# 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.

## Key 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.