Abstract: Objective: To investigate the effects of propranolol on the subcutaneous tumorigenesis of esophageal squamous cell carcinoma (ESCC) cells and the proliferation, migration, cell cycle, apoptosis and autophagy of ESCC cells and its possible molecular mechanisms. Methods: The cell proliferation was detected by MTT (methyl thiazol tetrazolium) assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS (Phosphate buffer saline) group (without propranolol) and treated groups (40, 60, 80, 100 μmol/L propranolol) were set up with 5 wells in each group. After treatment for 0, 24, 48, 72 h, 10 μl (5 mg/ml) of MTT was added to each well, and the absorbance was measured at 490 nm. The cell migration was tested by Transwell assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured, and PBS group (without propranolol) and treated groups (40, 60 μmol/L) were set up with 2 wells in each group. Photos were taken 40 h later, and the experiment was repeated for three times before statistical analysis. The cell cycle and apoptosis were detected by flow cytometry assay: ESCC Eca109, KYSE-450 and TE-1 cells were routinely cultured. PBS group (without propranolol) and treated group (80 μmol/L) were set up, fixed, stained, and fluorescence at 488 nm was detected. The protein levels were detected by Western blot: ESCC Eca109 and KYSE-450 cells were routinely cultured. PBS group (without propranolol) and treated groups (60, 80 μmol/L) were set up followed by gel electrophoresis, wet membrane transfer, and ECL imaging. The experiment was repeated for three times and then analyzed statistically. Subcutaneous tumor formation experiment in nude mice: 10 nude mice were assigned PBS group (without propranolol) and treated group (with propranolol). Five mice in each group were inoculated with 5×10⁶ cells/100 μl (Eca109) into the right underarm. The treated group was given a gavage of 0.4 ml/kg (6 mg/kg) every other day, and the tumor size was measured every other day for 3 weeks. After 20 days, the nude mice were dislocated and sacrificed to take tumor tissue. Result: The results showed that propranolol inhibited the proliferation of Eca109, KYSE-450 and TE-1 cells with IC₅₀ of around 70 μmol/L for 48 h. Eca109, KYSE-450 and TE-1 cell migration was inhibited by propranolol in a dose-dependent manner (P＜0.05); Propranolol blocked the cell cycle of Eca109 in G2/M phase, blocked the cell cycle of KYSE-450 and TE-1 in G0/G1 phase, and promoted apoptosis of three kinds of cells (P＜0.05). The results of cell fluorescence showed that LC3 fluorescence intensity of TE-1 was increased after 12 h, 24 h and 36 h treatment with propranolol (P＜0.05). Western blot results showed that compared with PBS group, the protein expressions of p-mTOR, p-Akt and cyclin D1 were down-regulated, while cleaved caspase 9 level was up-regulated (P＜0.05). The results of subcutaneous tumor formation in nude mice showed that the tumor weight of PBS group was (0.91±0.05)g, and that of the experimental group was(0.65±0.12)g, the difference was statistically significant (P＜0.05). Conclusion: Propranolol inhibits the proliferation, migration and cell cycle,promotes apoptosis and autophagy of ESCC cells, and inhibits subcutaneous tumor growth in nude mice. The mechanism might be related to the inhibition of PI3K/AKT/mTOR signaling pathway.

Key words: propranolol, ESCC, cell culture, proliferation, migration, cycle, apoptosis, autophagy, nude mice