Materials

The uracil analog U-359 was obtained according to the Horner-Wadsworth-Emmons methodology published earlier (Pięta et al. 2019). Tx was purchased from Sigma-Aldrich (St. Louis, MO, USA). For all experimental procedures, U-359 and Tx were solubilized in dimethyl sulfoxide (DMSO) and subsequently diluted in culture medium to achieve a DMSO concentration of less than 0.1%.

Cell culture

The MCF-7 breast cancer cell line, which is positive for hormone receptors (estrogen receptor, ER and progesterone receptor, PR), was purchased from the European Collection of Cell Cultures (ECACC). The cells were cultured in Minimum Essential Medium Eagle (MEME) supplemented with non-essential amino acids and antibiotics (streptomycin at 100 mg/mL and penicillin at 100 U/mL), along with 2 mM glutamine. All these materials were obtained from Sigma Aldrich, St. Louis, MO, USA. Additionally, 10% fetal bovine serum from Biological Industries, Beit-HaEmek, Israel, was added to the culture medium. The cells were maintained at 37 °C in a 5% CO2 atmosphere and allowed to grow until they reached 80% confluence.

Selection of Tx-resistant MCF-7 cells (MCF-7/Tx)

For the selection of Tx-resistant cells, MCF-7 cells underwent prolonged exposure to progressively increasing concentrations of Tx, ranging from 0.01 μM to 1 μM. The viability and proliferation of these cells were subsequently evaluated using the MTT assay. If their growth pattern closely resembled that of the original MCF-7 cells, the Tx concentration was further increased. This iterative process continued until the cells developed tolerance to a maximum Taxol concentration of 1 μM. Throughout all experimental procedures, the Taxol-resistant MCF-7 cells were consistently designated as MCF-7/Tx.

Determination of cell morphology using light microscopy

To determine morphological alterations in MCF-7 and MCF-7/Tx cells, we used Wright and Giemsa staining, sourced from Merck, Germany. Giemsa Dye was applied to impart varying colorations to the cytoplasm, ranging from orange to pink, while the nucleus was dyed in tones of blue to purple, contingent upon the cytoplasmic acidity.

In brief, the protocol initiated with the seeding of cells onto 6-well plates at a density of 4 × 105 cells per well. Subsequently, these cells underwent a 24-h incubation with U-359 and/or Tx at their respective IC50 concentrations, using untreated cells as a negative control. Following the incubation period, the medium was aspirated, and the cells were fixed in methanol for 5 min. Post-fixation, PBS was used for cell washing, followed by staining with Wright and Giemsa solution for 30 min. Subsequent to staining, the cells were subjected to two rinses with H2O and left to air-dry. Finally, the stained cells were photographed using a light microscope.

MTT-assay

The MTT assay was conducted following the established protocol by Mosmann (1983). In summary, cells (104 per well) were plated in 24-well plates with 100 µl of medium and allowed to grow. After 24 h, the cells were exposed to varying concentrations of the compounds under investigation, achieving final concentrations ranging from 0 to 100 μM, for an additional 24 h. Subsequently, the cells were incubated for 2 h at 37 °C with MTT solution [5 mg/ml in phosphate-buffered saline (PBS, Gibco, Invitrogen, Carlsbad, CA, USA)]. The resulting blue formazan product's absorbance was measured at 560 nm using an iMark Bio-Rad microplate reader (Hercules, CA, USA) and compared to the control (untreated cells). The IC50 values (the concentration at which 50% inhibition occurs) were then determined.

Quantitative real-time PCR assay

Real-time quantitative PCR (RT-qPCR) was used to assess the mRNA levels of ABC transporter and NF-κB genes in MCF-7 and MCF-7/Tx cells. Initially, MCF-7 and MCF-7/Tx cells were seeded onto 6-well plates at a cell density of 5.0 × 10^5 cells per well and left to grow. Following this, the cells were incubated with U-359, Tx, or U-359 + Tx at their respective IC50 concentrations for 24 h. The effects of the combination treatment were then compared with those observed when Tx was administered individually. To obtain total RNA, the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) was employed, following the manufacturer's protocol. The concentration of RNA was determined using a Nanophotometer, and a consistent concentration of 150 ng/µL was used for subsequent experiments. cDNA synthesis was carried out using the Transcriba Kit (A&A Biotechnology, Gdynia, Poland). Gene-specific primers (ABCB1, ABCG2, and NF-κB/p65) listed in Table 1 were used for amplification. Real-Time 2x-PCR SYBR Master Mix (A&A Biotechnology, Gdynia, Poland) was utilized for this purpose in the Stratagene MX3005P QPCR System (Agilent Technologies, Inc., Santa Clara, CA, USA), following the manufacturer's instructions. GAPDH was employed as the internal reference gene to standardize the expression of the investigated genes. The expression levels of the tested genes were determined using the 2-∆∆CT method (Winer et al. 1999).

