**Background:** Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality with limited treatment options. CPUL1, a synthetic phenazine derivative, has shown antitumor activity against HCC, but its mechanisms were poorly understood. This study aimed to comprehensively characterize CPUL1's anti-HCC effects and elucidate underlying molecular mechanisms using integrated omics approaches.
**Methods:** Three HCC cell lines (HUH-7, HepG2, BEL-7402) were treated with CPUL1 (0–14 μM) for 48 h. Cell viability was assessed by CCK-8 assay, and colony formation was evaluated over 14 days. Subcellular localization of CPUL1 was determined by LC-MS in BEL-7402 cells at 6, 24, and 48 h. In vivo antitumor activity was tested in a BEL-7402 xenograft nude mouse model (n ≥ 6 per group) with CPUL1 at 10, 20, and 40 mg/kg (i.v.), compared to sorafenib (20 mg/kg, i.g.) and cyclophosphamide (20 mg/kg, i.g.). RNA-seq transcriptomics and LC-QTOF/MS untargeted metabolomics were performed on BEL-7402 cells treated with 8 μM CPUL1 for 6 h and 48 h. Autophagy was assessed by Western blotting (LC3, p62, ATG5, ATG12, Beclin1, mTOR, AMPK, LAMP1, LAMP2, RAb7), transmission electron microscopy, immunofluorescence, and Lyso-Tracker Red staining. Co-treatment experiments with chloroquine (CQ, 20 μM) and 3-methyladenine (3-MA, 2 mM) were conducted.
**Key Results:** CPUL1 inhibited HCC cell proliferation with IC50 values of 4.39 μM (HUH-7), 7.55 μM (HepG2), and 6.86 μM (BEL-7402). Colony formation was nearly abolished at 5 μM (p < 0.001). CPUL1 accumulated predominantly in cytoplasm and mitochondria, with progressive nuclear and mitochondrial accumulation over time. In vivo, CPUL1 at 40 mg/kg significantly reduced tumor volume and weight, comparable or superior to sorafenib and cyclophosphamide (20 mg/kg), without significant body weight changes. Transcriptomics identified 989 DEGs at 6 h (598 down, 391 up) and 1207 DEGs at 48 h (874 down, 333 up). KEGG enrichment highlighted 'valine, leucine, and isoleucine biosynthesis', 'MAPK signaling', 'PI3K-Akt signaling', 'mitophagy-animal', and 'autophagy-animal' pathways. Metabolomics identified 229 DAMs at 6 h (59 up, 170 down) and 222 DAMs at 48 h (83 up, 139 down). Key metabolites showed severe depletion: glucose 6-phosphate (FC ≈ 0.11), phosphoenolpyruvate (FC ≈ 0.15), ATP (FC ≈ 0.34), glutathione (GSH declined, GSSG FC ≈ 2.67 at 48 h). Unsaturated fatty acids accumulated (oleic acid FC ≈ 7.35, linoleic acid FC ≈ 9.26 at 48 h). CPUL1 increased LC3-II conversion and p62 accumulation in a time-dependent manner across all three cell lines, indicating autophagic flux blockade. TEM showed autophagosome accumulation. Co-treatment with CQ potentiated LC3-II and p62 accumulation, while 3-MA showed partial additive effects. CPUL1 reduced LC3-LAMP1 colocalization, decreased Lyso-Tracker Red fluorescence (lysosomal alkalization), and downregulated LAMP1, LAMP2, and RAb7 protein expression in a time-dependent manner.
**Clinical Implications:** CPUL1 demonstrates potent anti-HCC activity through a dual mechanism: direct cytotoxicity (via mitochondrial dysfunction and ROS stress, as previously reported) and metabolic disruption exacerbated by autophagy inhibition. The compound blocks autophagic flux at the late stage by impairing lysosomal function and autophagosome-lysosome fusion, similar to chloroquine. This autophagy blockade likely sensitizes HCC cells to metabolic stress and nutrient deprivation, enhancing the compound's therapeutic efficacy. CPUL1's multifaceted mechanism—combining direct cytotoxicity with autophagy suppression—positions it as a promising lead compound for HCC therapy, potentially overcoming resistance mechanisms associated with cytoprotective autophagy. Further studies are warranted to evaluate its clinical potential and combination strategies.