Pharmacological Mechanisms of Kirenol Against Ovarian Carcinoma: A Network Pharmacology and Experimental Validation Study in vitro

Subsequently, the retrieved targets from these databases were amalgamated, and duplicate entries were meticulously eliminated to ensure a comprehensive and non-redundant collection of core targets for further analysis in the context of ovarian cancer therapy.Protein–protein interaction networkThe intersection of drug targets and disease targets presents a valuable approach to identify potential targets for kirenol in the context of ovarian cancer.

To construct the PPI network, the predicted targets were submitted to the String database (string-db.org/), specifying "Homo sapiens" as the species, and applying the following options: "high confidence > 0.9" and "hide disconnected nodes in the network." Default settings were maintained for other parameters during network construction. Subsequently, topological parameters of the PPI network, including betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), network centrality (NC), and local edge connectivity (LAC), were analyzed using the CytoNCA plug-in in Cytoscape 3.9.0 software.The topological parameters of the PPI network were further assessed based on the screening criterion, whereby values exceeding the median value of all nodes were considered.

Targets meeting this criterion were subjected to two consecutive screenings, resulting in the identification of core targets for kirenol's action in ovarian cancer. These core targets hold potential significance for further exploration and evaluation as therapeutic candidates in the treatment of ovarian cancer.Molecular dockingThe 2D structures of the core compounds were obtained from the PubChem database. The 3D structures of the core targets, namely MTOR (PDB ID: 1aue), EGFR (PDB ID: 1ivo), IL6 (PDB ID: 1alu), JAK2 (PDB ID: 2b7a), MAP2K1 (PDB ID: 1s9j), MMP9 (PDB ID: 1gkc), CDK4 (PDB ID: 2w96), and AR (PDB ID: 1e3g), were sourced from the Protein Data Bank (www.rcsb.org/) as predictive structures.The protein structures were prepared for molecular docking using PyMol 2.3.4 (pymol.org/).

The preparation involved the removal of water molecules, addition of nonpolar hydrogen atoms to the structures, and saving the processed files in PDBQT format. The binding energy, serving as a quantitative measure of the molecular compound's affinity to its target, was employed to evaluate the strength of the binding interactions.Molecular docking was carried out utilizing Autodock Vina 1.1.2 (vina.scripps.edu/), a computational method allowing the docking of ligands to their respective target molecules.

This approach offered significant insights into the potential binding interactions, shedding light on the possible molecular mechanisms underlying the observed effects of the core compounds on the selected targets.GO enrichment analysis and KEGG pathway enrichment analysisThe predicted targets were subjected to comprehensive analysis using the biological information annotation database DAVID (david.nifcrf.gov/).

This analysis involved conducting enrichment studies to elucidate the Gene Ontology (GO) function and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway associated with the identified targets. Through this approach, we aimed to gain insights into the biological processes and signaling pathways in which the predicted targets are implicated, shedding light on the potential mechanisms of action of kirenol in the context of ovarian cancer treatment.Cell experimentsCell cultureHuman ovarian cancer cell lines SKOV3 and A2780, as well as the human normal ovarian epithelial cell line IOSE-80, were procured from iCell Bioscience Inc (Shanghai, China) for experimental use.

A2780 and IOSE-80 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (BI) (Solarbio Science Technology Co., Ltd., Beijing, China) and 1% penicillin/streptomycin (P/S) at 37°C in a humidified atmosphere with 5% CO2. SKOV3 cells were maintained in McCOY's 5A medium (Boster Biological Technology Co., Ltd., Wuhan, China), while the other culture conditions remained consistent with the aforementioned cells.Kirenol (purity ≥99.6%) and LY294002 (purity ≥99.76%) were obtained from Tsbiochem (#T4S1943; #T2008).

These compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mmol/L. The cells were treated with various doses of Kirenol in fresh medium, with DMSO (at the same volume as the high dose group) serving as the solvent control group for comparison.Cell viability assaySKOV3, A2780 and IOSE-80 cells were separately seeded at a density of 2000,1000 and 1000 cells per well, respectively, in 96-well plates.

