Discussion On The Mechanism of Que In The Treatment of Breast Neoplasms And Immune Prevention And Treatment

doi:10.21203/rs.3.rs-819024/v1

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Discussion On The Mechanism of Que In The Treatment of Breast Neoplasms And Immune Prevention And Treatment

https://doi.org/10.21203/rs.3.rs-819024/v1

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Background: The precancerous disease of breast cancer is an inevitable stage in the emergence and development of breast neoplasms. Breast cancer (BC) is a common malignant tumor in female worldwide. A large number of literatures have proved that, as antitumor drugs, flavonoid compounds can promote proliferation and immune regulation of T cell. Many researchers believe that Quercetin (Que) has great potential in the field of anti-breast cancer. Besides that, γδ T cells are a class of non-traditional T cells, which have long attracted attention due to their potential in immunotherapy. Above all, JAK/STAT1 signaling pathway is closely related to the immunity.

Methods

In the experiment designed in this paper, we first used Que, one of the flavonoids, to screen the target gene. Then, MCF-10A, MCF-10AT, MCF-7 and MDA-MB231 BC cells were co-cultured with Que for 24h and 48h, apoptosis was found in some the cells. We then cultured Que with γδ T cells and found that Que can promote the proliferation of Vδ2 T cell subsets of γδ T cells, thus enhancing the killing effect of γδ T cells. Western blot was use to showed the change of JAK/STAT1 signaling pathway related proteins after the Que was co-cultured with MCF-10AT and MCF-7 for 48h.

Results

Network pharmacology has shown that Que related pathways include the JAK/STAT1 signaling pathway and are associated with precancerous breast cancers. Que induced apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 in a time and concentration-dependent manner. Most importantly, Que can promote the differentiation of γδ T cells into the Vδ2 T cell subpopulation, this means that Que and γδ T cells may play a synergistic role in killing tumor cells and cellular immune regulation. In addition, our results showed that Que can increase in protein levels of IFNγ-R, p-JAK2 and p-STAT1, while the concomitant decrease protein levels of PD-L1.

Conclusions

In conclusion, Que plays a synergistic role in killing BC cells and promoting apoptosis by regulating the expression of IFNγ-R, p-JAK2, p-STAT1, and PD-L1 in the JAK/STAT1 signaling pathway and promoting the regulation of γδ T cells. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of BC.

Cancer Biology

Oncology

General Cell Biology & Physiology

Que

Breast cancer

Breast precancerous lesion

γδ T cell

JAK/STAT1 signaling pathway

Precancerous lesions of breast cancer refers to some breast diseases that are not malignant tumors increased the possibility to develop into malignant tumors under the action of various factors. The World Health Organization (WHO) defines such lesions as precancerous because they are more than 20% likely to transform from non-neoplastic lesions to tumors [1]. The stage of continuous development of breast precancerous lesions is breast tumors. The incidence of BC is increasing year by year and has become the highest incidence of malignant tumors among women in the world [2, 3]. Selective estrogen receptor modulators and aromatase inhibitors have become common means of treatment of breast cancer precancerous lesions in modern medicine. However, these two drugs have anti-estrogen effects on breast, resulting in endocrine disorders and a series of adverse reactions. Researchers have been attempting to make the most scientific classification of BC according to its histopathological and molecular properties in order to understand its pathological diagnostic value more [4], but the theoretical community considers this to be an almost intractable task. Endocrine therapy, surgery and radiochemotherapy are the main therapy for BC, but these traditional treatment methods have low remission rate and high recurrence rate, which is not the best choice for patients longing for long-term survival. The combination therapy of new therapy represented by targeted therapy and immunotherapeutic combined with the above traditional therapies has made rapid progress in recent years with significant curative effect, indicating that immunotherapy provides new hope for patients with breast carcinomas.

