Danshensu Attenuated Epithelial-Mesenchymal Transformation and Chemoresistance of Colon Cancer Cells Induced by Platelets

Background : The interactions between platelets and tumor cells are well-known to play important roles in the progression of malignant tumors. Danshensu, a main water-soluble component of Salvia miltiorrhiza , can resist platelet aggregation and exert significant anti-tumor effects on various types of tumors. However, whether Danshensu could inhibit the progression of malignant tumors by suppressing the activities of platelets had not been reported. Methods : The effects of Danshensu on the platelet activity and epithelial-mesenchymal transformation (EMT)-like invasive phenotype of SW620 colon cancer cells were assessed by stimulating with the supernatants from co-cultured platelets and SW620 cells with direct contact (SCP). The expression and secretion of proteins were determined by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Hematoxylin and eosin (H&E) staining was performed to analyzed the histopathology of tumor tissues and immunohistochemical staining was conducted to examine the protein expression in tumors. Results : Co-incubation of SW620 cells with platelets directly or SCP both generated long spindle-shaped invasive phenotype. Pretreatment of platelets with Danshensu (25 µ M) inhibited the morphological changes of SW620 cells induced by SCP, which was associated with the inhibitory effects of Danshensu on platelet secretion. Danshensu diminished the secretion of a list of biological factors in SCP, including interleukin (IL)-6, tumor necrosis factor alpha (TNF- α ), IL-1 β and vascular endothelial growth factor (VEGF) that are all involved in tumor cell EMT and chemoresistance. Moreover, Danshensu up-regulated the expression of E-cadherin but down-regulated the levels of N-cadherin and Vimentin, resulting in the repression of SW620 cell migration. It was also shown that Danshensu enhanced the sensitivity of SW620 cells to oxaliplatin by suppressing the expression of MDR1. Furthermore, Danshensu could not only reduced the growth of subcutaneous tumors and liver metastasis that induced by SCP, but also down-regulated the expression of MDR1 in vivo . Mechanistic studies revealed that Danshensu suppressed the activation of the TGF- β /Smad signaling pathway. Conclusions : Danshensu attenuated EMT-like characteristics and chemoresistance by inhibiting secretion capability of platelets and activation of the TGF- β /Smad signaling pathway, suggesting that it may be optimized to be a therapeutic agent for fighting against colon cancer.


Introduction
Platelets have been associated with the progression of numerous types of solid tumors and poor prognosis in clinical practice [1]. Interestingly, there is a pathogenic feedback loop between platelets and tumor cells. In other words, tumor cells can activate platelets that play crucial roles in promoting the progression of cancer, including tumor growth, angiogenesis and metastasis [2]. Besides, there is a significant correlation between metastasis occurrence and the presence of superabundant platelets, which has often been found in colon cancer [3]. Tumor cells can activate platelets via a series of specific pathways involving integrin αVβ3 [4], FcγRIIa [5], ADP, CLEC-2 and podoplanin [6]. Moreover, aspirin exerts therapeutic and prophylactic effects on colon cancer metastasis potentially by inhibiting platelet activity [7]. Overall, suppressing platelet activation provides an effective and efficient route for the treatment of tumors [5,8].
Salvia miltiorrhiza has been employed to treat cardiovascular and malignant diseases for a long time [9]. Danshensu is one of the main active ingredients from Salvia miltiorrhiza that is traditional Chinese medicine widely used for antiplatelet aggregation and anti-tumor therapy [10]. As suggested by the screening of antiplatelet components from the aqueous extract of S. miltiorrhiza, Danshensu could significantly reduce platelet aggregation induced by multiple factors [11]. A large number of clinical and preclinical studies demonstrated that platelets could promote the develop-ment of tumor cells, and classic antiplatelet drugs including aspirin was able to prohibit tumorigenesis and metastasis [12]. In addition, we previously reported that Danshensu played an important role in inhibiting metastasis in spontaneous and experimental melanoma metastasis model as well as in non-small cell lung cancer models [13,14]. Moreover, Danshensu suppressed the progression of non-small cell lung cancer via inhibiting COX-2 activity and further attenuated metastasis in vivo. Of note, Danshensu could directly exert its inhibitory effect on tumor cell growth only at high concentration (100 µM Danshensu had a significant inhibitory effect on A549 cell proliferation after 48 h treatment) [14]. Although Danshensu was capable of inhibiting metastasis, whether the anti-metastatic activity was mediated by platelets remained unclear. In the present study, we aimed to clarify the role of Danshensu in tumor progression and to further elucidate whether it was associated with the inhibition of platelet activity.

