Exploring the Regulation Mechanism of Xihuang Pill, Olibanum and β-Boswellic Acid on the Biomolecular Network of Triple-Negative Breast Cancer Based on Transcriptomics and Chemical Informatics Methodology

Background Xihuang Pill (XHP) is mainly used to treat “Ru Yan (breast cancer)”. Evidence-based medical evidence and showed that XHP improves the efficacy of chemotherapy and reduced chemotherapy-induced toxicity in breast cancer patients. However, the mechanism of XHP against breast cancer is not clear. Methods The effect of XHP extract on cell half-inhibitory concentration (IC50) and cell viability of MD-MB-231 cells was detected by CCK-8 method. The cell inhibition rate of MDA-MB-453 cells were detected by MTT method. Apoptosis was detected by flow cytometry, cell transfer ability was detected by Transwell method, and cell proliferation ability was detected by colony formation assay. The expression of Notch1, β-catenin and c-myc mRNA in MDA-MB-453 cells were detected by real-time fluorescence quantitative PCR. Then, chemical informatics and transcriptomics methodology was utilized to predict the potential compounds and targets of XHP, and collect triple negative breast cancer (TNBC) genes and the data of Olibanum and β-boswellic acid intervention MD-MB-231 cells (from GSE102891). The cytoscape software was utilized to undergo network construction and network analysis. Finally, the data from the network analysis was imported into the DAVID database for enrichment analysis of signaling pathways and biological processes. Results The IC50 was 15.08 g/L (for MD-MB-231 cells). After interfering with MD-MB-231 cells with 15.08 g/L XHP extract for 72 h, compared with the control group, the cell viability, migration and proliferation was significantly decreased, while early apoptosis and late apoptosis were significantly increased (P < 0.01). After interfering with MDA-MB-453 cells with 6 g/L XHP extract for 72 h, compared with the control group, the cell inhibition and apoptosis rate increased, while the expression of Notch1, β-catenin and c-myc mRNA decreased. (P < 0.05). The chemical informatics and transcriptomics analysis showed that four networks were constructed and analyzed: (1) potential compounds-potential targets network of XHP; (2) XHP-TNBC PPI network; (3) DEGs PPI network of Olibanum-treated MD-MB 231 cells; (4) DEGs PPI network of β-boswellic acid -treated MD-MB 231 cells. Several anti-TNBC biological processes, signaling pathways, targets and so on were obtained. Conclusion XHP may exert anti-TNBC effects through regulating biological processes, signaling pathways, targets found in this study.