After 6 hours of incubation, various concentrations of Kirenol-containing medium (0, 100, 200, 300, 400, 500, 600, 700 μmol/L) were added to the cells, and they were further cultured for 72 hours. To assess cell viability, the CCK-8 assay kit (Abbkine Scientific Co., Ltd., Wuhan, China) was utilized, with a mixture of complete medium and CCK-8 at a ratio of 9:1. The cells were then incubated at 37°C for 1 hour.

Subsequently, the optical density (OD) was measured at 450 nm using a microplate reader to determine the IC50 value. Additionally, the cells were seeded onto a 96-well plate and allowed to adhere for 6 hours. The original medium was then replaced with Kirenol-containing medium at various concentrations (0, 100, 200, 300, 400, 500, 600, 700 μmol/L).

At 24, 48, and 72 hours, the original medium was replaced with a CCK-8 mixed solution. The 96-well plate was further incubated at 37°C for 1 hour, after which the optical density was measured at 450 nm.These experiments were conducted to evaluate the cytotoxic effect of Kirenol on SKOV3, A2780 and IOSE-80 cells and to determine the half-maximal inhibitory concentration (IC50) of the compound in these cell lines. The CCK-8 assay allowed for the assessment of cell viability at different time points and Kirenol concentrations, providing valuable insights into its potential anti-cancer activity.Colony formation experimentPrior to the clonogenic assay, SKOV3 and A2780 cells were subjected to a 72-hour pretreatment with varying concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells.

Following this pretreatment, the cells were plated at a density of 500 cells per Petri dish and allowed to grow for a period of 10–14 days.After the incubation period, the cells were fixed using 100% methanol for 30 minutes and then stained with 0.2% crystal violet solution for 1 hour. Subsequently, the cells were thoroughly washed and left to dry.The clonogenic assay was performed to assess the long-term proliferative potential and survival of SKOV3 and A2780 cells after treatment with different concentrations of Kirenol.

This experimental approach allows for the observation of colony formation and provides valuable information about the compound's ability to inhibit cell growth and colony formation, thereby contributing to the evaluation of its potential as an anti-cancer agent.Wound-healing assaySKOV3 and A2780 cells were cultured in 6-well plates, forming a monolayer. Subsequently, the cell monolayers were intentionally scratched using a 200 μl pipette tip to create wounds.

Afterward, the cells were carefully washed with PBS.Next, SKOV3 and A2780 cells were treated with different concentrations of Kirenol, namely 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells. The wound healing process was closely monitored and recorded at 0 hours (immediately after scratching) and at 48 hours using microscopy, and photographs were captured.The wound healing assay was performed to assess the migratory capability of SKOV3 and A2780 cells in response to Kirenol treatment.

By observing and documenting the closure of the scratched wounds over time, this assay provides valuable insights into the compound's potential to impede cell migration and motility, which are important aspects of cancer cell metastasis and invasion.Cell cycle by flow cytometrySKOV3 and A2780 cells were subjected to a 72-hour pretreatment with varying concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells.

After the pretreatment period, the cells were detached, centrifuged, and resuspended in pre-chilled PBS. Subsequently, they were fixed with 70% ethanol at -4°C overnight. For cell cycle analysis, the fixed cells were stained with PI/RNase staining buffer (Keygentec, JiangSu, China).

Each flow cytometer tube was loaded with 500 μL of cell suspensions and allowed to stain for 30 minutes in the dark. Flow cytometry, using BD Biosciences equipment, was employed to determine the cell cycle distribution. Through flow cytometry, the distribution of cells in different phases of the cell cycle (G0/G1, S, and G2/M) was assessed.