The research and development of food with anti-cancer properties is an important link in the field of anti-tumor. Quercetin (Que;3, 3’, 4’, 5’, 5, 7–Pc-ntabyolroxyflavonv) is a kind of flavonoids, widely exists in nature, mainly in the form of rutin, quercetin, hypericin and so on. Que can be obtained by acid hydrolysis reaction [5]. It is widely distributed in the leaves, flowers and fruits of fruits, vegetables and Chinese herbal medicine in nature, and has multiple biological activities and pharmacological effects. Therefore, more and more researchers begin to pay attention to the dietary substances rich in flavonoids such as Que. Current studies on Que have covered in vitro anti-inflammatory, anti-tumor, anti-platelet aggregation, protection of endothelial cells, anti-oxidation, inhibition of T cell activation and so on [5, 6]. Through previous research results, we found that Que can effectively prevent and treat BC through estrogen and anti-estrogen effects, reversing drug resistance and chemotherapy sensitization, inhibiting cell growth and inducing apoptosis, inhibiting tumor cell invasion and transformation, and inhibiting tumor angiogenesis [7, 8]. Which means that it has the potential to become an alternative or complementary drug for the treatment of BC. Firstly, Que is a native Chinese medicine monomer with estrogenic activity, which can activate ERβ in human breast carcinoma cells (T47D) to produce anti-estrogen effect, thereby affecting the proliferation of mammary cancer cells. Animal experiments have shown that Que reduces the risk of BC [9]. Secondly, Que can reverse the multi-drug resistance (MDR) problem of tumors [10], effectively improve the effect of chemotherapy, thereby improving the poor prognosis. Correlation studies have found that Que can regulate the unbalance of Th1/Th2 cell subsets, induce the body to produce immune response, and make it play a normal role in immune regulation [11]. In animal experiments, combined use of Que and doxorubicin can effectively inhibit tumor in BC (4T1) mice model, reduce cytotoxicity and improve therapeutic effect [12]. Moreover, Que can effectively inhibit the proliferation of tumor cells and even induce apoptosis by blocking the division cycle, interfering with the signal transduction of stem cells [13, 14], and regulating the expression of tumor-related genes and proteins [15]. In addition, we all know that the failure to effectively control tumor invasion and metastasis is the main cause of recurrence and death in BC patients, which is closely related to matrix metallo-proteinases (MMPs). Que can inhibit the proliferation and invasion of BC cells by down-regulating the expression of mRNA and protein in MMP9 [16]. In a mouse model of colorectal cancer, Que may inhibit the survival and metastasis ability of colon 26 (CT26) cells by regulating MMPs and tissue inhibitor of metalloproteinases (TIMPs), thereby inhibiting the metastasis of cancer cells to the lung, which confirmed that Que may be an effective treatment for metastatic colorectal cancer [17]. Nowadays, the high cost of treating cancer and the low success rate of most conventional treatments lead to an urgent need for cost-effective prevention and treatment methods. In this context, it is naturally the most appropriate direction to study the interaction between diet and cancer. Therefore, this study aims to elucidate the key properties of Que on breast cancer precancerous lesions, apoptosis of BC cells or regulation of immune cells, and consider that these properties are anti-tumor properties to improve the therapeutic effect of BC.

γδ T cells are innate immune cells, accounting for 0.5 ~ 5% of all peripheral T lymphocytes. It was found by observation that γδ T cells were mainly densely distributed in mucosal epithelium, belonging to a special class of immune cells between acquired immunity and natural immunity. Its subgroups are usually divided into Vδ1 T cell and Vδ2 T cell [18]. γδ T cells play an immune-regulatory role in the immune response to many infectious and immune diseases, known as the first line of defense against infection, autoimmune diseases and cancer [19]. Some studies have confirmed that γδ T cells play an active role in the anti-tumor immune response of nasopharyngeal carcinoma, colon cancer, renal cancer, non-small cell lung cancer [20–22]. Research staff found that γδ T cells isolated from BC, lung cancer, ovarian cancer, colon cancer and other patients could effectively kill tumor cells or primary tumor cells when interleukin-2 (IL-2) was amplified in vitro [23]. This indicates that γδ T cells have strong anti-tumor activity. It is noteworthy that many scholars have taken the presence of tumour-infiltrating γδ T cell in tumors as an important indicator to predict the recurrence and poor survival of BC patients [24]. γδ T cells have a good application prospect in tumor immunotherapy, and phase I/II clinical research is underway [21]. However, γδ T cells have complex biological activities, and γδ T cells in peripheral blood may also have functional subsets with different functions. In-depth study of these γδ T cell subsets can better use γδ T cells for the adoptive immunotherapy of BC and predict the prognosis of patients with breast cancer precancerous lesions.

As is known to all, JAK-STAT1 signaling pathway is usually involved in the process of tumor cell recognition and tumor-driven immune escape. Signals of cell can be transmitted from extracellular to intracellular through this pathway, activating related genes, and regulating cell growth and differentiation [25]. STAT1 is a growth inhibitor and apoptotic promoter, which can promote apoptosis and inhibit the growth and differentiation of cells in the JAK/STAT1 pathway [26]. Specifically in BC, loss of STAT1 in the breast epithelium has been associated with neuro-driven tumorigenesis and the development of spontaneous breast tumors in BALB/C mice, which are associated with loss of epithelial interferon regulatory factor (IRF1) and impaired T cell infiltration and killing capacity [27]. Since the co-inhibitory signal of programmed death receptor 1 (PD-1)/ programmed cell death 1 ligand 1 (PD-L1) co-mediated T cell activation inhibits the killing function of T cells, tumor cells can effectively avoid the recognition, killing and clearance of the immune system. PD-1/PD-L1 interaction plays a negative regulatory role in human immune response [28]. PD-1 can maintain immune tolerance in normal immune system. In the environment of virus infection or tumor, PD-1 reduces the proliferation and activation of T cells by binding to its ligand, resulting in weakened immune response [29]. At this point, the immune surveillance mechanism of the immune system on tumor cells fails and immune escape occurs. In the JAK-STAT1 pathway, the transcription factor STAT1 in tumor cells can regulate the expression of PD-L1, and is positively correlated with the expression of PD-L1 [30]. In addition, we observed that PD-L1 binds to PD-1 receptors on activated T cells, leading to a weakening of anti-tumor immunity by inhibiting T cell activation signals [31]. Therefore, by reducing the expression of PD-L1 and thereby decreasing the combination with PD-1, it plays an important role in the treatment of patients with breast carcinoma.