INTRODUCTION
Breast cancer is the leading cause of death among women worldwide, and the incidence rate has increased significantly in recent years, which seriously threatens women's health (Fan et al., 2014;Desantis et al., 2017). Breast cancer is currently divided into five subtypes by coding sequence microarray technology (Prat et al., 2015;Cejalvo et al., 2018): (1) Luminal-A type; (2) Luminal-B type; (3) human epidermal growth factor receptor 2 (HER2) overexpression type; (4) base-like type; (5) normal type. Basal-like breast cancer is non-specific invasive ductal carcinoma (Jiang et al., 2019), and its ER, PR and HER2 are negative, also known as triple negative breast cancer (TNBC). At present, TNBC accounts for 10 to 17% of all breast cancers (Warner et al., 2015;Kulkarni et al., 2019). The vast majority of TNBCs are highly invasive ductal carcinomas with nuclear polymorphism, high mitotic rate, and minimal tubule formation (Kulkarni et al., 2019). TNBC is generally classified into basal cell-like type 1, basal cell-like type 2, immunoregulatory, interstitial and mesenchymal stem cell types, and luminal androgen receptor type (Shi et al., 2018;Jiang et al., 2019). TNBC is a poorly differentiated tumor with strong invasive and metastatic ability, easy to invade blood vessels, and increased recurrence rate (Jiang et al., 2019).
At present, the management and treatment measures for triple-negative breast cancer are mainly: (1) local treatment: surgery is still the first choice for local treatment (Li et al., 2019a); (2) systemic therapy: combination chemotherapy of taxane and anthracycline is currently the common choice for TNBC neoadjuvant chemotherapy, but anthracyclines have irreversible toxicity to the heart (Mangone et al., 2019); (3) targeted therapy (Jhan and Andrechek, 2017). However, due to the ineffectiveness of traditional endocrine therapy and targeted therapy, patients with TNBC sometimes have tumor metastasis very early, which seriously affects their physical and mental health (McCann et al., 2019). At present, in order to find new chemotherapy or sensitizing drugs, plants and natural products are gradually becoming the source of new TNBC drug development (Szarc Vel Szic et al., 2017). The current study found that some traditional Chinese medicine compounds and natural medicines can inhibit the proliferation, metastasis, and drug resistance of TNBC cells in a variety of ways (Baraya et al., 2017;Szarc Vel Szic et al., 2017).
Xihuang Pill (XHP) is from the Wai Ke Quan Sheng Ji by Wang Weide in 1740, which is mainly used to treat "Ru Yan (breast cancer)" and so on. XHP is compose of Myrrha, Bovis Calculus, Olibanum and Moschus. Systematic reviews and metaanalysis showed that XHP combined with chemotherapy significantly enhanced tumor response in breast cancer patients, improved Karnofsky performance scores and reduced chemotherapy-induced toxicity (Guo et al., 2018;Mao et al., 2019). He et al. found that XHP-containing serum increased TP53 and Bax (P < 0.05), and decreased the ratio of Bcl-2/Bax in MDA-MB-435 cells . The mechanism of anti-TNBC of XHP has been reported in many studies, such as improving the immunosuppressive state of the tumor microenvironment and reversing immune escape, thereby inhibiting tumor growth. XHP reduces the number of Treg cells by inhibiting the expression of PI3K and AKT and upregulating the expression of AP-1 in Treg cells, thereby promoting Treg cell apoptosis (Li et al., 2018a). Other study found that the mechanism of XHP inhibition of tumors may be related to the up-regulation of gene and protein expression of MEKK1, SEK1, JNK1 and AP-1 in Treg cells in the tumor microenvironment (Su et al., 2018). Zheng et al. found that XHP can block the cell cycle of the Hs578T cell line and promote its apoptosis (Zheng et al., 2016).
Recent studies showed that the main anti-breast cancer herbs in XHP are Myrrha and Olibanum, especially Olibanum (Cheng et al., 2016;Hao et al., 2018). Although the above studies have described some of the mechanisms of XHP against TNBC, the mechanism remains unclear. In our previous studies, we successfully used multiple bioinformatics techniques and transcriptomics to analyze the mechanisms by which traditional Chinese Medicine interferes with different types of breast cancer (Zeng and Yang, 2017;Yang et al., 2018;Yang et al., 2019). Therefore, this study will use a multi-directional pharmacology strategy based on chemical informatics and transcriptomics to clarify the mechanisms by which XHP and Olibanum treat TNBC. The research process is shown in Figure 1. Preparation of XHP solution: XHP was immersed in DMEM medium pre-cooled at 4°C for 24 h (mass concentration 0.1 g/ mL) in a sterile sealed container; use ultrasonic vibration to help dissolve for 2 h and continue to soak for 48 h at 4°C. The supernatant was filtered through a 0.22 mm micropore filter to obtain an XHP leaching solution. The XHP solution is stored at 4°C (or −20°C); during the experiment, it was diluted to the desired concentration with DMEM medium.

Experimental Material Preparation
Preparation of XHP solution required for High Performance Liquid Chromatography (HPLC): 1.00 g of XHP powder was accurately weighed and placed in a 50 ml Erlenmeyer flask. Pipet 20 ml of methanol accurately, sonicate in an ice bath for 20 min, extract twice, and place at room temperature. Centrifuge at 4,000 r/min for 5 min. The supernatant was placed in a pear-shaped bottle and concentrated under reduced pressure. Reconstitute with methanol and transfer to a 25 ml volumetric flask. Finally, make up to volume with methanol and shake well.
Preparation of acetyl-11-keto-b-boswellic acid reference substance: Take an appropriate amount of acetyl-11-keto-bboswellic acid, accurately weigh it, place it in a measuring flask, add methanol to volume, and make it to a mass concentration of 1.22 mg/mL.

Cell Line
Human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and MDA-MB-453 were provided by the Cell Center of Xiangya School of Medicine, Central South University. While experimenting, the MD-MB-231 cells and MDA-MB-453 cells were cultured in high glucose DMEM medium containing 10% FBS in an incubator at 37°C, 5% CO2, and the medium was changed every other day. The group without XHP was the control group, and the group with XHP extract was the experimental group (XHP group), and each group had three duplicate wells.

