University of South China (Hengyang, China).

Tumor tissues (control group and DADS-treated group) were cut into 0.5-mm³ sections, fixed using 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and further cut into 5 µm thick tissue sections. The tissue sections were dewaxed in xylene and rehydrated in a descending (100, 95, 90, 80 and 75%) ethanol series, 3–5 min each time. Following washing with PBS, the sections were stained with hematoxylin and following a second wash, the sections were differentiated. The sections were subsequently stained with eosin following washing. Following dehydration with ethanol, the sections were mounted, and observed using light with a ×40 microscopy objective (Eclipse E200; Nikon Corporation, Tokyo, Japan).

The tumor tissues and cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (1:100; Thermo Fisher Scientific, Inc.), sonicated and clarified by centrifugation at 12,000 × g, 15 min at 4°C. All groups of protein were quantified using a bicinchoninic acid protein assay and equal amounts of total protein extracts (30 µg/well) were separated by 8–12% SDS-PAGE. Following electrophoresis for 2 h at 100 V, the proteins were electrically transferred onto a polyvinylidene fluoride membrane. The membranes were then blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with primary antibodies against TTP (1:500), uPA (1:1,000), MMP-9 (1:1,000) or β-actin (1:1,000) at 4°C overnight. Thereafter, the membranes were washed three times with TBST (20 mmol/l Tris base, pH 7.6, 150 mmol/l NaCl and 0.1% Tween-20), incubated with the HRP-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies for 50 min at room temperature, and washed three times with TBST. Protein visualization was performed using the ECL Plus Western Blotting Detection system (Tanon-6200, Tanon Science & Technology Co., Ltd., Shanghai, China) to collect the images and QuantityOne 4.5.0 software (Bio-Rad Laboratoris, Inc., Hercules, CA, USA) for analysis.

A total of 200/well MCF-7 and MDA-MB-231 cells were planted into 9 cm petri dishes. After treating cells with 0, 100, 200 or 400 µM DADS for 24 h (using 0 µM as the control group), cells were washed with PBS three times, then fresh serum-free DMEM was added. After 3 weeks, 5 ml 100% methanol was added to the dishes for 15 min to fix the cells at room temperature. Giemsa was used to stain cells for 20 min at room temperature. Following PBS washing of the cells and air-drying, the clone formation rate was calculated under microscope, clone formation rate (%)=(clone number/plated cell number) ×100%.

For the Transwell assays, 25 µg Matrigel was added to the upper side of porous filters (pore size, 8 µm) and the gel was allowed to form at 37°C for 2 h. Following rehydration of the coated filters with 100 µl medium, 1×10⁶ cells in 100 µl serum-free DMEM supplemented with 0.2% FBS were seeded into the upper part of each chamber, whereas the lower compartments were filled with 500 µl complete DMEM containing 10% FBS to serve as the chemoattractant agent. Cells were incubated at 37°C with 5% CO₂ for 24 h to allow for invasion. At 24 h, cells on the upper surface of the filters were removed using cotton swabs. Cells that had invaded to the lower surface of the filter were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet at room temperature. Images were captured using an epifluorescence inverted microscope (magnification, ×100). The total number of invaded cells was normalized to the number of non-targeting control cells and expressed as fold change.

MCF-7 and MDA-MB-231 cells (2×10⁵/well) were cultured in 6-well plates in DMEM containing 10% FBS. The cell monolayer in each well was scratched with a 200 µl plastic pipette tip to create a linear wound. The monolayer was washed twice with PBS to remove debris and detached cells, and the cells were then exposed to serum-free medium with or without various concentrations of DADS (100, 200 and 400 µM) for 24 h and using 0 µM as control group. The wound areas were then observed using an inverted microscope (magnification, ×100). The migration distance was measured, and migration rates are expressed as the ratio of the treated group value to the control group value.