Based on the above content, this paper focuses on the effect of Que on immune cells γδ T cells and how to synergistically play the role of anti-breast cancer.

Que Target Gene Identification

According to the structure of Que, the potential target genes of Que were predicted using PharmMapper database, TCMSP database and Swiss Target Prediction database.

Prediction of Precancerous lesions of BC-Related Que Target Genes by System Pharmacology Approach

Genes previously associated with breast cancer lesions were searched by GeneCards database (https://www.genecards.org/) with “Precancerous lesions” as the key word to obtain disease target information.

GO and KEGG Enrichment Analysis

The target of Que was intersected with the disease target, and the protein-protein interaction analysis and GO and KEGG enrichment analysis were performed by STRING database. Import PPI data into Cytoscape 3.6.1 for visualization.

Samples collection

After obtaining the consent of participants, peripheral blood samples were obtained from healthy controls (HC), and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hyperaque gradient centrifugation. All the above procedures were carried out according to the guiding principles of the Medical Ethics Committee of Guangdong Provincial Health Bureau, and the ethics involved was examined and approved by the Ethics Committee of the First Affiliated Hospital of Jinan University in Guangdong Province.

Cell culture

MCF-10A (a non-tumorigenic epithelial cells), MCF-10AT (breast cancer precancerous cells) and MCF-7, MDA-MB-231 (human breast cancer cells) (American Type Culture Collection ATCC, USA). The cells were maintained as monolayer cultures (25 cm² plastic culture flasks) in F12/DMEM and DMEM (1x) medium with 10% fetal bovine serum (Gibco South America) and Donor Equine Serum (HyClone, USA), Pen strep at 37°C in an incubator with an atmosphere containing 5% CO₂. The cells were cultured every 2 days.

Que was co-cultured with T cells

Peripheral blood of sterile anticoagulant normal subjects was collected and separated with lymphocyte separation solution to obtain peripheral blood mononuclear cells. Then resuspended in RPMI 1640 complete medium (containing 500µL double antibody + 10% fetal bovine serum), and coated in 24-well culture plate with 1µL (1 µg/mL) solid phase anti-human TCR γδ monoclonal antibody. Stimulated at 37℃ and 5%CO₂ for 3 days. Starting from the fourth day, the solution was changed by centrifugation every other day, supernatant was removed, and IL-2 (10ng/mL) was supplemented in time. Que working solution with concentrations of 0, 2.5, 5 and 10 µM was added into the culture medium (constant volume to 1mL system). The culture was again placed at 37℃ and 5%CO₂. γδ T cells were cultured for 16 days and Que was cultured for 13 days, it is used to detect γδ T cell subsets.

MTT assay

The IC₅₀ value of Que was detected by MTT method. In short, MCF-10AT, MCF-7 and MDA-MB-231 cells were seeded in 96-well plates for 48h. MTT solution ( sigma ) was incubated at 37℃ for 4h. A total of 140µL of dimethyl sulfoxide ( Sigma ) working solution was added to each well and shaken on a low-speed shaker for 10 min to completely dissolve the crystal. The IC₅₀ value of Que was determined by absorbance at 490 nm (Biotek, USA).

Apoptosis assay

The apoptosis rates were verified by the Annexin V staining. As negative controls, the untreated cells were used, and cells treated with staurosporine (eBioscience, USA), which was used to induce apoptosis, were used as positive controls. Cells were collected, the number of cells was about 3*10⁵/ mL, washed and centrifuged with PBS and the supernatant was discarded. The 195µL binding buffer with 5µL of annexin V-FITC (eBioscience, USA) then incubated for 15 min at room temperature. Resuspend the cells with 190µL binding buffer and 10µL of PI (20ug/ml) were added to the machine and then immediately put cells on the machine (Cytoflex S, Beckman Coulter, USA) to detect cell apoptosis, and data were analyzed by Flowjo software (Flowjo LLC, USA) (annexin ־/PI־ ,(early apoptotic cells (annexin+/PI־ ,(late apoptotic cells (annexin+/PI+), and necrotic cells (annexin ־/PI+).

