3.3. Antiproliferative Activity In Vitro

The growth inhibition activity was assessed according to the slightly modified procedure performed at the National Cancer Institute, Developmental Therapeutics Program [56].

Examined compounds were dissolved in DMSO (1 × 10⁻² M). The experiments were carried out on seven human tumor cell lines and two normal cell lines. The following cell lines were used: HeLa (human cervical adenocarcinoma; purchased from ATCC), CaCo-2 (human colorectal adenocarcinoma), HuT78 (T-cell lymphoma), THP-1 (acute monocytic leukemia), SW620 (colorectal adenocarcinoma, metastatic), MDA-MB-231 (human breast adenocarcinoma), HL60 (promyelocytic leukemia cell line), foreskin fibroblast cells (BJ) and MDCK1 (Madine–Darby canine kidney fibroblast like cells). MDCK1 cells were used between 24 and 26 passages.

Adherent cells were cultured in the Dulbecco’s modified Eagle medium—DMEM (Gibco, EU) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco, EU), 2 mM glutamine, and 100 U/0.1 mg penicillin/streptomycin. Cells on suspension were cultured in RPMI 1640 (Gibco, EU) medium supplemented with 10 % FBS (Gibco, EU), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES. Cells were grown in humidified atmosphere under the conditions of 37 °C/5% of CO₂ gas in the CO₂ incubator (IGO 150 CELLlife^TM, JOUAN, Thermo Fisher Scientific, Waltham, MA, USA). A erythrosin B (Sigma-Aldrich, St. Louis, MO, USA) dye exclusion method was used to assess cell viability before plating.

Adherent cells (HeLa, CaCo-2, MCF-7 and MDCK-1) were plated in 96-well flat bottom plates (Greiner, Frickenhausen, Austria) at a concentration of 2 × 10⁴ cells/mL. Suspension cells (THP-1 and HuT78) were plated in 96-well microtiter plates (Sarstead, Newton, USA) at a concentration of 1 × 10⁵ cells/mL. Twenty-four hours later, cells were treated with test agents in five 10-fold dilutions (10⁻⁷ to 10⁻⁴ M) and incubated for further 72 h. Working dilutions were freshly prepared on the day of testing. The solvent was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in working concentrations. After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay, which detects dehydrogenase activity in viable cells [57]. For this purpose, upon completion of the incubation period, growth medium was discarded and 50 μL of MTT was added to each well at a concentration of 5 mg/mL. After four hours of incubation at 37 °C, water insoluble MTT-formazan crystals were dissolved in 150 μL of dimethyl-sulfoxide (DMSO) for adherent cells, and in 10 % SDS with 0.01 M/L HCl for cells grown in suspension. The absorbance (OD, optical density) was measured on a microplate reader (iMark, BIO RAD, Hercules, CA, USA) at 595 nm.

Percent of life cells was calculated as follows: % = OD (sample)–OD (background)/OD (control)–OD (background) × 100.

Optical density (OD) of background for adherent cells is the OD of MTT solution and DMSO; OD (background) for suspension cells is OD of the culture medium with MTT and 10% SDS with 0.01 M/L HCl; OD (control) is the OD of the cells growth without tested compounds.

The results were expressed as GI₅₀, a concentration necessary for 50% of inhibition. Calculation of GI₅₀ value curves and QC analysis is performed by using the Excel tools and GraphPadPrism software (La Jolla, CA), v. 5.03. Briefly, individual concentration effect curves are generated by plotting the logarithm of the concentration of tested compounds(X) vs. corresponding percent inhibition values (Y) using least squares fit. The best fit GI₅₀ values are calculated using Log (inhibitor) versus normalized response—Variable slope equation, where Y Ľ 100/(1 ţ 10 ((LogIC₅₀ _ X) * HillSlope)). QC criteria parameters (Z0, S:B, R2, HillSlope) were checked for every GI₅₀ curve.

The HuT78 cells were plated in 6-well plates at a concentration of 5 × 10⁵ cells per well and treated 24 h and 48 h with selected compounds 36c, 42a, 42c, 45a, 45b, 45c and 46c at a concentration of 5 μM. After drug treatment, the cells were fixed with ice-cold 70% ethanol in phosphate-buffered saline (PBS) and incubated with 0.3 μg/mL propidium iodide for 30 min at room temperature. Before being analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, SAD), samples were treated with 0.4 μg/mL RNase A for 5 min at room temperature. The resultant DNA histograms were generated and analyzed using FlowJo 7.6 software (Treestar, Inc, Ashland, OR, USA). Experiments were done in duplicate and the quantitative data are reported as average value ± standard deviation. Comparisons between control (non-treated) and treated groups were done using one-way analysis of variance (ANOVA) with Tukey–Kramer’s post hoc test with MedCalc statistical program. P-value less than 0.05 was considered statistically significant.

Changes in the (∆Ψm) were measured using TMRE (Tetramethylrhodamine, Ethyl Ester, Perchlorate) dye. In brief, tested cells (HuT78) were plated in 6-well plates at a concentration of 5 × 10⁵ cells per well and treated with 5 μM of compounds 36c, 42a, 42c, 45a, 45b, 45c, and 46c. After 48 h of treatment, cells were collected, centrifuged 6 min at 1100 rpm, and stained with 200 nM TMRE dye according to the kit protocol (TMRE Mitochondrial Membrane Potential Assay Kit, abcam, Cambridge, UK). Positive control cells were treated with 20 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) for 10 min. Cells were analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, SAD) and FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Proapoptotic potential of compounds was tested on HuT78 cells using Alexa Fluor 488 annexin V and propidium iodide (Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were plated in 6-well plates at a concentration 5 × 10⁵ cells/well and treated for 24 and 48 h with 5 µM 36c, 42c, 45a, 45b, 45c, and 46c. After incubation, cells were collected and centrifuged at 1100 rpm for 6 min, stained according to the manufacturer’s protocol and analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA) using FlowJo software (FlowJo, LLC, Ashland, OR, USA).