Preparation of Platelets and Supernatant
Platelet-rich plasma for research use only was provided by Jiangsu Province Blood Center (Nanjing, China). Platelets were prepared as described previously [15]. The platelets (2 × 10 8 /mL) were then exposed to colon cancer cells (1 × 10 6 /mL) or thrombin (0.5 U/mL) for 15 min following treatment with Danshensu or solvent (basal medium) for 30 min at 37°C. The supernatant was filtered using a sterile 0.22-micron filter. Before incubation with the SW620 cancer cells, 10% FBS was added into the supernatants. The supernatants from the co-cultured platelets and colon cancer cells with direct contact (SCP) and the supernatant of platelets activated by thrombin (STP) were collected according to the protocol as described previously and stored at -80°C freezer (Supplementary Fig. 1). This study was approved by the Institutional Ethics Committee of Nanjing University of Chinese Medicine and conducted in accordance with the principles of the Helsinki Declaration. All patients have provided written informed consent before sample collection and subsequent analysis.

Cell Player™ Cell Morphology Monitoring
SW620 cells were incubated with platelets, STP or SCP collected by centrifugation and filtration. The cells were maintained in the supernatants and observed every 1 h by IncuCyte ZOOM™ Live Content Imaging System (Essen BioScience Inc. (Essen) IncuCyte™, Michigan City, Indiana, USA).

Detection of Multiple Inflammatory Factors in Supernatants
The levels of VEGF, IL-6, IL-1β and TNF-α in the supernatants were measured by ELISA kits (Peprotech, Rocky Hill, NJ, USA) according to the manufacturer's instructions (n = 3).

ATP Measurement
ATP levels in the supernatants were assessed using an ATP assay kit (Beyotime Biotechnology, China) (n = 3). In brief, the platelets (2 × 10 8 /mL) were exposed to colon cancer cells (1 × 10 6 /mL) or thrombin (0.5 U/mL) for 15 min after treatment with Danshensu or solvent (basal medium) for 30 min at 37°C. The supernatants were filtered using a sterile 0.22-micron filter after centrifugation. ATP detection working solution was added to a black 96-well culture plate and was incubated for 3 minutes at room temperature. Then, the supernatants were added to the wells, and the luminescence was measured immediately. The concentration of ATP in the sample was calculated based on the standard curve.

Cell Migration Assay
24-well Transwell migration chambers with 8 µm pore size were purchased from Corning incorporated (Kennebunk, ME, USA). Briefly, the treated cells were resuspended in 200 µL serum-free culture medium (1 × 10 6 /mL). Then, 800 µL of complete medium containing 10% FBS was supplemented to the lower chamber. After 24 h, the non-migrated and non-invasion cells were removed by a wet cotton swab. The migration and invasion of the SW620 cells were assessed using 0.1% crystal violet. The SW620 cells were counted in five randomly selected fields, and images were obtained under a microscope (200×) (Leica Microsystems, Watzlar, Germany). The number of cells was calculated through the membrane, and then the migration scores of SW620 cells were examined (n = 3).

