Tanshinone IIA promotes apoptosis by downregulating BCL2 and upregulating TP53 in triple-negative breast cancer

Tanshinone IIA (Tan IIA) was mainly used for cardiovascular disease treatment. Recent studies have demonstrated the role of Tan IIA for tumor treatment, but its mechanism remains unclear. At the first, the inhibitory effect of Tan IIA on 4T1 breast cancer cells was determined by CCK8 and colony formation assay. Then, a 4T1 BALB/c model of breast cancer was established to evaluate the anti-cancer effect of Tan IIA in vivo. Flow cytometry analysis and the TUNEL test were used to detect cell apoptosis in vitro and in vivo, respectively. The related targets and mechanisms of Tan IIA were predicted through network-based systems biology. At last, molecular docking and the molecular biological techniques were used to evaluate the predicted targets. Tan IIA displayed encouraging inhibitory influences on 4T1 cells after incubation for 24 h and showed a half-maximal inhibitory concentration (IC50) of 49.78 μM after 48-h incubation. After 23 days of treatment, the relative tumor volumes in the Tan IIA group were 65.53% inhibited compared with the control group. Furthermore, Tan IIA induced 4T1 cell apoptosis both in vivo and in vitro. The possible targets of Tan IIA for TNBC treatment were predicted with network-based systems biology, and results showed that TP53, NF-κB, AKT, MYC, and BCL-2 were the hub targets. The mechanism against breast cancer may be based on the P53 signaling pathway, the PI3K/Akt pathway, the MAPK signaling pathway, and the mTOR signaling pathways. Molecular docking analysis reveals that Tan IIA has a high affinity for p53, Bcl-2, and NF-κB1; the binding energies were − 6.92, − 6.07, and − 6.28 kcal/mol, respectively. The predicted proteins were further validated using Western blotting. Increased expression of phosphorylated p53 and p53 and decreased expression of Bcl-2 were found in Tan IIA-treated 4T1 cells. Tan IIA is potentially effective for the treatment of 4T1 breast cancer, and the molecular mechanism may be through enhancing the activity of p53 and decreasing Bcl-2 to suppress proliferation and promote apoptosis.


Introduction
Breast cancer is the malignant tumor with the highest incidence rate among women all over the world (Sung et al. 2021). Triple-negative breast cancer (TNBC) is the subgroup Jinfeng Liu, Chang Zhang, and Shuang Liu have contributed equally to this study and share first authorship. lacking ER, PR, and HER2 expression. It accounts for 15% of total breast cancer patients with a high biological aggressive nature and a high rate of proliferation and invasion (Waks and Winer 2019;Arnedos et al. 2012). Despite the improvement of treatment strategies, the prognosis of TNBC patients remains poor. Searching for effective regimens remains the top priority in TNBC research.
As one of the herbs with a long history of application, danshen (Salvia miltiorrhiza) is widely used in cardiovascular diseases. Tanshinone, one of the main lipophilic components of Salvia miltiorrhiza, has been proved to have diverse pharmacological activities including anti-inflammation, antioxidation, and anti-cancer. Tanshinone IIA (C19H18O3, Tan IIA, Fig. 1) is one of the important tanshinones. For the well-reported anti-inflammatory and antiangiogenic activities, the antitumor effects of Tan IIA have received more attention in gastric cancer, colorectal cancer, lung cancer, etc. (Sui et al. 2017;Xu et al. 2018;Wang et al. 2019;Li et al. 2020 Apr). The anti-TNBC effects of Tan IIA have been reported (Wu et al. 2018), but the mechanisms are currently unclear.
Network pharmacology, a new discipline of multi-target drug molecular design, reveals the synergistic effect of Chinese herbal medicine on diseases and its underlying mechanism by constructing a multi-component and multitarget network (Hopkins 2008;Hao and Li 2018;Xian et al. 2021). The aim of the present study was to investigate the effects and mechanisms of Tan IIA on the TNBC treatment. We found that Tan IIA is potentially effective for the treatment of 4T1 breast cancer, and the molecular mechanism may be through enhancing the activity of p53 and decreasing Bcl-2 to suppress proliferation and promote apoptosis.

