DATS/DADS/DAS (Helin Co., Ltd., China) was isolated from garlic extract (Helin Co., Ltd.) with the purity of 97% as determined by HPLC. It was dissolved at a concentration of 1 M in 100% DMSO as a stock solution, stored at −20°C, and diluted with culture medium before each experiment to a final DMSO concentration of 0.1%. IL-15 medium (Gibco, Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) was supplemented with 2% penicillin/streptomycin. Heat-inactivated fetal bovine serum (FBS) was obtained from Sijiqing Biotech Co., Ltd. (Hanzhou, China). Human platelets were purchased from Blood Center of Jiangsu, China. Thromboxane B2 (TXB2) and 6-keto-PGF1α radioimmunoassay (RIA) kits were purchased from Beijing North Institute of Biological Technology (Beijing, China). Transwell filter discs (8 µm) for migration assay by Corning were from Fisher Scientific (Nepean, ON, Canada). Rat tail collagen was prepared by the Galenical Pharmacy Institute of Nanjing University of Chinese Medicine, Nanjing, China. Recombinant human TGF-β1 protein was from PeproTech (Princeton, NJ, USA) and human TGF-β1 neutralizing antibody was from R&D Systems (Minneapolis, MN, USA).

The MDA-MB-231 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and was grown to a monolayer culture in IL-15 medium supplemented with 10% heat-inactivated FBS, penicillin/streptomycin at 37°C with 5% CO₂. The cells were not used >15 to 20 passages after the initiation of culture.

Freshly drawn venous blood from healthy volunteers was collected into 130 mM aqueous trisodium citrate anticoagulant solution (9:1). The donors claimed to not have taken drugs known to interfere with platelet functions during 2 weeks prior to blood collection and gave their informed consent. This study was approved by the Ethics Commitee of Nanjing University of Chinese Medicine. Citrated blood samples were centrifuged at 150 rpm for 15 min to obtain platelet-rich plasma, followed by a second centrifugation at 1,500 rpm for 15 min to obtain platelet-poor plasma (used as a blank value).

Dissolved DATS was prepared using a stock solution of 10 µM DATS with PBS. In the next step, 50 µl of the stock solution were incubated with 450 µl of platelet-rich plasma for 5 min. Platelet aggregation was then measured in a four-channel aggregometer (Chrono-Log Corporation, Havertown, PA,USA) using the turbidimetric method according to the manufacturer's instructions. Follwoing 5 min of pre-incubation at 37°C, the platelets were stimulated by the addition of ADP (10 µmol/l), PAF (5 nmol/l) or thrombin (0.1 U). The extent of platelet aggregation was determined by the area under the aggregation curve from 0 to 5 min following exposure to the stimulants, the platelet aggregation rate was expressed as a percentage of the area value, and the full platelet aggregation was always expressed as 100%. Due to the fact that DATS exerts a significant inhibitory effect on PAF-induced platelet aggregation, we therefore investigated the concentration-response curve of DATS from 0.01 to 10 µM.

The washed platelets (3.0×10⁸/ml), pre-incubated with DATS at 37°C for 30 min, were treated with PAF (5 nmol/l) for 5 min at 37°C. Incubation was terminated by the addition of 50 µM indomethacin and 2 mM EDTA, the mixture was centrifuged at 14,000 × g for 2 min at 4°C and the TXB2 and 6-keto-PGF1α contents of samples were determined using the [¹²⁵I]TXB2 and [¹²⁵I]6-keto-PGF1α RIA kits.

TGF-β1 levels were detected in the conditioned medium from tissue culture (40 h), washed platelets or platelet-rich plasma using the Quantikine TGF-β1 immunoassay kit (R&D Systems) following the manufacturer's instructions.

Whole-cell lysates were prepared with RIPA buffer containing protease and phosphatase inhibitors. Nuclear and cytoplasmic cell extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). Equal amounts of cell lysates (50 µg) were loaded on 8 or 10% SDS-PAGE and transferred onto PVDF membranes. After the membranes were blocked, they were incubated with monoclonal antibodies against p-Smad, Smad (1:1,000; Cell Signaling Technology, Danvers, MA, USA), GPADH (1:5,000; Bioworld Technology, Louis Park, MN, USA) followed by incubation with horseradish peroxidase-conjugated IgGs (1:10,000; Bioworld Technology). Target proteins were developed with an ECL detection agent (Millipore, Braunschweig, Germany) and visualized with the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

The MDA-MB-231 cells (5×10⁵) were seeded into a 6-well plate and allowed to grow to a confluent monolayer in complete medium. The medium was replaced with serum-free medium containing 1×10⁷ platelets treated with PAF (5 nmol/l) for 1 h at 37°C. The monolayers were disrupted (i.e., wounded) with P200 micropipette tips and any cellular debris present was removed by washing with sterile PBS.

Cell monolayers were then incubated with the medium containing various concentrations of DATS for 24 h at 37°C. Images of the exact wound areas were acquired using an inverted microscope (Zeiss, Jena, Germany) and the number of cells in the scraped zone of each well was counted at the indicated time points using an inverted microscope (Zeiss) (0 and 24 h after scraping). The number of cells in the scraped zone of each well was counted 3 times and the counts were averaged.

