Journal Article
PEGylated liposomal fluopsin C triggers cuproptosis and ferroptosis pathways and suppresses 3D tumor spheroid growth in NCI-H460 cells
Luan Vitor Alves de Lima; Matheus Felipe da Silva; Liana Martins de Oliveira; Maria Claudia Terkelli de Assis; Isabella Cristina Oliveira Carvalho; Isaura Maria Fuzinatto; Simone Cristine Semprebon; Renan Vinícius de Oliveira Nocetti; Danielle Lazarin-Bidoia; Celso Vataru Nakamura; Ingrid Felicidade; Sandra Regina Lepri; Phelipe Oliveira Favaron; Mickely Liuti Dealis; Luis Fernando Cabeça; Galdino Andrade Filho; Mário Sérgio Mantovani
Archives of Toxicology · Vol. 100, Issue 5 · pp. 1981-1994 · 2026
Abstract
Copper-based metallodrugs are promising anticancer agents, but their toxicity limits clinical translation. This study evaluated the toxicological and molecular effects of PEGylated Fluopsin C (PEG-FlpC) in in vitro human non-small-cell lung carcinoma (NCI-H460) cells cultured as 2D monolayer and 3D spheroids. Cells were exposed to PEG-FlpC at its IC 50 (1.0 µM) for 24 h. Flow cytometry revealed G₁-phase arrest and non-apoptotic cell death. PEG-FlpC markedly suppressed spheroid growth, with 2 × IC 50 -treated spheroids growing only 58.1% compared to 131.6% in the control. Ultrastructural analyses showed mitochondrial clustering and swelling, ER dilation, and cytoplasmic vacuolization. Gene profiling demonstrated downregulation of TP53 and BECN1 , with upregulation of TP73 and SQSTM1 , consistent with autophagy blockade and stress activation. PEG-FlpC modulated ferroptosis-related ( GPX4 , SLC7A11 , and TFRC ) and cuproptosis-related ( ATP7B , MTF1 , DLAT , and CDKN2A ) genes. Pharmacological inhibition confirmed a predominantly copper-mediated mechanism, as TTM (10 µM) restored 77.7% of metabolic activity. Combined inhibition with TTM and Fer-1 (1 µM) further increased viability to 89.4%, indicating that PEG-FlpC induces non-apoptotic regulated cell death through an interconnected cuproptosis–ferroptosis mechanism associated with mitochondrial dysfunction.