Balb c 3t3 clone a31

The COLO-38, Balb/3T3 Clone A31 and THP-1 cell lines were purchased from ATCC, Manassas, VA, USA. COLO-38 cells, derived from amelanotic melanoma, were cultured in RPMI 1640 1× medium supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin, 1% L-Glutamine and 25 mM HEPES (4-2-hydroxyethyl-1-piperazinyl-ethanesulphonic acid) buffer. Balb/c 3T3 clone A31 cells, murine fibroblasts, were maintained in DMEM with 10% Calf Bovine Serum (CBS; ATCC, Manassas, VA, USA) and 1% penicillin-streptomycin (10,000 unit/mL). THP-1 cells, derived from a monocyte cell line isolated from the peripheral blood of a patient with acute leukemia, were cultured in RPMI 1640 medium with 10% Fetal Bovine Serum (FBS), 1% Penicillin/Streptomycin and 0.05% mM β-Mercaptoethanol. All cell lines were maintained at 37 °C in modified air containing with 5% humidified CO₂.

The breast cancer cells MDA-MB-231 (ATCC: HTB-26) and MCF-7 (ATCC: HTB-22) were used to determine antiproliferative activity under in vitro conditions. The breast epithelial cells (MCF-10A, ATCC: CRL-10317) and mouse embryonic fibroblasts (BALB/c 3T3 clone A31, ATCC: CCL-163) were used as control. Cell cultures were purchased from American Type Cultures Collection (ATCC, Manassas, Virginia, USA). In cell culture, growth medium DMEM-high glucose, 10% (v/v) FBS and antibiotics (Sigma-Aldrich, Schnelldorf, Germany) were used. Cells were incubated at 37 °C, 5% CO₂ and 95% humidity. Cells were grown in plastic flasks with a surface of 75 cm² (Deltalab S.L., Barcelona, Spain).

All cell lines, breast cancer MCF-10A, MCF-7 and MDA-MB-231, and BALB/c 3T3 clone A31 (mouse embryonic fibroblasts) were purchased from the American Type Culture Collection—ATCC (Manassas, VA, USA). The MCF-7, MDA-MB-231 and BALB/c 3T3 clone A31 were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate and 1% MEM non-essential amino acids (NEAA) without antibiotics. The non-tumorigenic breast cell line (MCF-10A) was cultivated in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids (NEAA), 20 ng/mL human epithelia growth factor (hEGF), 10 μg/mL insulin and 0.5 μg/mL hydrocortisone without antibiotics. All cell lines were maintained in a humidified atmosphere with 5% CO₂ at 37 °C.

The human hepatoma cell line (HepG2) was purchased from the American Type Culture Collection (ATCC HB-8065). These cells were cultured in Minimum Essential Medium Eagle (MEME) (ATCC). The murine fibroblast cell line (Balb/c 3T3 clone A31) was purchased from the American Type Culture Collection (ATCC CCL-163), and the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC). The media were supplemented with 10% BCS (Balb/c 3T3), 10% FBS (HepG2), 1% l-glutamine, and 1% antibiotic solution. The cells were maintained in 75-cm² cell culture flasks (NUNC, Roskilde, Denmark) in a humidified incubator, NuAire (Plymouth, MN, USA) at 37 °C and 5% CO₂. The medium was refreshed every two days and the cells were trypsinized by 0.25% trypsin–0.02% EDTA after reaching 70–80% confluence. Then, cell suspensions at a density of 2 × 10⁵ cells/mL (HepG2) and 5 × 10⁴ cells/mL (Balb/c 3T3) were transferred to 96-well plates (100 μL/well) and incubated for 24 h before the exposure to the studied compounds.