siRNA duplexes for TTP were designed and produced by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences for TTP siRNA were sense, 5′-UCGCCACCCCAAGUACAAATT-3′ and antisense, 5′-UUUGUAGGGGUGGCGATT-3′. Scramble control RNAi was used as control siRNA, the sequence for the negative control siRNA was 5′-GCAAGCTGACCCTGAAGTT-3′. The TTP and control siRNA were transfected into MCF-7 and MDA-MB-231 cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, MCF-7 and MDA-MB-231 cells (1×10⁵/well) were inoculated into 6-well plates and cultured in DMEM with 10% FBS at 37°C until reaching 70% confluence. Then, 2 µl TTP or control siRNA (0.5 µg/µl) was mixed with 5 µl Lipofectamine 3000. The mixture was added to the cell culture, and cells were incubated for 12 h at 37°C. The cell solution was replaced with DMEM containing 10% FBS and cells were incubated for another 24 h.

The GSE42568 breast cancer microarray dataset was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Using the data from the GEO dataset normal breast (17 samples) vs. breast cancer (104 samples) were analyzed. P<0.05 was considered to indicate a statistically significant difference.

Statistical analysis was performed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA). Each experiment was repeated three times. All results are presented as the mean ± standard deviation or standard error of three independent experiments. The χ² test was applied for enumeration data. Student's t-test (unpaired) and one-way analysis of variance was used to identify statistically significant differences followed by the Student-Newman-Keuls test for further group comparison. P<0.05 was considered to indicate a statistically significant difference.

In breast cancer microarray datasets from the GEO database, a microarray dataset (GSE42568) was identified in which TTP mRNA levels were significantly downregulated in breast cancer compared with in normal breast tissue (Fig. 1A). In the same microarray dataset, uPA and MMP-9 mRNA levels were identified to be significantly upregulated in breast cancer compared with in normal breast tissue (Fig. 1B and C).

Compared with the control group, the mean volume and weight of the tumors from mice in the DADS-treated group were significantly decreased (Fig. 2). Protein was extracted from the xenografts, and western blot analysis was performed to detect the expression of uPA, MMP-9 and TTP. uPA and MMP-9 protein expression levels were significantly downregulated, whereas TTP expression was significantly upregulated in the DADS-treated group compared with the corresponding control groups, suggesting that DADS induced the expression of TTP in breast cancer xenografts (Fig. 3A and B). Furthermore, H&E staining revealed that MCF-7-derived tumors in the DADS-treated group were markedly smaller compared with the control group (Fig. 3C).

To examine the effect of DADS on cellular proliferation, a tablet cloning assay was performed on MCF-7 and MDA-MB-231 cells treated with DADS. It was demonstrated that the clone formation of cells was markedly decreased in a dose-dependent manner following DADS treatment (Fig. 4A). In addition, invasion and migration rates of MCF-7 and MDA-MB-231 human breast cancer cells were significantly decreased following DADS treatment, as determined using the Transwell (Fig. 4B) and wound healing assays (Fig. 5) compared with the untreated control groups.

Western blotting was used to examine the expression of TTP and proteins associated with invasion, including uPA and MMP-9 in breast cancer cells. As presented in Fig. 6, DADS treatment significantly affected the expression of TTP, uPA and MMP-9 in a dose- and time-dependent manner (P<0.05).

Following transfection of MCF-7 and MDA-MB-231 cells with TTP siRNA for 24 h, western blot analysis revealed that TTP siRNA significantly decreased the effect that DADS exhibited on TTP, uPA and MMP-9 expression (Fig. 7). The Transwell assay demonstrated that TTP siRNA significantly reversed the anti-invasive effect of DADS in breast cancer cells (Fig. 8). Furthermore, the wound healing assay revealed that TTP siRNA counteracted the suppressive effect of DADS on breast cancer cell migration (Fig. 9). All the aforementioned results indicate that DADS may inhibit the proliferation, invasion, and migration of breast cancer cells by upregulating TTP while downregulating the expression of uPA and MMP9 (Fig. 10) and TTP may be a novel target of DADS in the progress of breast cancer.