Abstract 2825: Oncosis-inudcing hydrazones complexes with selective anticancer activity

Abstract INTRODUCTION: The molecular hybridization of three pharmacophores—benzothiazole, cyclobut-2-ene-1, 2-dione, and hydrazone—has led to the development of novel compounds with diverse biological activities, including anticancer, anti-inflammatory, and antiviral properties. Among these, certain derivatives demonstrated potent and selective anticancer activity, particularly in CCRF-CEM and HCT116 cell lines. Some derivatives also emitted green fluorescence, enabling organelle-specific imaging by fluorescence microscopy. In U2OS cells, these fluorescent derivatives induced cytoplasmic vacuolization within 24 hours, suggesting a non-apoptotic cell death mechanism consistent with oncosis, characterized by energy depletion, increased membrane permeability, organelle swelling, and protein denaturation. METHODS: Various microscopy techniques were employed to explore the fluorescent features of target organelles in U2OS cells. Compound 6, with its stable green fluorescence, was selected for detailed analysis. Mitochondria were visualized using MitoTracker dye, with colocalization assessed via Pearson correlation. Mitochondrial membrane potential was evaluated using TMRM dye, which accumulates only in cells with intact membrane potential. Endoplasmic reticulum colocalization with cytoplasmic vacuoles was analyzed using mCherry-tagged calreticulin in a recombinant U2OS cell line. Lysosomal integrity and vacuole colocalization were examined with LysoTracker dye. To determine if cytoplasmic vacuolization is depended on caspase activation, cells were treated with compound 6 in the presence or absence of the pancaspase inhibitor Z-VAD-FMK, and caspase-3/PARP-1 cleavage was assessed by Western blot. The FUCCI reporter system was used for cell cycle analysis. RESULTS: Compound 6 strongly colocalized with mitochondria and induced mitochondrial dysfunction, ER vacuolization, and oncosis-like non-apoptotic cell death. This suggests that compound 6 exerts anticancer effects by targeting multiple cellular compartments, involving ER stress and mitochondrial disruption. The oncosis-like pathway is significant as it may bypass apoptosis resistance, a key hurdle in cancer therapy. Additionally, compound 6 reduced LysoTracker staining, indicating lysosomal dysfunction. Cell cycle analysis revealed a concentration-dependent G2/M arrest, further supporting its anticancer efficacy. ACKNOWLDEDGEMENT: This work was supported by the Technology agency of the Czech Republic project PERMED: T2BA (TN02000109) and the National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102). Infrastructure was supported by projects CZ-OPENSCREEN (LM2023052) and EATRIS-CZ (LM2023053). We also acknowledge support from Charles University projects [SVV260637; SVV260521; UNCE 204064; Cooperation ONCO] and IGA_LF_2024_038. Citation Format: Alzbeta Srovnalova, Robert Kakplanek, Zdenek Kejlik, Jan Hajduch, Pavel Martasek, Josef Srovnal, Sona Gurska, Katerina Jecmenova, Petr Dzubak, Marian Hajduch, Milan Jakubek. Oncosis-inudcing hydrazones complexes with selective anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2825.