Cell motility was tested in a Transwell Boyden chamber (Corning Costar, Cambridge, MA, USA) using a polycarbonate filter (8 µm pores). The MDA-MB-231 cells (3×10⁵) resuspended in 90 µl IL-15 medium medium containing various concentrations of DATS were carefully transferred into the upper chamber. The lower chamber was filled with 600 µl 10% FBS medium containing 3×10⁷ PAF-activated platelets to attract cells in the upper chamber. The Transwell Boyden chamber was then incubated at 37°C for 6 h. After the gentle remo val of the filter from the chamber, the cells on the upper side of the filter were removed by wiping with a cotton swab. The filter was fixed with 5% glutaraldehyde at 40°C for 10 min and stained with 0.1% crystal violet stain solution (c0121; Beyotime, Shanghai, China). The cells on the lower surface of the filter, which penetrated the pore of the filter, were fixed onto a glass slide. Cells in 5 randomly selected microscopic fields (using an inverted microscope; Zeiss) (magnification, ×400) of the lower surface were counted. This experiment was performed independently 3 times.

In vitro invasion assay was performed under the same conditions as the Transwell chamber motility assay except that the upper surface of the filter was coated with rat tail collagen. Rat tail collagen was maintained in a stock solution of 5 mg/ml and stored at -20°C. The rat tail collagen was mixed with 10× IL-15 medium and 1 M NaOH at a ratio of 1.37:0.22:0.1 at 40°C. A total of 70 µl of the complex was then added to the upper chamber and incubated at 37°C for 30 min. An additional 100 µl of IL-15 medium was added to the surface of the collagen and was incubated at 37°C for a further 30 min, after which the medium was removed. The MDA-MB-231 cells (3×10⁵) in 90 µl medium treated with various concentrations of DATS were carefully transferred onto the collagen in the upper chambers. The lower chamber was filled with 600 µl medium supplemented with 10% FBS to attract cells in the upper chamber. Following 24 h of incubation at 37°C, the filter of the chamber was gently removed and the cells on the upper side of the filter were wiped. The filter was then fixed with 5% glutaraldehyde at 4°C for 10 min and stained with 0.1% crystal violet staining solution. The stained cells were counted in 5 randomly selected microscopic fields (magnification, ×400). This experiment was performed independently 3 times.

The results were analyzed using a two-tailed Student's t-test using SPSS 11.0 software (Aspire Software International, Leesburg, VA, USA) and thye results were considered significant between two samples at a value of P<0.05.

To examine the effects of activated platelets on tumor cell migration and invasion, we used PAF as a platelet agonist. The MDA-MB-231 human breast cancer cells were incubated with activated platelets for 24 h and the levels of TGF-β1 in the platelet-tumor cell system were measured by ELISA. The results revealed that the activated platelets rather than PAF or resting platelets (RP) promoted the release of TGF-β1 in MDA-MB-231 cells (Fig. 1A and B). Moreover, the activated platelets induced the horizontal (Fig. 1C) and vertical migration (Fig. 1D and E) and invasion (Fig. 1F and G) of MDA-MB-231 cells, all of which were attenuated in the presence of the TGF-β1 neutralizing antibody. These results indicate that only activated platelets have the potential to trigger the malignant biological behaviors of tumor cells and the release of TGF-β1 plays an essential role in facilitating these malignant behaviors in the platelet-tumor cell system.

The number of sulfur atoms in allyl-sulfides is an important factor to determine the chemical and biological activities of garlic-derived organosulfides. As shown in Fig. 2A, DATS has more sulfur atoms compared with diallyl sulfide (DAS) and diallyl disulfide (DADS). In Fig. 1B, we demonstrated that MDA-MB-231 cells exposed to activated platelets stimulated by PAF secreted increased pro-metastatic factor TGF-β1. To this end, various concentrations of DATS (0–20 µM) and 10 µM DADS/DAS were added to the activated platelet-tumor cell system and incubated for 24 h at 37°C. It was found that DATS attenuated the activated TGF-β1 level in the cell culture supernatant in a dose-dependent manner. However, 10 µM DADS/DAS had no obvious effect on the release of TGF-β1 (Fig. 2B).

Since the level of TGF-β1 was decreased following treatment with DATS, we hypothesized that the MDA-MB-231 cells stimulated with the activated platelets would yield a net decrease in metastatic potential when treated with DATS. Indeed, we found that 10 µM DATS inhibited the horizontal (Fig. 2C) and vertical migration (Fig. 2D) and invasion (Fig. 2E) of MDA-MB-231 cells induced by activated platelets using wound healing and Transwell Boyden chamber assays. Furthermore, the addition of exogenous rTGF-β1 resulted in a reversed effect on the inhibition by DATS (Fig. 2C–F). Of note, it was shown that 10 µM DATS also suppressed the phosphorylation of Smad, a pivotal molecule of EMT that is closely associated with metastasis, induced by activated platelets and 10 ng/ml rTGF-β1 reversed this effect (Fig. 2G).

To determine whether DATS influences platelet function in the blood circulatory system, the platelets were incubated with various concentrations of DATS while being stirred. DATS inhibited platelet aggregation in a dose-dependent manner. Treatments of the cells with 5 and 10 µM DATS with the platelets for 10 min decreased platelet aggregation by 55 and 65% (Fig. 3A and B), respectively.

Thromboxane A₂ (TXA2) and prostacyclin I2 (PGI2) are two metabolites that are associated with platelet activation. An RIA kit was used to examine the effects of DATS on the levels of TXA2 (measured as TXB2), PGI2 (as 6-keto-PGF1α) and the thrombogenic ratio (TXB2/6-keto-PGF1α) of platelet excreta in human platelets activated by PAF. Significant changes in the levels of TXB2 and 6-keto-PGF1α and the TXB2/6-keto-PGF1α ratio were observed in the presence of DATS. DATS significantly reduced the level of TXB2, and increased the level of 6-keto-PGF1α in a dose-dependent manner, leading to a marked decrease in the TXB2/6-keto-PGF1α ratio (Fig. 3C–E).