In vivo anti-tumor efficacy studies

For tumorigenesis assessment, viable MDA-MB-231 cells (5 × 10⁶/100 μl PBS per mouse), as confirmed by trypan blue staining, were subcutaneously injected into the right flank of 7- to 8- week old female SCID mice. When the average tumor volume reached 100 mm³, mice were randomly divided into various treatment and control groups (4–6 mice per group). Body weights were recorded once every two days. After about two weeks, mice were sacrificed and the tumors were excised and stored at −80 °C until analyzed by western blotting. At the same time, some internal organs (liver, spleen, lung and kidney) were also collected for pathological study. The in vivo experiments were performed in accordance with relevant guidelines and regulations approved by the Committee on the Ethics of Animal Experiments of the Shenyang Pharmaceutical University.