Bioinformatics analysis:
The POLH gene expression and survival (based on all available RNA sequence data) in normal vs tumor samples were analyzed using TNM plot (23)
Virtual high throughput screening and molecular dynamics studies of small molecules:
As described earlier (14), three important compound libraries, namely. Nubbe library, Sellecchem, and Zinc database were carefully selected for this study, focusing on their potential anticancer activity against ovarian cancer. The determination of threshold counts was performed using OSIRIS, a Java layered library, while MOLVIS 8.0 facilitated energy minimization of these small molecules. Chem Draw Ultra 8.0 ver. was used to draw the structure of small molecules. Ligand preparation for docking studies involved pre-processing the small molecules through the PRODRG2 7.0 server to increase stringency. From the initial screening, 284 potent small molecules were selected for further molecular docking studies.
To study the interaction, polymerase eta (Pol ղ) (PDB ID No: 3TQ1) was retrieved from the Protein Data Bank (PDB). The visualization tool Discovery Studio 2020 (Dassault System BIOVIA) was employed to remove associated moieties, hetatoms, and inhibitors in the structures (24). The cleaned and optimized structure of the target was saved in PDB format for subsequent processing.
The ligands for the interaction were pre-processed using LigPrep module of Schrodinger suite 2019, ensuring geometry optimization, chirality, and desalting. The binding modes with best Glide G scores were selected. To analyze the results of docking studies, the XP visualizer of the Glide module was utilized (25). Stable small molecules were further subjected to ADMETox screening to assess the physicochemical properties and toxicity screening.
Molecular dynamics simulations were performed using Schrodinger suit 2019, Employing the GPU accelerated desmond tool (26). Energy minimization and molecular dynamics simulations were conducted, with the protein-ligand interaction, poses obtained from docking being solvated in water box at a distance of 12Å from the protein surface. The Langevin method was employed in this study, where the temperature was initiated from 0K moving up to 298K using 60ps simulations, with a pressure of 1.034 bars. An integration time step of 2 ns, followed by 1 ns, was conducted at 298K to relax the structure and ensure density periodically. The process was repeated to bring the temperature 310K, the addition of ions at pH 7.2 was performed through two step energy minimizations. The root means square deviation (RMSD) analysis was then carried out using 100 ns MD trajectory, and calculations were retrieved upon completion of the full trajectory.
Cell culture:
The ovarian cancer (Human) cell lines OVCAR3, OVCAR8, 2008, C13, SKOV3 and A2780 were kindly gifted by Dr. Qi-En Wang of the Ohio State University, Columbus, USA. All the cells were authenticated with the help of STR profiling and maintained in RPMI 1640 media (Life Technologies, Grand Island, NY, USA) with 10% FBS (Life Technologies) and 1% antibiotic/antimycotic solution supplement (Life Technologies). The OVACR3 cells with stable knockdown of Pol η (OVCAR3-shPOLH) were successfully established in our laboratory. CSC spheroids were maintained in the ultralow attachment dishes (Corning, Kennebunk, ME, USA) in DMEM-F12 medium (Life Technologies) containing 20% knockout serum replacement (Life Technologies), 20 ng/mL bFGF (Life Technologies), and 10 ng/mL EGF (Life Technologies). All the cells were maintained at 37°C humidified cell incubator containing 5% CO2. Chrysin (Sigma Aldrich Co, St. louis, MO, USA) stock solution was prepared in DMSO (Himedia Laboratories, Nashik, India).
Cisplatin treatment:
The stock solution of the cisplatin (Sigma Aldrich) was prepared freshly with PBS and further diluted to the desired concentration with culture medium for cell treatment.
Western blot:
Ovarian cancer cells and CSCs were lysed using RIPA buffer (Sigma Aldrich) with protease inhibitor mini tablets (Thermo Scientific, Rockford, IL, USA). An equal amount of proteins from cell lysate following protein quantification were loaded in 10% polyacrylamide gel and transferred to a PVDF membrane (Millipore), and membrane was blocked with BSA. Protein bands were detected by using anti-DNA polymerase eta primary antibody (Abcam, Cambridge, UK) and goat anti-rabbit secondary antibody (Invitrogen).
Colony formation assay:
Cells were trypsinized and seeded in 6 well plates at a density of 500 cells/well in triplicate. Cells were treated with chrysin for 24hrs followed by cisplatin treatment (10 µM for 12 hrs). Further, cells were grown for 10 days were fixed with 60% ethanol for 10 minutes and colonies were stained with 1% methylene blue. The percentage colony formation ability was calculated.
siRNA, shRNA, and gene transfection.
siGENOME human POLH siRNA SMARTpool, and non-targeting scramble siRNA (5’-UUCUCCGAACGUGUCACGUdTdT-3’) were procured from Dharmacon. Human POLH shRNA (Cat no: EcoliVB200131-1181xqd; product detail: pPB[shRNA]-Puro-U6 > hPOLH[shRNA#1]) was purchased from Vector Builder, USA. Lipofectamine 2000 (Life Technologies) was used to transfect siRNA and shRNA into ovarian cancer cells. To establish POLH-deficient stable cell lines, shPOLH plasmid was transfected into OVCAR3 cells. Puromycin (2 mg/mL) was used to select the transfected stable clones with Pol η knockdown were validated by western blotting (Fig S2B).
Real-time PCR analysis:
TRIzol reagent (Life Technologies), was used to isolate the total RNA from the cells. The cDNA was synthesized from 1µg of total RNA using the high-Capacity cDNA Reverse Transcription kit (Applied Biosystem). Further, Fast SYBR Green PCR Master Mix (Applied Biosystem) was used to amplify the cDNA in each 20 µL reaction in 96-well plate. PCR reactions were run on the ABI 7500 Fast Real-Time PCR system in the central instrumentation facility of the CSIR-Indian Institute of Chemical Biology. All primer sequences are given in (Fig S3).
