Astragaloside-IV Inhibits Pancreatic Cancer Cell Proliferation in Vitro and in Vivo by Inducing Cell Cycle Arrest and Apoptosis

Astragaloside IV (AS-IV) or 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcyl-cloastragenol is a bioactive saponin extract from the root of Astragalus membranaceus. It has been proven to have an anti-tumor effect in a variety of tumors by inducing cell apoptosis and inhibiting cell proliferation. Its effects on pancreatic cancer have not been investigated. This study investigated the effects of AS-IV on proliferation, apoptosis and migration of pancreatic cancer cells in vitro and in vivo and explored its underlying mechanism. Pancreatic cancer cell lines SW1990 and Panc-1were treated with different doses of AS-IV. Plate clonality, CCK-8, EDU and ow cytometry were used to explore the effect of AS-IV on pancreatic cancer cell proliferation and cell cycle in vitro. Wound healing was used to investigate the effects of AS-IV on pancreatic cell migration. The protein expression levels of Bax/Bcl2, caspase3/7, cyclin D1, cyclin E and CDK4 were analyzed by western blotting. The results showed that AS-IV signicantly inhibited tumor cell proliferation and cell cycle, induced apoptosis both in vitro and vivo on a dose-dependent basis and signicantly inhibited the growth of pancreatic cell xenograft tumor in nude mice. Wound healing assays indicated that AS-IV also inhibited the migration of pancreatic cancer cells in a dose-dependent manner. This research conrmed that AS-IV inhibited pancreatic cancer cell proliferation by blocking the cell cycle and inducing apoptosis. It was hypothesized from this experiment that the potential mechanism of AS-IV inducing apoptosis of pancreatic cancer cells may be understood by activating the Bcl2/Bax/Caspase-3/Caspase-7 signaling pathway.

At a speci ed time, 10 µL CCK8 solution were added to each well for four hours. The absorbance was determined with the wavelength of 450 nm using a MK3 ELISA reader (Thermo Fisher Scienti c, USA).

EDU assays
Panc-1 and SW1990 cells were inoculated into 96-well plates and treated with different concentrations of AS-IV. Cell proliferation rates were determined according to the instructions using a 5-ethynn-20 deoxyuridine (EdU) kit (RiboBio, Guangzhou, China). 100 µL culture medium containing 50 M EdU was added to each well and incubated for 12 hours. The cells were then xed with 4% paraformaldehyde for 30 minutes and followed by treatment of 0.5% Triton for 10 minutes and Apollo reaction cocktail (RiboBio, Guangzhou, China) for 30 minutes. The cells were then contaminated with DAPI for 30 minutes for DNA analysis and observed under a uorescence microscope (Olympus CX41-72C02, Tokyo, Japan).

Wound healing assay
Wound healing assays were used to assess cell migration. Initially 5x10 5 Panc-1 or SW1990 cells/Well were added to the six-well plate. The cells were cultured overnight to produce a fused monolayer. A 10µL pipette tip was used to make a direct scratch on the cell monolayer. The suspension cells were washed with phosphate buffered saline (PBS) three times. 1ml serum-free medium containing different concentrations of AS-IV was then added. The wound healing process was photographed at 0, 24 and 48 hours.

Plate clonality assays
Pancreatic cancer cells of Panc-1 and SW1990 were suspended in 2 ml complete medium and seeded into six-well plates at 1×10 3 cells per well. After different treatments 40 and 80 μM AS-IV , they were cultured at 37℃ in air containing 5% CO 2 for two weeks. Cultures containing different concentrations of AS-IV were changed every two days. After 14 days, the colonies were xed with 4% paraformaldehyde and then stained with 0.5% crystal violet for 20 minutes. The separate experiments involving Panc-1 and SW1990 were each conducted three times.

Cell cycle analysis
Panc-1 and SW1990 cells at the logarithmic growth phase were incubated. After being treated with the varying doses of AS-IV (0, 40 and 80 μM) for 48 h, cells were harvested and resuspended with cold 75% ethanol at -4˚C overnight. The ethanol was then removed and 150 μl propidium iodide (PI) was added and incubated at 4℃ for 30min in darkness (Mei Lun Bio, Dalian, China). Flow cytometry was used to measure cell cycle distribution ((BD, Franklin Lakes, NJ, United States).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay To explore the apoptotic in Panc-1 and SW1990 cells, apoptosis was examined using a one-step TUNEL apoptosis assay kit (Yeasen, Shanghai, China). Cell crawling were washed with phosphate buffered saline (PBS) three times and xed with 4% paraformaldehyde for 30 minutes. This was then washed with PBS three times and 100μL Proteinase K was added for 20 min at 37℃ . Equilibration buffer was then  added and the cells were incubated at room temperature for 20min and 50μl TDT enzyme incubation   buffer containing 34μl ddH2O,10ul 5×Equilibration Buffer, 5μl FITC-12-dUTP Labling Mix and 1 μl  Recombinant TdT Enzyme, was then added for 60min at 37℃. This was rinsed with PBS three times and   cells were contaminated with DAPI for 10