This analysis provides valuable information on how Kirenol influences the cell cycle progression and proliferation of SKOV3 and A2780 cells, offering insights into its potential as a regulator of cell growth and a candidate for ovarian cancer treatment.Apoptosis by flow cytometryThe Annexin V-FITC/PI Apoptosis Detection Kit (Keygentec, JiangSu, China) was employed for flow cytometry analysis to assess apoptosis. SKOV3 and A2780 cells were subjected to a 72-hour pretreatment with different concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells.

Following the pretreatment period, the cells were detached using EDTA-free trypsin, centrifuged at 800 rpm for 5 minutes, and subsequently re-suspended in pre-cooled PBS.Next, the supernatant was removed, and the cells were suspended in 300 μL of binding buffer, along with 3 μL of Annexin V-FITC and 3 μL of Propidium Iodide reagent. The cell suspension was then incubated in the dark for 15 minutes. Flow cytometry, utilizing BD Biosciences equipment, was employed to measure and analyze apoptosis.

This experimental setup enabled the detection and quantification of apoptotic events in SKOV3 and A2780 cells upon exposure to different concentrations of Kirenol. By utilizing Annexin V-FITC and Propidium Iodide, apoptotic cells can be differentiated from live and dead cells, providing valuable insights into the compound's pro-apoptotic effects and potential as a therapeutic agent for ovarian cancer.Western blotSKOV3 and A2780 cells were subjected to a 72-hour pretreatment with different concentrations of Kirenol, specifically 0, 100, 150, and 200 μmol/L for SKOV3 cells, and 0, 100, 200, and 300 μmol/L for A2780 cells.

In the subsequent response experiment, the cells were treated with LY294002 (10 μmol/L) alone or in combination with Kirenol (100 μmol/L) for an additional 48 hours.Cell lysates were prepared using RIPA lysis buffer (Beyotime Biotech, Jiangsu, China) supplemented with phenylmethylsulphonyl fluoride (PMSF; Solarbio, Beijing, China) in a ratio of RIPA to PMSF as 100:1. After cell lysis, the protein concentration was determined using a BCA Protein assay Kit (Beyotime Biotechnology Co., LTD., Shanghai, China).

Protein samples were then denatured by heating at 99°C for 15 minutes. Subsequently, polyacrylamide gel electrophoresis (PAGE) rapid preparation kit (Servicebio Technology Co., Ltd., Wuhan, China) was utilized to separate and resolve the protein samples, followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, California, United States). The PVDF membranes were blocked with 5% skimmed milk for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies, and subsequently incubated with secondary antibodies for 2 hours at room temperature.

Finally, the protein bands were visualized using a Tanon 5200 Chemiluminescence Imaging System, and ECL western blotting substrate (Tianneng Life Science Co., LTD., Shanghai, China) was employed for signal detection. ACTIN served as the internal control for protein normalization. The main antibodies included ACTIN (#YM3028;Immunoway, China), Matrix metalloproteinase (MMP)-2 (#ab92536; Abcam, Cambridge, United States), Bcl-2 (#T40056; Abmart, China), Bax (#T40051; Abmart, China), PI3K(#T40064; Abmart), Phospho-PI3K (Tyr467/199) (#T40065; Abmart), AKT(#T55561; Abmart), and Phospho- AKT (Ser473) (#T40067; Abmart), CCND1(#YM0176; Immunoway), CDK4(#ab108357; Abcam), and Phospho- RB (Ser 807) (#ab184796); Abcam).

Image J (version, 1.51J8) was used to determine blot’s intensity.This series of experiments facilitated the investigation of protein expression and alterations in response to Kirenol treatment, alone or in combination with LY294002, to elucidate potential molecular mechanisms underlying Kirenol's effects on SKOV3 and A2780 cells. Western blotting analysis allowed for the characterization of protein changes and signaling pathways involved in the response to treatment.Statistical analysisStatistical analyses were carried out using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, United States).

The data are presented as the mean ± standard deviation (SD). For comparisons between two groups, t-test was employed, while one-way ANOVA was used for comparisons among multiple groups. P-values less than 0.05 were considered statistically significant.

All experiments were performed in triplicate to ensure robustness and reproducibility of the findings.