Reagent and Instrument
DMEM medium and Transwell kit were purchased from Corning Inc.; Fetal bovine serun (FBS) was purchased from Ausbian Inc., Australia; CCK-8 kit and Giemsa dye solution were purchased from Sigma Inc., USA; The Annexin V-FITC/PI double-stained cell apoptosis assay kit was purchased from eBioscience Inc., USA.

Experimental Methods for MD-MB-231 Cell
Detection of XHP Half-Inhibitory Concentration (IC50) and Cell Viability Some 96-well plates were seeded at 3,000 cells (100 ml) per well. Nine (9) groups were set according to the concentration of added XHP, and the concentrations of XHP were 0, 1, 2, 5, 10, 20, 50, 75 and 100 g/L. After treatment for 72 h, 10 ml of CCK-8 reagent was added 2 to 4 h before the termination of culture, and the OD value was detected by a microplate reader at 450 nm. After logarithmic processing, a scatter plot was prepared to calculate the IC50 value of XHP. Using this IC50 value as the drug concentration of XHP intervention in MD-MB-231 cells in subsequent experiments (including cell viability detection).
After treatment with XHP extract for 72 h, the culture was continued for 5 d with the control group. 10 ml of CCK-8 reagent was added 2 to 4 h before the termination of the culture, and the cell viability was measured daily for 5 days via the above procedure.

MD-MB-231 Cell Apoptosis Detection by Flow Cytometry
Some 6-well plates were seeded at a minimum of 5 × 10 5 cells per well (2 ml) and plated for 24 h. Apoptosis detection was performed by flow cytometry according to the procedure in the instructions of Annexin V-FITC/PI double-stained cell apoptosis assay kit.

Transwell Detection
The cells were cultured in 24-well plates. About 100 ml of serumfree medium was added to each well of the Transwell's inner chamber, and 600 ml of medium containing 30% FBS was added to each well of the Transwell's outer chamber, and plated for 18 h at a cell number of 1 × 105 per well. Transfer cells, fix, stain with Giemsa stain, photograph with fluorescence microscopy, and count cells at a magnification of 200×.

Cell Clone Detection
Some 6-well plates were seeded at 800 cells (2 ml) per well, and continue to culture for 10 days, and change the solution once every 3 days. Cell clones were photographed under a fluorescent microscope before termination of the experiment. Fix the cells with 4% paraformaldehyde, crystal violet staining, photograph.

Determination of Cell Inhibition Rate by MTT Method
The MDA-MB-453 cells in the logarithmic growth phase were used for experiments. The cell concentration was adjusted to 1 × 10 5 cells/mL and inoculated in 96-well culture plates; 90 ml of cell suspension was added to each well, and then 10 ml of different concentrations of XHP (0,4,6,8,10,12,14 g/L) were added. Six (6) duplicate wells were set for each group, and after incubating in a cell incubator at 37°C and 5% CO2 for 72 h, 20 ml MTT (5mg/mL) was added to each well. After continuing the culture for 4 h, the supernatant was discarded, and DMSO solution (DMSO) 150 ml/well was added to each well. After shaking for 10 min to fully dissolve the crystals, the OD value of each well was measured with a microplate reader at a wavelength of 490 nm. The total RNA of each group of cells was extracted with Trizol according to the kit instructions. The OD260/OD280 ratio is calculated, and the ratio ≥1.8 means the purity and concentration of the RNA meet the experimental requirements. After detecting the integrity of RNA by agarose gel electrophoresis, the primers of Notch1, b-catenin, c-myc and internal reference GAPDH were added to amplify the corresponding target fragments. Finally, the real-time fluorescence quantitative PCR program was performed on the machine. The primers were designed by Primer 3.0 software and synthesized by Yuantai Bio-Technology Co., Ltd., see Table 1.

