Cell lines and cultures
Hepatocellular carcinoma cell line (HepG2) was a kind donation from The Nigerian Institute of Medical Research (NIMR). Cells from HepG2 were passaged and maintained in MEM media (Cytiva, US) enriched with 10% fetal bovine serum (FBS) (Cytiva, US) concurrently with 10,000 U/mL and 10,000 µg/mL pen-strep antibiotics (Life Technologies, US). The culture was regularly maintained at 37°C inside a humidified incubator supplied with 5% CO2. The cell line was routinely harvested with 0.25% trypsin-EDTA (Cytiva, US) at the exponential phase growth, usually at 70–80% confluence.
Flavokawain B (FKB)
FKB was identified and its purity (Supplementary material, Figure S6 – S8) was confirmed from the data reported elsewhere [13]. A stock solution of the bioactive compound or 5-fluorouracil (5FU) (MedChemExpress, US) having 1.824 mg in 20 µL dimethyl sulfoxide (DMSO) was prepared in a 1.5 mL Eppendorf tube. The solution was completely dissolved and kept at 2–8°C for future use. The final concentration of the DMSO per µL was calculated at 0.1% (v/v). Negative control (NC) containing 0.1% DMSO was used throughout the experimentation period.
Neutral red cytotoxicity assay
The anti-proliferative effects of FKB on HepG2 cells were assayed according to the neutral red uptake assay protocol [17] with minor modifications. Cell suspension was cultured in a 96-well microplate at approximately 1 × 105 cells/mL and incubated at suitable culture conditions for 24 h to allow absolute attachment to the culture plate. On day one, cells are treated in a serial dilution (400 µM, 200 µM, 100 µM, 50 µM, 25 µM and 12.5 µM) with FKB and allowed to incubate for 72 h. Control wells containing 5-Fluorouracil (5FU) and 0.1% (v/v) DMSO were treated as a positive and negative control, respectively. On day three, cells are washed with phosphate buffered saline (PBS) and incubated with a neutral red medium at suitable culture conditions for 2 h. After that, cells are washed with PBS and a 150 µL destain solution was added to each well containing cell. Subsequently, the plate was agitated gently for at least 10 min and the coloured formation was quantified at 540 nm. The half-maximal inhibitory concentration (IC50) was extrapolated from a dose-response inhibition curve.
Anti-metastasis assay
Cell scratch assay
The inhibitory effects of FKB on the migration of HepG2 cells were assayed according to the in vitro scratch assay protocol established elsewhere [18] with modifications. The cell suspension was cultured in a 6-well plate and incubated overnight to form an 80–90% confluent monolayer in suitable culture conditions. Using a sterile 200 µL pipette tip, the cell monolayer was scraped in a straight line to create a wound on the cells. Cells debris was removed by washing the cells with PBS and a fresh medium (supplemented with 2% FBS) with or without a test agent was added to the cells. The plate was placed under the phase-contrast inverted microscope and cell migration was observed during image acquisition at 0, 24, 48, and 72 h. The rate of cell migration (RM) was quantified as:
$${R}_{M}= \frac{{W}_{i}- {W}_{f}}{t}$$
Where RM is the rate of cell migration (mm/h), Wi is the initial wound width (nm), Wf is the final wound width (nm), and t is the duration of migration (h)
Cell exclusion zone assay
The effects of FKB on the migration of HepG2 cells were also performed using the cell exclusion zone assay. A single drop of 2 µL agarose gel dissolved in PBS was made into a 24-well plate and allowed to solidify in sterile conditions. The cell suspension was cultured into the barrier plate and incubated overnight to form a 100% confluent monolayer in suitable culture conditions. Carefully, the barrier was removed from the plate and cells were cultured in a fresh medium (supplemented with 2% FBS) with or without a test agent. Image acquisition at 0 and 72 h was obtained with the aid of a phase-contrast inverted microscope. The percentage wound area at time t (%At) was quantified as:
$${\%A}_{t}= \frac{{A}_{t0}- {A}_{t\varDelta } }{{A}_{t0}} \times 100$$
Where %At is the percentage wound area at time t, At0 is the initial wound area at time t (h), and At∆ is the wound area after time t (h).
Gene expression studies
Total RNA extraction
A purified total RNA from treated and untreated HepG2 cells was extracted using the HiYield Total RNA Mini Kit (RBC Real Biotech, Taiwan) in accordance with the manufacturer’s instruction manual. Briefly, a lysis buffer was directly added into the 6-well plate containing scraped cell monolayer from methods 2.4.1. The plate was incubated for 5 mins, scraped with a cell scraper and the lysates were transferred into the filter columns. After a brief centrifuge at low speed, 70% absolute ethanol was added to the filtrates and then with a vigorous shake. Next, sample mixtures were transferred into the retention columns and centrifuged at maximum speed for 2 mins. Samples were then washed with the initial wash buffer, followed by another wash buffer containing ethanol. After centrifuge, the samples were spin-dried at maximum speed for 3 min and incubated for 3 mins with 50 µL of RNase-free water. Purified RNA samples were collected after a brief centrifuge at maximum speed
Real-time PCR
A purified total RNA from HepG2 cells with or without test agents was extracted (supplementary material) using the HiYield Total RNA Mini Kit (RBC Real Biotech, Taiwan) in accordance with the manufacturer’s instruction manual. The Purified RNA templates were used to quantify the relative mRNA expressed in HepG2 cells using the SYBR Green Master Mix (ToroIVD, Shanghai, China). The one-step real-time PCR was performed on a Rotor-gene Q machine (QIAGEN, Düsseldorf, Germany). Briefly, a total of 10 µL reaction mixture was prepared in a PCR tube (Nest Biotechnology, Wuxi, China). The mixture comprised 5 µL of the one-step master mix, 0.5 µL of 50 mM Mn(OAc)2, 0.2 µL each of 10 µM forward and reverse primers, 2 µL of the RNA template, and 2.1 µL of PCR grade water. The cycling conditions used in template amplification include denaturation at 90°C for 30 sec, reverse transcription at 61°C for 20 min, pre-denaturation at 95°C for 1 min, 45 cycles of denaturation at 95°C for 15 sec, annealing ranging between 53–59°C for 15 sec and extension at 73°C for 30 sec. The primer sequences used for PCR amplification are presented in Table S1 of the supplementary data. Three independent biological replicates were performed and relative mRNA expression levels were quantified according to the 2−∆∆ct method using GAPDH as the reference gene.
Data analysis
All data obtained from the experimentation were analysed using the GraphPad Prism 5.0 (GraphPad Software, San Diego, USA). One-way ANOVA and Dunnett's multiple comparison test were used to compare treatment groups to the control group. Data are expressed as mean ± SD and results with p < 0.05 values were considered significant. Three independent biological replicates were performed for each treatment group.