Ccd 18co

The human colon cell line, CCD-18Co, and colon cancer cell lines, WiDr and HT-29, were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). WiDr and CCD-18Co cells lines were cultured in Eagle's Minimum Essential Medium and HT-29, in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO), 1% penicillin and streptomycin. All cell lines were cultured in a humidified incubator with 5% CO₂ at 37°C. All experiments were conducted on cell lines with passage number 1–10.

Authenticated CRC lines were obtained from ECACC: HT29 (ECACC 91072201), Colo205 (ECACC 87061208), Colo320DM (ECACC 87061205), SW480 (ECACC 87092801), and SW620 (ECACC 87051203). Authenticated primary human colonocytes were obtained from ATCC: CCD-18Co (ATCC CRL-1459) and CCD-841 CoN (ATCC CRL-1790). All lines were regularly monitored for mycoplasma contamination every 2 months by PCR^{40 (link)}, and only mycoplasma-free cells were used for studies. All cells were used for no more than 15 passages. All cells were grown at 37 °C in HEPA-filtered humidified air in 5% CO₂, in media supplemented with 10% heat-inactivated FBS (Gibco, Paisley, Scotland, UK, Cat. no. 10099-141), L-Glu (2 mM, Lonza, Burton on Trent, UK, Cat. no. 17-606E) and penicillin/streptomycin (100 U/ml, Gibco, Cat. no. DE17-602E). Colo205 and Colo320DM cells were grown in RPMI medium (Gibco, Paisley, Scotland, UK, Cat. no. 31870), HT29 were grown in McCoy’s medium (Lonza, Burton on Trent, UK, Cat. no. 12-688F), SW480 and SW620 were grown in L15 medium (Lonza, Burton on Trent, UK, Cat. no. BE12-700F), and HCT-116 (17) were grown in DMEM (Gibco, Paisley, Scotland, UK, Cat. no. 21885-025). CCD-18Co and CCD-841 primary cultures were grown in EMEM (Sigma-Aldrich, Saint Louis, MO, USA, Cat. no. M2279).

Human osteosarcoma cell lines HOS (ATCC, no. CRL-1543^TM, ATCC, Manassas, VA, USA) and Saos-2 (ATCC, no. HTB-85), both derived from primary bone tumors and human normal cells—colon fibroblasts CCD-18Co (ATCC, no. CRL-1459^TM) and fetus osteoblasts hFOB 1.19 (ATCC, no. CRL-11372^TM) were used in the present study. The cells were cultured in appropriate media: HOS and CCD-18Co—EMEM (Sigma Aldrich, St. Louis, MO, USA), Saos-2—McCoy’s (Sigma Aldrich), and hFOB 1.19—DMEM/F-12 without phenol red and with L-glutamine. All media were supplemented with fetal bovine serum (FBS, Sigma Aldrich). The HOS and Saos-2 culture media were supplemented with an antibiotic/antimycotic solution (Sigma Aldrich), whereas the hFOB1.19 medium was additionally supplemented with G418 (Sigma Aldrich); the CCD-18Co medium was used without antibiotics. The cells were cultivated in standard conditions at 37 °C, 95% humidity, and with 5% CO₂, except for the hFOB 1.19 cells, which required 34 °C.
All of the tested compounds (1–7) were synthesized with the methods described previously [23 (link),24 ]. The stock solutions of these compounds were prepared in DMSO, whereas their working solutions at the required concentrations were made by dilution in the appropriate culture medium.
The solutions of DL-Thiorphan (Sigma-Aldrich) were prepared as described previously [9 (link)].

HCT 116 and CCD-18co were obtained from American Type Culture Collection (ATCC) (Rockville, MD USA). HCT 116 cell line (ATCC Number: CCL-247™) was cultured in McCoy 5A media (1x) (Sigma Aldrich, USA) whereas the normal human colon cell line, CCD-18co (ATCC Number: CRL-1790™) was cultured in EMEM (Eagle’s Minimum Essential Medium) (1x) (Sigma-Aldrich, USA). Culturing of HCT 116 and CCD-18co were carried out in a sterile laminar flow chamber to avoid any possible contamination. McCoy 5A and CCD-18co media were enriched with 10 % fetal bovine serum. All incubations in this study were done at a high humidity environment of 5 % carbon dioxide (CO₂) and at a temperature of 37 °C. The cultured cells were observed and checked daily by using an inversion microscope to see the morphology and cell growth, cultured up to 70-90 % confluence of cells. To subculture the cells, the old media was removed from the flask and phosphate buffer saline (PBS) was used to rinse the excess media. Solution of trypsin-EDTA (0.25 % trypsin/0.03 % EDTA) was added to remove the cells from the surface of the tissue culture flask. Media was added to inactivate the trypsin solution and centrifuged at 1000 rpm for 3 min. The cells were collected and transferred to a new labelled flask with a fresh media. Subculture of cells was done every 2 to 3 days for HCT 116 cell line and 4 to 5 days for CCD-18co cells.