Huachansu triggers mitochondrial apoptosis and ER stress to inhibit hepatocellular carcinoma progression

Huachansu, derived from the dried skin glands of the Chinese toad (Bufo bufo gargarizans), has been used in traditional Chinese medicine for its anti-tumor properties, particularly in hepatocellular carcinoma (HCC). However, its molecular mechanisms remain unclear. This study investigates Huachansu's therapeutic potential by focusing on mitochondrial apoptosis and endoplasmic reticulum (ER) stress pathways. In vitro studies on HepG2 cells revealed that Huachansu (48-96 mg/mL for 24 h) disrupted mitochondrial function by elevating reactive oxygen species (ROS) generation (~ 83% and ~ 208% increase at 48 and 96 mg/mL, respectively) and reducing membrane potential. Molecular analyses showed upregulated pro-apoptotic Bax and ER stress effector CHOP alongside downregulated anti-apoptotic Bcl-2 and caspase family proteins, indicating PERK-ATF4 pathway activation. In vivo investigations using orthotopic and xenograft HCC mouse models demonstrated significant tumor volume (~ 25% at 2 g/kg and ~ 39% at 4 g/kg) and weight reduction with Huachansu treatment (2-4 g/kg/day, oral gavage for 18-21 days). Hepatic function improvements were evidenced by decreased serum ALT, AST, and LDH levels alongside increased superoxide dismutase (SOD) activity. Histopathological analyses confirmed reduced tumor progression and organ toxicity, while inflammatory cytokine profiling revealed diminished pro-inflammatory mediators. Mechanistically, Huachansu activated ER stress markers (PERK, ATF4) in tumor tissues without inducing systemic toxicity. These findings demonstrate that Huachansu exerts potent anticancer effects in HCC through concurrent induction of mitochondrial damage and ER stress activation. This study provides a mechanistic basis for Huachansu's traditional use and highlights its potential as a promising therapeutic agent for HCC treatment.