Cell lines and cultures
Murine triple-negative breast cancer 4T1 cell lines and the normal breast cell line (MCF10A) were kindly provided by Procell Life Science & Technology Co., Ltd. Due to the growth and metastatic properties, tumor-bearing mice with 4T1 cells are an animal model of human stage VI breast cancer. 4T1 cells were expanded in a humidified incubator at 37 °C with 5% CO 2 in RPMI-1640 medium (Gibco, USA), supplemented with 100 U/mL penicillin (Gibco, USA) and 100 mg/mL streptomycin (Gibco, USA) and 10% fetal bovine serum (Gibco, USA). MCF10A cells were cultured in DMEM medium (Gibco, USA) supplemented with 10 μg/mL insulin (Thermo Fisher Scientific, USA), 20 ng/ mL human epidermal growth factor (HEGF) (Thermo Fisher Scientific, USA), 0.5 µg/mL hydrocortisone (Thermo Fisher Scientific, USA), 5% horse serum (Thermo Fisher Scientific, USA), and 5% penicillin-streptomycin (Gibco, USA). The cells were also incubated in a humidified incubator with 5% CO 2 under 37 °C.

Cell proliferation assay
CCK-8 viability assay was performed to evaluate the effects of Tan IIA on cell proliferation. For this, 4T1 and MCF10A cells, suspended with 0.25% trypsin (Gibco, USA), were inoculated into 96-well plates at a density of 3 × 10 3 cells per well. Subsequently, 4T1 cells were treated with Tan IIA at concentrations of 10, 20, 30, 40, 50, 60, 70, or 80 μM for 24, 48, and 72 h, respectively. And MCF10A cells were treated with the highest concentration of Tan IIA (80 μM). Finally, 96-well plates incubated with 10 µL of CCK-8 solution for 60 min were detected by a microplate reader (IMark, Bio-Rad). Record the absorbance (Abs) at 450 nm and calculate the cell inhibition rate (Xue et al. 2019). All experiments were performed in six replicates and repeated three times.

Colony formation assay
Transfected cells were reseeded into 6-well plate with 500 cells per well by treating with Tan IIA for 48 h. Cells were further treated with 0 (DMSO), 12.5, 25, or 50 μM Tan IIA, and 3 wells were used per group. Then, 6-well plates cultivated at 37 °C for approximately 12 days were washed with 1 × PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Finally, natural colonies stained with 0.5% crystal violet for approximately 20 min were imaged and quantified. All experiments were performed in triplicate and repeated three times.

Apoptosis assay by flow cytometry
The Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA) was used to detect apoptotic cells following the manufacturer's protocol. 4T1 cells were treated with Tan IIA in serial concentrations (0, 12.5, 25, and 50 μM) at 37 °C for 24 h. After washing with PBS, cells were trypsinized and subsequently incubated in FITC-conjugated Annexin-V binding buffer containing PI in the dark for 15 min. After incubation, cell apoptosis was assessed using FCS Express software (De Novo Software) on EXFLOW (Dakewe Biotech Co., Ltd.).

Docking exercises of ingredients binding to main targets
Compound structures were obtained from TCMSP Database and Analysis Platform and saved as docking ligands in MOL2 format. The crystal structure of p53, Bcl-2, and NF-κB1 was obtained from the Protein Data Bank (PDB ID: 3D06 (Suad et al. 2009), PDB ID: 6GL8 (Casara et al. 2018), and PDB ID: 1MDI (Qin et al. 1995)). Other targets' crystal structures from Protein Data Bank include EGFR (5UG9), CASP3 (4JJE), KIT (3G0E), AKT2 (1O6L), and Bcl-2l1 (3SP7) (Planken et al. 2017;Vickers et al. 2013;Gajiwala et al. 2009;Yang et al. 2002;Zhou et al. 2012). The crystal structures of candidate proteins were modified using the Autodock 4.2 software (Morris et al. 2009) to remove ligands, add hydrogen, remove water, and patch amino acids. The docking binding free energy was scored, and the docking models with the lowest binding affinity were displayed by PyMOL (3D).

