The Plant Protease Inhibitor EcTI Suppresses Melanoma Progression In Vivo

Melanoma dissemination depends on tumor cell plasticity, extracellular matrix remodeling, and adaptive signaling pathways that promote survival, migration, invasion, and therapeutic resistance. In this study, we investigated the antitumor effects of the plant-derived Kunitz-type protease inhibitor EcTI using both in vitro B16F10-Nex2 melanoma cells and an in vivo murine melanoma model, focusing on adhesion-dependent signaling, autophagy, mitochondrial dysfunction, and regulated cell death. EcTI was efficiently internalized by melanoma cells and showed partial colocalization with lysosomal and mitochondrial compartments, suggesting intracellular trafficking toward these organelles. Treatment reduced cell adhesion to extracellular matrix proteins, particularly fibronectin and laminin, and inhibited migration, invasion, and angiogenic signaling. These effects were associated with modulation of the adhesion-dependent FAK/Src/ERK signaling axis and decreased MMP-9 activity. EcTI also disrupted autophagy, as indicated by accumulation of acidic vesicular organelles, increased LC3-II levels, and modulation of ULK1, Ambra1, and Beclin-1 signaling. In parallel, EcTI induced mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential, intracellular Ca²⁺ dysregulation, and increased reactive oxygen species production. These alterations triggered regulated cell death involving apoptotic and necroptosis-like mechanisms. Importantly, EcTI significantly suppressed tumor growth in vivo without detectable systemic toxicity and modulated inflammatory mediators associated with tumor progression. Overall, these findings demonstrate that EcTI exerts broad antitumor activity by modulating multiple signaling pathways associated with melanoma progression and represents a promising therapeutic candidate for melanoma treatment.