Role of AMPK-mTOR signaling in anticancer effects of silibinin on esophageal squamous cell carcinoma

Abstract

Esophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of cancer-related death. The prognosis for advanced esophageal cancer is very poor due to the limitations of surgery and emergence of chemoresistance. Identification of non-toxic, naturally occurring anticancer agents which may be used in combination with conventional chemotherapeutic drugs may help improve patient survival. Silibinin, a flavonoid isolated from the milk thistle, Silybum marianum, had been shown to have anticancer effects on several types of cancer cells. However, its effect on esophageal cancer has not been reported. Furthermore, although silibinin is known to downregulate the expression of mammalian target of rapamycin (mTOR) in cancer cells, little is known about the upstream signaling events targeted by silibinin. In this study, we investigated the effects of silibinin, alone and in combination with chemotherapy drugs, on human esophageal squamous carcinoma (ESCC) cells in vitro and on ESCC tumor xenografts in nude mice, and explored novel signaling pathways targeted by silibinin. We found that silibinin inhibited cell proliferation, colony-formation, and anchorage-independent growth of ESCC cells. The effects were attributed to silibinin-induced G2/M phase cell cycle arrest and apoptosis. Wound healing assay and invasion chamber assay showed that silibinin had suppressive effect on the migration and invasion ability of esophageal cancer cells. Western blot analysis showed that silibinin increased the expression of phosphorylated AMP-activated protein kinase (p-AMPK) and decreased the phosphorylation of its downstream targets, mTOR and acetyl-CoA-carboxylase (ACC). The anti-proliferation and anti-invasion effects of silibinin on ESCC cells were abolished by treatment with the AMPK inhibitor, compound C. In addition, silibinin increased the sensitivity of esophageal cancer cells to the traditional chemotherapeutic drugs 5-fluorouracil and cisplatin in vitro. In vivo, orally administered silibinin significantly inhibited the growth of ESCC tumor xenografts in immunodeficient mice, and exerted synergistic effects with chemotherapy drugs. In summary, our data demonstrate that silibinin has suppressive effects on ESCC cells, and that these effects may be mediated through the AMPK/mTOR pathway. Silibinin may be a potentially useful drug in the treatment of esophageal cancer.