Western blotting
Total protein from Tan IIA-treated cell lysis was quantified according to the manufacturer's BCA protein guideline, separated on 10% SDS-PAGE, and transferred to PVDF membranes. After blocking with 5% defatted milk dissolved in Tris-buffered saline containing 0.1% Tween-20, membranes were incubated with appropriate dilutions of primary antibodies (BCL2, TP53, phosphor-TP53, and β-actin (Abcam, Cambridge, UK)) followed by secondary antibody. Finally, quantification was performed using an image acquisition and analysis system (CHEMIDOC XRS + , Bio-Rad, USA) and ImageJ (Su 2018).

In vivo xenografts
2 × 10 6 4T1 cells were injected subcutaneously into the upper middle groin of 5 BALB/c female mice. Tumor volumes were determined every day (tumor volume = length*width 2 /2). When the average length of the subcutaneous tumor reached 10 mm, the mice were sacrificed in a humane manner, and then the tumors were collected. The collected tumors were cut into small pieces with a length of 2 mm in the clean area and planted in the fat pads of the fourth pair of left breasts of the anesthetized (Isoflurane, Rodent Anesthesia Machine, provided by Hebei Lanmeng Bio Pharmaceutical Technology Co., Ltd.) BALB/c female mice. Three days later, the mice were randomly divided into 3 groups (n = 18) and injected intraperitoneally every other day. The dosage was 2 mg/kg, the dosage of Tan IIA was 10 mg/kg (Lin et al. 2013), and the control group was given the same amount of normal saline. Starting from the 8th day, three mice were randomly selected from each group every 3 days and sacrificed in a humane manner. The tumors of the mice were isolated and stored at low temperature. All experiments were approved by the Experimental Animal Ethics Committee of Nankai University.

Immunohistochemistry (IHC) and TUNEL assay
After fixation and permeabilization, immunohistochemical sections of tumor tissue were prepared according to the instructions of the KI67 Cell Proliferation Kit (IHC) (Sangon Biotech (Shanghai) Co., Ltd., China) to check the proliferation status. In addition, apoptosis in tumor tissues was detected using the In Situ Cell Death Detection Kit (Roche Diagnostic Mannheim, Germany) according to the manufacturer's instructions. Representative images were obtained using a confocal microscope (Olympus)  after the final overlay with DAPI-containing mounting medium.

Statistical analysis
Statistical significance was determined using SPSS 12.0. All experimental data were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni correction for multiple comparisons.

Effect of Tan IIA treatment on the proliferation of TNBC 4T1 cells
The effects of Tan IIA on the viability of 4T1 were evaluated with CCK-8 assay. 4T1 cells were exposed to various concentrations (10, 20, 30, 40, 50, 60, 70, or 80 μM) of Tan IIA for 24, 48, and 72 h, respectively. When treated with more than 40 μM of Tan IIA for 24 h, sustainable and stability-inhibitory effects were shown ( Fig. 2A, B). Tan IIA inhibited 4T1 cell proliferation in concentration-and time-dependent manners as the IC50 values at 24, 48, and 72 h were of 81.89, 49.78, and 41.60 μmol/L, respectively. Moreover, Tan IIA had no obvious toxicity to normal breast cells (P > 0.05, Fig. 2A). Due to the specificity of 4T1 cells, we chose the IC50 concentration via calculating at 48 h as the highest dose concentration for subsequent in vitro experiments (Fig. 2C).
Next, we determined the effect of Tan IIA on the ability of 4T1 to form colonies. Similarly, Tan IIA significantly reduced the colony formation by 67.73%, 31.87%, and 13.94% after treated with 12.5 μM, 25 μM, and 50 μM of Tan II for 48 h (Fig. 2D). The underlying mechanism of cell viability inhibition was further studied by determining the apoptotic effects using the Annexin-V/PI method. The apoptotic cells (D2 + D3) increased after treated with Tan II with a concentration-dependent manner (P < 0.01, Fig. 2E). Consistent with the cell viability assays, these results again indicated the ability of Tan II to induce growth inhibition.