Triple Negative Breast Cancer Biological Network
To construct the biological network of TNBC, the TNBC-related genes were collected from OMIM database (http://omim.org/) and Genecards (http://www.genecards.org) (Zeng and Yang, 2017;Yang et al., 2018;Yang et al., 2019). Finally, one thousand and two hundred and twenty-one (1220) TNBCrelated genes were obtained. These TNBC-related genes will be used for subsequent biological network construction and network analysis (see Table S2).
The networks were analyzed by the plugin MCODE to obtain cluster. The definition and the methodology of acquisition of clusters were described in our previous work (Zeng and Yang, 2017;Yang et al., 2018;Yang et al., 2019), such as "Exploring the pharmacological mechanism of Yanghe Decoction on HER2positive breast cancer by a network pharmacology approach" (Zeng and Yang, 2017) and "Investigating the regulation mechanism of baicalin on triple negative breast cancer's biological network by a systematic biological strategy" .

XHP's Fingerprint
XHP and acetyl-11-keto-b-boswellic acid reference substance were analyzed and compared according to the steps of HPLC Detection. The fingerprint was shown in Figure S1.

Experimental Results for MD-MB-231 Cell
Inhibition Effect of XHP on the Growth of MD-MB-231 Cell Different concentrations of XHP were applied to MD-MB-231 cells for 72 h, and the OD value of each group was detected by CCK-8 kit. The OD value could indirectly reflect the number of viable cells. The results are shown in Table 2. The XHP extract concentration value was logarithmically processed. The OD value of each concentration group was compared with the OD value of the non-medicated solvent group, and the cell inhibition rate was calculated. Draw a scatter plot with Log (concentration value) and cell inhibition rate ( Figure 2). The IC50 value of XHP extract intervention in MD-MB-231 cells for 72 h was 15.08 g/L.

Effect of XHP on the Viability of MD-MB-231 Cells
The CCK-8 reagent contains WST-8, which is reduced in the cell mitochondria to a highly water-soluble yellow formazan product, the number of which is proportional to the number of viable cells. The OD value measured at a wavelength of 450 nm can indirectly reflect the cell proliferation. The cells were intervened for 72 h with XHP extract at a final concentration of 15.08 g/L. Compared with the control group, the cell viability was significantly decreased (P < 0.01), as shown in Figure 3.

Effect of XHP on apoptosis of MD-MB-231 cells
The cells were intervened for 72 h with XHP extract at a final concentration of 15.08 g/L. Compared with the control group, the early apoptosis and late apoptosis of the cells after XHP intervention were significantly increased (P < 0.01) ( Table 3 and Figure 4).

Effect of XHP on Migration Ability of MD-MB-231 Cells
The cells were intervened for 72 h with XHP extract at a final concentration of 15.08 g/L. The effect of XHP on cell metastatic ability was determined by observing the migration of cells in the Transwell chamber to serum-containing media. Compared with the control group, the migration ability of the cells after XHP intervention were significantly decreased (P < 0.01) ( Figure 5).

Effect of XHP on Cell Cloning Ability
The cells were intervened for 72 h with XHP extract at a final concentration of 15.08 g/L. After 10 days of continuous culture, the clone formation rate was calculated to quantitatively analyze the proliferation ability of breast cancer cells. Compared with the control group, the cloning ability of the cells after XHP intervention were significantly decreased (P < 0.01) ( Figure 6).

Experimental Results for MDA-MB-453 Cell
Inhibition Effect of XHP on the Growth of MDA-MB-453 Cell The results of MTT also showed that XHP had a proliferationinhibiting effect on MDA-MB-453 cells in a dose-dependent manner. When the concentration of XHP is lower than 6g/L, the inhibition rate of MDA-MB-453 cells is ≤20%, and there is no cytotoxicity. Therefore, 6 g/L was selected as the non-cytotoxic concentration of XHP for subsequent MDA-MB-453 experiments. The results are shown in Figure 7.

Effect of XHP on apoptosis of MDA-MB-453 Cell
After 72 h intervention with 6 g/L XHP, the results of flow cytometry showed that the apoptosis rate in the test group was significantly higher than that in the control group (normal saline serum group) (P < 0.05) (Figure 8).

