Ovarian cancer chemoresistance: the role of inflammasomes in cisplatin resistance and the impact of mitochondrial reprogramming in parp inhibitor resistance
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Data
2025-12-17
Autores
Souza, Josiany Carlos de
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Universidade Federal do Espírito Santo
Resumo
Epithelial ovarian cancer (EOC) is the most lethal gynecological neoplasm, characterized by high recurrence rates and resistance to cisplatin (CDDP) and PARP inhibitor treatments. Understanding the mechanisms related to these phenotypes is urgent. Therefore, in the first chapter of this study, the role of NLRP1 and NLRP3 inflammasomes and pyroptosis in CDDP chemoresistance is investigated. Cell viability of A2780, ACRP, and OVCAR3 cell lines were measured by the MTT assay, and IC50 values were determined. Cytokine levels in the supernatant were measured by ELISA. Caspase-1 activation was detected by the Caspase-Glo® 1 assay. The cell death profile was determined by flow cytometry (Annexin V/PI), and membrane pore formation was measured using PI. A cell migration assay was performed combining CDDP and the caspase-1 inhibitor. The IC50 of primary cells (PtAF) and viability after treatment with NLRP3 inhibitor (MCC950)+CDDP were determined by Cell Counting Kit-8 (CCK-8). The IC50 values indicated different CDDP resistance profiles in the cell lines. Secretion of inflammatory cytokines, caspase-1 activation, lytic cell death profile, and pore formation were higher in cells treated with CDDP. Caspase-1+CDDP inhibition reduced cell migration. PtAF treated with MCCC950+CDDP decreased cell viability and proliferation. The data suggest that inflammasome activation and pyroptosis are mechanisms associated with EOC chemoresistance to CDDP, contributing to the devastating scenario of this disease. Targeting these pathways may represent a promising treatment strategy. In the second chapter, the effect of the PARP inhibitor (Rucaparib) on mitochondrial reprogramming and its contribution to resistance is explored. Using primary cells: PtAF, PtD, and PtAD, viability assays with Rucaparib and Fludarabine Phosphate were performed by CCK-8. Oxygen consumption and extracellular acidification were detected by Seahorse. Mitochondrial content and mitochondrial membrane potential were determined by immunofluorescence with Mitotracker deepened and TMRE. NADH content was identified by autofluorescence microscopy (FLIM). The viability of cells treated with mitochondrial complex inhibitors was determined by crystal violet. The results indicated increased mitochondrial content, oxygen consumption, extracellular acidification especially in PtAF and increased NADH levels in the other cell lines. The combination of fludarabine phosphate with rucaparib had an additional cytotoxic effect on PtAF and PtD. These data suggest that PARPi affects mitochondrial metabolism and mass of cell lines differentially and that NAD transport (via SLC25A51) may play a key role in resistance, representing a potential target. Taken together, these data contribute to understanding the mechanisms underlying treatment resistance in EOC, highlighting inflammatory and mitochondrial targets as potential pathways for new therapeutic approaches.
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Palavras-chave
NADH , NLRP1 , NLRP3 , Piroptose , Metabolismo mitocondrial