4-Methoxydalbergione Induces Dual Activation of Apoptosis and Autophagy-Dependent Cell Death via ROS-MAPK Signaling...

Neuroblastoma, the predominant extracranial solid malignancy in the pediatric population, remains a major clinical challenge due to pronounced intratumoral heterogeneity and intrinsic therapeutic resistance. 4-Methoxydalbergione (4-MD), a benzoquinone derivative isolated from Dalbergia odorifera, has demonstrated anticancer activity in several tumor models; however, its effects and underlying cell death mechanisms in neuroblastoma remain unclear. Here, we investigated the cytotoxic effects of 4-MD in human neuroblastoma cells using cell viability assays, flow cytometry, immunoblotting, and fluorescence microscopy. 4-MD reduced cell viability in a dose- and time-dependent manner and induced caspase-3 cleavage accompanied by MAPK activation, indicating apoptotic cell death. Concurrently, 4-MD promoted autophagosome accumulation, as evidenced by LC3-II accumulation, acidic vesicular organelle formation, ATG5 upregulation, and p62 degradation, in association with activation of the AMPK/mTOR/ULK1 signaling axis. Pharmacological inhibition of autophagy significantly attenuated 4-MD-induced cytotoxicity without affecting caspase-3 activation, demonstrating a caspase-independent, pro-death role of autophagy. Reactive oxygen species (ROS) acted as a critical upstream mediator, as antioxidant treatment suppressed both apoptotic and autophagic signaling. Moreover, inhibition of Na⁺,K⁺-ATPase with ouabain selectively reduced autophagy-dependent cell death, implicating autosis as an additional mechanism. Notably, 4-MD exhibited minimal toxicity toward primary cortical neurons. Collectively, these findings demonstrate that 4-MD engages multiple, non-redundant cell death pathways through coordinated ROS-MAPK-AMPK/mTOR/ULK1 signaling, highlighting its potential to overcome therapeutic resistance in heterogeneous neuroblastoma cells.