Mulberrofuran G Induces Apoptosis and Cell Cycle Arrest Through Suppression of the JAK2/STAT3 Pathway by Direct JAK2 Inhibition and Reactive Oxygen Species Induction in the NCI-H460 Cell Line.

BACKGROUND: Large cell lung carcinoma (LCLC) is an aggressive non-small cell lung cancer subtype. Mulberrofuran G (MG) from Morus alba L. possesses pharmacological properties, but its anticancer mechanisms in LCLC remain unexplored. This study aimed to validate the antitumor activity of MG against NCI-H460 cells and elucidate its dual mechanism involving Janus kinase 2 (JAK2) inhibition and reactive oxygen species (ROS) induction. METHODS: This study was conducted at the Nanjing University of Chinese Medicine and China Pharmaceutical University, Nanjing, China, from 2023 to 2025. Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Flow cytometry using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) measured apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Western blot analyzed p21, cyclin B1, caspases3/7/9, cleaved caspases3/7/9, poly(ADP-ribose) polymerase (PARP), cleaved PARP, JAK2, p-JAK2, signal transducer and activator of transcription 3 (STAT3) and p-STAT3. Interactions were confirmed via docking, cellular thermal shift assay, and ADP-Glo. In vivo efficacy was evaluated in zebrafish. Data analysis was performed using GraphPad Prism 8.0. Statistical significance was determined using Student's t test or one-way/two-way ANOVA followed by Tukey's post hoc test (P<0.05). RESULTS: MG suppressed proliferation (IC50=9.90 μM), induced G2/M arrest, and triggered mitochondrial apoptosis. MG inhibited JAK2/STAT3 via direct kinase inhibition (IC50=17.17 μM) and ROS-mediated suppression. N-acetylcysteine (NAC) reversed these effects. Zebrafish tumor growth decreased by 19.7% (P<0.05). CONCLUSION: MG exerts anticancer effects against LCLC by inducing ROS accumulation and dual suppression of the JAK2/STAT3 pathway, highlighting its potential as a therapeutic candidate.