2.8. Tumor Establishment and In Vivo Treatments

SMMC-7721 cells were trypsinized and resuspended with PBS. 1×10⁷ cells were injected subcutaneously into each male BALB/c nude mice (6-8 weeks old, 20 ± 2 g, Shanghai SLAC Laboratory Animal Co. Ltd., Shanghai, China) to produce implanted tumors. Then the mice were randomly divided into three groups: Control, 50 μg/kg·d melittin, and 100 μg/kg·d melittin groups. Melittin (50 or 100 μg/kg·d) was intravenously injected via tail vein daily. The same volume of saline was used for the control mice. Then, the volumes of tumors were measured with a slide caliper and were evaluated by the following equation: a (the larger diameter) × b (the smaller diameter)²/2. After 11 days of treatment, mice were anaesthetized with overdose of Pentobarbital sodium (1%) through intraperitoneal injection and were sacrificed. All procedures were performed in accordance with the Helsinki Declaration. The experiment was approved by the Ethics Committee of Second Military Medical University.