Compounds 1 −5 were dissolved as described in the previous section. The cytotoxicity assay was initially performed against the cell lines L929 (mouse fibroblasts), as well as KB 3.1 (human papillomavirus-related endocervical adenocarcinoma), as described previously [16 (link)]. A detailed protocol, as well as sources of the cell lines, is given in the Supporting Information.
After incubating the cell lines with a serial dilution of the test compounds (final range: 37 to 0.6 × 10−3 µg/mL) for five days, the cells were dyed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), which is only converted to its purple formazan derivative by living cells. Then, the intensity of the purple derivative in relation to cells without additive (set to 100% viability) for each concentration of a test compound was quantified. For this, the absorption at 595 nm was measured using a microplate reader to calculate the percentage of cell viability. From this, the half-maximum inhibitory concentration (IC50, in µM) was calculated.
If an inhibition of cell viability with an IC50 < 50 µM was observed, further cell lines were subjected to the test compounds: PC-3 (human prostate adenocarcinoma), SK-OV-3 (human ovary adenocarcinoma), MCF-7 (human breast adenocarcinoma), A431 (human squamous carcinoma) and A549 (human lung carcinoma).
After incubating the cell lines with a serial dilution of the test compounds (final range: 37 to 0.6 × 10−3 µg/mL) for five days, the cells were dyed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), which is only converted to its purple formazan derivative by living cells. Then, the intensity of the purple derivative in relation to cells without additive (set to 100% viability) for each concentration of a test compound was quantified. For this, the absorption at 595 nm was measured using a microplate reader to calculate the percentage of cell viability. From this, the half-maximum inhibitory concentration (IC50, in µM) was calculated.
If an inhibition of cell viability with an IC50 < 50 µM was observed, further cell lines were subjected to the test compounds: PC-3 (human prostate adenocarcinoma), SK-OV-3 (human ovary adenocarcinoma), MCF-7 (human breast adenocarcinoma), A431 (human squamous carcinoma) and A549 (human lung carcinoma).
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