Study on the Anticancer Effect of an Astragaloside- and Chlorogenic Acid-Containing Herbal Medicine (RLT-03) in Breast Cancer

Background Although surgery, chemotherapy, radiotherapy, and endocrine therapy are widely used in clinical practice for breast cancer treatment, herbal medicines (HMs) are considered as an alternative to palliative treatments because of their coordinated intervention effects and relatively low side effects. Astragaloside (AS) and chlorogenic acid (CGA) are major active ingredients of Radix Astragali and Lonicera japonica, which have shown antitumorigenic properties in certain cancers, but the role of HMs containing both AS and CGA remains unclear in breast cancer. In this study, we explored an AS- and CGA-containing HM (RLT-03) extracted from Radix Astragali, Lonicerae Japonicae Flos, Trichosanthin, and Rhizoma imperatae. Methods RLT-03 was extracted using water and n-butanol, and the AS and CGA ingredients in RLT-03 were identified by high-performance liquid chromatography (HPLC) and evaporative light-scattering detector (ELSD). 4T1, EMT6, BT-549, and MDA-MB-231 breast cancer cell lines were used, and an EMT6 xenograft model was established. Cell proliferation, migration, and apoptosis were measured in vitro, and tumor volume and weight were observed in vivo. The expression of VEGF, EGF, IL-10, TGF-β, and CD34 and cell apoptosis in tumors were examined. Results RLT-03 inhibited cell viability and induced apoptosis in a dose- and time-dependent manner. In vivo, tumor volume and weight were reduced, and the expression of VEGF, EGF, IL-10, TGF-β, and CD34 was suppressed in the tumor microenvironment, while cell apoptosis was induced. Conclusion RLT-03 exhibited therapeutic effects against breast cancer by regulating the expression of ligands of receptor tyrosine kinases (RTKs) and inflammatory factors. Thus, RLT-03 represents a potential supplementary HM that can be used in breast cancer therapy.


Introduction
Breast cancer is one of the most common malignant tumors in women. About 1.3 million women are affected by breast cancer every year worldwide, and more than 330,000 women die from it [1,2]. Surgery, chemotherapy, radiotherapy, and endocrine therapy are widely used in clinical practice, but such therapies have limitations [3,4]. erefore, finding a way to suppress cancer and improve the quality of life of patients is particularly important in the treatment and prevention of breast cancer, especially for late-stage patients and those with a poor performance status. Currently, herbal medicines (HMs) are considered as capable of inducing immunomodulatory effects. ey protect cancer patients from complications and improve quality of life and survival time [5,6]. For example, KIOM-C and Ganoderma lucidum have been used in the treatment of malignant HT1080 sarcoma and ovarian cancer [7,8].
Astragaloside (AS) and chlorogenic acid (CGA) are major active ingredients derived from Radix Astragali and Lonicera japonica, which have shown antitumorigenic properties in certain cancers. In previous studies, Astragalus-based HMs have been shown to enhance the efficacy of platinum-based chemotherapy and improve platinum-derived toxicities for late-stage non-small cell lung carcinoma (NSCLC) [9]. In addition, they have been shown to inhibit the viability and invasive potential of MDA-MB-231 breast cancer cells by suppressing the activation of the mitogenactivated protein kinase (MAPK) pathway and downregulating matrix metalloproteases (MMPs)-2 and -9 [10]. Furthermore, several studies have indicated that CGA may act against cancer [11,12] by regulating the expression of apoptosis-associated genes and causing cell-cycle arrest [13][14][15][16]. However, the role of an HM containing both AS and CGA in breast cancer remains unclear. erefore, the present study aimed to explore the possible function and mechanism of the astragaloside-and chlorogenic acid-containing HM (RLT-03) in breast cancer progression in vitro and vivo.

RLT-03 Medicine Preparation.
RLT-03 is a HM developed by the Pharmaceutical Department of Chengdu Fuxing Hospital and has been used for the treatment of breast cancer. e theoretical guides behind RLT-03 formula were the traditional Chinese medicine theory "Jun, Cheng, Zuo, and Shi (Monarch, Minister, Assistant, and Guide)" and the Malignant Tumor Projection eory (MTPT).
is HM is prepared mainly from the extracts of Radix Astragali (Chinese name: Huang Qi), Lonicerae Japonicae Flos (Chinese name: Jin Yinhua), Trichosanthin (Chinese name: Tian Huafen), and Rhizoma imperatae (Chinese name: Bai Maogen) by water and n-butanol. e extract was dried in rotary evaporators and a vacuum drying chamber; the dried fractions were stored at 26°C in a dryer.

