Tanshinone IIA Inhibits Triple-Negative Breast Cancer Cells MDA-MB-231 via G Protein-Coupled Estrogen Receptor- (GPER-) Dependent Signaling Pathway

Due to the lack of classic estrogen receptors, there has been a shortage of targeted therapy for triple-negative breast cancer (TNBC), resulting in a poor prognosis. However, the newly discovered G protein-coupled estrogen receptor (GPER) has been found to be expressed in TNBC cells. Salvia miltiorrhiza (Danshen) is an essential Chinese medicine for gynecological disorders, and its component tanshinone IIA (Tan IIA) exerts an anticancer effect. Therefore, this study attempted to investigate whether GPER is involved in the inhibitory effect of Tan IIA on TNBC. We applied various databases and GO pathway analysis to predict the possible mechanism of Tan IIA. We identified 39 overlapping targets, including c-Jun, c-Fos, and caspase-3, and enriched cell cycle-related pathways. Next, we demonstrated the strong binding ability of Tan IIA to GPER by molecular docking assay. In the subsequent validation tests, Cell Counting Kit-8 (CCK8) assay showed that Tan IIA inhibited proliferation of MDA-MB-231 cells time and dose dependently without affecting normal cells. Using Transwell plate, flow cytometry, and Western blot assays, we showed that Tan IIA inhibited migration and induced apoptosis of MDA-MB-231 dose dependently. Importantly, protein expressions of GPER, epidermal growth factor receptor (EGFR), extracellular regulated protein kinases (ERK), c-Fos, and c-Jun were all decreased by Tan IIA dose dependently. Administration of GPER inhibitor partly abolished these effects. Furthermore, nuclear translocation of c-Fos and c-Jun as well as cell cycle-related proteins was downregulated by Tan IIA dose dependently. In summary, Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce apoptosis, and the possible mechanism may be the regulation of GPER-mediated pathways, suggesting that GPER could be a therapeutic target for TNBC.


Introduction
Triple-negative breast cancer typically occurs in premenopausal women aged 40 to 55 years old and is defined by the absence of receptor estrogen (ER), progesterone (PR), and human epidermal growth factor receptor-2 (HER2) [1]. It is highly aggressive and resistant to chemotherapeutic agents. Approximately 46% of TNBC patients develop distant metastases with a 5-year survival rate less than 30% [2].
It is intractable to treat TNBC due to the lack of a specific targeted therapeutic regimen. GPER is a recently identi-fied novel estrogen receptor. It is expressed in TNBC cells and is closely associated with the development of breast cancer [3]. It was reported that GPER plays a critical role in tumorigenesis through Hippo/YAP/TAZ pathway [4]. In addition, GPER and downstream PKA/BAD signaling maintain the stemness of breast cancer stem cells [5]. GPER could mediate the activation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) [6]. Among them, EGFR is thought to be correlated with multiple types of tumors, and it is upregulated in TNBC with an indication of poor prognosis [7]. In breast cancer cells, GPER agonists were shown to increase survival of tumor cells through activating EGFR/ERK pathway [8].
Traditional Chinese medicine (TCM) has a long history of application in the prevention and treatment of complex diseases [9], and in clinical practice, Salvia miltiorrhiza could be used in breast cancer therapy [10]. It contains a number of diterpene quinone and phenolic acid derivatives, such as Tanshinone, cryptotanshinone, isocryptotanshinone, miltirone, tanshinol, and salviol [11]. Among them, Tan IIA was reported to exhibit a remarkable antibreast cancer effect [12][13][14][15]. Our previous study demonstrated that GPERmediated PI3K/AKT signaling pathways are involved in the inhibitory effect of cryptotanshinone on the proliferation of breast cancer SKBR-3 and MCF-7 [16,17]. In this context, it can be questioned whether GPER is also involved in the antitumor property of Tan IIA.
Network pharmacology is a drug research tool and has been extensively employed in TCM to uncover the molecular mechanisms underlying their efficacy [18]. In the current study, we applied this method to predict the possible mechanisms of Tan IIA against breast cancer cells, and in vitro tests were subsequently performed for validation.

Protein-Protein
Interaction. Common targets were imported into Cytoscape software to map the "componenttarget-disease" interaction network. On the other hand, common targets were also imported into STRING (https:// string-db.org/) [27] to construct a protein-protein interaction (PPI) network model, with the species limited to "Homo sapiens" and a confidence score > 0:4 [28].

