Macranthoside B Suppresses the Growth of Adenocarcinoma of Esophagogastric Junction by Regulating Iron Homeostasis and Ferroptosis through NRF2 Inhibition

Background: Macranthoside B (MB) is a saponin compound extracted from hon-eysuckle that has been reported to exhibit significant medicinal values, particularly anti-tumor activities. This study aimed to evaluate the anticancer efficacy of MB in treating adenocarci-noma of the esophagogastric junction (AEG) and elucidate its underlying mechanisms. Methods: Three AEG cell lines and normal gastric epithelial cells were used to assess the an-ticancer activity of MB in vitro. A series of experiments, including RNA sequencing (RNA-seq) analysis, transmission electron microscopy (TEM), immunofluorescence, and western blot assay, were conducted to validate the molecular mechanisms by which MB may mediate these physiological changes. Finally, we used shRNA assays to silence the key gene driving these changes and examined the expression of molecules involved in the affected pathways. Results: MB exhibited significant anti-AEG cell activity with IC50 values ranging from 9.5 to 12.7 μM. RNA-seq results indicated that MB treatment in AEG cells significantly altered mRNA levels of autophagy- and ferroptosis-related genes. Further experiments revealed that MB treatment led to the up-regulation of lipid reactive oxygen species (Lip-ROS), oxidative stress-related pathway genes, and LC3B-labeled autophagic vesicles in AEG cells. Moreover, MB mediated NCOA4-dependent ferritinophagy, disrupting iron homeostasis and causing subsequent ferroptosis. We further confirmed that the intrinsic connection between autophagy and ferroptosis was due to the inhibition of NRF2 by MB. The inhibition of NRF2 by MB triggered transcriptional repression of its downstream effector molecules HERC2 and VAMP8, thus stabilizing NCOA4. Conclusion: This study demonstrated MB to inhibit AEG cell growth by regulating iron ho-meostasis and inducing ferroptosis through the inhibition of NRF2, providing a basis for the development of novel drugs for AEG treatment