Flow Cytometry

PBMCs were isolated from samples of HC and then were incubated with the following antibodies: CD3-FITC (clone SK7), TCR γδ-PE (clone IP26), Vδ1- PE-Cy7 (clone TS8.2), Vδ2-PerCP (clone B6), PE-Cy7 isotype Control (clone P3.6.2.8.1), PE isotype control (clone eBM2a), FITC isotype control (clone MOPC-21), Percp isotype control (clone MOPC-21) (Biolegend, SanDiego, USA; BD, Biosciences, San Jose, USA; Abcam Cambridge, UK). PBMCs were stained with different combinations of anti-human TCR γδ, Vδ1, Vδ2, and CD3 antibodies at room temperature in the dark room for 20 min. A total of 30,000–50,000 CD3 + T cells were collected with a Cytoflex S flow cytometer (Beckman Coulter,USA), and data were analyzed by Flowjo software (Flowjo LLC, USA).

Co-culture of γδ T cells and MCF-10A, MCF-10AT, MCF-7, MDA-MB-231 cells proliferating treatment and CFDA SE (CFSE) assay

Subconfluent (80%) monolayers of MCF-10A, MCF-10AT, MCF-7,MDA-MB-231 cells were trypsinized (Invitrogen,USA) adjusted for 2x10⁶ cells/mL. 1mL resuspended related cell lines were taken out and placed in a 1.5mL centrifuge tube, and 0.4µL CFSE (concentration was 5mM) was added to make the final concentration of the sample 2µM. Then, the trypsinized cells were pre-incubated with γδ T cells were diluted in RPMI 1640 medium with 10% fetal bovine serum (Ausgenex, Australia), Pen strep (Life Technologies, USA) at 37°C for 4–5 h in a humid atmosphere containing 5% CO₂. After this period, co-culture with γδ T cells, the cells were taken out and stained with 0.5µL PI, and the killing efficiency of γδ T cells was detected by Cytoflex S (Beckman Coulter, USA) immediately.

Western blot Analysis

Extract total protein according to protein extraction kit instructions. Protein concentration (Beyotime, China) was determined by BCA method. After electrophoresis, the protein was transferred to PVDF membrane (Millipore, Germany) and blocked with 5% skimmed milk powder for 2h at room temperature. The membrane was incubated overnight with primary antibody (diluted according to manufacturer instructions) at 4°C. On the second day, the horseradish peroxidase-conjugated secondary antibody diluted by 1:5000 was incubated at room temperature for 1h. After a lot of washing with TBST, PVDF membrane was detected by gel automatic exposure imaging system. Image-Pro Plus software was used for gray value analysis in Western blotting.

Statistical analysis

SPSS 13.0 statistical software was used to analyze the data, the mean ± standard deviation was used to represent the data, and the Wilcoxon test method for comparison of two independent samples of the original data was used to analyze whether the data were statistically different. One-Way ANOVA was used for comparison between groups. The inspection standard shall be as per P < 0.05 indicates significant statistical difference, * indicates P & lt; 0.05, ** means P ≤ 0.01, *** means P ≤ 0.001. The experimental data and related statistical data were graphed by GraphPad Prism 5 software.

Prediction of the Que Target Genes

The chemical structure of Que was obtained from the PubChem database as shown in Figure 2A. Based on its structure, PharmMapper database, TCMSP database, and Swiss Target Prediction database were used to predict the potential target genes of Que. The targets obtained from these databases were combined and duplicates were removed to obtain 482 potential target genes.

Prediction of Precancerous lesions of breast cancer-Related Que Target Genes by System Pharmacology Approach

By GeneCards database (https://www.genecards.org/), with "Precancerous lesions," as the keyword search genes associated with breast cancer lesion before, get 1602 disease targets information. Intersecting Que 482 potential targets genes with 1602 disease targets resulted in 314 genes, as shown in Figure 2B, and these target genes were not only Precancerous lesions related genes but also the drug targets. The PPI network graph of target contains 157 nodes and 589 edges, in which the nodes represent targets and the edges represent the interaction between targets. Import the PPI data into Cytoscape 3.6.1 software for visualization. As shown in Figure 2C, the top 20 targets for the degree value of target are MAPK1, SRC, HRAS, AKT1, HSP90AA1, MAPK8, RHOA, MAPK14, ESR1, EGFR, IGF1, RXRA, JAK2, CASP3, AR, PTK2, IL-2, STAT1 and MMP9.

Functional Enrichment Analysis for the Que Target Genes

In order to explore the relationship between these 314 potential target genes, the GO and KEGG enrichment analysis were done by the STRING database. A variety of GO enrichment terms were enriched, including 1980 biological processes, 100 cellular components, and 223 molecular functions. We found that the biological processes such as cellular process and metabolic process, cellular components like binding and catalytic activity, and molecular function like cell and intracellular (Figure 2D), were enriched, which may be involved in the biological activity in the Que treatment process. In addition, 176 KEGG pathways were enriched (Figure 2E), among the major Que related pathways screened by KEGG, the pathways were mainly associated with cancer pathway and PI3K-Akt signal pathway, however, JAT/STAT ranked fourth among the pathways screened for immunity to Que (Figure 2F). It is suggested that Que may improve the progression of breast cancer from precancerous lesions to BC or improve the prognosis of breast cancer patients through JAK/STAT1 signaling pathway.