Pathway and Functional Enrichment
Analyses. Key target gene GO functional enrichment analysis was performed based on R software using Bioconductor bioinformatics package with P value < 0.05 and Q value < 0.05. Results were output as bar graphs and bubble plots.
2.6. Cell Culture. Human breast cancer cell line MDA-MB-231 (National Infrastructure of Cell Line Resource, Beijing, China) was cultured in L15 glucose medium supplemented with 10% fetal bovine serum at 37°C in a non-CO 2 atmosphere. HEK293 cells (National Infrastructure of Cell Line Resource, Beijing, China) were cultured in DMEM glucose medium supplemented with 10% fetal bovine serum at 37°C in CO 2 atmosphere. 100 μg/mL streptomycin and 100 U/mL penicillin were added to avoid contamination.

Cell Proliferation Assay.
Cells were seeded at the density of 2 × 10 4 cells per well in 96-well plates and grew overnight. MDA-MB-231 cells were treated with Tan IIA at the concentrations of 0, 1, 5, 10, 50, or 100 μM, respectively, for 24 h, 48 h, and 72 h. HEK293 cells were treated with Tan IIA at the concentration of 5, 10, or 50 μM for 48 h. 0.2% DMSO was used as a control. After drug administration, 100 μL CCK8 (1 mg/mL) was added and incubated for 2 h in the dark. Subsequently, absorbance was measured at 450 nm using a multifunctional enzyme marker (Multiskan GO, Thermo Fisher Scientific, USA).

Migration
Assays. Cell migration ability was assayed by Transwell plates (Corning, NY, USA). Cells at logarithmic growth phase were centrifuged and resuspended in medium containing 5% FBS, and the cell density was adjusted to 1 × 10 5 cells/mL. 100 μL of cell suspension was inoculated in the upper chamber of the Transwell, and 750 μL of medium containing 10% FBS was added to the lower chamber. After attached, the cells were incubated with Tan IIA at concentrations of 0, 5, 10, G15+10, or 50 μM in each well. The cells were incubated for 48 h, then the upper chamber was aspirated, and 4% paraformaldehyde (Solarbio, Beijing, China) was added and fixed at room temperature for 20 min. 0.1% crystal violet staining solution (Solarbio, Beijing, China) was added and stained at room temperature for 20 min. The upper chamber was gently rinsed with running water and soaked three times for decolorization and staining, the upper chamber was aspirated, and the residual water in the upper chamber was gently wiped off with a cotton swab. The experiment was repeated three times.
2.9. Apoptosis Rate Analysis. Cells were seeded overnight in 6-well plates at a density of 3 × 100000 per well for adhesion. Changed the medium with 0, 5, 10, G15+10, or 50 μM Tan IIA and incubated for 48 h. Collect cells and added 500 μL 2.11. Molecular Docking. In order to further verify the interaction between active components of Tan IIA and GPER, computer molecular docking technology was carried out. The conformation of GPER was retrieved from the UniProt database. The resulting conformation was a seven-helix structure. Subsequently, the crystal structure of GPER obtained from GPCR-I-TASSER was optimized by PyMOL and AutoDock tool software for the later docking. The structure of Tan IIA is built by PubChem on its chemical structure, respectively. A flexible docking was carried out by AutoDock Vina, and the grid box was covered on the protein to make a blind docking. Visualization of docking results was built using PyMOL and Discovery Studio.
2.12. Statistical Analysis. Data was analyzed using SPSS 20.0. Results of three independent tests were presented as mean ± SD. One-way ANOVA followed by the LSD-t multiple comparison test was performed among multiple groups. Unless stated otherwise, P values < 0.05 were considered significant.

Network Pharmacology Predicted the Possible
Mechanisms of Tan IIA on TNBC. 85 drug targets were retrieved from both SwissTargetPrediction and TCMSP databases, and 1212 TNBC genes were obtained. Overlapping 39 targets were identified (Figure 1(a)) and further imported into Cytoscape software for "component-targetdisease" interaction network construction. Purple color represents Tan IIA, blue color represents 39 common targets, and red color represents the diseases (Figure 1(b)).
Protein-protein interactions were also identified by STRING database, where the size, color, and shade of the nodes represent the degree value. Caspase-3, c-Fos, and c-Jun were selected for further in vitro experiments (Figures 1(c) and 1(d)). 39 common targets were run through R software, and then, GO analysis was performed. The mainly enriched pathways include cell cycle-related pathways (Figure 1(e)).

