3.4. Antiproliferative Assay

Breast cancer cell line MCF-7 (ATCC^®® HTB-22) and the drug-resistant subline of the human breast cancer MCF-7 (ECACC 86012803; KCR) were purchased from LGC Promochem (Teddington, UK). Both cell lines were cultured in Eagle’s Minimal Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. In every third passage, 0.56 µg/mL doxorubicin was added to the medium in order to maintain the ABCB1 (P-glycoprotein) expression in KCR cells. A2780 human ovarian cancer cell line (ECACC European Collection of Authentical Cell Culture, Sigma Cat. no. 93112519) and the cisplatin-resistant human ovarian cancer cell line A2780cis (ECACC European Collection of Authentical Cell Culture, Sigma Cat. no. 93112517) were purchased from Merck KGaA (Darmstadt, Germany). The human ovarian cancer cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The RPMI 1640 medium of the cisplatin-resistant cell line A2780 was supplemented with 1 µM cisplatin. HeLa (ATCC^® CCL-2™) human cervix carcinoma, HTB-26 breast adenocarcinoma, T-47D (ATCC^® HTB-133™) ductal carcinoma, and MRC-5 human embryonal lung fibroblast cell lines (ATCC^® CCL-171) were purchased from LGC Promochem (Teddington, UK). The cells were cultured in Eagle’s Minimal Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. HTB-26 cell line was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. T-47D cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM Na-pyruvate, and 100 mM Hepes. All of the cells were incubated at 37 °C, in a 5% CO₂, 95% air atmosphere.

The antiproliferative effect of the compounds was determined on the human breast (MCF-7, KCR, T-47D, HTB-26), cervical (HeLa), and ovarian (A2780, A2780cis) cancer cells, and on MRC-5 (human embryonic lung fibroblast) cell line. The adherent cells were cultured in 96-well flat-bottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine serum or RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, respectively. The density of the cells was adjusted to 6 × 10³ cells in 100 μL per well, the cells were seeded for 24 h at 37 °C, 5% CO₂, then the medium was removed from the plates and fresh medium (100 μL per well) was added to the cells. The effects of increasing concentrations of compounds on cell proliferation were tested in 96-well flat-bottomed microtiter plates. The compounds were diluted in the appropriate medium, the dilutions of compounds were performed in separate plates and then added to the cells. The starting concentration of the compounds was 100 μM, and two-fold serial dilution was performed (concentration range: 100-0.19 μM). The culture plates were incubated at 37 °C for 72 h; at the end of the incubation period, 20 μL of MTT (thiazolyl blue tetrazolium bromide, Sigma) solution (from a stock solution of 5 mg/mL) was added to each well. After incubation at 37 °C for 4 h, 100 μL of sodium dodecyl sulfate (SDS) (Sigma) solution (10% in 0.01 M HCI) were added to each well and the plates were further incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Mean IC₅₀ values were obtained by best fitting the dose-dependent inhibition curves in GraphPadPrism5 program (GraphPad Software version 5.00 for Windows, San Diego, CA, USA) from four parallel experiments for each cell line. Results are expressed in terms of IC₅₀, defined as the inhibitory dose that reduces the proliferation of the cells exposed to the tested compounds by 50% [14].