Ziyu II induces autophagy via inhibiting Akt/mTOR pathway
Akt/mTOR is an important signaling pathway for cell survival, which is closely associated with cancer progression and development [29]. It has been previously reported that Ziyu II treatment decreased the phosphorylation level of Akt in human umbilical vein endothelial cells [30]. Therefore, we aimed to test whether Akt/mTOR signaling was involved in Ziyu II-induced autophagy in CRC cells. As shown in Fig. 5A, Ziyu II treatment significantly inhibited Akt/mTOR signaling pathway, as evidenced by decreased phosphorylation levels of Akt, mTOR, p70S6K, and 4E-BP1. To further confirm these results, we transfected a constitutively active form of Akt to rescue Ziyu II-induced Akt/mTOR inhibition. As expected, Akt reactivation markedly reduced Ziyu II-induced LC3-II turnover and LC3 puncta accumulation (Fig. 5B-G), suggesting an important role of the Akt/mTOR signaling pathway in Ziyu II-induced autophagy. Moreover, most of Ziyu II-treated xenografts displayed reduced p-Akt staining (Fig. 5H and I). Taken together, these findings suggest that Ziyu II induces autophagy by inhibiting Akt/mTOR signaling pathway in CRC cells.