Demethylzeylasteral alleviates myeloid leukemia through PERK/eIF2α/ATF4/CHOP-mediated cell apoptosis.

BACKGROUND: Myeloid leukemia, as a malignant tumor of hematologic system, urgently requires discovery of novel therapeutic drugs. Demethylzeylasteral (DML), a natural terpenoid primary extract from Tripterygium, has demonstrated antitumor properties across various cancer types. However, its functions in myeloid leukemia have not been clearly elucidated. This study aimed to explore the effects of DML on the proliferation and apoptosis of myeloid leukemia cells, and elucidate its mechanism. METHODS: Cell viability was detected by Cell Count Kit-8 (CCK-8) method. Post-DML treatment, cell cycle, apoptosis, intensity of unfolded proteins, adenosine triphosphate (ATP), intracellular calcium, and reactive oxygen species (ROS) levels were assessed by flow cytometry. Additionally, transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted to uncover pathways linked to gene enrichment in the DML treatment group, which were validated using small molecular inhibitors, short hairpin RNA (shRNA) interference, flow cytometry, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. In vivo, the anti-leukemia activity of DML was evaluated in a NB4 cell-derived xenograft model using NOD-Prkdc (scid) Il2rg (em1)/Smoc (M-NSG) mice. RESULTS: A library of 65 terpenoids was screened against myeloid leukemia cell lines (K562, NB4, THP-1), identifying DML through screening and validation using CCK-8 assays. Comprehensive in vitro analyses revealed that DML treatment significantly reduced cell viability while inducing G0/G1 phase arrest and apoptosis, accompanied by characteristic endoplasmic reticulum (ER) stress markers including elevated intracellular calcium, ROS generation, ATP depletion, and unfolded protein accumulation. Transcriptomic profiling combined with mechanistic validation through shRNA knockdown and pharmacological inhibitors demonstrated that DML activated the PERK/eIF2α/ATF4/CHOP signaling axis, initiating an irrecoverable unfolded protein response (UPR) that culminated in apoptotic cell death. The therapeutic relevance of these findings was substantiated in vivo, where DML administration significantly prolonged survival in an M-NSG mouse xenograft model engrafted with NB4 leukemia cells. CONCLUSIONS: These results systematically establish that DML induces apoptosis in myeloid leukemia cells directly through PERK/eIF2α/ATF4/CHOP signaling pathway in response to ER stress.