Chiliadenus iphionoides ethanolic extract suppresses hepatocellular carcinoma cell survival through ROS-associated mitochondrial dysfunction and intrinsic apoptotic signaling in p53-null Hep3B cells

Abstract

Background and aims Hepatocellular carcinoma (HCC) is a cause of cancer-related mortality, and therapeutic efficacy is constrained by treatment resistance and toxicity. This study evaluated the anticancer activity and underlying molecular mechanisms of the Chiliadenus iphionoides ethanolic extract in hepatocellular carcinoma cells. Methods Hep3B cells were treated with the C. iphionoides ethanolic extract (10–100 µg/mL) for 24 h. Cell viability, tumor-associated markers, intracellular reactive oxygen species (ROS), oxidative DNA damage, apoptosis, mitochondrial membrane potential, intrinsic apoptotic signaling, cell-cycle regulatory genes, and cell-cycle distribution were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), ELISA, RT-qPCR, fluorometric assays, and flow cytometry. ROS dependency was examined by N-acetyl-L-cysteine (NAC) pretreatment. Key findings were evaluated in HepG2 and Huh-7 HCC cells, and non-tumorigenic THLE-3 hepatocytes. Results C. iphionoides reduced Hep3B viability in a concentration-dependent manner (IC50 = 35.2 µg/mL; 95% CI, 31.85–38.47) and suppressed the expression of alpha-fetoprotein, GPC-3, and VEGF-A. Treatment-induced ROS accumulation was accompanied by increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) and γH2AX levels, consistent with oxidative DNA damage. Mitochondrial depolarization was associated with Bax activation, increased caspase-9 activity, enhanced cleavage of caspase-3 and PARP-1, reduced BCL-2 expression, and increased Annexin V-positive apoptotic cells, indicating the activation of the intrinsic mitochondrial apoptotic pathway. Cyclin D1 mRNA was downregulated, whereas p21 mRNA increased despite the TP53-null background of Hep3B cells. Concordantly, G0/G1-phase accumulation increased from 50.1 ± 0.9% to 76.5 ± 1.4%, while S-phase cells decreased from 31.5 ± 0.7% to 11.2 ± 0.9%. NAC pretreatment substantially attenuated ROS accumulation, oxidative DNA damage, mitochondrial dysfunction, and apoptotic signaling, supporting an upstream role for ROS. Comparable responses were observed in HepG2 and Huh-7 cells, whereas THLE-3 hepatocytes were less affected, indicating the preferential rather than absolute sensitivity of malignant hepatocytes. Conclusion C. iphionoides ethanolic extract exhibits anticancer activity in HCC cells in vitro through ROS-associated oxidative DNA damage, mitochondrial apoptosis, and p53-independent G0/G1 arrest.