Involvement of Mitochondrial Damage and Oxidative Stress in Apoptosis Induced by Betulin Plus Arsenic Trioxide in Neuroblastoma Cells

Abstract

Background/aim: Arsenic trioxide (As₂O₃), a potent toxin in traditional Chinese medicine, has been utilized as an anticancer agent in Chinese culture for over a millennium. Betulin, commonly extracted from the bark of birch trees, has been identified for its pharmacological properties, including antibacterial, anti-inflammatory, antitumor, and antiviral activities. The aim of this study was to determine the efficacy and underlying anticancer signaling cascade induced by As₂O₃ and betulin in neuroblastoma cells.

Materials and methods: SK-N-SH cells were treated with As₂O₃ with or without betulin. Cell viability and apoptotic signaling were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, measurement of mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS), and quantitative western blotting analysis. Student's t-test in addition to one- or two-way analysis of variance was used to examine significant differences between comparison groups.

Results: The combined treatment of As₂O₃ plus betulin was more effective than single treatments in suppressing cell viability and induction of apoptosis, which correlated well with elevated ROS levels. The apoptotic signaling cascade of As₂O₃ plus betulin was revealed as ROS elevation and relative loss of MMP, leading to the cleavage of caspase-3 and -9. As₂O₃ plus betulin treatment also reduced the expression of BCL2 apoptosis regulator, BH3-interacting domain death agonist, and BCL2-like-1.

Conclusion: The novel combination of As₂O₃ plus betulin has the potential to serve as a practical anti-neuroblastoma drug.

Keywords: Arsenic trioxide; betulin; cytotoxicity; neuroblastoma.