Sn 38

HT-29, T84, HRT-18, SW480 and SW620 human CRC cells and the additional cell lines mentioned were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, without antibiotics) supplemented with 5 % (HT-29, T84, and HRT-18) or 10 % (SW480 and SW620) ^v/_v heat-inactivated newborn calf serum (NCS) and maintained as stocks in 75-cm² flasks at 37 °C in a humidified atmosphere of 90 % air/10 % CO₂. Cells for use in binding assays or for measurements of DPPIV enzyme activity were seeded into 48-well plates at 50,000 cells/well and allowed to adapt to culture for 48 h. The cells were then cultured in medium containing 1 % NCS for a further 48 h. For flow cytometry, cells were seeded into 6-well plates and allowed to adapt for 48 h prior to treatment. Cells for migration assays were cultured and treated in 10-cm dishes. Anticancer agents used were: 5-fluorouracil (5-FU), irinotecan (IT), cisplatin (Cis), vinblastine (Vin), and methotrexate (MTX) from Mayne Pharma Canada; oxaliplatin (Ox) from Sanofi Canada; and SN-38 from Toronto Research Chemicals. Where single drug concentrations are used, these were defined as optimal (typically, just maximal) based upon the response of the cells at that passage level and the lot(s) of drug as obtained from the supplier(s).

A reference standard, irinotecan hydrochloride, was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). CPT (purity ≥ 98%), which was used as the ISTD, was purchased from Shanghai Yuanye Biological Technology Co. Ltd. (Shanghai, China). SN-38 and SN-38 glucuronide (purity ≥ 98%) were purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Irinotecan hydrochloride for injection was obtained from Hengrui Medicine Co. Ltd. (Jiangsu, China). Sterilized saline for injection was purchased from Shijiazhuang No. 4 Pharmaceutical Co. Ltd. (Hebei, China). D-saccharic acid 1, 4-lactone, alamethicin, and uridine 5′-diphosphoglucuronic acid (UDPGA) triammonium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA).
HPLC-grade acetonitrile and methanol were obtained from Honeywell Burdick & Jackson Company (Mexico City, Mexico). Formic acid (HPLC grade) was purchased from ROE Scientific Inc. (Beijing, China). Deionized water for the HPLC analysis was prepared using a Milli-Q water purification system (Milford, MA, USA). All of the other reagents were of analytical grade.