Colony Formation Assay
The SW620 cells were cultured in DMEM with 10% FBS in the 6-well plates, and 500 SW620 cells were seeded into each well of the plates (n = 3). The plates were incubated at 37°C for 14 days, and the colony formation of the cells was assessed using 0.1% crystal violet, after which the formed colonies were photographed (Leica Microsystems, Watzlar, Germany).

Histopathological and Immunohistochemical Staining
All tissue specimens were embedded in paraffin according to standard histological procedures, sectioned (4 µm), and stained with hematoxylin and eosin (H&E) or MDR1 antibody (Cell Signaling Technology, 1:500) as described previously [16]. Then, the sections were examined and quantitatively analyzed using PerkinElmer MANTRA microscope (Mantra, PerkinElmer, USA). The mean DAB staining intensity was quantified by Mantra quantitative pathology workstation (Mantra, PerkinElmer, USA) (n = 4).

Detection of ALT and AST in Serum
The whole blood sample was placed at room temperature for 2 h, centrifuged at 2500 R/min for 10 min under the condition of 4°C, and the upper serum was separated. The full-automatic biochemical analyzer (Hitachi 7020) was used. After debugging, according to the glutamic oxaloacetic transaminase determination kit and alanine aminotransferase assay kit (Wako Pure Chemical Industries, Ltd, R1: el157; R2: el158; R1: ej715; R2: eh029) (n = 4).

Animal Study
All procedures were approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine, and performed in accordance with the use of laboratory animal guidelines of Nanjing University of Chinese Medicine (Nanjing, China, Approval No. ACU170905). The SW620 cells were pre-incubated in the SCP or the supernatants collected from co-cultured tumor cells and platelets that treated with 25 µM Danshensu for 24 h, after which the residues were removed by centrifugation. Six-week-old male BALB/c nu/nu mice were supplied by Model Animal Research Center of Nanjing University (Nanjing, China) and housed under specific pathogenfree (SPF) conditions. Subsequently, 2 × 10 6 SW620 cells suspended in a mixture of 100 µL PBS and 100 µL Matrigel (BD Bioscience, USA) were subcutaneously injected into each nude mouse. Control animals were subcutaneously injected with untreated SW620 cells. One week later. The mice were intraperitoneally administered with 3 mg/kg OXA on a weekly basis for 4 weeks. The length and width of tumors were measured using a digital caliper every 4 days for 28 days following tumor cell injection. The mice were sacrificed on day 28 after subcutaneous injection of SW620 cells, and the tumor and liver tissues were fixed in formalin and prepared for histopathological examination.

Statistical Analysis
The data were represented as mean ± standard deviation (SD) and statistically analyzed using one-way ANOVA or Student's T-test. All results were analyzed by SPSS 22.0 software (SPSS Inc., Chicago, USA). p < 0.05 was considered statistically significant.

Platelet-Colon Cancer Cell Interactions Played a Pivotal Role in EMT-Like Invasive Phenotype
It has been well accepted that platelets can induce EMT-like transition and promote metastasis in vitro [17]. We observed the morphological changes of SW620 cells by IncuCyte Zoom, a real-time dynamic imaging system. SW620 cells underwent EMT-like morphological changes following the treatment of SCP, STP or platelets for 24 h (Fig. 1A). SW620 cells incubated with SCP had pronounced EMT-like phenotypic changes compared with those incubated with STP. Further, the expression of E-cadherin was down-regulated while the levels of N-cadherin and Vimentin in the SW620 cells were significantly up-regulated following the treatment of SCP for 24 h compare with control (Fig. 1B,C). These data indicated that SW620 exerted significant effects on activating secretion ability of platelets and platelet secretion stimulated the formation of an EMTlike invasive phenotype.

