The AS-IV powder (C41H68O14) with a molecular weight of 784.97 and purity exceeding 98% was purchased from ShanghaiyuanyeBio Co, batch number: S31401. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (MA, USA). Antibodies against Bax (50599-2-Ig), Bcl-2 (26593-1-AP), caspase3 (19677-1-AP), Caspase7 (27155-1-AP), cyclin D1 (60186-1-Ig), cyclin E (11554-1-AP), CDK4 (11026-1-AP), Ki-67 (27309-1-AP) and β-actin (66009-1-Ig) were obtained from Proteintech (Proteintech, Rosemont, USA). The haematoxylin and eosin (H&E) staining kit (G1120), was purchased from Slarbio (Slarbio, Beijing, China).
Cell lines and cell culture
The Panc-1 and SW1990 cell lines used in the study were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in Dulbecco’s modified Eagle’s media containing one mg/mL D-glucose with 10% fetal bovine serum (FBS) (Gibco, USA) and supplemented with penicillin/streptomycin (Sigma, USA) at 37°C in an atmosphere of 5% CO2 and 95% air.
The AS-IV was dissolved in DMSO for the treatment of cell lines. The final concentration of DMSO was less than 0.1% (v/v).
Cell viability was determined by CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan) after exposure to different concentrations of 0, 20, 40 and 80 μM of AS-IV. The Panc-1 and SW1990 cells were placed on a 96-well culture plate at a density of 5 x103 cells/well and then exposed to increasing doses of AS-IV for 24, 48 and 72h.
At a specified 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 Scientific, USA).
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 fixed 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 fluorescence microscope (Olympus CX41-72C02, Tokyo, Japan).
Wound healing assay
Wound healing assays were used to assess cell migration. Initially 5x105 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 1×103 cells per well. After different treatments 40 and 80 μM AS-IV , they were cultured at 37℃ in air containing 5% CO2 for two weeks. Cultures containing different concentrations of AS-IV were changed every two days. After 14 days, the colonies were fixed 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 fixed 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 minutes. The TUNEL stained cells were observed under a fluorescence microscope (Motic BA410T, China).
JC-1 prob assay
The mitochondrial depolarization of Panc-1 and SW1990 cells with different treatments were determined by JC-1 probe (Beyotime, Jiangsu, China). Cells exposed to different concentrations of AS-IV in a six-well plate were incubated with an equal volume of JC-15 μg/ml staining solution at 37℃ for 20 minutes and then washed twice with JC-1 staining buffer. After treatment, cells were resuspended with JC-1 staining buffer and the ratio of green to red fluorescence intensity was measured with an FACS Calibur flow cytometer (BD, Franklin Lakes, NJ, United States).
Cell apoptosis was assessed by flow cytometry after Annexin‐V‐fluorescence 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 flow 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. Total proteins were extracted with RIPA cleavage buffer, separated by 10% sodium dodecyl sulfate SDS-PAGE, and transferred to polyvinylidene fluoride (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 flank of 1×106 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 (D2 × 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 mm3 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 sacrificed 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 was performed similarly to the procedure reported previously (Camellia oil (Camellia oleifera Abel.) Attenuates CCl4-induced liver fibrosis via suppressing hepatocyte apoptosis in mice). Immunohistochemical studies were performed on paraffin 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 fields randomly selected from each section.
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 significance of the variance between two or more groups was evaluated using student’s t-test or ANOVA. P<0.05 had statistical significance.