Effects of Tan II on xenograft tumor models
To further evaluate the anti-tumor efficacy of Tan IIA, BALB/C mice bearing 4T1 tumor were treated orally with Tan IIA. Doxorubicin (Dox) was used for positive control. After the 23 days of treatment, no death or adverse events occurred in all groups. No significant differences in body weight change were observed in the three groups. Treatment with Tan II and Dox induced significant growth delay of 4T1 tumor compared to untreated controls, as shown in Fig. 3A. The tumor weight was reduced by 34.47% and 51.00% in Tan II-treated and Dox-treated groups, respectively. On the 23rd day, the relative tumor volumes of Tan IIA and Dox were 877.67 ± 250.80 mm 3 and 656.33 ± 187.35 mm 3 , while the tumor volume of the control group was 1339.33 ± 56.58 mm 3 (Fig. 3B).
Ki-67 expression analysis was performed to evaluate cell proliferation by immunohistochemistry. On the 23rd day, all the three groups showed Ki-67 strong staining. Ki-67 staining in the treated groups showed slightly weakened (P < 0.05, Fig. 3D). Apoptosis levels in situ were analyzed by the TUNEL assay, and the distribution of the TUNELpositive nuclei was higher after the treatment of Tan IIA (P < 0.05, Fig. 3E).

Predicted targets and mechanisms of Tan IIA for breast cancer treatment based on network pharmacology
After searching the TTD and CTD databases, we obtained 1344 TNBC-related target candidates. Furthermore, 215 drug targets were predicted from BATMAN-TCM and TCMSP databases. The potential TNBC targets for Tan IIA treatment were matched by combining the disease and drug targets. Forty-five common targets were obtained to construct an ingredient-target (cI-cT) network using Cytoscape (Fig. 4A). We further put the screened 45 targets into the STRING website to get the protein-protein interactions (PPI) among these proteins with the conditional effect score that was set as > 0.9. After removing the targets without interactions, a PPI with 39 targets was obtained (Fig. 4B), which indicated that these targets tend to form close associates among each other. TP53, NF-κB, AKT, MYC, and BCL2 were the hub targets among the PPI network. The functions of 39 hub targets were further analyzed with the DAVID website; we found these targets majorly involved in cancer-related biological processes and pathways, such as cell apoptosis, P53 signaling pathway, PI3K/Akt pathway, the MAPK signaling pathway, and the mTOR signaling pathway (Fig. 4C, D).
To ascertain the interaction between Tan IIA and their hub targets, computational docking exercises were conducted to mimic the binding characteristics. It is generally believed that the binding energy value less than − 5.0 kcal/mol indicates good binding activity, and less than − 7.0 kcal/mol indicates strong binding activity. The binding energies of Tan IIA with these hub targets P53,  Table 1. Binding affinities to the first three targets were − 6.92, − 6.07, and − 6.28, respectively, indicating a good or strong binding activity. Docking results were visualized in the PyMOL software. Figure 4E shows Tan IIA docking with P53, BCL-2, NF-κB, EGFR, CASP3, KIT, AKT2, and BCL-2L1, which are mainly related with the cell apoptosis.

