4-Methoxydalbergione Induces Dual Activation of Apoptosis and Autophagy-Dependent Cell Death via ROS–MAPK Signaling in Human Neuroblastoma Cells

TL;DR

4-Methoxydalbergione kills neuroblastoma cancer cells by triggering both apoptosis and autophagy through ROS and MAPK signaling, with minimal harm to healthy neurons.

Contribution

4-MD induces dual cell death mechanisms in neuroblastoma via ROS–MAPK and AMPK/mTOR/ULK1 signaling, offering a novel therapeutic strategy.

Findings

4-MD reduces neuroblastoma cell viability and activates apoptosis through caspase-3 and MAPK.

4-MD induces autophagy via AMPK/mTOR/ULK1 signaling and autophagosome accumulation.

ROS mediates both apoptotic and autophagic effects, and autosis is implicated as an additional mechanism.

Abstract

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.

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