Elements Analysis of RLT-03.
High-performance liquid chromatography (HPLC) and an evaporative light-scattering detector (ELSD) were used to identify AS and CGA in RLT-03. Ten microliters of a sample of RLT-03 (5.07 mg/ml) and standard samples were injected into the HPLC system (Waters 2695 and Waters 2996 Diode Array Detector, USA) and ELSD (Sedere Sedex75, France), respectively.
2.6. Cell Scratch Assay. Cell migration ability was quantitated by the cell scratch assay. Approximately 2 × 10 4 cells were aliquoted into each well of a 6-well plate, and a microscope was used the following day to confirm that each well was coated with cells. A 1 mL pipette tip was used to scratch cells from the bottom of the well, and the plates were washed with phosphate-buffered saline three times to remove the displaced scratched cells. Cells were exposed to different concentrations of RLT-03 (2.0 and 3.0 mg/ml for 4T1 and BT-549 cells; 1.25 and 2.0 mg/ml for MDA-MB-231 cells; 1.5 and 2.0 mg/ml for EMT6 cells). Cells were cultivated simultaneously in an incubator at 37°C under 5% CO 2 . Images of the samples were captured at 0, 24, and 48 h by Nikon TS100 (Nikon, Japan). is procedure was repeated thrice.

Xenograft Breast Cancer Model and Treatment.
Female SPF-Balb/c mice weighing 21 ± 1.2 g were obtained from the Chengdu Dossy Experimental Animals Co. Ltd. (Chengdu, China). is animal study was approved by the Animal Care and Use Committee. EMT6 cells (1 × 10 6 cells/ mouse) were transplanted into the right dorsal side of each mouse. Sixteen tumor-bearing mice were divided into two groups (n � 8 each). Saline was administered to the blank control group by oral gavage, while 20.0 mg/g of RLT-03 was administered to the sample group by oral gavage for 21 days. e tumor volume and weight were recorded at three-day intervals. Tumor volume was calculated using the following formula: volume (mm 3 ) � width 2 × length/2.

Enzyme-Linked Immunosorbent Assay (ELISA).
e ELISA kits (20140603DE and 20140603DV, Abcam) were used to detect the levels of the IL-10 and IL-12 cytokines in the serum. Cytokine levels were analyzed by measuring the absorbance at 450 nm on Multiskan Mk3 ( ermo Fisher, USA).

Hematoxylin-Eosin Staining Assay.
Tumor tissues were dissected from the implanted mice, fixed in 10% buffered formalin for 24 hours, and embedded in paraffin. Tissue sections were cut at 5 μm thickness and stained with hematoxylin-eosin. Images were acquired by Motic BA400 (Motic, China).

TUNEL Assay.
TUNEL assay (Roche, 10279600 kits) was performed to evaluate apoptosis in tumor sections. Tumor sections were dewaxed at 26°C. Sections were treated with 3% H 2 O 2 and incubated with equilibration buffer and terminal deoxynucleotidyl transferase enzyme. Finally, sections were incubated with antidigoxigenin-peroxidase conjugate. Tissue peroxidase activity was evaluated through DAB application. Images were acquired by Motic BA400 (Motic, China).

Western Blot Assay.
Tumor tissues were lysed in lysis buffer and then centrifuged at 15,000 rpm for 15 min at 4°C. Protein concentration was determined using the BCA kit (Beyotime). A total of 50 µg of protein was subjected to 8-10% SDS-PAGE and transferred to a PVDF membrane (Merck Millipore, USA). e membranes were blocked for 1 hour at 26°C with 5% bovine serum albumin containing 0.1% Tween-20 and incubated with the primary antibodies (EGF, VEGF, and TGF-β) (Beyotime) (diluted by 1 : 1000) overnight at 4°C. en, the membranes were washed with TBST three times and incubated with the corresponding secondary antibody (diluted by 1 : 5000) at 37°C for 2 hours. e membranes were then washed again, and the proteins were visualized using an enhanced chemiluminescence assay kit (Beyotime). Images were captured by JY-Clear ECL (JUNYI, China).

Flow Cytometry Assay.
In vitro, 4T1, EMT6, and BT-549 cells (5 × 10 5 cells/well) were seeded in 6-well plates. Cells were exposed to RLT-03 (dose � IC 50 ) for 24 hours before being collected. Cells were stained with propidium iodide and an Annexin V kit (Yeasen, China) for 15 min in the dark. Samples were examined using a Guava ® EasyCyte plus flow cytometer (Merck, USA).

Statistical Analysis.
Results are expressed as means. e difference between mean values was assessed by the t-test using Prism GraphPad 8.0. A p value of <0.05 was considered to indicate a statistically significant difference.

