BALB/c nude mice (6-week-old) were purchased from Junke biology Co., Ltd (Nanjing, China). All mice were individually housed and had free access to food and water. Then, 58?F and CNE1 cells (5x10⁶ cells in 200 l medium) were inoculated into right flank of mice. Mice were divided into 2 groups: sham group and 10 mg/kg lupeol group (5 mice per group). Specifically, mice of the 10 mg/kg lupeol group were intraperitoneally administrated with lupeol (10 mg/kg, every 3 days) referring to previous studies [Citation23,Citation24]. Mice in the sham group received an equivalent volume of PBS. At day 40 after lupeol treatment, mice were euthanized by cervical dislocation after anesthetizing with 3% isoflurane. The tumor volume and weight were recorded, respectively. Animal experiments were conducted following the approval of the Ethics Committee of Shenzhen Peoples Hospital (No. SYXK(YUE)2017-0174).

The animal experiment was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University. BALB/c nude mice (5 weeks old, female, Guangdong Medical Laboratory Animal Centre, China) were used for animal experiments. Approximately 4 million CNE1 cells were injected subcutaneously into the right flank of each mouse. Palpable solid tumours developed within a month after tumour cell inoculation, and mice were randomly allocated to four different groups (five mice /group) as follows: control (PBS) group, CuB treatment groups [0.5 mg/kg (low-dose) group and 1 mg/kg (high-dose) group] and gemcitabine (GEM, 25 mg/kg) group. Mice received intraperitoneal injections of PBS, CuB, and gemcitabine 3 times weekly.

6-8 week old male balb/cnude micewere purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Nasopharyngeal carcinoma CNE2 cells were collected during logarithmic growth and rinsed with PBS twice and resuspended to a density of 1 x 10⁷ cells/ml using fresh and cooled DMEM/F12 medium (free of FBS and antibiotics). Each mouse was inoculated subcutaneously with a 0.1 ml cell suspension on the right-side flank. Tumor-bearing mice were used for in vivo anticancer studies 8 days after inoculation when the average tumor volume reached ~200 mm³.