Flow cytometry
Cell apoptosis was assessed by ow cytometry after Annexin-V-uorescence isothiocyanate (FITC)/ PI staining. The Panc-1 and SW1990 cells were exposed to differing doses of AS-IV for 48 hours respectively and then stained with 5 μL of FITC Annexin V and 5 μL PI for 10 minutes at room temperature in the dark and analyzed by ow cytometry according to the manufacturer's instructions (KeyGEN BioTECH, Jiangsu, China). Three independent experiments were conducted.

Western blotting assays
Western blot assays were performed similarly to the procedure reported previously (Transient receptor potential vanilloid-type 2 targeting on stemness in liver cancer [21]. Total proteins were extracted with RIPA cleavage buffer, separated by 10% sodium dodecyl sulfate SDS-PAGE, and transferred to polyvinylidene uoride (PVDF) membranes purchased from Millipore (MA, USA). The membrane was sealed with 5% skim milk, incubated with a primary antibody and then incubated with a secondary antibody. β-Actin was used as an internal reference.

Animal work and experimental protocols
Four-week-old male nude mice were provided by Hunan SJA Laboratory Animal Technology, Inc. (Hunan, China). Each nude mouse was given a single subcutaneous injection on the right ank of 1×10 6 Panc-1 cells, suspended in a matrix glue (BD Biosciences, CA). After seven days, the tumor size was measured twice weekly using a digital caliper and was calculated as (D 2 × d) / 2, where D is the large diameter and d is the small diameter of the tumor. Mice were randomly divided into two groups when the tumors were up to 100 mm 3 as a control group treated only with DMSO and an AS-IV treatment group receiving 0.1mg/10g/day AS-IV dissolved in DMSO. Each mouse was gavaged daily for 21 days. After anesthesia, tumor-bearing mice were sacri ced and the tumors were removed for further study. Animal experiments were conducted in accordance with the guidelines for animal care and use issued by the National Laboratory of the United States, and the experimental program was approved by the Animal Care and Use Committee of South China University (Hengyang, China).

Immunohistochemistry (IHC)
Immunohistochemistry was performed similarly to the procedure reported previously (Camellia oil (Camellia oleifera Abel.) Attenuates CCl4-induced liver brosis via suppressing hepatocyte apoptosis in mice) [22]. Immunohistochemical studies were performed on para n sections using anti-ki67 antibody developed using a biotinylated alkaline phosphatase-conjugated secondary antibody and diaminobenzidine (DAB) substrate kit according to standard methods in routine pathology. The positive cells were evaluated using ImageJ software and all measurements were made in three microscope elds randomly selected from each section.

Statistical analysis
Data is shown as mean ± standard deviation (SD). Statistical analysis was conducted using the SPSS (Chicago, IL, USA) and GraphPad Prism 8 (San Diego, CA) software. The signi cance of the variance between two or more groups was evaluated using student's t-test or ANOVA. P<0.05 had statistical signi cance.

AS-IV inhibited cell proliferation and induced cell cycle arrest in pancreatic cells
To uncover the functional roles of AS-IV in pancreatic cells, Panc-1 and SW1990 cells were treated with a series of AS-IV concentrations for 24, 48 and 72h. The CCK-8 data showed that AS-IV inhibited the proliferation of Panc-1 and SW1990 cells in a dose-dependent manner (Fig. 1a). Colony formation assays and the EDU experiment also showed that the clonogenic ability of Panc-1and SW1990 cell lines were signi cantly decreased after being treated with differing concentrations of AS-VI ( Fig. 1b and c).
The regulation of the cancer cell cycle plays a signi cant role in cancer cell proliferation. To explore the impact of AS-IV on pancreatic cancer cell cycle regulation, ow cytometry assays of Panc-1 and SW1990 cell lines were conducted. As shown in Fig. 2a, the data indicated that AS-IV induced an accumulation of cells in G1 phase, accompanied by a decrease of cells in the S phase and the effect was dose-dependent.
Previous research had shown that mammalian cells encode three D cyclins that coordinately function as allosteric regulators of cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) to regulate cell cycle transition from G1 to S phase [23]. These experimental results showed that the expression levels of cyclin D1, cyclin E and CDK4 decreased signi cantly after treatment with differing concentrations of AS-IV. This data con rmed that AS-IV blocked the transition from G1 to S phase by decreasing the expression of cyclinD1, cyclin E and CDK4 (Fig. 4).

