Ht 29

Human CRC Cell lines including SW480, HT-29, SW1463 and HCT116, as well as FHCs (fetal human cells), were obtained from ATCC. SW480 and SW1463 cells were seeded in ATCC-formulated Leibovitz’s L-15 medium. HT-29 and HCT116 cells were cultured in ATCC-formulated McCoy’s 5a medium modified. FHCs were maintained in DMEM:F12 medium. All the cell lines were incubated under 37 °C with 5% CO₂ in air atmosphere, and routinely subcultured every 3 days.

The cell line HT-29 was purchased from ATCC (Manassas, VA, USA). The HT-29 cell line was cultivated in DMEM (ATCC) supplemented with 10% FBS (Biowest, Kansas City, MO, USA) and 10 mM HEPES (pH = 7.3, Promega Madison, WI, USA). The conditions of incubation were 37 °C, 4% CO₂, and 95% RH. Assays were carried out in 12-well microtiter plates (MTP) or single vials. Cover slides were placed on them when necessary, and 1 mL of DMEM with 50 µg/mL of gentamicin was then added. A total of 4 × 10⁴ cells were inoculated for 24 h to let them attach.

All the human cell lines [H460 (ATCC^® HTB-177, Manassas, VA, USA); A549 (ATCC^® CCL-185); Caco-2 (ATCC^® HTB-37™); HT-29 (ATCC^® HTB-38™); MCF-7(ATCC^® HTB-22); MDA-MB-231 (ATCC^® HTB-26); Jurkat, T-cell leukemia (DSMZ ACC 282, Braunschweig, Germany) and Ramos B-cell lymphoma (ATCC^® CRL-1596™)] were cultured at 37 °C in a humidified CO₂ incubator (5% CO₂) in complete RPMI media (hematological cell lines Jurkat and Ramos) or DMEM media (adherent cells H460, A549, Caco-2, HT-29, MCF-7, and MDA-MB-231) (both supplemented with 10% (v/v) FBS and 1% (v/v) penicilin–streptomycin). When the cells reached 80% confluence, they were subcultured. For this purpose, adherent cells were washed with PBS and treated with 1 mL 0.25% trypsin-EDTA to detach them. Once collected, cells were centrifugated at 1200 rpm for 5 min. Suspension cells were collected directly from the cell culture plate and centrifugated in the conditions mentioned above. When necessary, the cells were counted using a Neubauer counting chamber and dyed with Trypan Blue.

HCT-116, Colo205, HT-29 and Caco2 cell lines were purchased from ATCC and maintained under standard conditions in culture medium (HCT-116 and HT-29 – McCoy’s 5A Medium; Colo205 – RPMI-1650 medium; Caco2 – DMEM medium) supplemented with 10% FBS (Fetal Bovine Serum). Cells were made quiescent by lowering FBS concentration to 0.5% FBS for 24 h prior to harvest.

Gastric adenocarcinoma (AGS; ATCC CRL-1739) and carcinoma (NCI-N87; ATCC CRL-5822) cells, colorectal adenocarcinoma (HT-29; ATCC HTB-38) and carcinoma (HCT-116; ATCC CCL-247) cells were purchased from ATCC (Manassas, VA, USA). AGS cells were maintained in F-12K medium (ATCC), NCI-N87 were maintained in RPMI-1640 medium, HT-29 and HCT 116 cells were maintained in McCoy’s 5A modified medium. All the culture media were complexed with 10% fetal bovine serum (ATCC). All the cells tested were placed at 37 °C in a humidified atmosphere of 5% CO₂ to retain the proliferating condition.

Human liver cancer cell lines HepG2, Bel-7404, and human umbilical vein endothelial cells (HUVECs) (ATCC, Manassas, VA, United States) were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotic. Human renal proximal tubular epithelium HK-2 and human colorectal cancer cell line HT-29 (ATCC, Manassas, VA, United States) were grown in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotic. All the cells were maintained at 37°C with 5% CO₂ under a humidified atmosphere. For the cytotoxicity assay, cell viability was tested using the Cell Counting Kit-8 (CCK-8, DojinDo, Japan). The cells were seeded in 96-well plates with 5,000 cells (100 μL) per well, allowed to attach for 6–8 h, and then exposed to CD437 at different concentrations (100 μL), prepared in PBS or 0.1% DMSO (negative control), for 24 h. CCK-8 (10 μL) was then added to each well and the plates were incubated at 37°C for 1–4 h. The absorbance was measured at 450 nm, and the cytotoxicity was calculated using the following formula:
The 50% inhibitory concentration (IC₅₀) value was determined using GraphPad Prism 8 (GraphPad Software Inc., CA, United States). The assay was carried out in triplicate.

The cell lines used in this study were the murine lymphoma line L5178Y-R (ATCC CRL-1722), the human colorectal adenocarcinoma line HT-29 (ATCC HTB-38), the human breast cancer line MCF-7 (ATCC HTB-2), and the monkey kidney epithelial cell line MA-104 (ATCC CRL-2378.1). Peripheral blood mononuclear cells (PBMCs) were obtained from 20 mL to 30 mL (three experiments were performed) samples of blood from a healthy volunteer donor, using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Cells were maintained in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% antibiotic–antifungal solution (Life Technologies), whereas MCF-7 cells were grown in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% FBS and 1% antibiotic–antifungal solution (Life Technologies) (complete culture medium). Cells were cultured at 37 °C in an atmosphere of 5% CO₂.