Chemicals and reagents
Kaempferol, silver nitrate, dimethyl sulfoxide (DMSO), Dimethylthiazolyltetrazolium bromide (MTT), and 2,7-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium was obtained from Gibco Life Technologies. Heat-inactivated fetal bovine serum (HFBS) was procured from Invitrogen Co., (Waltham, MA, USA). Lactate dehydrogenase (LDH) CytoTox Non-radioactive cytotoxicity assay kit was supplied by Promega (Madison, Wisconsin, USA). ELISA kits for B-cell lymphoma 2 (Bcl-2), Bcl-2-like protein 4 (Bax), cytochrome-c (Cyt-c), tumor suppressor protein (P53), and Caspase-3 (Cas-3) and annexin V-FITC were obtained from Abcam (Cambridge, UK). Dulbecco’s modified eagle’s medium (DMEM) diluted Matrigel was purchased from BD Biosciences (USA). All other chemicals used were of the highest purity grade available from different commercial sources. Milli-Q water was used as a solvent for silver. DMSO was used to prepare stock solutions of kaempferol at a concentration of 1 mg/mL and stored in -20 °C for further use.
Hepatocellular carcinoma cell line (HepG2) was obtained from ATCC. HepG2 cells were cultured at 37 °C in 5% CO2 incubator in RPMI-1640 medium supplemented with 1% (v/v) penicillin–streptomycin and 10% (v/v) HFBS. When the confluency of cells reached 80%, trypsinization was used to detach and subculture cells. HepG2 cells were seeded at a density of 15,000 cells per well in 96-well plates for the treatments.
Kaempferol-coated AgNPs synthesis
A total volume of 10 ml of kaempferol (stock solution) and 90 ml of 0.1 M AgNO3 solution was incubated together for 24 h at room temperature, turning the solution into the dark brown color of AgNPs. Then, the solution was heated and stirred at 90 °C for 15 min and centrifuged later at 9000 rpm at room temperature.
Kaempferol-coated AgNPs characterization
Fourier transmission infrared (FTIR) and UV–VIS spectral analysis were used to detect the optical density of kaempferol-coated AgNPs. X-ray diffraction (XRD) was used to know about the crystalline structure of AgNPs. Zetasizer (Nano series, ZEN 3600, Malvern, U.K.) was used to determine the average size. In addition, transmission electron microscopy (TEM) was used to examine morphology and aggregation.
Cells viability/cytotoxicity assay
MTT colorimetric assay was applied to detect the cytotoxicity of kaempferol-coated AgNPs. Briefly, HepG2 cells were incubated after seeding in 96-well plates with different concentrations of kaempferol-coated AgNPs and doxorubicin (DOX) for 24 h at 37 °C in a humidified incubator. Cells were then washed with 1× phosphate buffer saline (PBS) and treated with 20 μl of MTT (5 mg/ml) at 37 °C for 30 min. The formed crystals of formazan were dissolved using 200 μl of DMSO and re-incubated at 37 °C for another 30 min. The differences in color intensities were recorded at 570 nm using a microplate reader (Othman et al. 2021a).
LDH leakage assay was used to assess the cytotoxic effect of kaempferol-coated AgNPs. For this purpose, a colorimetric assay was used according to the manufacturer's instructions. Released LDH by cell lysis is measured in the supernatants with an enzymatic assay by converting the tetrazolium salt into red formazan. The intensity of the formed color is proportional to the number of lysed cells. The color intensities (absorbance) differences were recorded at 490 nm using a microplate reader. In addition, the relative LDH leakage (%) was calculated and recorded related to the control cells (Braydich-Stolle et al. 2005).
Determination of apoptotic markers
ELISA kits were employed to analyze the protein levels of apoptotic biomarkers. Pro-apoptotic markers including Bax, Cyt-c, P53, and Cas-3 were measured along with the anti-apoptotic marker Bcl-2. As follow, 2 × 106 HepG2 cells were treated with kaempferol-coated or AgNPs (concentrations of 1/2 LC50 and 1/3 LC50). Furthermore, DOX at LC50 (11.02 μm/ml) as a positive control and cells with the vehicle as negative. After 24 h of incubation, cells were collected by centrifugation at 1800×g for 5 min to remove the medium and washed twice with PBS. The pellets were then lysed by pipetting in 50 µl cold lysis buffer. Next, the cell lysate was centrifuged at 12,000×g for 1 min at 4 °C, and the supernatant was collected. Protein levels of apoptotic markers were analyzed using the Bradford method in each lysate. Cell extraction buffer PTR was used for dilution of lysates if the level of protein exceeds 4 g/l. In the end, the resultant color of the samples was recorded at 405 nm in a microplate reader (Biotech, Inc., USA).
Migration and invasion assay
Corning Transwell 8.0-μm pore membranes (Corning, USA) were used to evaluate cell migration and invasion. Briefly, 1 × 105 HepG2 cells were seeded onto the upper chamber of each transwell. For invasion evaluation, the upper chamber of transwell was coated with 100 μL of 1:8 DMEM-diluted Matrigel before the seeding of HepG2 cells. Lower chambers of transwell were contained fresh medium as a chemo-attractant with the presence or absence of kaempferol-coated AgNPs or DOX. After incubation for 48 h, the cells remaining at the upper surface of the membrane were removed using a cotton swab, whereas those cells that had migrated or invaded to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The number of HepG2 cells migrated and invaded through the filter was photographed and counted using a light microscope at a magnification of 200x.
HepG2 cells at a 2 × 105 cell/ml concentration were cultured in T25 cell culture flasks and treated with kaempferol-coated AgNPs or DOX. After 24 h, cells were colected and resuspended in a binding buffer. Then, annexin V-FITC (Cat. No: ab14085) was added and incubated at 30 °C for 10 min in the dark. Cell cycle distribution was analyzed by flow cytometry.
Oxidative stress determination
HepG2 cells at a 2 × 105 cell/ml concentration were cultured in T25 cell culture flasks and treated with kaempferol-coated AgNPs or DOX. After 24 h, cells were harvested and lysed in a lysis buffer. The lysates were then spun down by centrifugation at 12,000×g at 4 °C for 1 min, and the supernatant was collected. The Bradford method was used to determine the protein levels in each cell lysate. Green fluorescence strain DCFH-DA was used to detect ROS levels in the cell lysates following the previous report (Othman et al. 2021a). In addition, lipid peroxidation (LPO) was analyzed using the colorimetric methods of Ohkawa et al. (1979). In addition, Glutathione (GSH) protein level as an antioxidant marker was measured following Ellman (1959).
All data are expressed as mean ± standard deviation (S.D.). One-way ANOVA and post hoc Tukey's test were used to compare the results between groups. The statistical program Graph Pad Prism 9.3.1 was employed in the analysis. Significant differences were considered if P values < 0.05.