In vivo anticancer assay (xenograft nude mice model)

The 6‐week‐old male nude mice (BALB/cAnN.Cg‐Foxn1nu/CrlNarl) were purchased from the National Laboratory Animal Center. Mice were maintained in the animal facility in the Department of Life Science, National Dong Hwa University, Hualien, Taiwan. OC2, OCSL and SAS cells (2 × 10⁶ cells/mice) were separately injected subcutaneously (s.c.) into the right flank of the nude mice, but only the SAS xenograft nude mice model was successfully established. The experiment protocol was conducted as follows: Six‐week‐old male nude mice were randomized into three groups (three mice/group in each experiment); 2 × 10⁶ cells of SAS were injected s.c. at day 0. After 10 days post‐injection, and the tumour was growth at less 1 mm³, mice were treated with DMSO or honokiol. Control group mice were orally fed with DMSO, and the experimental group mice were orally fed with honokiol 5 mg/kg or 15 mg/kg at days 1, 4, 7, 10, 13, 16, 19 and 22, Tumour size was measured at above days. Tumour volume was estimated by the equation (L × S ²/2, L as the longest diameter, S as the shortest diameter). Closing the experiment on day 35, the nude mice were killed, and the tumour was harvested and weighed. Two independent experiments were conducted. The experimental protocol was complied with Taiwan's Animal Protection Act and was approved by the Laboratory Animal Care and Use Committee of the National Chiayi University (IACUC Approval No. 104016).