Effect of XHP on the Expression of Notch1, b-Catenin and c-myc mRNA
Compared with the control group (saline saline group), the expressions of Notch1 mRNA, b-catenin mRNA and c-myc mRNA in the XHP group were significantly reduced (P < 0.01) ( Figure 9).
This experiment showed that XHP can promote apoptosis, inhibit cell proliferation, metastasis and vitality, and can downregulate the expression of Notch1 mRNA, b-catenin mRNA and c-myc mRNA at the cellular level. In addition, other studies have also demonstrated the anti-TNBC effect of XHP. Zheng et al. (2016) found that XHP significantly inhibited the viability of Hs578T cell line in a dose-and time-dependent manner. The mechanism may be that XHP induced apoptosis through the inherent Bcl-2 dependent pathway and cell cycle arrest. Su et al. (2018) found that XHP may promote Treg cell apoptosis in the tumor microenvironment and further inhibit the tumor growth of breast cancer in 4T1 mice. The mechanism may be that XHP upregulated the MEKK1, SEK1, JNK1 and AP-1 gene and protein expression in Treg cells in the tumor microenvironment. Li et al. (2018a) further research indicates that XHP promotes apoptosis of Treg cells by inhibiting PI3K/AKT/AP-1 signaling pathway, thereby reducing the number of Treg cells, weakening the immunosuppressive state of the tumor microenvironment, reversing the mainstream immune escape, and inhibiting the growth of tumors. However, the molecular mechanisms of XHP's anti-TNBC effects, such as regulatory pathways and targets, need further study. Based on this, this study will further study the mechanism of XHP reversing the multidrug resistance of tumors, and provide a theoretical basis for the development of new drugs and clinical combination drugs.

XHP's Potential Targets and TNBC Genes
After the potential target prediction, a total of 1,178 potential targets were obtained. Myrrha contains 550 potential targets; Bovis Calculus contains 680 potential targets; Olibanum contains 390 potential targets; Moschus contains 845 potential targets. Some of the targets contained in different herbs overlap ( Figure   S2). In the outer circle, red, blue, green, purple stand for potential targets of Bovis Calculus, Moschus, Myrrha, Olibanum, respectively. In the inner circle, the greater the number of purple links and the longer the dark orange arc, the more overlap between the input target lists. The blue link indicates the amount of functional overlap between the input target lists. The relationship among potential compounds and potential targets was shown in Figure 10. This network consists of 81 potential compounds, 1,175 potential targets and 6,057 edges. The targets near the center are regulated by more compounds than ones in the peripheral. For example, the targets in the center are: AR (55 edges), CYP19A1 (52 edges), ESR1 (36 edges), NR3C1 (36 edges), ESR2 (35 edges), PTPN1 (35 edges), MAPT (34 edges), HSD11B2 (31 edges), NR1H4 (29 edges); the targets in the peripheral (AXL, CAMK2B, CXCR1, NEK2, NEK6) are regulated only by one compound. In addition, some important targets related to TNBC are regulated by more XHP compounds: EGFR (four edges), PARP1 (eight edges), PARP10 (three edges), PARP14 (one edge), PARP15 (one edge), PARP2 (two edges), VEGFA (two edges), PIK3CA (11 edges), AKT1 (eight edges), AKT2 (one edge). After searching, a total of 1,221 TNBC-related genes were obtained. These TNBC genes will be combined with XHP's predicted targets to construct an XHP-TNBC PPI network so as to observe the association between XHP and TNBC.
It can be seen from the potential compounds-potential targets network of XHP that the herbs in XHP have multi-component and multi-target effects in anti-TNBC. Many compounds can act on one or more targets at the same time, and some targets can be regulated by multiple compounds. They may be the main active ingredient and target of XHP against TNBC. Currently, the study   found that TNBC can also be subdivided into six subtypes: two basal-like-related (BL1 and BL2), two mesenchymal related subtypes [mesenchymal (M) and mesenchymal stem-like (MSL)], an immunoregulatory subtype (IM) and a tubular androgen receptor type (LAR) (Waks and Winer, 2019). LAR subtype is sensitive to androgen receptor (AR) inhibitors due to high expression of AR. Enzalutamide (an AR inhibitor) is an advanced prostate cancer drug approved by the US FDA (Traina et al., 2018). The current study on Enzalutamide is a phase II clinical study of patients with locally advanced or metastatic AR +TNBC; The study showed that approximately 55% of patients with TNBC were AR-positive, and enzalutamide had a good effect on the LAR subtype TNBC (Traina et al., 2018).
Recent studies have shown that tamoxifen may be effective against certain subtypes of TNBC, which is associated with ESR2 (Mukhopadhyay et al., 2019). Studies at the Roswell Park Comprehensive Cancer Center in the United States have shown that TP53 status is a determining factor in the duality of estrogen receptor-beta (ESR2) function (Mukhopadhyay et al., 2019). ESR2 and mutant TP53 can be combined to predict survival in patients with TNBC (Mukhopadhyay et al., 2019). Current research shows that miRNA genetic variation is associated with the expression of important receptors such as ER and HER2, and the single nucleotide polymorphism (SNP) site present in the estrogen receptor alpha gene (ESR1) may be involved in the development and progression of TNBC (Zhang, 2011).

