Apoptosis induction and inhibition of invasion and migration in gastric cancer cells by Isoorientin studied using network pharmacology | BMC Complementary Medicine and Therapies

Reagents

Isoorientin (Cat# 4261–42-1) and Paclitaxel (Cat# 33069–62-4) was purchased from Chengdu Herbpurify co., LTD (Chengdu, China). HGC27 and NCI-N87 cells were purchased from Shanghai Shaijie co., LTD (Shanghai, China). The cell counting kit-8 (CCK-8) (Cat# E606335) was purchased from Sangon Biotech (Shanghai, China). Primary antibodies for phosphoinositide 3-kinase (PI3K) (Cat# AF6241), p-PI3K (Cat# AF3242), protein kinase B (AKT) (Cat# AF0836), p-AKT (Cat# AF6261), B cell lymphoma 2 (BCL-2) (Cat# AF6139), Bcl-2-associated X protein (BAX) (Cat# AF0120), caspase-3 (Cat# AF6311) and β-actin (Cat# AF7018) were purchased from Affinity (Jiangsu, China). cleaved-caspase-3 (Cat# ab32042) were purchased from abcam (Shanghai, China). Cell culture medium was purchased from GibcoTM Invitrogen (Thermo Fisher, New York). Mitochondrial membrane potential assay kit with JC-1 (Cat# M8650) was purchased from Solarbil (Beijing, China).

Network pharmacology analysis

Target intersection analysis of C. florida and GC was performed as follows. 1) the components of C. florida were searched in CNKI, Wanfang Database, and PubMed. The target proteins of bioactive components in C. florida were retrieved from the traditional Chinese medicine systems pharmacology (TCMSP) database (http://tcmspw.com/), PubChem, and the Swiss Target Prediction database. 2) We searched for disease-related targets in the DisGeNET database using the keyword “gastric carcinoma.” 3) The C. florida bioactive component and GC disease targets were imported into the Wayne analysis software after removing duplicates.

Protein–protein interaction (PPI): The targets obtained through the method above were subjected to PPI analysis using the STRING database (https://string-db.org/). The PPI network was analyzed using Cytoscape (http://www.cytoscape.org/, version 3.9.0), and the core targets were filtered based on the degree value.

Pathway enrichment analysis: Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of critical targets was built using MetaScape software. The top 20 biological processes, molecular function, cellular component, and KEGG pathways with P < 0.05 were identified. Finally, visualization processing was carried out on the Bioinformatics platform.

Cell culture and treatment

Gastric cancer cells were cultured at 37 °C under 5% CO₂ in Dulbecco’s modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Hyclone, South America) with 100 IU/mL streptomycin and 100 IU/mL penicillin (Amresco, USA) in a Thermo incubator. Cells were divided into the following groups: (1) Control; (2) 200 nM Paclitaxel as a positive control; (3) 35 µM Isoorientin; (4) 70 µM Isoorientin; and (3) 140 µM Isoorientin.

Cell viability

Cell viability was measured using the CCK8 assay. Isoorientin was dissolved in dimethylsulfoxide and prepared as a 100 mM storage solution. Gastric cancer cells were seeded in 96-well culture plates overnight. Various concentrations of Isoorientin were added to the wells. After treatment, 10 µL CCK8 reagent was added to each well, followed by incubation at 37 °C for 1 h, and the absorbance was measured at 450 nm. The results were expressed as the relative percentage of the control group.

Colony formation assay

For the colony-formation assay, 500 HGC27 cells were seeded in 6-well plates with the medium changed every three days for 3 weeks. The cells were fixed with 4% paraformaldehyde for 15 min, the fixative was aspirated, and the cells were washed once in phosphate-buffered saline. Then dye with crystal violet for 5 min. Colony counting is performed and analyzed using Prism software.

Cell cycle analysis

HGC27 cells were treated with Paclitaxel or Isoorientin for 48 h. The cell cycle was examined using a DNA content detection kit (Cat# CA1510 Solarbio, Beijing, China). Cells were collected and fixed in cold 95% ethanol for 2 h. Subsequently, cells were centrifuged at 1500 rpm for 5 min, and the supernatants were discarded. The cells were washed twice with cooled PBS, stained with 0.4 mL of propidium iodide for 30 min, and measured using flow cytometry.

Cell invasion and migration analyses

The cell invasion assay used Millicell inserts (Costar, USA) coated with Matrigel (Cat# 356237, Corning, USA). HGC27 cells were treated with Paclitaxel or Isoorientin, and 2 × 10⁴ cells were seeded in the upper chambers in FBS-free DMEM, while the lower chambers were loaded with DMEM containing 10% FBS. The non-migrating cells on the upper cavity were removed 48 h later with a cotton swab, and the cells invading the lower side of the membrane through the Matrigel layer were fixed, stained, and counted.

For the cell migration assay, cells were treated with Paclitaxel or Isoorientin and cultured as confluent monolayers. A defect in the monolayer was created using a 1000-μl pipette tip. Cells were cultured for 48 h, and migration was recorded under an inverted microscope.

Cell apoposis analysis

Apoptosis was measured using an Annexin V- FITC/PI Kit apoptosis detection kit (Cat# FXP018, 4A Biotech, China) and flow cytometry. After treatment, cells were harvested, washed in cold PBS, and labeled with Annexin V- FITC for 30 min at 4 °C in the dark, followed by incubation with propidium iodide. Flow cytometry analysis was performed on a flow cytometer and analyzed using FlowJo software (BD Biosciences, USA).

Mitochondrial membrane potential assay

After treatment, cells were washed once with PBS, added 1 mL of JC-1 staining reagent working solution and the same volume of RPMI 1640 culture medium mixture, culture at 37 °C for 20 min. After incubation, aspirate the supernatant, wash twice with JC-1 staining buffer, add 2 ml of cell culture solution, and observe the fluorescence intensity of mitochondrial membrane potential under laser confocal scanning microscope.

RNA Extraction and qPCR analysis

Total RNA was isolated from HGC27 cells using the TRIzol reagent (Cat# 10296028, Invitrogen, USA) according to the manufacturer’s instructions, and 1 μg total RNA was reverse-transcribed using ReverTrace (Cat# 11752050, ABI-invitrogen, USA). SYBR green-based real-time quantitative PCR using a High Fidelity PrimeScriptTM RT-PCR Kit (Cat# 4472920, ABI-invitrogen, USA) was performed using a CFX96 connect instrument (Bio-Rad, USA). β-actin rRNA served as a control. The primers are as following:

Western blots analysis

Protein concentrations for cell lysates were determined using a BCA protein assay kit (Cat# MD913053, MDL). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk for 1 h at room temperature. Proteins were then detected using primary antibodies incubated overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. The primary antibody were diluted as following: β-actin 1:1000, PI3K 1:500, p-PI3K 1:1000, AKT 1:1000, p-AKT 1:1000, BCL-2 1:1000, BAX 1:1000, Caspase3 1:1000, and cleaved-caspase-3 1:1000.

Statistical analysis

The data were expressed as mean ± standard deviation. Significant differences were expressed as ^*
P < 0.05, ^**
P < 0.01, and ^***
P < 0.001.