Clinacanthus nutans Induced Reactive Oxygen Species‑dependent Apoptosis and Autophagy in HCT116 Human Colorectal Cancer Cells

Objective: Our study aims to investigate the apoptosis‑ and autophagy‑inducing effects of C. nutans in HCT116 human colorectal cancer cells. Materials and Methods: Cytotoxicity of ethanol extract, hexane, ethyl acetate, and aqueous fractions of C. nutans against various cancer cell lines was determined via MTT assay. Apoptosis assays including annexin V, mito‑ID, and Hoechst 33342/propidium iodide staining were carried out. The level of intracellular reactive oxidative species was determined using flow cytometry. Western blot analysis was carried out to assess the protein expression in C. nutans ethyl acetate fraction (CNEAF)‑treated HCT116 cells. Results: CNEAF was found to exert the strongest cytotoxic effect against HCT116 cells (IC50 = 48.81 ± 1.44 µg/mL). CNEAF‑induced apoptosis was evidenced by nuclear morphological alterations, phosphatidylserine externalization, dissipation of mitochondrial membrane potential, and elevation of intracellular reactive oxygen species (ROS) level. Dissipation of mitochondrial membrane potential was attributed to the upregulation of Bax and Bak accompanied by downregulation of Bcl‑2 and Bcl‑xL, leading to caspase‑3, ‑9, ‑8, and ‑10 activation. Interestingly, an upregulation of death receptor 5 was detected, suggesting involvement of intrinsic and extrinsic pathways. In addition, the occurrence of autophagy by CNEAF was supported by LC‑3 accumulation and p62 degradation. The reduction of intracellular ROS level by N‑acetylcysteine showed that the apoptosis and autophagy induced by CNEAF is ROS dependent. Conclusions: CNEAF induced ROS‑dependent apoptosis and autophagy on HCT116 cells.