Caco 2

The human colon adenocarcinoma cell line Caco-2 (86010202, Sigma, St. Louis, MO, United States) was cultured in Eagle’s minimum essential medium (EMEM; 30-2003, ATCC, Manassas, VI, United States)) supplemented with 10% fetal bovine serum (FBS; 16140-071, Gibco, Gaithersburg, MD, United States), 1% non-essential amino acids (NEAA; 11140-050, Life Tech, Carlsbad, CA, United States), and 1% penicillin/streptomycin (P4333, Sigma). The human colon cell line HT29-MTX-E16 (86010202, Sigma) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; D6546, Sigma) supplemented with 10% FBS (16140-071, Gibco), 1% NEAA (11140-050, Life Tech), 1% Glutamax (35050-061, Gibco), and 1% penicillin/streptomycin (P4333, Sigma). The human acute monocytic leukemia cell line THP-1 (Tib-202, ATCC) was cultured in Roswell Park Memorial Institute (RPMI) 1640 (R0883, Sigma) supplemented with 10% FBS (16140-071, Gibco), 1% Glutamax (35050-061, Gibco), and 1% penicillin/streptomycin (P4333, Sigma). The cells were tested for mycoplasma contamination and found negative.

The LoVo and Caco-2 were obtained from American Type Culture Collection (ATCC), USA. Cells were grown in DMEM (Sigma) media containing 10% FBS and 10000 U/ml antibiotic and then kept at 37°C in 5% CO₂ incubator. LoVo and Caco-2 were treated with 1 μM 5-aza-2′-deoxycytidine (Sigma) for 72 hours, and a medium was replaced every 24 hours. Control cultures had an equal volumes of drug solvent (DMSO) [12 (link)].

The human colon adenocarcinoma cell lines of HCT-15, SW-480 and Caco-2 were obtained from the American Tissue Culture Collection (ATCC) Manassas, VA, USA. HCT-15 and SW-480 cell lines were maintained in RPMI 1640 medium (SAFCO) (Sigma Aldrich, Castle Hill, NSW) supplemented with foetal calf serum (FCS, 10 %) (Hyclone Quantum Scientific, Clayton South, VIC), glutamine (10 mM), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES 10 mM) and penicillin (100U/ml)/streptomycin (100 μg/ml) (Sigma Aldrich, Castle Hill, NSW). The Caco-2 cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich, Castle Hill, NSW) supplemented with 20%FCS and penicillin (100U/ml)/streptomycin (100 μg/ml), 2 mM/L glutamine, 0.1 mM non-essential amino acids. Cells were grown at 37 °C in 5 % CO₂ humidified atmosphere.

The colon carcinoma HT29 (ATCC® HTB-38™) and Caco2 (ATCC® HTB-37™) cell lines were both purchased from American Type Culture Collection. HT29 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Massachusetts, USA) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, Massachusetts, USA). Caco2 cells were maintained in Minimum Essential Medium Eagle (MEM, Sigma-Aldrich, Missouri, USA) supplemented with 20% FBS, 1% Penicillin-Streptomicin and L-Glutamine. HT29 and Caco2 differentiation was achieved by spontaneous post-confluence growth arrest. Briefly, 5x10⁴ cells per well were seeded in a 6 multi-well plate. Cells were harvested at day 3 (pre-confluence), day 7 (confluence), day 14 (post-confluence) after seeding and processed for RNA extraction. Each single experiment was conducted in triplicate. Results are expressed as mean of 3 independent experiments.

Because of the known cytotoxicity of capsaicin [12 (link), 13 (link)] (a main substance responsible for the hot and spicy taste in the chili), capsaicin (305.41 g/mol molecular weight; Sigma-Aldrich, St. Louis, MO, USA) were tested with enterocytes (Caco-2 and HT-29 cell lines). As such, the human colorectal adenocarcinoma cells, Caco-2 (ATCC HTB-37) and HT-29 (ATCC HTB-38), from the American Type Culture Collection (Manassas, VA, USA) were maintained in supplemented Dulbecco’s modified Eagle medium (DMEM) and McCoy’s 5a modified medium, respectively, at 37°C under 5% CO₂ and sub-cultured before use in the experiments. Then, capsaicin in the different concentrations (0.02, 0.2 and 2 mM) was incubated with the enterocytes for 24 h before the determination of cell viability using tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) solution (Thermo Fisher Scientific, Rockford, IL, USA) [38 (link)]. The activated cells were incubated with 0.5 mg/mL of MTT solution for 2 h at 37°C in the dark and diluted by dimethyl sulfoxide (DMSO; Thermo Fisher Scientific) before measurement with a Varioskan Flash microplate reader at absorbance of optical density (OD) 570 nm.

The human colonic carcinoma cell line Caco-2 (ECACC, Salisbury, UK) was routinely cultured in Dulbecco's modified Eagle's minimal essential medium DMEM (Sigma), supplemented with 10% (w/v) foetal bovine serum (FBS, Sigma), 1% (w/v) nonessential amino acids solution (Sigma), and antibiotic solution (100 U/mL penicillin, 100 μg/mL streptomycin). Cells were maintained in T-75 culture flasks at 37°C in a 5% CO₂ atmosphere. For adhesion assay, the Caco-2 cells were seeded at a concentration of 10⁵ cells/well in 6-well tissue culture plates (Falcon) to obtain confluence and cultured for 20 days prior to use in adhesion assay. The cell culture medium was changed on alternate days and replaced by fresh DMEM supplemented with 2% (w/v) FBS and without antibiotic at least 1 h before the adhesion assay. A 1 mL aliquot of Lactobacillus suspension (10⁸ ufc/mL in phosphate buffered saline, PBS) was added to each well of the tissue culture plate and incubated at 37°C in 5% CO₂ atmosphere for 3 h. Afterwards, the cells were washed three times with 1 mL of PBS in order to remove nonadherent bacteria and lysed by addition of Triton X 100 (0.05% solution) for 10 min; then appropriate dilutions were plated on MRS agar. Adhesion was expressed as the percentage of bacteria adhered to Caco-2 cells compared to the initial amount of bacteria.