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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.<\/jats:p>","DOI":"10.3390\/molecules31162829","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T17:01:35Z","timestamp":1786640495000},"page":"2829","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Plant Protease Inhibitor EcTI Suppresses Melanoma Progression In Vivo"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9324-8424","authenticated-orcid":false,"given":"Camila Ramalho","family":"Bonturi","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruno Ramos","family":"Salu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen Chwen Ming","family":"Lie","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0949-2200","authenticated-orcid":false,"given":"M\u00e1rcia","family":"Bonini","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8747-6316","authenticated-orcid":false,"given":"Rita de Cassia","family":"Sinigaglia","sequence":"additional","affiliation":[{"name":"Electron Microscopy Center, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7986-923X","authenticated-orcid":false,"given":"Miryam Paola","family":"Alvarez-Flores","sequence":"additional","affiliation":[{"name":"Centre of Excellence in New Target Discovery (CENTD), Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7717-7013","authenticated-orcid":false,"given":"Ana Marisa","family":"Chudzinski-Tavassi","sequence":"additional","affiliation":[{"name":"Centre of Excellence in New Target Discovery (CENTD), Instituto Butantan, S\u00e3o Paulo 05503-900, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2372-7814","authenticated-orcid":false,"given":"Heloisa Sobreiro","family":"Selistre-de-Araujo","sequence":"additional","affiliation":[{"name":"Department of Physiological Sciences, Universidade Federal de S\u00e3o Carlos, Rodovia Washington Luis, Km 235, S\u00e3o Carlos 13565-905, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7346-6328","authenticated-orcid":false,"given":"Maria Luiza Vilela","family":"Oliva","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Universidade Federal de S\u00e3o Paulo (UNIFESP), S\u00e3o Paulo 04044-020, SP, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2113","DOI":"10.1001\/jama.2025.13074","article-title":"Cutaneous melanoma: A review","volume":"334","author":"Joshi","year":"2025","journal-title":"JAMA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/S0140-6736(23)00821-8","article-title":"Cutaneous melanoma","volume":"402","author":"Long","year":"2023","journal-title":"Lancet"},{"key":"ref_3","first-page":"12","article-title":"Cancer statistics; 2024","volume":"74","author":"Siegel","year":"2024","journal-title":"CA Cancer J. 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