Preparation of plant extract
Plant samples (wild type) were collected from the Southern part of Nigeria, and was authenticated by Prof. J. D. Olowokudejo, at the Dept. of Botany, University of Lagos, Nigeria, with the Voucher Number 7491. The fresh leaves of B. coccineus were air dried and the powdered sample was extracted by maceration in ethanol. The filtrate was collected and evaporated to dryness using the evaporator at reduced pressure, to get the ethanol extract. Stock concentration of 20mg/mL was prepared by dissolving 200mg of ethanol leaf extract in 1ml of DMSO and 9mL of PBS. Stock solution was stored at -20 °C.
All chemicals used for this study were of analytical grade. All the primary and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signalling Technology (Danvers, MA). Annexin V Apoptosis Detection Kit with PI was purchased from Bio-legend (San Diego, CA). The two-human epithelial, adherent ovarian cancer cell lines OVCAR3, and SW626 were obtained from ATCC (Manassas, VA).
The OvCa cell lines, OVCAR-3, and SW6262 were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA). Following to the ATCC cell culture method with a small modification, OVCAR3 cells were grown in RPMI media supplemented with 20% Fetal Bovine Serum (FBS) (Fisher scientific, PA, USA), 0.01 mg/ml bovine insulin, 1% of Non-essential amino acid solution and 1% mL of penicillin/streptomycin solution (Fisher Scientific, PA, USA). However, SW626 cells were grown in L-15 media (Fisher Scientific, PA, USA), supplemented with 10% FBS, and Penicillin/Streptomycin and penicillin/streptomycin solution. All cell cultures were maintained in a humidified incubator at 37 °C and 5% CO2.
Cell proliferation assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Fisher Scientific, PA, USA) assay was carried out to determine the viability of OVCAR3 and SW626 cells after B. coccineus ethanol leaf extract treatment. Both growing cells were trypsinized with 0.25% Trypsin-EDTA (Fisher Scientific, Pittsburgh PA), and seeded in 96-well plates at a cell density of 10,000 cells/well in triplicates. Cells were treated with different concentrations of B. coccineus ethanol leaf extract ranging from 0.020 mg/ml - 5 mg/ml each well per concentration, and incubated for 24, 48 and 72 h at 37℃, and 5% CO2 incubator. DMSO (0.1%) used as a negative control. Next, 20µL of MTT (5 mg/ml in PBS) reagent was added to each well and incubated for 2-3 h. The formazan crystals were solubilized in 100µL of DMSO, and the optical density was measured at 570 nm using a microplate reader (Spectramax M5, Molecular Devices, Sunnyvale, CA) . The optimal IC50 (half-maximum inhibitory concentration) values were calculated for both cell lines OVCAR-3 and SW626.
Live/Dead cell staining
Twenty-five thousand OvCa cells were seeded in a 48-well plate overnight at 37℃, and then treated with the plant extracts for 48h. Following the manufacturer's protocol (Thermo Fisher Scientific, Carlsbad, CA, USA), cells were washed and incubated with 2drops/mL of cell viability imaging probes for 15 min. The cells were then imaged using EVOS FL fluorescent microscope at 10X objective.
Annexin V/PI apoptosis detection
Apoptosis assay detection kit with propidium iodide (PI) (Biolegend, San Diego, CA) was used to investigate if plant extract could induce cell apoptosis in OvCa cells. Both (OVCAR3 and SW626) cells were seeded (1x 106) in a six- well plate for 24h and treated with an IC50 value of plant extract for 48 h. Further, cells were washed, harvested with 0.25% trypsin, and counted using the hemocytometer (Countess II FL, Life Technology, CA, USA). Subsequently, cells were washed twice with an ice -cold Annexin V binding buffer, and an equal number of cells (1x 105) were stained with Annexin V (conjugated with FITC ) (5 μL) and PI (10 μL) in dark for 15 min at room temperature, according to manufacturer instructions. The Guava Flow cytometer (EMD Millipore, Brillarica, MA, USA) was used to acquire (50, 000 events/ sample) the frequency of apoptotic cells and analyzed using FlowJo 10.0.06 software.
