Autophagy-dependent apoptosis induction by oridonin are mediated by ROS-dependent AMPK-mTOR-ULK1 pathway in colon cancer.

Abstract

Oridonin, a bioactive diterpenoid isolated from Rabdosia species, exhibits broad-spectrum anticancer activity across various tumor types. However, its impact on colon cancer and the underlying molecular mechanisms remains poorly understood. Our study revealed that oridonin significantly suppressed the proliferation of HCT8 and HCT116 colon cancer cells by inducing G2/M phase cell cycle arrest. Moreover, oridonin triggered apoptotic cell death, as indicated by elevated levels of cleaved caspase-3 and PARP. Simultaneously, it activated autophagy, as evidenced by increased expression of Beclin 1 and LC3-II, along with decreased LC3-I and p62 levels. In addition, inhibiting autophagy with 3-methyladenine (3-MA) reduced cell apoptosis, whereas blocking apoptosis using Z-Val-Ala-Asp(OMe)-FMK (Z-VAD-FMK) enhanced autophagy. Furthermore, oridonin also induced the accumulation of reactive oxygen species (ROS), which contributed to apoptosis; this effect was largely reversed by the ROS scavenger N-acetyl-L-cysteine (NAC). Mechanistically, oridonin increased phosphorylation of AMP-activated protein kinase (AMPK) and suppressed phosphorylation of mammalian target of rapamycin (mTOR) and Unc-51-like kinase 1 (ULK1). Silencing AMPK with siRNA blocked oridonin's effects on the AMPK/mTOR pathway, as well as its regulation of autophagy and apoptosis. Moreover, co-treatment with NAC almost completely blocked activation of the AMPK-mTOR-ULK1 signaling pathway. In vivo, oridonin significantly suppressed tumor growth in a xenograft model, accompanied by elevated expression of LC3-II and cleaved caspase-3. Collectively, these findings demonstrated that oridonin could exert potent anti-tumor effects in colon cancer by inducing cell cycle arrest and promoting autophagy-dependent apoptosis via ROS-mediated activation of the AMPK-mTOR-ULK1 signaling pathway.