Danshensu Inhibited Platelet Activation Mediated by Colon Cancer Cells and Prevented EMT-Like Invasive Phenotype
Danshensu was reported to suppress platelet adhesion and aggregation by inhibiting cyclooxygenase-2 (COX-2) and thromboxane B2 [10] (Fig. 2A). We first explored the effects of Danshensu on the proliferation and membrane permeability of SW620 cancer cells by MTT and LDH re- lease assays (Supplementary Fig. 2 and Fig. 2B). Strikingly, 25 µM Danshensu attenuated the ATP secretion from platelets induced by SW620 colon cancer cells, whereas 25 µM Danshensu did not have obvious impacts on the cytotoxicity of SW620 cells (Fig. 2B,C, Supplementary Fig.  2). Subsequently, we observed the morphological changes of SW620 cells after incubation with SCP and Danshensu using IncuCyte Zoom, and detected the expression levels of EMT-related proteins by Western blot assay (Fig. 2D). After SW620 cells were stimulated with SCP, the invasive ability of the cells, as well as the expression levels of N-cadherin and Vimentin were all increased (Fig. 3A,B). However, the levels of E-cadherin were up-regulated while the expression of N-cadherin and vimentin were significantly downregulated in Danshensu treated group compared with those in the SCP group (Fig. 3C,D). Taken together, the EMT phenotype was reversed in the presence of Danshensu. We next explored the effect of Danshensu on cell migration using Transwell assay in the SW620 cells. The results showed that the platelets treated with Danshensu was able to attenuate the migration of SW620 cells induced by SCP (p < 0.001, Fig. 3A,B).

Danshensu Diminished SCP-Induced Clonogenic Survival and Chemoresistance
Chemoresistance is a common problem in cancer treatment. Fischer et al. [18] reported that EMT phenotype increased tumor cell chemoresistance, eventually leading to tumor development and difficulty in cancer treatment. OXA is used for colorectal cancer treatment, but the development of chemoresistance is inevitable in clinical practice [19]. Drug efflux protein MDR1 is a main marker for the drug chemoresistance in the colorectal cancer cells [20]. Therefore, we examined the role of SCP in the chemoresistance of SW620 cells. Compared with the control group, SCP group significantly increased the clonogenic survival of SW620 cancer cells. However, prophylactic adminis-tration of Danshensu onto platelets significantly decreased the clonogenic survival (p < 0.001, Fig. 4A,B). MTT assay showed that the resistance of SW620 cells to OXA was increased following the stimulation of SCP (Fig. 4C). Nevertheless, intervening with platelets with Danshensu alleviated SCP-induced chemoresistance and MDR1 protein expression ( Fig. 4C-E).

Danshensu Antagonized EMT and Chemoresistance Through Limiting Tumor Cell-Induced Platelet Secretion and Repressing the Activation of TGF-β Signaling Pathway
It has been known that platelets contain numerous growth factors and cytokines, and the secretions of cocultured tumor cells and platelets play a crucial role in the process of EMT. Notably, we previously reported that a variety of secreted factors from platelets were involved in the malignant progression of tumor cells [15]. In the present study, we examined the levels of various cytokines in the supernatants by ELISA assay. Our results demonstrated that the levels of IL-6, TNF-α, IL-1β and VEGF were increased following the co-culture of tumor cells and platelets. We uncovered that Danshensu administration inhibited the production of TNF-α, IL-6, and IL-1β in the co-cultured tumor cells and platelets, which was associated with the inhibition of the TGF-β/Smad signaling pathway (Fig. 5). Additionally, inactivation of the TGFβ/Smad signaling pathway suppressed EMT and chemoresistance. To reveal the mechanisms underlying Danshensu limited EMT and chemoresistance, we further investigated whether the TGF-β/Smad signaling pathway was regulated by SCP. Compared with those in the control group, the levels of TGF-β, p-Smad2 and p-Smad3 in the SW620 cells were significantly increased in the SCP group (Fig. 5E,F). More interestingly, prophylactic administration of Danshensu onto platelets decreased the levels of TGF-β, p-Smad2, and p-Smad3 in the SW620 cells (Fig. 5E,F).

