The aim of this assay is to evaluate the in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) activity of the samples being tested. A colorimetric assay, using a sulforhodamine B (SRB) reaction, has been adapted to provide a quantitative measurement of cell growth and viability [12 (link),13 (link)]. This form of assay employs 96-well cell culture microplates. All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC), unless otherwise indicated, and derived from different types of human cancer: A-549 (ATCC CCL-185), lung carcinoma; HT-29 (ATCC HTB-38), colorectal carcinoma; MDA-MB-231 (ATCC HTB-26), breast adenocarcinoma; and PSN-1, pancreatic adenocarcinoma [14 (link)]. Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (for A-549, HT-29 and MDA-MB-231) or RPMI (for PSN-1) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin, at 37 °C, 5% CO2 and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.
Cells were seeded in 96-well microtiter plates at 5 × 103 cells per well in aliquots of 150 μL and allowed to attach to the plate surface for 18 h (overnight) in drug-free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 μL aliquots of 4× concentrated compound stock solutions made in complete culture medium) using ten serial dilutions (concentrations ranging from 10 to 0.000262 μg/mL) and triplicate cultures (final concentration of DMSO being 1%). After 72 h treatment, the antitumor effect was measured using the SRB methodology: briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS and stained in 0.4% SRB solution for 30 min at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm. Effects on cell growth and survival were estimated by applying the NCI algorithm [15 (link)]. Doxorubicin and DMSO (solvent) were used as the positive and negative controls, respectively, in this assay. Prism 9.1.0. from GraphPad was used for the statistical analysis of the cell growth inhibition results. Using the mean ± SD of triplicates, a dose–response curve was automatically generated using nonlinear regression analysis to a 4-parameter logistic curve. Three reference parameters were calculated (NCI algorithm) by automatic interpolation: GI50 = compound concentration that produces 50% cell growth inhibition, as compared to control cultures; TGI = total cell growth inhibition (cytostatic effect), as compared to control cultures and LC50 = compound concentration that produces 50% net cell killing (cytotoxic effect).
Cells were seeded in 96-well microtiter plates at 5 × 103 cells per well in aliquots of 150 μL and allowed to attach to the plate surface for 18 h (overnight) in drug-free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 μL aliquots of 4× concentrated compound stock solutions made in complete culture medium) using ten serial dilutions (concentrations ranging from 10 to 0.000262 μg/mL) and triplicate cultures (final concentration of DMSO being 1%). After 72 h treatment, the antitumor effect was measured using the SRB methodology: briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS and stained in 0.4% SRB solution for 30 min at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm. Effects on cell growth and survival were estimated by applying the NCI algorithm [15 (link)]. Doxorubicin and DMSO (solvent) were used as the positive and negative controls, respectively, in this assay. Prism 9.1.0. from GraphPad was used for the statistical analysis of the cell growth inhibition results. Using the mean ± SD of triplicates, a dose–response curve was automatically generated using nonlinear regression analysis to a 4-parameter logistic curve. Three reference parameters were calculated (NCI algorithm) by automatic interpolation: GI50 = compound concentration that produces 50% cell growth inhibition, as compared to control cultures; TGI = total cell growth inhibition (cytostatic effect), as compared to control cultures and LC50 = compound concentration that produces 50% net cell killing (cytotoxic effect).