Cell proliferation experiments in MCF-10AT, MCF-7 and MDA-MB-231 cells

The effects of Que the proliferation of MCF-10AT, MCF-7 and MDA-MB-231 cells were determined by MTT assay and the results were given in Figure 3C. The half maximal inhibitory concentration (IC₅₀) values of Que on MCF-10AT, MCF-7 and MDA-MB-231 cells were 52.39µM, 53.76µM and 64.23μM, respectively.

The apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 cells was induced by Que at different concentrations at different time periods

To evaluate the apoptotic properties of Que, induced cell death, we performed the Annexin-V binding assay. We detected the apoptosis of MCF-10A, MCF-10AT, MCF-7, MDA-MB-231 cells treated with Que at 5μM, 20μM, 80μM, and 120μM at 24 h and 48 h, respectively (Figure 3A and 3B). First, Que was treated with mammary gland cells for 24h. We compared MCF-10AT with MCF-10A, MCF-7 and MDA-MB-231 cell lines at Que concentration of 5μM. The total apoptotic cell population % was determined as 2.85±0.21% and 3.27±0.14%, 2.84±0.21%, 2.85± 0.21% (P=0.006, P=0.109, P=0.004, respectively), there was significant statistical significance. MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 20μM concentration of Que. The total apoptotic cell population % was determined as 7.60±0.21% and 3.31±0.12%, 12.76±0.37%, 20.17± 2.90% (P=0.004, P=0.002, P=0.004, respectively), there was significant statistical significance. MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 at 80μM concentration of Que. The total apoptotic cell population % was determined as 35.63±0.60% and 1.64±0.05%, 25.34±5.06%, 29.80± 0.98% (P=0.002, P=0.004, P=0.004, respectively), there was significant statistical significance. MCF-10AT was compared with MCF-10A, MCF-7 and MDA-MB-231 cell lines at 120μM concentration of Que. The total apoptotic cell population % was determined as 40.25±0.71% and 3.40±0.07%, 29.38±2.50%, 29.40± 2.51% (P=0.002, P=0.002, P=0.002, respectively), there was significant statistical significance. Next, We found that MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 5μM concentration of Que, the total apoptotic cell population % was determined as 1.75±0.33% and 1.34±0.15%, 9.63±1.70%, 9.58± 0.32% (P=0.240, P=0.004, P=0.004, respectively). MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 20μM concentration of Que, the total apoptotic cell population % was determined as 8.44±1.54% and 0.84±0.10%, 11.12±1.38%, 14.23±1.62% (P=0.004, P=0.016, P=0.004, respectively), there was significant statistical significance. MCF-10AT was compared with MCF-10A, MCF-7 and MDA-MB-231 cell lines at 80μM concentration of Que, the total apoptotic cell population % was determined as 72.36±1.77% and 3.52±0.14%, 61.95±1.22%, 74.73±0.77% (P=0.004, P=0.004, P=0.009, respectively), there was significant statistical significance. MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 120μM concentration of Que, the total apoptotic cell population % was determined as 79.19±1.76% and 5.52±0.30%, 61.95±1.22%, 74.37±0.80% (P=0.004, P=0.004, P=0.002, respectively), there was significant statistical significance (Figure 3D). These results indicate that Que can induce apoptosis on MCF-10AT, MCF-7, MDA-MB-231 and other pre-breast cancer cells and BC cells at different times and at different concentrations, showing a time and concentration dependence. However, Que has a stronger apoptosis effect on MCF-10AT cells with pre-breast cancer lesions. However, MCF-10A cells had no apoptosis effect. Apoptosis trends at different concentrations at 24h and 48h were shown in the figure (Figure 3E).

Que can promote γδ T cell subsets and their amplification

In vitro induction of peripheral blood mononuclear cells from healthy volunteers in complete medium containing TCR γδ monoclonal antibody and cytokine IL-2. After adding 0μM, 2.5μM, 5μM, and 10μM Que on day 3, the cell morphology was observed under microscope and the proportion of γδ T cell subsets was determined by flow cytometry after 10-12 days. We found that the number of γδ T cells increased with the increase of the number of days in vitro. Flow cytometry showed that the γδ T cells could be effectively amplified, and the purity of γδ T cells could reach more than 60%, and the γδ T cells could be expanded to 90% at the concentration of 2.5μM to 5μM. Vδ2 T cell subsets were dominant at 2.5μM Que, Vδ1 T cell subsets were dominant at 5μM Que, and Vδ2 T cell killing subsets were dominant at 10μM Que (Figure 4A and B). These results indicate that γδ T cells have better killing and immunomodulatory effects.

