Celltiter 96 proliferation kit

HOb cells were cultured as was described in Cell culture section and seeded in 48‐well plates at 1 × 10⁴ cm⁻². Cell proliferation was measured using the CellTiter 96® Proliferation kit (MTS; Promega, Madison, WI). The proliferation was evaluated following the manufacturer's instructions, measuring the absorbance at 490 nm in a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA) after 1, 3, and 7 days of culture. The HOb mineralization capability was assessed using Alizarin Red S (ARS) to quantify calcium deposits after 7 and 14 days of culture. The ARS was extracted with cetylpyridinium chloride in 10 mM Na₂HPO₄ (pH 7.0) and measured at 540 nm. Three independent samples were analyzed for each condition in each assay.

The compounds were evaluated for their cytotoxic activity in human cancer cells: PC3 (prostate), Hep3B and HepG2 (hepatocellular), MCF7 (breast), A549 (lung) and HeLa (cervical). In addition, a cell line of immortalized human hepatocytes (IHH) was included as a control of non-cancerous cells. PC3 cells were cultured in RPMI-1640 medium, while Hep3B, HepG2, IHH, MCF7, A549 and HeLa in DMEM and supplemented with fetal bovine serum 10% and with 2 mM glutamine. All cultures were incubated at 37 °C in a 5% CO₂ atmosphere (Basu et al., 2006 (link)). The amount of viable cells without proliferation was determined using the Cell Titer 96® proliferation kit, following the manufacturer's instructions (Promega, Madison, WI, USA). Cell viability was determined by absorbance at 450 nm using an automated ELISA reader. The concentrations used were 100, 10, 1, 0.1, and 0.01 μg/mL to obtain a dose/response curve. The experiments were performed in triplicate in three independent experiments. Paclitaxel was used as reference. The data were analyzed in the Prism 5.0 statistical program. IC₅₀ values were determined by regression analysis.
Selectivity Index was determined only against the hepatocellular carcinoma lines. This allowed determining the selectivity of the cytotoxic activity in them. SI was calculated as followed (Badisa et al., 2009 (link)): $SI=\frac{I{C}_{50}IHHcell}{I{C}_{50}cancercell}$