2.1. Cell culture and treatment
OVCAR-3 and A2780/CP70 ovarian cancer cell lines were provided by Dr. Jiang at the West Virginia University, and the A2780/wt ovarian cancer cell line was kindly provided by Dr. Kenneth Tew at the Medical University of South Carolina. IOSE 364, normal ovarian surface epithelial cells from healthy women, but immortalized with SV40 T/t, were courtesy of Dr. Auersperg at University of British Columbia, Canada. All cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) at 37 °C with 5% CO2. A stock solution of kaempferol (Sigma) and cisplatin (Sigma) were prepared in dimethyl sulfoxide (DMSO) at 100 mM and stored at -20 °C. Different concentrations of kaempferol and cisplatin were prepared in a RPMI 1640 medium with FBS for cell treatments, and DMSO was included in the preparations to ensure equal concentrations of DMSO in each treatment.
2.2. Cell proliferation assay
As kaempferol has a yellow colour, a colour gradient appeared in serial dilutions of kaempferol in the RPMI 1640 medium, which interferes with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)-based colour absorbance assays (unpublished data). Repeated washing of cells with PBS, effectively removed colourful treatments, but caused an appreciable and an uneven loss of cells (unpublished data). For this reason, genomic DNA abundance was measured to estimate cell numbers after each treatment. Ovarian cancer cells were seeded in 96-well plates at 2000 cells/well and incubated overnight before treatment with 0-160 μM kaempferol for 24 hours in triplicates. The medium was removed, and the plates were freeze-thawed to lyse cells. Each well was added with 200 μl 1x CyQUANT cell lysis buffer (Invitrogen) containing 5x SYBR Green I (Invitrogen) and incubated at room temperature (RT) for 5 minutes. The reaction (50 μl) was transferred to PCR strip tubes and the fluorescent signal was measured at 90 °C with a real-time Chromo4™ PCR instrument (Bio-Rad, Hercules, CA). To ensure that cell proliferation assays were performed within a linear range of cell numbers, a standard curve was generated by seeding different amount of OVCAR-3 cells (based on counting with a hemacytometer) in a 96-well plate, and measuring genomic DNA abundance after overnight incubation. Three independent experiments were performed and data was pooled for statistical analysis.
2.3. Cytotoxicity assay
Ovarian cancer cells were seeded in 96-well plate at 5000 cells/well, incubated overnight, and treated in triplicates with 100 μl kaempferol for 24 hours. Culture medium (20 μl) was sampled to measure free lactate dehydrogenase (LDH) levels. Lysis Solution (10x) was then added into cells and incubated at 37 °C for 1 hour to release all LDH into culture medium, which is sampled again (20 μl) to measure total LDH levels. A CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) was deployed to measure the free and total LDH levels from each well, and non-cell wells were measured as a medium for background control. LDH levels contained in intact cells were derived by subtracting the free LDH levels from total LDH levels, and normalized to the total LDH of the control samples. Two to three independent experiments were carried out and the results pooled for statistical analysis.
2.4. Apoptosis assay
To time apoptosis in ovarian cancer cells, A2780/CP70 Ovarian cancer cells were seeded in 96-well plates at 10,000 cells/well, incubated overnight, and treated in triplicates with 0- or 80-μM kaempferol for 0, 2, 4, and 8 hours. No-cell wells were included for background correction. At each time point, a plate of the culture medium was removed and frozen in -80 °C until final analysis. Cells were thawed, lyzed with Passive Lysis Buffer (Promega), and analyzed for caspase 3/7 activities with a Caspase-Glo 3/7 Assay (Promega) and the total protein levels with a BCA assay (Pierce) as per the instructions. Caspase 3/7 activities were normalized by total protein levels, and the levels of kaempferol-treated cells were expressed as percentages of controls for statistics. To analyze kaempferol-induced apoptosis in ovarian cells, all 3 lots of ovarian cancer cells and 1 lot of immortalized normal cells were seeded in 96-well plates, incubated overnight, and treated with various concentrations of kaempferol for 2 hours. A positive control, cisplatin, was also included in the cancer cell treatments for confirmation and comparison. Caspase 3/7 activities and total protein levels were analyzed as described above.
2.5. Caspase-9 inhibition experiment
OVCAR-3 cells were seeded in 96-well plates at 10,000 cells/well and incubated overnight. Cells were treated with a caspase-9 inhibitor (Z-LEHD-FMK) (0-10 μM) or 10 μM negative control (Z-FA-FMK) (Biovision, Mountain View, CA) in triplicates for 24 hours, and treated with 0 or 80 μM kaempferol for 2 hours. Caspase 3/7 activities were measured with a Caspase-Glo 3/7 Assay (Promega) and normalized by cell numbers, which were measured with a CellTiter 96 Aqueous One Solution Cell proliferation Assay (Promega) following the manufacturer’s instructions. Data from two independent experiments were pooled for statistical analysis.
2.6. Western Blot
Ovarian cancer cells were seeded in 60-mm dishes, incubated overnight, and treated with 0-80 μM kaempferol for 24 hours. The cells were harvested with M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (Pierce) as per the instructions, subject to SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% non-fat milk in TBST, and probed with different primary antibodies targeting human p53, Bad, Bax, or Bcl-xL (Santa Cruz, Santa Cruz, CA). Membranes were incubated with Goat-anti-Mouse-Poly-HRP (Pierce) and visualized with x-ray film exposure. Membranes were further stripped with Restore PLUS Western Blot Stripping Buffer (Pierce) and re-probed with a GAPDH antibody (Santa Cruz) to normalize target protein abundances. Film images were quantitated with NIH “ImageJ” software and 2-3 independent experiments were pooled for statistical analysis.
2.7. Statistical analysis
All replicates within an experiment were averaged and the mean values from different experiments were pooled for statistical analysis. One-way ANOVA followed by a Dunnett’s test or t-test was performed, as appropriate, to determine the differences between groups using SPSS software and a p-value of less than 0.05 is considered to be significant.