Cytotoxicity of healthy human γδ T cells against MCF-10A, MCF-10AT, MCF-7, MDA-MB-231 cell lines

After amplification in vitro, the effector cell: target cell (E/T) was 10:1, the killing rates of γδ T cells against MCF-10A, MCF-10AT, MCF-7, MDA-MB-231 were 61.44±4.70, 55.52±3.10, 53.94±2.74, 53.28±1.73 (P=0.114, P=0.486, P=0.343, respectively) (Figure 4C). There was no significant difference between the groups. These results indicate that γδ T cells have a certain killing effect on both precancer and BC cells.

Que and γδ T cells have synergistic cytotoxic effects on mammary gland cell

In order to investigate the killing effect of Que and γδ T cells on mammary gland cell, we used 5μM Que to detect E/T (1:1, 5:1, 10:1) respectively to investigate the specific killing effect. We found that the cell killing rates of MCF-10A in E/T (1:1, 5:1, 10:1) were 24.12±4.34, 51.93±6.47, 64.94±3.61. The cytotoxicity rates of MCF-10AT in E/T (1:1, 5:1, 10:1) were 19.38±5.30, 33.45±5.49, 64.96±5.45，and MCF-10A > MCF-10AT (P=0.222, P=0.008, P=0.917, respectively). The cell killing rates of MCF-7 at E/T (1:1, 5:1, 10:1) were 13.23±2.68, 24.39±3.13, 55.59±5.98, compared with MCF-10AT, the killing rate of MCF-7 versus MCF-10AT was statistically significant at 5:1 (P=0.095, P=0.032, P=0.056, respectively). The cell killing rates of MDA-MB-231 at E/T: 1:1, 5:1, 10:1 were 12.77±3.64, 22.7±1.39, 59.04±5.67, respectively. Thus, compared with MDA-MB-231, MCF-10AT was also statistically significant at 5:1 (P=0.056, P=0.016, P=0.222, respectively). In addition, with the increase of effector cell proportion, Que concentration was still 5μM, and MCF-10A > MCF-10AT > MCF-7 > MDA-MB-231, 1:1 < 5:1 < 10:1 (Figure 4D). These results indicated that Que combined with γδ T cells had a certain specific killing effect on both precancerous breast cancer cells and BC cells, and the strongest killing effect on precancerous breast cancer cells and BC cells was found when the Que concentration was 5μM and E/T (10:1).

Effect of Que on IFNγ-R, Phospho-JAK2 (p-JAK2), Phospho-STAT1 (p-STAT1) and PD-L1 in MCF‐10AT and MCF-7 cell lines protein expression

Western blotting of IFNγ-R, p-JAK2, p-STAT1 and PD-L1protein were performed. The concentrations of Que were 0μM, 5μM, 20μM, 80μM and 120μM, and the cells were co-cultured with MCF-10AT and MCF-7 cells for 48 h, respectively. Our results showed that when MCF-10AT and MCF-7 cells were treated with Que concentration of 80μM and 120μM, respectively, a substantial increase in protein levels of IFNγ-R, p-JAK2 and p-STAT1 were significantly increased (P<0.0001), while the concomitant decrease protein levels of PD-L1 (P<0.0001, P=0.0005) (Figure 5A and B).

BC is a malignant tumor, mostly in female. Since most patients failed to pay attention to breast precancerous lesion, the number of BC patients increased year by year. Precancerous lesions of breast cancer are proliferative lesions with abnormal breast tissue morphology, proliferation or differentiation. After follow-up observation, it is possible to develop into cancer. As a stage of tumor development, most of these lesions are in an unstable state and can undergo malignant changes under the continuous action of certain factors. However, if the carcinogenic factors are removed, it is possible to maintain stability or degeneration or reversal, return to normal or close to normal. In addition, for precancerous lesions of breast cancer, the main therapeutic drugs are estrogen receptor modulators or aromatase inhibitors, which can cause endocrine disorders and increase the incidence of endometrial cancer or uterine fibroids and sarcomas [32]. Because T cell immune function is closely related to anti-tumor effect, it affects the disease progression and prognosis of patients with BC [33]. In-depth study of cellular immune status in patients with pre-cancerous breast cancer can help patients with pre-breast cancer control the progression of the disease by improving their autoimmune system. For BC patients, their own immune status determines the outcome and prognosis of the disease, which undoubtedly brings new hope to BC patients.