Biological Processes of XHP-TNBC PPI Network
The XHP-TNBC PPI network were analyzed by MCODE to obtain the clusters. The clusters of this network were shown in Figure S3 and Table 4. The genes and targets in clusters 1-10 were put into DAVID database to undergo GO enrichment analysis as an example.
After the GO enrichment analysis, a lot of biological processes of each cluster were return. Cluster 1 is associated with chemokines and their receptor-mediated signaling pathways, immune responses, ERK1/2 signaling pathways, angiogenesis,  T cell chemotaxis. Cluster 2 is involved in apoptosis and cell proliferation, hypoxia induction, cell cycle, estrogen-mediated biological processes, canonical Wnt signaling pathway, ERK1/2 signaling pathway, angiogenesis, T cell-mediated immune response. Cluster 3 is involved in ERK1/2 signaling pathway, MAPK signaling pathway, PI3K signaling pathway, angiogenesis, and inflammatory response. Cluster 4 is involved in cell migration and adhesion to angiogenesis, extracellular matrix, hypoxia-induced, PI3K signaling pathway, MAPK signaling pathway, ERK1/2 signaling pathway, and endoplasmic reticulum stress. Cluster 5 is associated with tumor necrosis factor-mediated signaling, apoptosis, cell cycle, angiogenesis, T cell receptor signaling pathway, Wnt signaling pathway, and endoplasmic reticulum-mediated endogenous apoptotic signaling pathway. Cluster 6 is involved in transcriptional regulation, apoptosis, cell matrix adhesion, T cell receptor signaling pathway, angiogenesis, Wnt signaling pathway, cell cycle, immune cell chemotaxis and T cell costimulation. Cluster 7 is associated with apoptosis, extracellular matrix, negative regulation of Wnt signaling pathway, NF-kB signaling pathway, and angiogenesis. Cluster 8 is related to redox, steroid metabolism, androgen and estrogen-mediated biological processes, oxidative stress. Cluster 9 is associated with redox, chemokine generation and mediated biological processes, steroid hormone-mediated signaling, angiogenesis, T cell selection, cell migration. Cluster 10 is involved in the transmission of intracellular signals. The details of each cluster and biological process was described in Table S3.
Since Cluster 1 contains many classic biological processes, bubble chart is created using the main biological process data contained in Cluster 1 ( Figure 12A).

Signaling Pathways of XHP-TNBC PPI Network
The XHP targets combining with TNBC genes were put into DAVID database for pathway enrichment analysis. After this, thirty-four (34) anti-TNBC-related signaling pathways were returned ( Figure 13).

Transcriptome Analysis of Olibanum-Treated MD-MB 231 Cells
Differentially Expressed Gene of Olibanum-Treated