HPRT assay:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutagenesis assay was accomplished according to the protocol earlier described by Zhu et al., 1998 (27). Briefly, OVACR-3 shControl (POLH proficient) or shPOLH (POLH deficient) cells were pre-treated with chrysin followed by cisplatin for 12 days and then selected with 6-thioguanine (6-TG) for 10 days. The number of 6-TG-resistant clones was counted.
Gamma H2AX foci formation assay
Cells were seeded on lysine-coated coverslip in a 6-well plate at a density of 1x105 cells/well and incubated for 24 hours. Chrysin, cisplatin and combination of both were treated to relevant wells. Coverslips were gently washed with PBS, and cells were fixed with 4% PFA for 15 minutes. Cells were permeabilized with 0.2% Triton-X for 10 minutes, blocked with 10% normal goat serum (Millipore) for 1 hour. Incubation with anti-gamma H2AX (phospho S139) primary antibody (Abcam) was performed at 4°C overnight. Cells were washed with saline, incubated with goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor®488 (Abcam) for 2 hours, counter stained with Hoechst 33342 (Invitrogen) was performed, mounted on a microscopic slide, and visualized under a confocal fluorescent imaging system (Zeiss, Germany).
Flow cytometry analysis:
For double positive population analysis, ovarian cancer cells were trypsinized, washed with PBS, and incubated with anti-CD44-FITC and anti-CD117-PE antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 minutes at 4°C in the dark. Cells were analysed on BD LSRFortessaTMflow cytometer at the central instrumentation facility of the CSIR-Indian Institute of Chemical Biology. For live-dead analysis, CSC spheroids were washed with PBS, stained with propidium iodide (Sigma Aldrich), and analyzed immediately on flow cytometer. FCS express and flow jo software were used to analyze the data.
CFDA-SE/PI live dead cell imaging of CSC spheroids:
The CSC spheroids were washed twice with warm PBS and incubated with 25 µM CFDA-SE (Invitrogen) at 37°C in water bath for 30 minutes under dark condition. Spheroids were then washed with PBS, added 50 µg/ml PI and incubated for 4–5 minutes at room temperature, followed by PBS wash. Pre-warmed serum free culture media was added to the wells, and spheroids were visualized under a fluorescent cell imager (Bio Rad, Hercules, CA, USA).
Animal experiments:
The animal study conducted in this research was approved by the institutional animal ethical committee and animal care was taken according to the guidelines of CSIR-Indian Institute of Chemical Biology. To establish human xenograft tumor model, 6–8 weeks-old female CrTac: NCr-Foxn1nu mice were purchased from Vivo Bio Tech (Hyderabad, India) and housed in the institutional animal house facility. Approximately 2×106 OVCAR3 cells were mixed with Matrigel matrix (Corning) in equal volume and subcutaneously injected into the flanks of the mice. Once the tumor volume reached 0.5 cm in size, the animals were randomized into four groups (n = 5). The treatment protocol involved pre-treatment with chrysin (10mg/kg body weight, administered twice a week) followed by cisplatin i.p. thrice (5 mg/kg, administered weekly) for a duration of 3 weeks. Tumor growth was monitored by measuring the tumor dimension using a caliper, and tumor volumes were calculated using the formula V = (a×b2)/2, where ‘a’ represents the longest diameter and ‘b’ represents the shortest diameter of the tumor. Following euthanasia, tumor weights were recorded, and xenograft tumor cells were isolated using RBC lysis buffer for subsequent analysis.
Hematology
Blood samples from the mice were collected in EDTA vials (BD-Falcon). Hematological parameters such as WBC, RBC, hemoglobin levels, and platelet counts were analyzed using an automated Yumizen H500 hematology analyzer (Horiba, Kyoto, Japan).
Immunofluorescence and immunohistochemistry of xenograft tumor tissue:
OCT-embedded tumor tissues were cut into 5–10 µM sections with cryotome (Leica, Wetzlar, Germany) on poly-lysine coated adhesive microscopic slides and stored at -80°C refrigerator for future use. Air-dried sections were fixed with 4% PFA followed by PBS wash. Antigen retrieval was performed using 0.01M sodium citrate buffer (pH = 6.0) at 95°C in a water bath for 5 min. Non-specific antigen blocking was performed using 10% normal goat serum. Slides were incubated overnight at 4°C with primary antibody followed by secondary antibody conjugated with Alexa Fluor®488 (Abcam) for 2 hours at room temperature. Further slides were counter-stained with DAPI, and tumor tissue sections were mounted with Fluoromount™ aqueous mounting medium (Sigma Aldrich) and visualized under a fluorescent imaging system (Zeiss). Immunohistochemistry was executed using IHC Kit (Abcam) as per protocol given by the manufacturer, counter-stained with hematoxylin, and slides were visualized under an inverted microscope (Olympus, Tokyo, Japan). Following antibodies were used: anti-Ki-67 primary antibody (Abcam) and anti-gamma H2AX (phospho S139) primary antibody (Abcam).
Hematoxylin and eosin staining:
Tissue sections were washed with distilled water followed by staining with hematoxylin. Subsequently, the sections were washed with running water. Next, the sections were incubated in 70% ethanol and stained with eosin solution. After dehydration with ethanol, the tissue sections were mounted with DPX and observed under an inverted microscope (Olympus).
Statistical analysis:
ANOVA or two sample t tests were carried out for data analysis. All experiments were performed in triplicate and values are given as mean ± SD.