Dld 1

Colon cancer cell lines DLD-1 and HT-29 were acquired from Cell Bank, China Academy of Sciences (Shanghai, China). Immortalized human colonic epithelial cell NCM460 was kindly provided by BeNa Culture Collection (Henan, China). DLD-1, HT-29, and NCM460 cells were cultured in RPMI-1640, McCoy's 5A, and DMEM (Gibco, Thermo Fisher Scientific), respectively. Each media contained 10% (v/v) fetal bovine serum (FBS), and the cells were maintained in a humidified incubator at 37 °C with 5% CO₂. Each cell line was recently authenticated by STR profiling and tested for mycoplasma contamination. For the bacterial infection study, cells were infected with S. moorei anaerobically or E. coli MG1655 at MOI (multiplicity of infection) of 100 for 2 h every 24 hours. Following incubation, then removed the bacteria and the bacteria-containing medium was substituted by a cell culture medium that contained 10% FBS, 20 µg/mL gentamycin, and 1% penicillin-streptomycin (PS) continue to culture cells in the carbon dioxide incubator. Cells were co-cultured with bacteria three times within 72 hours.

The female kidney cancer line A498, female ovarian cancer line OVCAR-8, male colon cancer line DLD-1 (ATCC), male kidney cancer line ACHN, female chronic myelogenous leukemia line K562 (ATCC), male acute T cell leukemia line Jurkat, male acute lymphocytic leukemia line Nalm-6 (DSMZ), male acute lymphocytic leukemia line SUP-B15 and male acute myeloblastic leukemia line Kasumi-1 were grown in L-glutamine containing RPMI (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich) and 1% Penicillin/Streptomycin (Sigma Aldrich) (cRPMI). The female breast cancer line MCF-7 (ATCC) was grown in L-glutamine containing RPMI supplemented (Gibco) with 10% heat-inactivated fetal bovine serum (Sigma Aldrich), 1% Sodium Pyruvate (Sigma Aldrich) and 1% Penicillin/Streptomycin (Sigma Aldrich). The female cervical cancer line HeLa (ATCC) was grown in L-glutamine containing DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich) and 1% Penicillin/Streptomycin (Sigma Aldrich). The male neuroblastoma line IMR-32 was grown in L-glutamine containing IMDM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich) and 1% Penicillin/Streptomycin (Sigma Aldrich) (cIMDM). A549.Histone.mKate2 (A549), DLD-1-RFP and K562-GFP were cultured in cRPMI supplemented with 1 μg/mL Puromycin (Thermofisher). Adherent cell lines were split every 2–4 days using Trypsin/EDTA (Sigma Aldrich). Cell culture medium was renewed every 48 h in all cultures and experiments. The cell lines used in the study have not been authenticated.

The antiproliferative potential of EO and ES extracts against the colorectal adenocarcinoma cell line DLD-1 (CCL-221™, purchased from the American Type Culture Collection and provided by Dr. Eva Fisher-Fodor and Dr. Olga Șoritau at the Oncological Institute “Prof. dr. Ion Chiricuță” from Cluj-Napoca) was investigated using the CCK8 assay. DLD-1 cells were cultured in RPMI-1640 medium (Gibco Life Technologies, Paisley, UK) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% glutamine (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotics–antimycotics (Gibco Life Technologies, Paisley, UK). The cultures were maintained at 37 °C with 5% CO₂ and 60% humidity. DLD-1 cells (1 × 10⁴ cells/well) were exposed to each sample at five different concentrations (0.27 μmol GAE, 0.54 μmol GAE, 0.81 μmol GAE, 1.08 μmol GAE and 1.35 μmol GAE for EO and 0.22 μmol GAE, 0.44 μmol GAE, 0.66 μmol GAE, 0.88 μmol GAE and 1.10 μmol GAE for ES). These concentrations were calculated based on the TP concentration expressed in μmol GAE/μL. The positive control consisted of doxorubicin (reference compound) at a concentration of 20 μg/mL, while the negative control comprised cells maintained in standard culture medium. The potential inhibitory effect was also evaluated for the solvent used for preparing hydroalcoholic products. The extracts were incubated for a further 24 h. Following 24 h of incubation, CCK-8 solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well, and the cell cultures were further incubated for 4 h at 37 °C in the dark. CCK-8 contains a water-soluble tetrazolium salt that is reduced by viable cells to produce a colored formazan dye. The amount of formazan dye produced is directly proportional to the number of viable cells in the sample [53 (link),54 (link),55 (link)]. Subsequently, the absorbance of each well was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). All experiments were performed in triplicate and expressed as mean  ±  SD. The calculation of cell survival (%) was performed based on the optical densities correlated with the optical density of the control.

The influence of CFA, CA, CTA, and CY was studied in relation to a colorectal adenocarcinoma DLD-1 cell line, which was obtained from the American Type Culture Collection (ATCC). DLD-1 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humified atmosphere of 5% CO₂ in the air. The cells viability in tested cell lines was examined at the concentrations of 0.5 µM, 1 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 300 µM, and 500 µM for every studied compound.