MD-MB 231 Cells
The transcriptome data comes from GSE102891. When performing the analysis, select "Boswellia Serrata Extract 128 ug/ml" as the experimental group, and select "control" as the control group to obtain gene expression data. Gene with a P value of <0.05 and Log2FC>1 or <−1 is considered to be a differentially expressed gene (DEG) ( Figure S5). PCA plot showed that the results of Olibanum group, b-boswellic acid group and control group were significantly different ( Figure S6A). The different clustering between Olibanum group and b-boswellic acid group were shown in Figure S6B.
A total of 227 genes were identified as DEGs and were used to construct DEGs PPI network of Olibanum-treated MD-MB 231 cells ( Figure 14A). The details of each DEG was shown in Table S5.
In Figure 14, the size of each node is related to its Degree; the bigger nodes have the larger value of Degree. The width of line is associated with its Edge Betweenness; the wider lines have the larger value of Edge Betweenness. In this network, the top 21 targets are: HSPA5 (33 edges), DDIT3 (28 edges These targets are considered to be the core targets of this network. In the central node of the network, HSPA5, DDIT3, DNAJC3, ATF3, XBP1, PPP1R15A, DNAJB9, TRIB3, PDIA3, ASNS, HERPUD1, DNAJB1, PDIA4, TXNRD1, DUSP1, ERN1 are genes related to endoplasmic reticulum stress, which is essential for regulating the control of protein quality by endoplasmic reticulum and maintaining the balance of redox state (Xu and Park, 2018;Amen et al., 2019;Gao et al., 2019;Kim E. K. et al., 2019). Current research shows that Endoplasmic Reticulum Associated Unfolded Protein Response (UPR) and endoplasmic reticulum stress can affect the migration and FIGURE 13 | Signaling pathway of XHP-TNBC PPI network (Red diamond stands for signaling pathway; Purple circle stands for XHP-TNBC; Blue circle stands for TNBC genes; Pink circle stands for XHP targets).
invasion characteristics of breast cancer cells. The mechanisms include extracellular matrix (ECM) remodeling, cell adhesion modification, chemoattraction, epithelial-mesenchymal transition (EMT), regulation of signaling pathways associated with cell migration, and cytoskeletal remodeling, which in turn promotes breast cancer cell migration and invasion (Cook et al., 2016;Han and Wan, 2018;Sisinni et al., 2019). Hence, in the future, targeting UPR and breast cancer stress may be potential targeted therapeutic strategies for the treatment of breast cancer.
HSPA5, as a gene related to endoplasmic reticulum stress, plays an important role in the biological processes of breast cancer; inhibition of HSPA5 can inhibit TNBC cell migration and invasion (Chen et al., 2015). The endoplasmic reticulum stressrelated gene DDIT3 also participates in the development of TNBC by regulating autophagy and apoptosis of TNBC cells (Singha et al., 2013). In vitro and in vivo showed that LYN-1604 exerts an anti-TNBC effect by targeting ULK1-regulated autophagic death; its induced autophagic death is closely related to these key genes such as ATF3, RAD21 and caspase3 (Ouyang et al., 2017;Zhang et al., 2017). The heat shock protein HYOU1 (also known as Orp150) plays an important role in hypoxia/ischemia and angiogenesis, which is overexpressed in certain invasive breast cancers, and its overexpression appears to be associated with poor prognostic indicators (Li et al., 2019b). In metastatic breast and ovarian cancer, HSPA1 has a different lysine methylation, and unmethylated HSPA1 shows potential as a prognostic marker in potentially highly serous carcinomas (Jakobsson et al., 2015).

Enrichment Analysis of DEGs PPI Network of Olibanum-Treated MD-MB 231 Cells
After the enrichment analysis, several biological processes and signaling pathways are obtained. The results of enrichment in the DAVID database indicate that these DEGs are primarily involved in biological processes associated with endoplasmic reticulum stress response, such as cellular response to topologically affected protein, MAPK signaling pathway, FoxO signaling pathway and so on. The main biological processes and signaling pathways were shown in Figures 15A, B. The details of each biological processes and signaling pathway were shown in Table S6.
Metascape is a portal that provides genetic annotation and analysis resources to help biologists understand one or more gene lists (http://metascape.org/gp/index.html#/main/step1) (Zhou et al., 2019). Metascape's enrichment analysis of DEGs adds new biological processes and signaling pathways; compared to DAVID, the Reactom pathway is added, and the background annotation genes are more complete ( Figure S7). Interestingly, the results of pathway enrichments indicate that Ferroptosis has the highest enrichment, and current studies show that induction of Ferroptosis has become an important strategy for the treatment of tumors (Galluzzi et al., 2018); especially in breast cancer, ferroptosis induces tumor cell death by activating the -ATF4 pathway of GCN2-eIF2a in TNBC cells (Chen et al., 2017). This suggests that the Olibanum extract may contain components that potentially induce Ferroptosis. The details of the biological processes, signaling pathways and Reactome pathways were shown in Table S7.

