AMPK/mTOR/ULK1 Axis-Mediated Pathway Participates in Apoptosis and Autophagy Induction by Oridonin in Colon Cancer DLD-1 Cells

Background: Oridonin has been demonstrated to exert strong antitumor activities in various types of human cancers. Our previous study established that oridonin induced the apoptosis of and exerted an inhibitory effect on colon cancer cells in vitro and in vivo. However, the mechanisms behind the antitumor effects of oridonin on colorectal cancer are not clearly known. This study explored whether autophagy was involved in antitumorigenesis effects caused by the usage of oridonin in colon cancer and examined whether the AMPK/mTOR/ULK1 signaling pathway was involved in this process.

Methods: Cell viability was determined using CCK-8 assay. The distribution of cell apoptosis was evaluated using flow cytometry. RT-PCR and Western blotting analysis were conducted to identify the key target genes and proteins involved in the AMPK/mTOR cascade. AMPK siRNA was used to disturb AMPK expression. A DLD-1 cell orthotopic transplantation tumor model was established to explore the anti-cancer effects in vivo.

Results: Oridonin exhibited a suppressive effect on DLD-1 cells in a concentration- and time-dependent manner. Additionally, in a dose-dependent manner, oridonin induced cell apoptosis via inducing the protein expression levels of cleaved caspase-3, cleaved PARP and stimulated autophagy by increasing protein expression levels of Becin1, LC3-II, decreasing protein expression levels of LC3-I, p62, which were respectively attenuated and elevated by autophagy inhibitor 3-MA. Furthermore, oridonin upregulated the expression level of p-AMPK and downregulated the expression levels of p-mTOR, p-ULK1 in the DLD-1 cells in a dose-dependent manner. Moreover, knockdown of AMPK by a specific siRNA reversed the expression levels of proteins involved in the AMPK/mTOR pathway, autophagy and apoptosis. In addition, outcomes from the in vivo experiments also showed that oridonin treatment significantly repressed tumorigenic growth of DLD-1 cells without any side effects, which was accompanied by the upregulation of p-AMPK, LC3-II, active caspase-3 protein expression levels and the downregulation of p-mTOR and p-ULK1 protein expression levels.

Conclusion: This study demonstrated that oridonin induced apoptosis and autophagy of colon cancer DLD-1 cells via regulating the AMPK/mTOR/ULK1 pathway, which indicated that oridonin may be used as a novel therapeutic intervention for patients with colorectal cancer.