Danshensu Retarded the Progression of SCP-Stimulated SW620 Tumors in vivo
To demonstrate the role of Danshensu in the progression of SW620 tumors in vivo, SCP-stimulated SW620 cancer cells were subcutaneously injected into mice, and the growth of tumors after intraperitoneal administration of oxaliplatin (OXA) was monitored accordingly (Fig. 6A). It was observed that SCP-stimulated SW620 cancer cells were insensitive to OXA and the volume of SCP-stimulated tumors were significantly increased compared to that of the control group (Fig. 6B). However, prophylactic administration of Danshensu boosted the sensitivity of SW620 cancer cells to OXA, and the tumor volume was strikingly decreased following the intervention of Danshensu compared with SCP alone (p < 0.01, Fig. 6B). Moreover, all mice in the SCP group displayed remarkable liver metastasis and the percentage of liver metastasis was significantly upregulated compared with that of control group (p < 0.001, Fig. 6C,D). Surprisingly, prophylactic administration of Danshensu onto platelets prevented the liver metastasis of SCP-stimulated SW620 cancer cells (p < 0.001, Fig. 6C,D). Additionally, MDR1 levels in the primary tumors were detected by immunohistochemistry. It was revealed that the expression of MDR1 was profoundly impaired following the treatment of Danshensu, in comparison to SCP alone (p < 0.001, Fig. 6E,F). In addition, liver metastasis can cause the increase of ALT and AST (Fig. 6G,H). Therefore, SW620 cells incubated with SCP can induced chemoresistance and facilitated metastasis in vivo, which could be reversed in the presence of Danshensu.