Wound healing assay
To investigate the effect of plant extract on OVCAR3 and SW626 cell lines migration, the wound- healing assay method was performed. Forty thousand (4x 104) cells/well were seeded in a 24well plates, and placed in the 37℃ and 5% CO2 incubator for 24h.; when 90% cell confluency was achieved, a monolayer of the cells were scratched with a 10 μL plastic pipette tip to create a uniform wound. Subsequently, the cells were rinsed with PBS and incubated with B. coccineus extract (IC50 value) for 24, 48, and 72 h. To assess the migration from every scratch, a phase-contrast microscope (EVOS XL Core) (Thermo Fisher Scientific, Carlsbad, CA, USA), at 10X magnification was used.
Clonogenic or colony formation assay is based on the cells ability to form a colony from a single cell. The effect of the plant extract on OVCAR3 and SW626 colony formation was investigated. To determine, 1600 cells/well were seeded in 24 well plate and place in the incubator for 24h. Cells were treated with extract and incubated at 37 °C for10 days. After washing with PBS, the cells were fixed with methanol for 20 min, stained with 0.1% crystal violet, and visualized using a phase-contrast light microscopy at 4X magnification, the formation of 50 cells and above were regarded as a colony.
Total RNA was isolated from OVCAR3 and SW626 cells treated with plant extract to determine its effect on apoptosis, cell cycle regulation and immunomodulation. The cells were treated for 24 h and 48 h, followed by washing with cold PBS, and lysed with lysis buffer. The binding, washing and elution of cells were performed as per manufacturer instructions using PureLink RNA Mini Kit (Thermo Fisher Scientific, USA). Next, RNA was suspended in nuclease-free water and quantified using the instrument Nanophotometer (Implen, Munich, Germany). cDNA was synthesized in a final reaction volume of 20 µL using 1.0 µg RNA and reverse transcription supermix for RT-qPCR according to the manufacturer protocol (Bio-Rad, Hercules, CA, USA). The primers sequences of all the genes used in this study were synthesized from the National Center for Biotechnology Information (NCBI) gene bank database. The primer sequences for MCL-1, BCL-2, BID, BAD, BAX, BAK, PARP, Cyclin D1, CDK2, P21, TNFα, IL10, P53 and 18S are shown in Table1. SYBR® Green PCR master mix reagents (Biorad, Hercules, CA, USA) were used for RT-PCR and gene expression was analyzed by CFX-manager software (CFX96 Real-Time System; Bio-Rad). All experiments were carried in triplicates.
Western blot analysis
To determine the expression of pro- and anti- apoptotic proteins, OVCAR3 and SW626 cells were treated with the plant extract for 24 h and 48 h time points. Cells were then washed with PBS, harvested, collected, and lysed with a RIPA buffer containing 1X protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). Following the standard protocol of protein isolation (Singh et al. 2019), the concentration of the total proteins was determined using BCA (bicinchoninic acid) protein assay kit (Thermo Scientific, Rockford, IL). The protein samples (30 μg) were denatured by heating in a Laemmli buffer and resolved on 12% SDS- PAGE. The separated proteins were transferred to PVDF membranes and incubated in blocking buffer (5% non-fat dry milk in TBS-T i.e. Tris-Buffered Saline containing 0.1% Tween 20) (Biorad, USA) for 1 h at room temperature. The membrane was then probed with primary antibodies (1:1000 dilution in 5% non-fat milk with TBS-T) against pro-apoptotic (BAD, BID), and anti-apoptotic (BCL-Xl, MCL-1) genes at 4 °C overnight followed by three wash with TBST. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) dilution for 2 h at room temperature. The primary and secondary antibodies were purchased from (Cell Signaling Technology MA, USA). GAPDH was used to ensure equal loading. The chemiluminescent reagent (Thermo Fisher Scientific, Rockford, IL) was added to the membrane to detect and visualize the proteins using Image Quant LAS4000 (GE Healthcare- Biosciences, Pittsburgh, PA) .
Statistical analysis was done by using a one-way analysis of variance (ANOVA) and unpaired t-tests. Results are expressed as standard errors of means (±SEM). P values less than 0.05 were considered statistically significant.