A. Nude female mice bearing MTT-Luc tumors were treated with 24 mg/kg/day PL (n=9) for 28 days. The control group was treated with vehicle (n=10). Tumor growth was assessed once a week. The graph shows a significant inhibition of tumor growth at all time points of treatment in the treated group (green) compared to control (red) animals. The graph represents data from 9 (treated) and 10 (vehicle) mice assessed at specific time points as mean +/− SEM. Statistical analysis was performed by Mann Whitney, U-test, at each time point. B. Representative tumors from control mice compared to the tumors from animals treated with PL. Scale bar: 1cm. C. Lungs were resected and subjected to bioluminescence imaging. The presence of lung metastases in treated mice was 46% lower than in control group. D. mRNA expression levels of Pou5f1, Twist1, Vegfa, Mmp9, and Nanog in tumors from both treated and control groups were assessed by quantitative real-time PCR. The target gene transcript levels were normalized to Actb. The box and whiskers graphs represent data from control (n=10) and treated (n=9) groups. *P<0.05; ** P<0.01; ***P<0.001, Mann Whitney, U-test. PL: piperlongumine; Mmp9: matrix metallopeptidase 9; Pou5f1: POU domain, class 5, transcription factor 1.