3.2.
Tan IIA Inhibited MDA-MB-231 Cell Growth. We tested five concentrations and three time points of intervention and found that Tan IIA inhibited breast cancer cells in a dose-and time-dependent manner (Figure 2(a)). Considering the effectiveness of inhibition as well as cell morphology, we chose 5, 10, and 50 μM concentrations of Tan IIA as the low, medium, and high doses and 48 h as the intervention duration for subsequent experiments. To test the toxicity of Tan IIA, we administrated 5, 10, or 50 μM of Tan IIA to HEK293 cells for 48 h. The results showed no significant difference in the proliferation rate ( Figure 2(b)), indicating that selected doses have no obvious toxicity. Next, to demonstrate that the inhibitory effect of Tan IIA is mediated by GPER, we introduced GPER specific inhibitor G15. The inhibitory capacity of Tan IIA was attenuated after addition of G15 in the middle dose group (Figure 2(c)). Figure 2(d) is the representative images of cells.

Tan IIA Inhibited
Migration of MDA-MB-231. We used Transwell assay to assess the effect of Tan IIA on the migration. Cells were treated with 0, 5, 10, G15+10, or 50 μM of Tan IIA and then were allowed to migrate in the Transwell chamber for 48 hours. Tan IIA increased the number of nonimmigrated cells in a dose-dependent manner compared to the control group (P < 0:01), while the number of nonimmigrated cells in the G15+10 μM Tan IIA group was significantly lower compared to that in the 10 μM Tan IIA group (P < 0:01) (Figure 4). These results suggested that inhibited migration of MDA-MB-231 cells by Tan IIA may be mediated by GPER.   Previous study has shown that nuclear translocation of c-Fos and c-Jun affects cell migration [30]. As shown in Figure 6, in MDA-MB-231 cells, the levels of c-Jun and c-Fos proteins in the nucleus were significantly reduced by Tan IIA treatment (Figures 6(a), 6(c), and 6(d)), whereas increased levels of c-Jun and c-Fos proteins were found in cytoplasmic extracts of Tan IIA-treated cells (Figures 6(b), 6(e), and 6(f)).

3.7.
Tan IIA Regulated the Expression of Cell Cycle-Associated Proteins. Based on network pharmacology, we   Tan IIA for 24 h, 48 h, or 72 h, and cell proliferation was tested using CCK8 assay kits; cell inhibition rate = 1cell proliferation rate. * * represents P values < 0.01 compared to 24 h of intervention, ## represents P values < 0.01 compared to 0 μM Tan IIA, △△ represents P values < 0.01 compared to 48 hours of intervention, and n.s represents no statistical difference compared to 48 hours of intervention. (b) Tan IIA did not affect proliferation of normal cells. (c) Addition of the specific GPER inhibitor G15 attenuated the inhibitory effect of Tan IIA on MDA-MB-231 cells. * * represents P values < 0.01 compared to control, and ## represents P values <0.01 compared to 10 μM Tan IIA. (d) MDA-MB-231 cell morphology under inverted microscope (magnification, ×200). Scale bar represents 100 μm. All results were presented as means of three independent tests ± SD. Each independent experiment was repeated six times. 5 Disease Markers tested the expression of proteins associated with G1 and S phases by Western blot. The results showed that following Tan IIA treatment for 48 h, the expressions of cyclin A2, cyclin D1, CDK2, CDK4, and CDK6 were all decreased in a dose-dependent manner (Figure 7).
3.8. Tan IIA Could Bind to GPER. Molecular docking showed that the affinity of Tan IIA-GPER was -8.7 kcal/ mol, which indicates that Tan IIA could strongly bind to GPER (Figure 8(a)). Visualization results showed that Tan IIA could bind to amino acid residues PHE146, THR149, All results were presented as means of three independent tests ± SD. Each independent experiment was repeated three times. 6 Disease Markers TRP150, LEU180, ILE181, LEU221, VAL225, and ILE229 on GPER as hydrophobic bonds and to THR149 and TRP150 on GPER as conventional hydrogen bonds (Figures 8(b)  and 8(c)).

