Selenoesters and Selenoanhydrides as Novel Agents Against Resistant Breast Cancer

Materials and Methods

Compounds. The eleven Se-compounds tested (selenoanhydride 1 and selenoesters 2-11, Figure 1) were kindly provided by Dr. Enrique Domínguez-Álvarez (Spanish National Research Council, Madrid, Spain) and by Professor Dr. Carmen Sanmartín (University of Navarra) (9). Se-compounds 1-11 were stable and their purity was assessed through spectroscopic techniques (elemental analysis, nuclear magnetic resonance, mass spectrometry and infrared spectroscopy). Compounds 12-15 were purchased from Sigma-Aldrich (Steinheim, Germany), respectively, to be used as non-selenium (12) isostere of selenoanhydride (1) and as inorganic chalcogen salts (13-15), for comparing their activity with the selenoesters. The compounds were dissolved in dimethyl sulfoxide (DMSO).

Cell lines. Breast cancer cell line MCF-7 (ATCC® HTB-22) was purchased from LGC Promochem (Teddington, Middlesex, UK). The MCF-7 cell line and its drug-resistant subline KCR were grown 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. The cell lines were incubated at 37°C, in an atmosphere of 5% CO₂ and 95% air. On every third passage, 0.56 μg/ml doxorubicin (Teva Pharmaceuticals, Budapest, Hungary) was added to the medium in order to maintain ABCB1 expression in KCR cells.

Cytotoxicity assay. The cytotoxic effects of the Se-compounds were determined on MCF-7 and KCR breast cancer cell lines. The effects of increasing concentrations of Se-compounds on cell growth were tested in 96-well flat-bottomed microtiter plates. The compounds were diluted in 100 μl of medium.

The adherent breast cancer cell lines were cultured in 96-well flat-bottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine serum. The density of the cells was adjusted to 1×10⁴ cells in 100 μl per well, the cells were seeded for 24 h at 37°C, with 5% CO₂ prior to the assay, then the medium was removed from the plates containing the cells, and dilutions of Se-compounds were previously made in a separate plate and added to the cells in 200 μl.

The culture plates were incubated at 37°C for 24 h; at the end of the incubation period, 20 μl of thiazolyl blue tetrazolium bromide (MTT; Sigma) solution (from a stock solution of 5 mg/ml) were added to each well. After incubation at 37°C for 4 h, 100 μI of sodium dodecyl sulfate (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). Inhibition of the cell growth was determined according to the formula below: Results are expressed in terms of IC₅₀, defined as the inhibitory dose that reduced the growth of the cells exposed to the tested compounds by 50%.

The selectivity was calculated using the selectivity index (SI), which is defined as the quotient of the IC₅₀ value determined for the non-tumorous MRC-5 cell line described previously (6) to the IC₅₀ value for the respective cancer cell line (MCF-7 or KCR). Following the criteria reported in bibliography (6), we considered a compound to be strongly selective when its SI was 6 or higher. Compounds with SI values of 1-3 and 3-6 were regarded as slightly and moderately selective, respectively.

Checkerboard combination assay. A checkerboard microplate method was applied to study the effect of drug interactions between the Se-compounds 1-11 and the chemotherapeutic drug doxorubicin. The assay was carried out on MCF-7 and KCR breast cancer cell lines. The adherent breast cancer cell lines were cultured in 96-well flat-bottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine serum. 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 with 5% CO₂ prior to the assay and then the medium was removed from the plates containing the cells.

The final concentration of the Se-compounds and doxorubicin used in the combination experiment was chosen in accordance with their cytotoxicity towards these cell lines. The dilutions of doxorubicin were made in a horizontal direction in 100 μl, and the dilutions of the Se-compounds vertically in the microtiter plate in 50 μl volume. The plates were incubated for 72 h at 37°C in 5% CO₂ atmosphere. The cell growth rate was determined after MTT staining. At the end of the incubation period, 20 μl of MTT (Sigma) solution (from a stock solution of 5 mg/ml) were added to each well. After incubation at 37°C for 4 h, 100 μl of SDS (Sigma) solution (10% in 0.01 M HCI) were added to each well and the plates were further incubated at 37°C overnight. Optical density (OD) was measured at 540/630 nm with Multiscan EX ELISA reader (Thermo Labsystems). Combination index (CI) values at 50% of the growth inhibition dose (ED50) were determined using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA) to plot four to five data points at each ratio. CI values were calculated by means of the median-effect equation, according to the Chou–Talalay method, where CI<1, CI=1, and CI>1 represent synergism, additive effect (or no interaction), and antagonism, respectively (11, 12).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2.

Interactions of the active Se-compounds with doxorubicin in KCR and MCF-7 cells. The figure indicates, at the most effective interaction ratio (doxorubicin:Se-compound), the concentration of the Se-compound in the presence of doxorubicin at the concentration indicated; furthermore the type of interaction (antagonism, additive effect and synergism) is also presented.

Apoptosis induction. The ability of the Se-compounds to induce apoptosis was determined on breast cancer cell lines. The apoptosis assays were performed using Annexin V-FITC Apoptosis Detection Kit from Calbiochem (EMD Biosciences, Inc. La Jolla, CA, USA), following the instructions provided by the manufacturer. This assay enables the quantification of early and late apoptotic events, as well as necrosis and cell death in the cell population exposed to the Se-compounds. The density of the cell suspension was adjusted to 1×10⁶ cells/ml. The cell suspension was distributed into 0.5 ml aliquots (5×10⁵ cells) to a 24-well microplate and incubated overnight at 37°C in 5% CO₂. On the following day, the medium was removed, and fresh medium was added to the cells. The cells were then incubated in the presence of Se-compounds at 2 μM for 3 h at 37°C. 12H-Benzo[α]phenothiazine M627 (13), which is a known early apoptosis inducer, was used as positive control. The samples were washed in PBS and fresh medium was added to the cells, followed by the incubation of the plate for 24 h at 37°C, in 5% CO₂. After the incubation period, the cells were trypsinized. The harvested cells were centrifuged at 2,000 × g for 2 min. The cells were then re-suspended in fresh serum-free medium. Thereafter, the apoptosis assay was carried out according to the rapid protocol of the kit and the fluorescence was analyzed immediately using a ParTec CyFlow flow cytometer (Partec, Münster, Germany).