4.2.5. Cell Growth Inhibition Assay (SRB Assay)

The potential cytotoxicity of the compounds was tested using an assay based on a protein-binding dye, the sulforhodamine B (SRB), according to the protocol [31]. Briefly, cells were seeded at an appropriate concentration previously optimized (5.0 × 10⁴ cells per mL for MDA-MD-231 and MDA-MB468 cells) by adding 100 μL of cells per well and then incubated at 37 °C for 24 h. All experiments were performed in two 96 well-plates, a T0 plate to be analyzed at 0 h (time of treatment with the compounds) and a T48 plate to be analyzed 48 h later. Then, cells were treated with five serial dilutions of each compound (ranging from 150 µM to 9.4 µM) by adding 100 μL of compound per well, and incubated at 37 °C for further 48 h. The effect of the vehicle (DMSO) on cell growth was also evaluated (control) by exposing untreated cells to the maximum concentration of DMSO (always lower than 0.25%). The maximum concentration tested for each compound was 150 mM. However, for some compounds, precipitation was detected within the tested range of concentrations. In those cases, the GI₅₀ concentration was not possible to determine and was indicated as “higher than” the highest concentration tested without causing precipitation. Doxorubicin (ranging from 100 nM to 6.25 nM) was used as positive control. Following 48 h incubation of cells with the compounds in the T48 plate (or 0 h in the T0 plate), cells were fixed by adding 10% (w/v) of cold TCA (w/v, final concentration) during 1 h at 4 ^°C. Subsequently, after washing with water, air-dried cells were stained with 0.4% of SRB (in acetic acid, w/v) for 30 min in the dark at room temperature (RT). At the end, cells were washed with 1% (v/v) of acetic acid and the bound dye was solubilized with 10 mM Tris base solution. The absorbance was measured at 510 nm in a microplate reader (BioTek’s SynergyTM Mx) using the Gen5 software (BioTek) [19,20]. The GI₅₀ concentrations for each compound (concentrations that inhibited cell growth by 50%) were assessed from the dose-response curves, determined in each cell line and presented as mean ± standard error (S.E.M) from at least three independent experiments.

Compounds 2e, 2f, 2h were also tested against the non-malignant MCF-12A human breast epithelial cells. For that, cells (seed at 5.0 × 10⁴ cells per mL) were incubated with the GI₅₀ concentrations of each of the three compounds obtained in the corresponding tumor cell lines for 48 h, followed by removal of the compound, addition of new medium to the cells and then 5 more days in culture. At the end of the 7 days in total, the sulforhodamine B (SRB) assay was performed, as previously described [30].