Ginsenoside Rh4 Induces Apoptosis Through ROS-Mediated Fas Signaling in Human Nasopharyngeal Carcinoma NPC/HK1 Cells

<h3>Background/Aim:</h3> Nasopharyngeal carcinoma (NPC) is a highly metastatic malignancy with a distinct geographic distribution and an urgent need for more effective therapies. Ginsenoside Rh4 (Rh4), a bioactive ginseng-derived compound, has shown antitumor activity in several cancers; however, its effects on NPC remain unclear. This study investigated the anticancer effects of Rh4 in NPC cells and the underlying mechanisms <i>in vitro</i>. <h3>Materials and Methods:</h3> Human NPC/HK1 cells and non-tumorigenic NP69 nasopharyngeal epithelial cells were used. Cell viability, colony formation, cytokeratin-18 fragment release, protein expression, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) production were assessed using MTT, colony formation, ELISA, immunoblotting, and MMP and ROS assays, respectively. <h3>Results:</h3> Rh4 significantly reduced NPC/HK1 cell viability at 2, 4, and 8 μg/ml (IC₅₀=3.02 μg/ml) and suppressed colony formation, while showing no significant cytotoxicity toward NP69 cells under the experimental conditions used in this study. Moreover, Rh4 increased cytokeratin-18 fragment release and cleaved PARP expression without significantly altering Beclin1 or LC3-II expression, indicating apoptosis rather than autophagy. Rh4 also upregulated Fas, cleaved caspase-8, 3, and 7, whereas cleaved caspase-9 remained unchanged. Consistently, ZB4, a Fas-neutralizing antibody, attenuated Rh4-induced cytokeratin-18 fragment release. Notably, Rh4 did not significantly affect MMP depolarization or Bax expression, suggesting selective activation of the extrinsic rather than intrinsic apoptotic pathway. Furthermore, Rh4 increased ROS production, and ROS scavenging reduced ROS accumulation, apoptosis, and Fas/caspase activation. <h3>Conclusion:</h3> Rh4 induces apoptosis in NPC/HK1 cells through ROS-associated Fas signaling and may serve as a promising therapeutic candidate for NPC.