Calycosin induces apoptosis in adenocarcinoma HT29 cells by inducing cytotoxic autophagy mediated by SIRT1/AMPK-induced inhibition of Akt/mTOR

Abstract

Autophagy promotes cell survival or induces apoptosis in cancer cells. While SIRT1 and AMPK induce autophagy in both normal and cancer cells, Akt and mTOR can inhibit it. Calycosin, a methoxyisoflavone, protects against several types of solid tumours including colorectal cancer. However, the mechanisms behind the antitumour effect of Calycosin remain largely unknown. This study investigates if autophagy mediates the anti-tumourigenesis effect afforded by Calycosin and examines if this effect involves activation of SIRT1 and/or AMPK. Human colorectal (HT29) carcinoma cells were cultured under normal conditions with Calycosin (50 μmol/L) in the presence or absence of chloroquine (10 μmol/L), EX-527 (100 nmol/L, SIRT1 inhibitor), or IGF-1 (100 ng/mL, Akt/mTOR activator) for 48 hours. Calycosin inhibited cell growth, proliferation and invasion and increased protein levels of Beclin-1 and LC3II, markers of autophagy. It significantly increased protein levels of cleaved caspase-3, Bax, and SIRT1, and activity of AMPK and reduced those of Bcl-2. These effects were parallel with concomitant reduction in protein levels p-src, integrin-β1 and Cyclin-D1 and activities of Akt and mTOR. Inhibition of autophagy by CQ reversed all these effects except cell invasion. Interestingly, co-incubating the cells with either EX-527 or IGF-1 completely prevented Calycosin-induced autophagy and all other associated effects and increased cell invasion. Also, blockade of SIRT-1 prevented the activation of AMPK, Akt, and mTOR, suggesting it to be an upstream regulator of these markers. In conclusion, Calycosin stimulates CRC cell apoptosis and inhibits their invasion by acting as SIRT1 activator which induces activation of AMPK-induced inhibition of Akt/mTOR axis.

Keywords: AMPK; mTOR; Calycosin; SIRT1; autophagy; colorectal cancer.