In the previous network pharmacology analysis of Que, we used relevant databases to screen 482 potential genes related to Que, and then screened genes related to breast precancerous lesions or BC. Studies have found that these genes are not only breast precancerous lesions or BC-related target genes, but also Que-related target genes. Then, we found that the biological processes such as cellular process and metabolic process, cellular components like binding and catalytic activity, and molecular function like cell and intracellular, were enriched, which may be involved in the biological activity in the Que treatment process. However, in KEGG enrichment analysis, JAT/STAT1 ranked fourth among the pathways screened for immunity to Que. Que-related pathways included JAK/STAT1 pathway, suggested that Que may improve the progression of breast cancer from precancerous lesions to BC or improve the prognosis of breast cancer patients through JAK/STAT1 signaling pathway.

The immune system plays an important role in immune defense, immune surveillance and immune homeostasis. When the body's immunity is reduced, the body cannot timely remove aging damaged mutant cells and invasive pathogens, resulting in tumor and infection. Therefore, improving the immune function of the body is of great significance for maintaining health and resisting diseases. Que widely exists in nature. It not only has anti-inflammatory, anti-tumor, anti-platelet aggregation, protection of endothelial cells and antioxidant effects, and it has a regulatory effect on immune cells. Studies have proved that Que has a two-way regulatory effect on immune cells. Under its influence, the differentiation of T cell subsets in normal mice was promoted, and pathological CD4 + T cells were inhibited to achieve the therapeutic effect on autoimmune myocarditis [34]. In addition, Que also enhanced the immune function of Th2 cells in immunocompromised mice [35]. Other studies have shown that flavonoids can promote immune cell proliferation and enhance immunity [36]. Some scholars found in the OVA-induced mouse asthma model experiment that Que could regulate the expression of T-bet and GATA-3 genes and affect the production of Th1/Th2 cytokines, resulting in the decrease of IL-4 level of Th2 cytokines and the increase of IFN-γ level of Th1 cytokines [11]. This study found that Que could induce breast precancerous lesions and apoptosis of breast cancer cells in a time and concentration-dependent manner. At 24 h, 120µM Que had the most obvious apoptosis effect on MCF-10AT cells, which was shown as MCF-10AT > MCF-7 > MDA-MB-231 > MCF10A. At 48 h, 80µM Que had apoptosis effect on breast cancer precancerous cells and BC cells. At the same time, it was consistent with the IC₅₀ value of Que. In addition to Que itself has a pro-apoptotic effect, this study also found that Que has a regulatory effect on immune cells, and has a promoting effect on the proliferation of γδ T cells and their subsets. When the concentration of Que is 5µM, The Vδ1 T cells regulatory subsets of γδ T cells are dominant, and γδ T cells play an immunomodulatory role. However, with the increase of the concentration, the killing subpopulation of Vδ2 T cells gradually became dominant. The results showed that Que could promote the differentiation of γδ T cells into Vδ2 T cells, and make them play a killing role. This suggests that Que has a stimulating effect on the body's immune system.

It is well known that γδ T cells have antiviral and anti-tumor effects, and have good transformation and application prospects in subsequent immunotherapy. However, due to the obvious heterogeneity of γδ T cells in patients, different subsets may have different functions. Previous studies have shown that γδ T cells can play the role of Th1 (type 1 helper T lymphocytes) by producing IL-2 and IFN-γ when intracellular bacteria are infected. When extracellular parasites and other infections occur, interleukin-10 (IL-10) and interleukin-4 (IL-4) are produced and play a role in Th2 (type 2 helper T lymphocytes) [37]. With the further research of γδ T cells, people have realized gradually that γδ T cells are related to the occurrence and development of diseases. Noguchi et al found that the number of γδ T cells in patients with solid tumors such as BC, lung cancer and gastric cancer was significantly lower than that in healthy controls because chemotherapy inhibited the proliferation of γδ T cells [21]. In the experiment, we found that when the ratio of γδ T cells to tumor cells was 10:1, γδ T cells had the largest killing effect on tumor cells. Then, we co-cultured Que with γδ T cells and tumor cells, and found that when the concentration of Que was 5µM and E/T was 10:1, the killing effect on MCF-10 A, MCF-10 AT, MCF-7 and MDA-MB-231 reached the maximum. These results indicated that Que and γδ T cells could further enhance the killing effect on tumor cells, and the Que and γδ T cells may have synergistic effect. These results show that Que indeed has a positive regulatory effect on cellular immune function. Therefore, people in daily life to eat rich in Que hawthorn, honey, juice and other food, to regulate the immunity function of the body is very useful. However, due to the complex biological characteristics of γδ T cell subsets, it is necessary to conduct more in-depth research on their functional subsets, need provide more basic research data for individualized immunotherapy. So as to comprehensively understand the cellular immune status of breast precancerous lesions and BC patients.

