A Novel 3‐Benzyloxychromone From Celastrus orbiculatus Thunb. Exhibits Anticancer Effects on Non–Small Cell Lung Cancer Cells via GSK‐3β–Dependent c‐Jun/ATF2 Pathway

ABSTRACT Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3‐oxygen–substituted isoflavone analogue (3‐benzyloxychromone, 3‐Boc) from the herb. Initially, we established cell line–derived xenograft mouse model using H1299 non–small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3‐Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3‐Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V‐FITC/PI apoptosis assays demonstrated that 3‐Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure‐based target prediction and MD simulation suggested that 3‐Boc potentially suppressed the activity of glycogen synthase kinase‐3β (GSK‐3β) by interacting with the ATP–binding site. Western blot analysis indicated that 3‐Boc triggered the phosphorylation of Serine 21 of GSK‐3α or Serine 9 of GSK‐3β in a time‐ and dose‐dependent manner. To investigate the dependency of GSK‐3β, we established GSK‐3β knockout in H1299 cells. Depletion of GSK‐3β enhanced 3‐Boc–induced cytotoxicity compared with wild‐type counterparts through activated c‐Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3‐oxygen–substituted chromone from the herb, which may have important implications for screening promising modulators of GSK‐3β and related signaling pathways in the treatment of cancer.