Mycosubtilin suppresses HepG2 proliferation, binds p38α in vitro, and engages ER stress-associated MAPK signaling.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy with limited durable responses to current therapies. This study evaluated the effects of mycosubtilin (Myco), a Bacillus subtilis-derived cyclic lipopeptide, on HepG2 cell proliferation and xenograft tumor growth, and investigated its anti-HCC mechanisms using temporal transcriptomic profiling, molecular simulations, and surface plasmon resonance (SPR). Myco significantly suppressed HepG2 proliferation in a dose- and time-dependent manner and inhibited xenograft tumor growth, with preliminary tolerability indicated by stable body weight and histopathological examination of selected organs. The antiproliferative effect of Myco was further confirmed in Huh7 human hepatocellular carcinoma cells, with IC50 values of 6.07 μg/mL at 24 h, consistent with the trend observed in HepG2 cells. Temporal transcriptomic analyses suggested an association between early endoplasmic reticulum (ER) stress responses and later MAPK and apoptosis-associated transcriptional programs. Molecular docking and molecular dynamics simulations predicted preferential interaction of Myco with p38α, and SPR analysis supported direct, reversible binding of Myco to immobilized p38α under the tested in vitro conditions. Myco-induced apoptosis was accompanied by ROS accumulation, mitochondrial membrane depolarization, cytosolic Ca2+ disturbance, BAX/BCL2 imbalance, and caspase activation. These findings identify p38α as a direct in vitro binding target of Myco and support Myco as a candidate for further evaluation in additional HCC models. JNK1 engagement remains computationally predicted, and validation in additional HCC models, pathway perturbation experiments, in vivo pathway analysis, and systematic pharmacokinetic/toxicological studies remain necessary.