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

Bin Shao¹, Heqi Bu¹, Ganglei Li¹

¹Department of Colorectal Surgery, Beilun District People's Hospital (Beilun Branch of The First Affiliated Hospital of Zhejiang University School of Medicine) Ningbo 315800, Zhejiang, The People's Republic of China.

American Journal of Cancer Research

|May 15, 2025

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.

Insights

Oridonin effectively combats colon cancer by halting cell growth and triggering programmed cell death (apoptosis) and autophagy. This natural compound activates the AMPK/mTOR pathway, leading to significant anti-tumor effects.

Area of Science:

Molecular Biology
Pharmacology
Oncology

Background:

Oridonin, a diterpenoid from Rabdosia species, shows anticancer potential but its role in colon cancer is unclear.
Understanding oridonin's molecular mechanisms is crucial for developing new colon cancer therapies.

Purpose of the Study:

To investigate the anti-tumor effects of oridonin on colon cancer cells.
To elucidate the molecular pathways, including cell cycle arrest, apoptosis, autophagy, and ROS generation, involved in oridonin's action.
To explore the role of the AMPK/mTOR signaling pathway in oridonin-induced anti-cancer effects.

Main Methods:

Cell proliferation assays using HCT8 and HCT116 colon cancer cell lines.
Flow cytometry to analyze cell cycle distribution and apoptosis.
Western blotting to detect key proteins involved in apoptosis, autophagy, and signaling pathways (caspase-3, PARP, Beclin 1, LC3, AMPK, mTOR, ULK1).
Reactive oxygen species (ROS) detection and manipulation using N-acetyl-L-cysteine (NAC).
In vivo studies using a colon cancer xenograft model.

Main Results:

Oridonin inhibited colon cancer cell proliferation by inducing G2/M phase arrest.
Oridonin promoted apoptosis via caspase-3 and PARP cleavage.
Oridonin activated autophagy, evidenced by increased Beclin 1 and LC3-II/LC3-I ratio.
Autophagy inhibition reduced oridonin-induced apoptosis, while apoptosis inhibition enhanced autophagy.
Oridonin-induced ROS accumulation contributed to apoptosis and was reversed by NAC.
Oridonin activated the AMPK/mTOR/ULK1 pathway, which was critical for its effects on autophagy and apoptosis.
In vivo, oridonin suppressed tumor growth and increased LC3-II and cleaved caspase-3 levels.

Conclusions:

Oridonin exhibits significant anti-colon cancer activity.
Oridonin induces cell cycle arrest, apoptosis, and autophagy through ROS-mediated activation of the AMPK/mTOR-ULK1 pathway.
These findings highlight oridonin as a potential therapeutic agent for colon cancer.

Read full analysis

Keywords:

Colon cancer Oridonin apoptosis autophagy reactive oxygen species (ROS)

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