3.2. Biological Activity

The colorectal adenocarcinoma HCT116 cells (HCT116 p53^+/+) and its p53-null isogenic derivative (HCT116 p53^−/−) were given by Prof. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, USA); the normal human fibroblasts HFF-1 cell lines were from ATCC (Rockville, MD, USA). Human cells were cultured in RPMI-1640 with UltraGlutamine (Lonza, VWR, Portugal) with 10% FBS (Gibco, Alfagene, Portugal) at 37 °C with 5% CO₂. The SH-SY5Y cells were cultured in DMEM/F-12 (Lonza) and 10% FBS and 2mM L-glutamine (Gibco). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂. Mycoplasma was routinely checked using the MycoAlert™ PLUS kit (Lonza).

In the sulforhodamine B (SRB) assay, the cells were seeded in 96-well plates at 5.0 × 10³ (HCT116, and HFF-1) cells/wells for 24 h, and the GI₅₀ (the concentration that causes 50% of maximal growth inhibition) values of compounds were obtained as described [21]. In the yeast targeted screening assay, Saccharomyces cerevisiae (strain CG379) expressing human wt p53 alone and combined with human MDM2 were used as described [69]. For the expression of human proteins (routinely grown in minimal selective medium), the cells were diluted to 0.05 OD600 in the selective induction medium with 2% (w/w) galactose, 1% (w/w) raffinose, 0.7% (w/w) yeast nitrogen base without amino acids from Difco (Quilaban, Sintra, Portugal), and all the amino acids necessary for yeast growth (50 g/mL) except leucine and tryptophan. The yeast cells were incubated at 30 °C under continuous orbital shaking (200 rpm) with 0.1–50 M compounds or 0.1% DMSO only for over 42 h. Yeast growth was analyzed by counting the number of colony-forming units (CFUs) after 2 days of incubation at 30 °C on Sabouraud Dextrose Agar from Liofilchem (Frilabo, Porto, Portugal).

The 1.5 × 10⁵ HCT116 p53^+/+ cells/well were seeded in 6-well plates and then treated with 5 µM 17 for 48 h. For the apoptosis analysis, an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) was used following the manufacturer’s instructions. An AccuriTM C6 flow cytometer and the BD Accuri C6 software were used for data acquisition.

For Western blotting, 1.5 × 10⁵ cells (per well) of HCT116 p53^+/+ cells were seeded in six-well plates and allowed to adhere for 24 h, followed by treatment with 5 μM 17 for 48 h. Protein extracts obtained from the human cancer cells were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Taper, Sintra, Portugal), according to the manufacturer’s instructions. The proteins were then run in SDS-PAGE and transferred to a Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). Proteins were detected using the primary antibodies anti-p53 (Santa Cruz Biotechnology, Dallas, TX, USA, SC-126, 1:5000), anti-BCL-2 (Santa Cruz Biotechnology, SC-7382, 1:200), anti-PARP (Cell Signaling, #9542, 1:1000), and anti-GAPDH (Santa Cruz Biotechnology, SC-32233, 1:10,000), followed by HRP-conjugated secondary antibodies anti-mouse (Santa Cruz Biotechnology, SC-2005, 1:2500) and anti-rabbit (Santa Cruz Biotechnology, SC-2006, 1:2500). GAPDH was used as a loading control. The signal was detected with an ECL Amersham kit from GE Healthcare (VWR) and the ChemiDoc™ MP Imaging System (Bio-Rad, Amadora, Portugal).

Data analyzed using GraphPad Prism 7.0. Statistical tests were used according to the dataset; p values < 0.05: statistically significant.