Acid-Treated MD-MB 231 Cells
Differentially Expressed Gene of b-Boswellic Acid-Treated MD-MB 231 Cells The transcriptome data comes from GSE102891. When performing the analysis, select "3-O-Acetyl-b-boswellic acid 46 ug/ml" as the experimental group, and select "control" as the control group to obtain gene expression data. Gene with a P value of <0.05 and Log2FC >1 or <−1 is considered to be a differentially expressed gene (DEG) ( Figure S8). A total of 950 genes were identified as DEGs and were used to construct DEGs PPI network of b-boswellic acid -treated MD-MB 231 cells ( Figure 14). In Figure 14B, the size of each node is related to its Degree;  Table S8. FOS, HSPA5, SIRT1, PTGS2, HDAC3, TIMP1, TGFB1, ATF3, CEBPB, CLPP are involved in endoplasmic reticulum-related biological processes such as endoplasmic reticulum stress response and endoplasmic reticulum unfolded protein response (Wilkinson, 2019;Limia et al., 2019;Omidkhoda et al., 2019), which mediate the development of breast cancer. Recent studies have shown that the cell surface adhesion receptor CD44 is a key positive regulator of PD-L1 expression in TNBC and non-small cell lung cancer (NSCLC) (Kong et al., 2019). The Notch-mediated tumorinterstitial-inflammatory network promotes TNBC invasiveness and CXCL8 expression (Liubomirski et al., 2019). High expression of UBE2C is a potential factor for poor prognosis of TNBC. Furthermore, loss of BRCA1 function results in increased expression of UBE2C and chemoresistance to doxorubicin in breast cancer cells (Qin et al., 2017). The up-regulation of Serpine2 promotes breast cancer cell metastasis and reduces patient survival (Jin et al., 2017). Meanwhile, recent studies have shown that CHIP/STUB1 ubiquitin ligase is a negative chaperone molecule of HSP90/HSC70, which is reduced or lost in breast cancer. The absence of CHIP reshapes the cellular transcriptome and releases key cancer-promoting factors, such as the matrix degrading enzymes of the cathepsin family (Luan et al., 2018). In addition, downregulation of TUFM can induce epithelial-mesenchymal transition, and analogs of resveratrol HS-1793 can down-regulate its expression and increase anticancer activity against MCF-7 cells (Jeong et al., 2012).

Enrichment Analysis of DEGs PPI Network of b-Boswellic Acid-Treated MD-MB 231 Cells
After the enrichment analysis, several biological processes and signaling pathways are obtained. The results of enrichment in the DAVID database indicate that these DEGs are primarily involved in biological processes associated with endoplasmic reticulum stress response, such as PERK-mediated unfolded protein response, negative regulation of apoptotic process, and so on. The signaling pathways are protein processing in endoplasmic reticulum, TNF signaling pathway, Antigen processing and presentation, Estrogen signaling pathway, ECM-receptor interaction. The main biological processes and signaling pathways were shown in Figures 15C, D. The biological processes in the Metascape database are similar to those in the DAVID database, in a different order ( Figure S9). The details of the biological processes, signaling pathways and Reactome pathways were shown in Table S9.
In summary, through further analysis of transcriptomics data, it is found that the TNBC-related biological processes regulated  by Olibanum extracts and b-boswellic acid mainly include endoplasmic reticulum stress response, oxidative stress, angiogenesis, inflammatory response, cell migration and adhesion, hypoxia induction, and autophagy. Of particular importance, the endoplasmic reticulum stress response is thought to be the primary upstream mechanism by which Olibanum extracts and b-boswellic acid play a role in TNBC cells. Hence, endoplasmic reticulum stress is a potential target in cancer therapy due to its important role in cancer development.

CONCLUSION
XHP may exert anti-TNBC effects through regulating biological processes, signaling pathways, targets found in this study. Meanwhile, the ability of Olibanum extracts and b-boswellic acid to induce endoplasmic reticulum stress and subsequently activate tumor cell death programs confirms that they are a promising class of anticancer agents. In addition, the Olibanum extracts may be used as an inducer of TNBC's Ferroptosis in the future.

DATA AVAILABILITY STATEMENT
All datasets for this study are included in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
LZ and KY dominated the concept and carried out a comprehensive design. LL, XX, AG, and TB were participants in the concept and design. KY, LZ, AG, TB, and LL are responsible for data analysis and interpretation in the chemical informatics section. TX and XX, and LL are responsible for data analysis and interpretation in experiments. KY, LZ, AG, and TB drafted the paper. LL and XX supervised the study. All authors participated in the analysis and interpretation of data and approved the final paper. KY, LZ, AG and TB should be considered joint first author. LL is the first corresponding author because she supervised the study.