RLT-03 Contains Astragaloside and Chlorogenic Acid.
e elements of RLT-03 were analyzed by HPLC and ELSD. e retention time of the peaks in the HPLC and ELSD corresponded to those of astragaloside and chlorogenic acid, respectively ( Figure 1).

Discussion
Breast cancer is one of the most common malignant tumors in women. WHO statistics have shown that about 330,000 patients worldwide die from breast cancer each year [1]. In China, the incidence of breast cancer presents a gradually increasing trend [2]. erefore, discovering effective drugs for late-stage breast cancer is of utmost importance. Although current therapies for breast cancer, such as chemotherapy, radiotherapy, and targeted therapy, are effective, these therapies have various side effects, are not suitable treatments for late-stage patients, and cannot be used in palliative care [3,18]. Recently, HMs have been suggested as suitable anticancer drugs for palliative care. us, based on the traditional Chinese medicine theory (TCM) and the combined application of multiple components of Compound Chinese Traditional Medicine (CCTM) [19], the combination of nearly 100,000 compounds has been used for clinical prevention and treatment of various diseases [20,21], and the effects of combined HMs for treating breast cancer has been suggested [22,23]. Furthermore, modernization of  Figure 1: RLT-03 contains astragaloside (AS) and chlorogenic acid (CGA). (a) ELSD analysis of RLT-03 HM. AS was identified in RLT-03. S1 represents AS at the retention time of 19.788 min, which is consistent with the astragaloside standard sample. (b) HPLC analysis of RLT-03 medicine. S1 represents CGA at the retention time of 6.197/6.613 min which is consistent with the chlorogenic acid standard sample (S1, the RLT-03 sample; S2, the AS standard sample; S3, the CGA standard sample). 4 Evidence-Based Complementary and Alternative Medicine   Chinese medicine is not just its westernization. In this study, we applied the Jun, Cheng, Zuo, and Shi (Monarch, Minister, Assistant, and Guide) theory and Malignant Tumor Projection eory (MTPT) as a theoretical guide behind the formulation of RLT-03 and explored the functions of its chemical components by applying molecular biology experiments in vitro and vivo.
Recently, the inhibitory function of the HM on cancer growth, invasion, and metastasis have been shown to be mediated through angiogenesis, cell differentiation, cell apoptosis, cytotoxic function, and immunity regulation [20,24,25]. For example, Kanglaite injection, Kushen injection, and Jinlong capsule have shown clinical efficacy on preventing disease progression with low toxicity [26]. Meanwhile, AS and CGA are major active ingredients from Radix Astragali and Lonicera japonica, respectively, which have shown antitumorigenic properties in certain cancers [27,28]. Individually, AS has been reported to have anti-inflammatory, anticancer, antioxidative, and immune-regulatory effects [29,30]. Consistent with previous studies, AS ameliorated cancer-associated inflammation, decreased the expression of inflammatory factors such as TGF-β and IL-10, and suppressed M2 macrophage polarization when administered to treat lung cancer [29]. Moreover, CGA has also been independently reported as having potential for tissue protection and cancer treatment. Studies have indicated that CGA could induce the Nrf2/ARE antioxidant system in hepatic cells [31,32] and protect the JB6 cell line against environmental carcinogen-induced carcinogenesis though NF-kappa B and MAPK pathways [33].
However, the literature lacks investigations focused on the direct effects of an HM that contains both AS and CGA on breast cancer. As expected, RLT-03 inhibited the growth of breast cancer in vitro and in vivo, which were demonstrated by MTT, scratch, and flow cytometry assays, as well as H&E and TUNEL staining. Besides, western blot and immunohistochemistry assays indicated that RLT-03 significantly inhibited the expression of VEGF, EGF, CD34, IL-10, and TGF-β related to the angiogenesis and inflammatory regulation of breast cancer. Moreover, compared to chemotherapy, RLT-03 showed limited pathological toxicity. Recently, RLT-03 has been approved for clinical use by Sichuan Food and Drug Administration, China.

Conclusion
RLT-03 exhibited prominent therapeutic effects on breast cancer mainly via the regulation of cell proliferation, apoptosis, and the tumor-microenvironment through the downregulation of the expression of RTK ligands and inflammatory factors. us, RLT-03 represents a useful and an alternative supplementary medicine for the late-stage and poor performance breast cancer patients, who could not tolerate or refuse second-/third-line therapy. e potential therapeutic effects of RLT-03 may be further ascertained in clinical trials.

Data Availability
No raw data were used from published articles.

Conflicts of Interest
ere are no conflicts of interest to declare.