AS-IV suppressed the migration of Panc-1 and SW1990 cells
To evaluate the effects of AS-IV on pancreatic cells of Panc-1 and SW1990 migration, wound healing assays were performed. The results showed that AS-IV signi cantly suppressed the wound healing in the experiments with both Panc-1 and SW1990 cells line compared with the control group (Fig. 2b).
AS-IV promoted apoptosis and mitochondrial events in Panc-1 and SW1990 cells Flow cytometry analysis, JC-1, TUNEL assay, caspase-3 and caspase-7 were used to explore the potential effect of AS-IV on the apoptosis of Pan-1 and SW1990 cells. As shown in Fig. 3, those results showed that after treatment with differing doses of AS-IV for 48h, the apoptosis rate of Panc-1 and SW1990 cells increased signi cantly in a dose-dependent manner.
The ratio of Bax/Bcl-2 in Panc-1 and SW1990 cells was increased by AS-IV (Fig. 4). Since mitochondria play a crucial role in the transduction of apoptotic signaling, the expression of mitochondria related apoptogenic proteins was further examined. The results showed that AS-IV increased the release of caspase 3 and caspase 7 in a dose-dependent manner and promoted the occurrence of apoptosis.
Previous research suggested that Bcl-2 family proteins could play a signi cant role in regulating cell growth and death. In this study western blot analysis proved that the expression of Bax protein was activated but the protein level of Bcl-2 were suppressed. Caspase-3, caspase-7, Bcl-2 and Bax are closely correlated with mitochondrial pathway mediated apoptosis. In this study, the expression of Bax/Bcl2, caspase-3 and caspase-7 were activated after treatment with differing concentrations of AS-IV (Fig. 4).

AS-IV suppressed pancreatic cancer tumor formation and growth in vivo
To con rm whether the AS-IV can affect tumorigenesis in vivo, Panc-1 cells were subcutaneously injected into four-week-old male nude mice separately. After 17 days post-inoculation treatment of AS-IV signi cantly suppressed the growth of pancreatic xenografts as shown in Fig. 5a and b. In addition, the expression levels of Bax, Bcl2, caspase3/7, cyclin D1, cyclin E and CDK4 in vivo were detected by western blot. The results showed that the expressions of Bcl2, caspase7, cyclin E and CDK4 were signi cantly reduced after AS-IV treatment in mice ( Fig. 5c and d). Immunohistochemical staining indicated a decreased proliferative index Ki67 expression in treatment of AS-IV group (Fig. 5e).