Discussion
In this study, we first used network pharmacology method to preliminary explore the possible mechanism of action of Tan IIA on breast cancer and identified c-Fos and c-Jun as the potential targets. Since our previous study has shown that cryptotanshinone could inhibit MCF-7 and SKBR-3 cells through GPER pathway and c-Fos as well as c-Jun are downstream of GPER, we intend to investigate whether GPER is also involved in the efficacy of Tan IIA on another breast cancer cell line MDA-MB-231. Through molecular docking assay, we found that Tan IIA could bind closely to GPER. Through CCK8, Western blot, flow cytometry, and Transwell tests, we demonstrated that Tan IIA could inhibit the proliferation and migration of MDA-MB-231 cells and induce their apoptosis, and GPER/EGFR/ERK signaling pathway may be involved. One of the main benefits of Chinese medicine is that it has relatively fewer toxic side effects [31][32][33]. In this study, Tan IIA successfully inhibited tumor cells and did not affect the proliferation rate of normal cell HEK293, confirming the advantages of Chinese medicine at the cellular level. The physiological homeostasis between cell apoptosis and proliferation is critical for tumor growth, and here, we demonstrated that Tan IIA could inhibit tumor cells from both sides. Besides, caspase-3, a critical biomarker for apoptosis, was predicted as a target, and the following in vitro test verified that Tan IIA could increase its expression. The important reasons for the poor prognosis of TNBC are its highly aggressive nature, rapid metastasis, and high recurrence rate [34]. Here, we showed that Tan IIA could also inhibit the migration of TNBC cells.
Then, we explore the involvement of GPER. Through extensive Western blot assay, we found that Tan IIA decreased GPER pathway-related proteins, including EGFR and ERK in a dose-dependent manner. Of note, all these effects were attenuated after the addition of GPER specific inhibitor G15, indicating a GPER-dependent mechanism. Indeed, the discovery of GPER brought a new angle to explain the effects of estrogen or estrogenic agents on cell growth and migration, especially for TNBC, in which GPER is overexpressed and associated with high recurrence rate [35]. According to a study, after 36-month follow-up, 90.5% of the TNBC patients with low GPER expression were still alive, while for GPER highly expressed patients, only 77.8% survived [36]. GPER/EGFR/ERK transduction signaling triggers c-Fos and c-Jun expressions [37][38][39], which could induce chromatin-activated protein 1 (AP-1) complex to promote cancer cell migration [40]. According to network pharmacology results, c-Fos and c-Jun are also potential targets The results were means of three independent replicates ± SD. Each experiment was repeated three times. * * P < 0:01 or * P < 0:05 was considered as statistically significant compared with the control group. 9 Disease Markers of Tan IIA. Consistently, the results of the subsequent in vitro experiments validated that Tan IIA decreased the expression of these two proteins as well as their nucleus translocation. Therefore, GPER may be a promising drug target for TNBC.
Furthermore, the network pharmacology and in vitro experiments are consistent with that Tan IIA could influence the cell cycle, especially the G1/S transition. Cell cyclerelated proteins could be potential targets for cancer therapy.  (c-f) The quantification of the two proteins. Data were expressed as mean ± SD of three independent experiments. * P < 0:05 and * * P < 0:01 compared with the control. 10 Disease Markers For instance, CDK4/6 inhibitors, such as palbociclib and ribociclib, have been approved by FDA for the treatment of breast cancer [41,42]. However, for TNBC, these inhibi-tors are ineffective, probably due to the high expression of CDK2, which leads to a greater shift of cells from S phase [43]. We chose G1/S phase-related proteins for Western blot Charts revealed that the protein expression of cyclin A2, cyclin D1, CDK2, CDK4, and CDK6 decreased significantly with the increase of Tan IIA concentration. Data were expressed as mean ± SD of three independent experiments. * P < 0:05 and * * P < 0:01 compared with control.

Disease Markers
test and showed that CDK2, CDK4, CDK6, cyclin A2, and cyclin D1 were all downregulated by Tan IIA. Cyclin A2 dominates the longest period of time in the late G1 phase and could bind to CDK2. Previous study has demonstrated that overexpression of cyclin A2 promotes TNBC proliferation, invasion, and migration while inhibiting apoptosis [44]. Cyclin D1 has been proven as an oncogenic factor since upregulation of cyclin D1 was related to poor cancer prognosis [45]. Taken together, Tan IIA showed significant inhibitory effects on a variety of cell cycle-related proteins.

Conclusions
This study suggests that Tan IIA could inhibit MDA-MB-231 cells. Importantly, these effects may be achieved through GPER/EGFR/ERK (1/2)/c-Fos and c-Jun signaling pathways. Our results provide a basis for applying Salvia miltiorrhiza in clinical practice.

Data Availability
The data used to support the findings of this study are included within the article.  The protein structure is shown in green, blue, and yellow (ribbon); Tan IIA is shown in yellow carbon. The blue lines represent the hydrogen bond between Tan IIA and GPER; the grey line represents the hydrophobic bond between Tan IIA and GPER.