Discussion
Platelets are small, enucleated cells which are abundant in blood, participating in various physiological and pathological processes of organisms, such as hemostasis, wound healing, inflammatory response, thrombosis, organ transplant rejection and tumor development [21]. Platelet granules, including α-granules and dense granules, store hundreds of mediators that essentially regulate platelet functions and secrete them after platelet activation to be involved in disease development [22]. In tumor microenvironment, platelets and their granular contents influence cancer progression, and result in abnormal secretion of platelets [23]. Labelle et al. [17] reported that direct platelet-tumor cell contact synergistically promoted tumor cell transition to an invasive mesenchymal-like phenotype. However, whether such EMT phenotype is induced by direct contact between platelets and tumor cells or secretions from the co-cultured platelets and tumor cells remains elusive. Our study compared the results of direct incubation of tumor cells with platelets to those of incubation with SCP based on real-time dynamic monitoring of tumor cell morphologies. Interestingly, SW620 cancer cells underwent obvious EMT-like morphological changes after the stimulation of SCP compared with the control. In addition, the impacts of platelet secretion on tumor cells was evaluated using thrombin as a platelet activator. Given that thrombin-activated platelet secretion also promoted tumor cell EMT, inhibition of platelet secretion might play a pivotal role in suppressing EMT process.
As an aromatic carboxylic acid, Danshensu is one of the most abundant active phenolic acids in the dried root of S. miltiorrhiza Bunge (Lamiaceae), which is a widely used traditional Chinese medicine. The effects of Danshensu on platelet aggregation and thrombosis have been well documented [10], and it exerts potential antitumor and anti-angiogenic effects by inhibiting platelet adhesion and promoting tumor microcirculation [13,24]. Our group also demonstrated the inhibitory effects of multiple components from S. miltiorrhiza on tumor progression [25]. More specifically, Danshensu significantly prevented tumor metastasis in vivo [14]. Although Danshensu presented remarkable antiplatelet and antitumor effects, whether it was involved in platelet-mediated development of malignant tumors still required in-depth studies.
In the present study, we found that Danshensu inhibited the effects of SCP on the EMT and migration of tumor cells by intervening with platelets (Fig. 3), and the secretions of VEGF, IL-6, IL-β and TNF-α in the co-cultured platelets and SW620 were reduced after Danshensu intervention (Fig. 5). Additionally, Danshensu prohibited SCPinduced tumor cell chemoresistance, and the expression of MDR1 was decreased after the treatment of Danshensu compared with the SCP group (Fig. 4).
It was reported that EMT enhanced the chemoresistance of tumor cells [18]. The lever of TGF-β was shown to be closely correlated with EMT, chemoresistance and poor survival outcomes of colon cancer patients [26]. In our study, TGF-β was decreased after Danshensu treatment compared with SCP group. Of note, the expression of TGF-β was required for the development of cancer, and the decreased level of TGF-β was related to Danshensu inhibiting platelet-mediated EMT and chemoresistance in the SW620 cancer cells. The TGF-β/Smad signaling pathway participated in the regulation of cell prolif-eration, survival, angiogenesis and chemoresistance to antitumor therapy [27]. In addition, the Smad2/3 phosphorylation also dramatically contributed to the induction of EMT phenotype of cancer cells [28]. We herein found that the phosphorylation of Smad2/3 was significantly attenuated after following the treatment of Danshensu compared with SCP alone (Fig. 5). We also uncovered that the coculture supernatant-stimulated tumor cells showed conspicuous chemoresistance both in vitro and in vivo (Figs. 4,6). Besides, the expression of MDR1 was significantly increased in the primary tumors compared with control group. However, the intervening effects of Danshensu on platelets reduced the chemoresistance of SW620 cancer cells mediated by SCP, which was consistent with what we observed in vitro (Fig. 6). Notably, the supernatant-stimulated tumor cells were more prone to metastasize to livers in vivo. Nevertheless, in the presence of Danshensu, the metastatic foci in the liver were significantly reduced compared with control (Fig. 6). Furthermore, Danshensu contributed to profound decrease in the growth of tumors and the expression of MDR1 in tumors following the treatment of OXA.
Aspirin is currently used in clinical tumor prevention and incorporated into clinical oncology guidelines due to its striking antiplatelet activity. Moreover, it was also documented to inhibit platelet-induced EMT of circulating tu-  mor cells [29]. Since aspirin could significantly inhibit the malignant development of colon cancer in the case of longterm and low-dose administration, antiplatelet activity was thought to be extremely important for the adjuvant treatment of malignant tumor development [30]. Targeted inhibition of platelet activity has certain inhibitory effects on the EMT and chemoresistance of tumor cells [31,32].
In our study, although 25µM of Danshensu could not directly inhibit the invasion of SW620 cacner cells, it was able to interfere with the activation of platelets by SW620 cancer cells and achieved the effects of reducing plateletinduced SW620 cancer cell migration and chemoresistance. The pretreatment of 25 µM Danshensu attenuated the release of IL-1β, IL-6, VEGF and TNF-α in the co-cultured platelet-tumor cell system, which reduced the effect of platelets on EMT and drug resistance of tumor cells. The underlying mechanism that Danshensu interfered with the interactions between platelets and tumor cells may be associated with the decreased activation of TGF-β/Smad signaling pathway. Therefore, Danshensu may emerge as an effective compound that potentially exerts anti-tumor activity through interfering with platelets.

Conclusions
In conclusion, our study showed that Danshensu resulted in the reduced production of a series of proinflammatory cytokines (e.g., IL-1β, IL-6, TNF-α and VEGF) stimulated by tumor cells. These pro-inflammatory factors could promote the EMT process of cancer cells, leading to the progression of cancer cells. Taken together, Danshensu suppressed tumor metastasis by inhibiting the interactions between platelets and tumor cells, as well as blocking the TGF-β/Smad signaling pathway induced by SCP (Fig. 7).

Author Contributions
AW and YZ conceived, designed and led the project. YC, KL, YX and YW performed the experiments. YC and AW analyzed the data. AW contributed reagents, materials, and analysis tools. YZ and YC wrote the manuscript with input from all authors. All authors have read and approved the final manuscript.