Changes in the level of major proteins in Tan IIA-treated 4T1 cells
To investigate the mechanism by which Tan IIA induced apoptosis, Western blotting was utilized to detect Bcl-2, p53, and phosphorylated p53 proteins in 4T1 cells. Western blot results showed that the levels of Bcl-2 gradually reduced in response to increasing Tan IIA concentrations in vitro compared with the control group after Tan IIA treatment (P < 0.05 for all, Fig. 5A, B). The levels of p53 and phosphorylated p53 also showed an upward trend. These data showed that Tan IIA could inhibit tumor growth by inducing apoptosis. Taken together, we mapped the hub targets to the canonical signaling pathways according to the results of the network-based systems and molecular-biological analyses. Tan IIA mediated its anti-cancer effects by reducing the

Fig. 5
The changes and mechanisms of target proteins in Tan IIA-treated 4T1 cells. A, B Changes in the level of major proteins in Tan IIAtreated 4T1 cells. C Potential pro-apoptotic mechanism of Tan IIA. *P < 0.05, **P < 0.01 expression of the Bcl-2 and upregulating the expression of P53 through the NF-kB signaling pathway (Fig. 5C).

Discussion
In recent years, with the in-depth research of bioinformatics, more and more herbal ingredients with anticancer effects have been discovered. Tan IIA, one of the active ingredients of Salvia miltiorrhiza, has been demonstrated to have inhibitory effects on multisystem tumors. In human colorectal cancer, Tan IIA effectively inhibits proliferation and metastasis by reducing HIF-1α levels and inhibiting the secretion of VEGF and bFGF (Sui et al. 2017). Tan IIA enhanced the cell cycle, apoptosis, and autophagy of drug-resistant gastric cancer cells by reducing MRP1, contributing to the anticancer effect of doxorubicin in gastric cancer (Xu et al. 2018 Cell apoptosis is an important manifestation of cell death, which is strongly associated with tumorigenesis and tumor progression. Bcl-2 is one of the anti-apoptotic proteins, which plays a significant role in mitochondrial-mediated apoptotic pathways. Bcl-2 can inhibit the pro-apoptotic protein Bax (Kale et al. 2018). In addition to playing an important role in regulating cell apoptosis, Bcl-2 can also regulate tumor migration, invasion, autophagy, and angiogenesis (Trisciuoglio et al. 2013). In our experiments, we found Tan IIA could inhibit the 4T1 proliferation and induce the apoptosis by reducing the expression of the Bcl-2. The protein encoded by TP53 responds to diverse cellular stresses to regulate metabolic changes, cell cycle, aging, apoptosis, or DNA repair. In 65-80% of TNBC cases, TP53 is the most commonly mutated gene (Shah et al. 2012). Recent studies have shown that TNBC patients with reduced p53 protein function have a reduced overall survival and an increased risk of metastasis (Powell et al. 2016). There is evidence that if DNA damage stress is strong enough, the phosphorylation of ser46 of p53 increases, and further induces apoptosis (Taira et al. 2007). According to the predicted results of the networkbased systems, p53 was one of the hub targets of Tan IIA for TNBC treatment. Similarly, under the influence of Tan IIA, the expression of TP53 and the phosphorylation of its related sites increased both in vivo and in vitro.
In conclusion, our present results demonstrated that Tan IIA inhibited cell proliferation and tumor growth in vitro and in vivo by inducing cell apoptosis, while the downregulation of BCL2 and the upregulation of TP53 were the possible mechanisms of Tan IIA in TNBC cell apoptosis. This study further supports the application evidence of Tan IIA in cancer clinical treatment. However, network pharmacological analysis found that the effect of Tan IIA is more than this, and its anticancer mechanism still requires further investigation.
Author contribution JH and XW conceived and designed research. JL, CZ, and SL conducted experiments and wrote the manuscript. XW analyzed data. All authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.
Funding This work was supported by the National Natural Science Foundation of China (Grant No. 81903934) and the National Natural Science Foundation of China (Grant No. 81473441).

Data availability
The datasets generated and/or analyzed during the current study are not publicly available due to subsequent experiments that have not been completed but are available from the corresponding author on reasonable request.

Declarations
Ethics approval All experiments were approved by the Experimental Animal Ethics Committee of Nankai University.

Competing interests
The authors declare no competing interests.