Hesperetin inhibits Eca-109 cell proliferation and invasion by suppressing the PI3K/AKT signaling pathway and synergistically enhances the anti-tumor effect of 5-fluorouracil on esophageal cancer in vitro and in vivo

Previously, we reported that hesperetin exhibited pro-apoptotic activity against esophageal cancer in vitro and in vivo. Here, we examined whether hesperetin inhibits cell proliferation and invasion and synergistically enhances the anti-tumor effect of 5-fluorouracil (5-FU) in esophageal cancer. Flow cytometry, EdU staining, and transwell invasion assays using Eca-109 cells showed that hesperetin induced cell cycle arrest at the G0/G1 phase and inhibited cell proliferation and invasion significantly. Moreover, hesperetin suppressed the expression of phosphorylated PI3K/AKT, cyclin D1, MMP-2, and MMP-9 and increased phosphorylated PTEN and p21 in Eca-109 cells. Hesperetin combined with 5-FU inhibited cell growth more effectively than did either drug alone in Eca-109 cells and in a xenograft mouse model. Hoechst 33258, Annexin V-PE/7-ADD double staining and TUNEL assay showed more apoptotic cells in the combination treatment group than in either individual treatment group. The combination down-regulated protein levels of Bcl-2 and up-regulated those of Bax, cleaved caspase-3, and cleaved caspase-9 more effectively than did either drug alone. These data suggest that hesperetin inhibited esophageal cancer cell proliferation and invasion by suppressing the PI3K/AKT signaling pathway. In conclusion, 5-FU and hesperetin exerted synergistic antitumor effects in vivo and in vitro and could constitute a novel therapeutic approach for esophageal cancer.