Chemicals and reagents

Tamoxifen (TAM) was acquired from Amria Pharmaceuticals Company (Alexandria, Egypt). DMSO was used to dissolve tamoxifen, and 2-ME was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in salt water. Both TAM and 2-ME were stored in a stock solution at −20 °C. Both TAM and 2-ME were serially diluted in RPMI1640 immediately before use, with final concentrations ranging from 2.5 to 100 M and 1.25 to 10 M, respectively.

Sigma Aldrich Chemical (St. Louis, MO, USA) was the exclusive supplier of all components, solvents, and reagents. (Milan, Italy-based) Sigma was kind enough to provide the RIPA lysis buffer. Thermofisher (Waltham, Massachusetts, United States) was where we got our fetal bovine serum, PBS, penicillin/streptomycin solution, RPMI-1640, and trypsin–EDTA.

Cell lines

Human BC cell line MCF-7 was obtained from the American Type Culture Collection (ATCC; USA) and cultivated at the National Cancer Institute (NCI; Egypt) in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Robert Clarke (Georgetown University Medical Center, Washington, DC, USA) generously supplied the tamoxifen-resistant MCF-7, LCC2. [18]. Then the resistance was confirmed by cytotoxicity [19]. Every cell line was maintained in a constant humid 37 °C incubator with 5% CO2.

Cytotoxicity assay

The anticancer activities of TAM and 2-ME on BC cells were evaluated using the Sulphorhodamine-B (SRB) assay [20]. Cells were sown in 96-well microtiter plates at a density of 3 × 103 cells per well. Prior to drug incubation, a 24-h period was allocated for cell attachment. Subsequently, the cells were subjected to different doses of TAM ranging from 2.5 to 15 µM for MCF-7 cells and 10 to 100 µM for LCC2 cells, as well as 1.25 to 10 µM of 2-ME for both MCF-7 cells and their counterpart. This exposure lasted for a duration of 48 h. A control vehicle consisting of 0.1% v/v of DMSO was used. The cells were subjected to staining using a 0.4% solution of SRB dye, followed by fixation using a 20% concentration of trichloroacetic acid after the incubation period. The optical density (OD) of each well was measured spectrophotometrically at a wavelength of 570 nm using a TECAN sunriseTM ELISA reader from Germany.

The survival fraction may be calculated by dividing the OD of treated cells by the OD of control cells. The determination of the IC50 (concentration that inhibits cell growth by 50%) for each medication was performed using GraphPad Prism 8, via the use of sigmoidal dose–response curve-fitting models. Various concentrations of 2-ME were combined with the quarter and half of the IC50 value of TAM for the combination (ranging from 1.25 to 10 µM). Furthermore, the CompuSyn program was used to compute the combination index (CI) [21,22,23] to assess the extent of interaction between TAM and 2-ME on LCC2 and MCF-7 cells. Following the calculation of CI, the lowest concentrations given the smaller CI value were used for subsequent experiments. Therefore, this work conducted mechanistic studies by using a concentration of 2.5 μM TAM (half of the IC50) and/or 2.5 μM 2-ME on MCF-7 cells, as well as 35 μM TAM and/or 1.25 μM 2-ME on LCC2 cells, for a duration of 48 h.

Assay of caspases 3, Bcl2, and Bax

In accordance with the guidelines provided by the manufacturer, colorimetric assay kits (Cloud-clone Corp, Houston, TX, USA) were used to evaluate the expression levels of apoptosis-related markers, namely Bcl2, caspases 3, and Bax, in cell lysate. The measurements were conducted at a wavelength of 450 nm using specific product numbers for each marker: SEA626Hu for Caspase 3, SEA778Hu for Bcl2, and SEB343Hu for Bax. Following a 24-h period, a total of 8*105 cells were introduced into individual wells of 6-well plates. The cells were treated with TAM, 2-ME, and a combination of both compounds for a duration of 48 h. Following the incubation time, the cell pellets were lysed using protein lysis buffer, and subsequently, the apoptotic markers were evaluated. The identification of changes in Bcl2, caspases 3, and Bax was achieved by a comparison of the data with the untreated control level. The experiment was conducted in triplicate, with each trial being performed independently.

Determination of protein concentration

The cells were subjected to trypsinization, followed by collection and subsequent storage at a temperature of −80 °C. This was done after exposing the cells to TAM and/or 2-ME for 48 h. Subsequently, cellular lysis was achieved by using a RIPA lysis buffer supplemented with protease inhibitors. The RIPA lysis solution consisted of 25 mM Tris HCL at pH 7.6, 150 mM NaCl, 1% triton X-100, 1% sodium deoxycholate, and 0.1% SDS. The protein contents in the cell lysate were measured using the Bradford assay kit obtained from Pierce (Rockford, IL, USA) [24].

Western blot analysis

In summary, the RIPA buffer was used for cell lysis, followed by the transfer of the lysed cells to an Eppendorf tube and subsequent centrifugation at a speed of 13,000 revolutions per minute for a duration of 15 min. The proteins extracted were separated on a PVDF membrane using SDS-PAGE with a 12 percent acrylamide gel. The membranes were probed using the mouse monoclonal anti-HIF-1α primary antibodies (Santa Cruz Biotechnology, Inc., CA, USA) at a dilution of 1:500. Following an overnight incubation period, the membranes were subjected to a washing step and then incubated at room temperature for one hour. During this incubation, an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novus Biologicals, LLC, Littleton, CO, USA) was used at a dilution of 1:5000. The commercially available kit was used to detect the antibody that is bound to the membrane. The program Win Image Studio Lite 5.2.5 was used to quantify band intensities. β-actin (Santa Cruz Biotechnology, Inc., CA, USA) was employed as a loading control at a dilution of 1:500.

Determination of triglycerides (TG) and cholesterol

In the present study, LCC2 or MCF-7 cells were cultured in 6 well plates with a density of 8 × 105 cells per well. After administering TAM and/or 2-ME therapy, the levels of total cholesterol and TG were analyzed using the BIOLABO kit (Les Hautes Rives, France) by a colorimetric method, following the directions provided by the manufacturer. The absorbance at a wavelength of 340 nm was measured using a spectrophotometer manufactured by Milton Roy Co., TX, USA. The amount of cholesterol and triglycerides was determined by performing calculations using the corresponding protein content.

Statistical analyses

Three replicates of each experiment were performed independently. The data were presented as means ± SD and were subjected to analysis using GraphPad Prism 8. Post hoc tests were used in conjunction with ANOVA to assess the statistical disparities among the tested parameters. A significance level of P < 0.05 was considered to indicate statistical significance. The CompuSyn program was used to compute the combination index (CI) for quantifying the extent of interaction between 2-ME and TAM. The phenomenon of synergism is represented by CI value that is less than 1, while an additive effect is shown by a CI value equal to 1, and antagonism is characterized by a CI value greater than 1.