Table 1 Primer sequences for real-time PCR reactionAssessment of ABCB1 and ABCG2 protein levels by ELISA-based method

The protein levels of ABCB1 and ABCG2 in MCF-7 and MCF-7/Tx cells were determined using an ELISA-based method, including ABCB1 (Multidrug resistance protein 1 kit) and ABCG2 (ATP-binding cassette sub-family G member 2 kit), provided by Finetest BT LAB (Shanghai, China).

Briefly, MCF-7 and MCF-7/Tx cells were seeded on 6-well plates at the density of 5.0 × 105 cells per well and left to grow for 24 h. Subsequently, the cells were incubated with U-359, Tx, or U-359 + Tx at their respective IC50 concentrations for 24 h. Following the incubation period, the cells were washed with PBS and harvested by centrifugation at 200xg for 5 min. Cell lysates were prepared following the provided protocol. For the ELISA assay, appropriately diluted protein extracts (50 μg) and standards were added to the wells of 96-well plates pre-coated with specific antibodies for ABCB1 and ABCG2. The specific ABC transporter proteins in the tested samples are bound to the immobilized antibodies on the plate surface. A secondary antibody, conjugated with horseradish peroxidase (HRP), was then added. This HRP conjugate enabled a sensitive colorimetric readout. Finally, a stop solution was added to each well, and the optical density (OD) of the resulting yellow solution was measured spectrometrically at a wavelength of 450 nm to quantify the protein levels of ABCB1 and ABCG2 in the samples.

Multidrug resistance assay kit

To detect the multidrug-resistant phenotype in breast cancer cells, we used the Multidrug Resistance Assay Kit from Sigma-Aldrich (St. Louis, USA) and followed the manufacturer's guidelines.

Briefly, MCF-7 and MCF-7/Tx cells were seeded in 96-well plates at a density of 8.0 × 104 cells per well and left to grow for 24 h. The cells were incubated with U-359, Tx, or U-359 + Tx at their respective IC50 concentrations for 24 h. Subsequently, 100 μL of the MDR Dye Loading Solution was added to each well. The plates were then incubated at 37 °C in a humidified environment with 5% CO2 for a duration of 15 min. The analysis of fluorescence intensity was carried out using a Flexstation 3 instrument.

Assessment of NF-κB protein level by ELISA-based method

NF-κB/p65 activity was assessed in cellular protein extracts (10 μg) using the Human Nuclear factor NF-kappa-B p65 subunit ELISA Kit (BT LAB, Shanghai, China).

Briefly, MCF-7 and MCF-7/Tx cells were seeded in 6-well plates at a density of 5.0 × 105 cells per well and left to grow for 24 h. The cells were then incubated with U-359 (specific inhibitor), Tx (test substance), or a combination of U-359 and Tx at their respective IC50 concentrations for 24 h. Subsequently, the cells were washed with PBS and collected by centrifugation (200xg, 5 min), followed by the collection of cell culture supernatants.

Nuclear extracts were obtained through repeated freeze–thaw cycles to release the intracellular components. These extracts were then analyzed using a kit comprising a 96-well plate with immobilized oligonucleotides for the NF-κB subunit. NF-κB present in the sample bound to antibodies coated on the wells. Biotinylated human NF-κB antibody was added, binding to NF-κB in the sample. Subsequently, streptavidin-HRP was added, binding to the biotinylated NF-κB antibodies. After a washing step to remove unbound streptavidin-HRP, substrate solution was added, resulting in color development proportional to the amount of NF-κB. The reaction was terminated by the addition of acidic stop solution, and absorbance was measured at 450 nm.

Statistical analysis

Statistical analysis was performed using Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). The study collected data from at least three independent experiments, each conducted in triplicate, and the results were expressed as the mean ± standard error of the mean (SEM). To determine the statistical significance, a one-way analysis of variance (ANOVA) was initially applied, followed by a post-hoc multiple comparison Student–Newman–Keuls test or test T. Levels of significance were denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001 (compared with control) or #p < 0.05, ##p < 0.01, and ###p < 0.001 (compared with Tx) indicating the statistical significance of the findings.