4.3. In Vitro Anticancer Activity Assay

The tumor cell lines U–251 (glioblastoma), MCF7 (breast), NCI/ADR–RES (resistant ovary), 786–0 (kidney), NCI–H460 (lung), PC–3 (prostate), OVCAR–3 (ovary), HT–29 (colon), and K–562 (leukemia) were kindly provided from the National Cancer Institute at Frederick, Maryland, USA. The non–tumor cell line HaCaT (keratinocytes) was kindly provided by FOP/Unicamp. Cells were cultured in complete medium (RPMI–1640) supplemented with 5% heat fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO₂.

Antiproliferative activity was assessed by the sulforhodamine B (SRB) colorimetric assay as previously reported [30]. First, the stock solution of Coronarin D (0.3 mM) was prepared aseptically using DMSO followed by serial dilution in complete medium. The cells were seeded in 96–well plates (3 × 10⁴ cells/mL, 100 μL/well), incubated for 24 h and treated with Coronarin D at final concentrations of 0.79, 7.9, 78.5, and 785.0 μM (100 μL/well), in triplicate, and then incubated for 48 h at 37 °C in 5% CO₂. A second plate, named T0, was prepared to infer the absorbance value of untreated cells at the sample addition moment. The antineoplastic agent doxorubicin hydrochloride was used as a positive control, at final concentrations of 0.0431, 0.431, 4.31, and 43.1 μM (100 μL/well), in triplicate. The final concentration of DMSO (≤0.25%) did not affect cell viability [46]. Subsequently, the cells were fixed with 50% trichloroacetic acid and stained with SRB protein dye (0.4%). Determination of protein content was performed using a microplate reader (Molecular Devices^®, VersaMax model) at 540 nm. Using the absorbance values, the cell growth (%) for each cell line, at each sample concentration, was calculated considering at 100% of cell growth the difference between the absorbances of untreated cells after 48 h incubation (T₁) and at the sample addition moment (T₀). The curve cell growth vs. sample concentration was plotted and the effective concentration TGI (concentration required for total cell growth inhibition) was calculated by sigmoidal regression using the Origin 8.0 software (OriginLab Corporation, Northampton, MA, USA).