BACKGROUND: Non-small cell lung cancer (NSCLC) remains a major cause of cancer-related mortality worldwide. While mitophagy, a process that selectively removes damaged mitochondria, typically promotes cell survival, its excessive activation can trigger apoptosis, offering a novel therapeutic avenue. In this study, we report for the first time the identification of prosapogenin CP4 (PCP4), a natural compound isolated from Anemone rivularis, which exerts its anticancer effects by exacerbating mitophagy-mediated apoptosis in A549 cells through dual modulation of the AMPK-mTOR and PINK1/Parkin pathways.
METHODS: PCP4 was purified using activity-guided fractionation and structurally characterized via LC-MS and NMR. Antiproliferative activity was assessed through MTT and LDH assays, while migration and invasion were evaluated using wound healing and transwell assays. Mitochondrial integrity was assessed using confocal microscopy, 10-NAO, TMRM, and MitoSOX staining. Western blotting, GFP-LC3 puncta, tandem fluorescence-tagged LC3 (tf-LC3), and Mito-QC assays were employed to monitor mitophagy and autophagic flux. Apoptosis was analyzed via Annexin V-FITC/PI staining and caspase-3 activation. Antitumor efficacy was validated in vivo using an A549 xenograft model.
RESULTS: PCP4 significantly inhibited proliferation, migration, and invasion of A549 and H1299 cells, while inducing mitochondrial fragmentation, depolarization, and oxidative stress. GO/KEGG analyses highlighted enrichment of PI3K-AKT, AMPK, and PINK1/Parkin pathways. Mechanistically, PCP4 enhanced autophagic flux and mitophagy via AMPK-mTOR and PINK1/Parkin signaling; blocking mitophagy partially abrogated its pro-apoptotic effects. Molecular docking confirmed the strong binding affinity of PCP4 to AMPK, PIK3R1, and PINK1. In vivo, PCP4 suppressed tumor growth without systemic toxicity and promoted mitophagy-associated apoptosis in tumor tissues.
CONCLUSION: This study introduces PCP4 as a first-in-class mitophagy-exacerbating natural product that targets both the AMPK-mTOR and PINK1/Parkin axes to induce apoptosis in A549 cells. Its dual-pathway action, potent anticancer efficacy, and low toxicity profile underscore its therapeutic potential and mechanistic novelty compared to existing autophagy modulators.
