Xenograft model

Five-week-old male Balb/c nudemice were obtained from SLC (SLC; Tokyo, Japan) Five mice made up the control group while five other mice made up the experimental and delivery groups for each concentration. For tumor induction, HCT116 human colon cancer cells (2.5 × 105 cells/0.1 ml) were subcutaneously injected into the left flank of the mice (each group had 10 animals). One week after the injection of cells, the mice were co-treated with AAE20, 40 mg/kg/day and 0.2cm³ PBS/DMSO for 21 days. Tumor size was measured by taking two perpendicular diameter measurements, using a caliper, every 2 days. The tumor volume was calculated using the following formula: V = 1/2 (length x width). The body weight of each animal was measured at a set time, once per week. After the 3-week treatment, the tumor was removed and frozen in liquid nitrogen for western blot analysis or fixed with formalin for immunohistochemistry, TUNEL and H&E staining. All of the animal experiments were approved by the Ethics Committee for Animal Experimentation of Hannam University (Daejeon, Korea, HNU 2016–9).