Discussion
Pancreatic ductal adenocarcinoma is the most common type of pancreatic cancer [24]. Over the past few decades, with the advancement of surgical technology and the improvement of neoadjuvant therapy, remarkable progress has been made in treating pancreatic cancer. However, pancreatic carcinoma has high malignancy affecting the digestive system with insidious onset, invasive fast-growth, high recurrence rate and fatality. The treatments of pancreatic cancer are often refractory, but diagnosis is often made at advanced stages of the disease, making few patients eligible for surgical intervention [4]. The development of new antineoplastic drugs will offer opportunities for the treatment of pancreatic cancer by new 'traditional Chinese medicine'.
Several studies have shown that AS-IV could play an important role in controlling immunode ciency and reducing the side effects of anti-tumor drugs [25]. Other studies have indicated that AS-IV showed antiproliferation and anti-migration activities in vitro and vivo and provides the experimental basis for preparing a new antitumor medicine [19,[26][27][28]. However, its pharmacological effect on pancreatic cancer remains unclear. This study demonstrated that AS-IV inhibited the proliferation, apoptosis and migration of pancreatic cells in vitro and inhibited the growth of pancreatic cancer xenograft tumor in vivo. Further studies indicated that cell apoptosis induction and cell cycle arrest were affected by AS-IV as part of its antitumor activity in pancreatic cancer. In this study, it was demonstrated that AS-IV signi cantly impaired the cell cycle in Panc-1 and SW1990 cells by inducing a G1 phase arrest and therefore a reduction in the percentage of cells progressing to S phase.  [29]. Studies have also shown that AS-IV can inhibit epithelial-mesenchymal transition (EMT) through the Akt/GSK-3β/β-catenin pathway, inhibiting the metastasis and invasion of pancreatic cancer cells [27]. To detect the effect of AS-IV on pancreatic cell metastasis, this study performed a wound healing assay that con rmed that AS-IV inhibited the metastasis of Panc-1 and SW1990 cells in a concentration-dependent manner in vitro.
Accumulating evidence suggests that the imbalance between apoptosis and proliferation plays a signi cant role in the occurrence and development of cancer [30]. This study proved that pancreatic cancer cells multiplication capacities were affected after treatment with varying concentrations AS-IV.
Previous studies have shown that D-type cyclins D1, D2 and D3 associate with CDK4 or CDK6 to form heterodimeric complexes, which control the progression of G1 phase (G1-to-S phase) and initiate DNA replication [31][32][33]. The oncogenic capacity of cyclin D1 has been established in assorted studies. Cyclin D1 is essential for G1 progression and inhibiting the expression of cyclin D1 can block the cellular entry into S phase [34,35]. In addition, cyclin D1 and cyclin E limit the rate progress of G1 in early and late G1 phase, respectively [34,36]. In this study, ow cytometric analysis was used to explore the change of cell cycle after treatment with differing concentrations of AS-IV. The results showed that the cell cycle was arrested in the G1 phase in a dose-dependent manner and the western blot research further con rmed that the expression of cyclin D1, cyclin E and CDK4 signi cant decreased after treatment with differing doses of AS-IV. These results proved that AS-IV blocked the pancreatic cancer cell cycle from G1 phase to S phase and inhibited the expression of cyclin D1, cyclin E and CDK4.
The blockage of cell apoptosis is one of the important mechanisms needed to control abnormal cell proliferation in carcinogenesis. In this study, TUNEL, JC-1 and ow cytometric analysis were used to detect Panc-1 and SW1990 cells apoptosis rate after different doses of AS-IV in vitro. Those experiments also found that with increases in the dose of AS-IV, the rate of apoptosis also increases.
Some past studies have found that Bax, Bcl2 and caspases3/7are associated with the mitochondrial apoptosis pathway [37]. Of these, Bcl2 is an antiapoptotic molecule that inhibits cell apoptosis by reducing reactive oxygen species [38] and it has been also observed that the down-regulation of Bcl2 or over-expression of Bax can promote tumor cell apoptosis [39,40]. Bax promotes cell apoptosis by enhancing the permeability of mitochondria and mediating the synthesis of apoptotic complexes [41][42][43]. This study found that AS-IV treatment increased the ratio of Bax/Bcl-2, which further demonstrated the role of the mitochondria in AS-IV induced apoptosis. Bcl2 and Bax regulates the release of caspases by changing mitochondrial membrane potential and permeability. Several studies strongly suggested that caspase-3 and caspase-7 have critical roles in apoptosis cell death and in normal development [44,45]. They can trigger the cascade of cell apoptosis and therefore play pivotal roles in regulating apoptosis [46][47][48]. In this study, the results of western blot experiments showed that AS-IV elicited the expression of Bax, caspase-3 and caspase-7 and down-regulated Bcl-2 synthesis in a dose dependent manner.

Conclusion
It is hypothesized from this study that the potential mechanism of AS-IV inducing apoptosis of pancreatic cancer cells may be the activation of the Bcl2/Bax/Caspase-3/Caspase-7 signaling pathway. This study con rmed that AS-IV inhibits the pancreatic cell proliferation in vivo and vitro by blocking the cell cycle and inducing apoptosis. However, there are still limitations that make it impossible to be absolutely certain and the underlying regulatory mechanisms of AS-IV in pancreatic cancer need to be further investigated.

Declarations • Author Declarations
Ethics approval and consent to participate Animal experiments were conducted in accordance with the guidelines for animal care and use issued by the National Laboratory of the United States, and the experimental program was approved by the Animal Care and Use Committee of South China University (2019111008012).

Consent for publication
All authors have read and consented to publish.

Availability of data and materials
All data generated or analysed during this study are included in this published article.