JAK/STAT1 pathway is almost involved in the entire immune regulation process, including immune surveillance and immune escape. STAT1 is a growth inhibitor and proapoptotic factor, which can promote cell apoptosis, inhibit cell growth and differentiation, and play an important role in inhibiting the occurrence and development of tumors, especially the interferon (IFN) system [38]. STAT1 dependent IDO overexpression blocked the activation of T cells, which is related to the increase of PD-L1 expression in high-level triple negative breast cancer (TNBC) cells [39]. In many cancer diseases, STAT1 overexpression also promotes tumor cell survival and immune depletion by prolonging IFN-γ signaling. Some cytokines act on the receptor IFN γ-R on tumor cells. After phosphorylation by JAK1/2, they promote tumor immunogenicity by up-regulating MHC and immune checkpoint proteins (such as PD-L1 and B7-1) through STAT1 signal of transcription factor [40]. Studies have shown that the pathogenesis of BC is closely related to the significant increase in the expression levels of inflammatory factors such as TNF-α and IL-6, while in TNBC, it is closely related to the increase in PD-L1 [30]. The expression of these cytokines may be regulated by IFNγ-R, nuclear transcription factor-STAT1 and JAK1/2. Our results showed that after MCF-10AT and MCF-7 cells were treated with 80µM or 120µM at Que for 48 h, the levels of IFNγ-R, p-JAK2 and p-STAT1 increased, and the level of PD-L1 decreased. These results indicate that Que may further increase the phosphorylation of JAK2 on the JAK/STAT1 pathway by activating IFNγ-R on tumor cells, and increase the nucleation of p-STAT1, thus reducing the expression of PD-L1 on the precancerous cells of breast cancer. It further reduce the binding of PD-L1 and PD-1 on the T cell receptor, avoiding to a weakening of anti-tumor immunity by T cell activation signals, so that the immune cells of the body can play a normal immune role. Thus, the JAK-STAT1 pathway becomes an attractive therapeutic target during precancerous lesions of breast cancer and BC development and progression.

Summary, Que as a naturally occurring flavonoids in nature, its anti-tumor and immune regulation should be widely used in clinical, so in daily life need to appropriate consumption of foods rich in Que to prevention and treatment of disease. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of BC. Therefore, the use of Que in daily life to improve the immune function of the body to enhance the ability to fight disease has received more attention.

Availability of data and materials

The data sets analyzed in the present study are publicly available data from the PubChem database, PharmMapper database, TCMSP database, Swiss Target Prediction database and GeneCards database. The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgements

Part of the data in this study were obtained through the PubChem database, PharmMapper database, TCMSP database, Swiss Target Prediction database and GeneCards database. We are grateful for the source of data used in our research.

Funding

This work was supported by the National Natural Science Foundation of China (nos. 82074430, 81803979, 81673979); the Natural Science Foundation of Guangdong Province, China (nos. 2018A030313393 and 2016A030313114); Science and Technology Program of Guangzhou, China (nos. 201803010051, 201707010245, and 201704020117) and the Fourth Batch of TCM Clinical Outstanding Talent Program of China (no. 444258).

Author information

Dan Qiu, Xianxin Yan and Xinqin Xiao contributed equally to this work

Affiliations

College of Traditional Chinese Medicine of Jinan University, Jinan University, No.601, West Huangpu Avenue, Guangzhou, 510632, Guangdong, China.

Dan Qiu, Xianxin Yan, Xinqin Xiao, Yanqiu Wang, Ruirui Ma, Shouyi Hong & Min Ma

The First Affiliated Hospital of Jinan University, Jinan University, Guangzhou, 510632, Guangdong, China.

Guijuan Zhang & Jingyu Cao

Contributions

Dan Qiu: Writing - original draft, Writing - review & editing.

Xianxin Yan, Xinqin Xiao: Resources, Writing - original draft.

Guijuan Zhang, Yanqiu Wang, Jingyu Cao, Ruirui Ma, Shouyi Hong: Participate in article writing.

Min Ma: Conceptualization, Writing - original draft, Writing - review & editing, Project administration, Funding acquisition.

Corresponding authors

Correspondence author. Min Ma, Jinan University, No.601, West Huangpu Avenue, Guangzhou 510632, Guangdong, China. E-mail addresses: [email protected] and tmamin@jnu. edu. cn.

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Not involved in the relevant ethical research.

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None.

Competing interests

The authors declare no conflicts of interest.

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Discussion On The Mechanism of Que In The Treatment of Breast Neoplasms And Immune Prevention And Treatment

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Abstract

Figures

Background

Materials And Methods

Que Target Gene Identification

Prediction of Precancerous lesions of BC-Related Que Target Genes by System Pharmacology Approach

GO and KEGG Enrichment Analysis

Samples collection

Cell culture

Que was co-cultured with T cells

MTT assay

Apoptosis assay

Flow Cytometry

Co-culture of γδ T cells and MCF-10A, MCF-10AT, MCF-7, MDA-MB-231 cells proliferating treatment and CFDA SE (CFSE) assay

Western blot Analysis

Statistical analysis

Result

Discussion

Conclusions

Declarations

References

Status:

Version 1