Epitulipinolide diepoxide induces apoptosis in bladder cancer cells by inhibiting the ERK/MAPK signaling pathway and promoting autophagy

Epitulipinolide diepoxide has demonstrated antitumor effects in the previous studies. This paper investigates its role in inhibiting the proliferation of bladder cancer cells and explores the underlying mechanisms. Bladder cancer cell lines (T24, 5637, and J82) were treated with different concentrations of Epitulipinolide diepoxide, and the IC50 values were determined at 24, 48, and 72 h. Cell proliferation, apoptosis, migration, and invasion were assessed using flow cytometry, Transwell assays, and clonogenic assays. For the T24, 5637, and J82 cell lines, cell viability and clone formation were evaluated by CCK-8, while cell proliferation, apoptosis, migration, and invasion were further analyzed using flow cytometry, Transwell, and scratch assays. The Epitulipinolide diepoxide target-pathway network was investigated using network pharmacology, molecular docking, and kinetic simulation. Western blotting was used to evaluate the protein levels in the ERK/MAPK pathway (ERK/PERK/JUK/P38), as well as autophagy markers (LC3/ATG5/P62). Autophagy was further assessed by measuring these protein levels again using Western blot. To validate the role of autophagy in the regulation of Epitulipinolide diepoxide on cell behavior, chloroquine (Chq), an autophagy inhibitor, was applied. Additionally, a combination of ERK agonists (C16-PAF) was used to explore the involvement of this pathway. Epitulipinolide diepoxide significantly inhibited the proliferation of T24, 5637, and J82 bladder cancer cell lines, as well as the invasion of T24 cells and the healing of scratches. After treatment with Epitulipinolide diepoxide, the levels of ERK, JNK, and P38 in the ERK/MAPK pathway were reduced, while the levels of PERK protein increased. Moreover, CHq and C16-PAF partially reversed the Epitulipinolide diepoxide-induced malignant behavior in T24 cells.