Abstract LB103: Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells

Background: Nuclear events play a well-established role in the cytotoxicity of doxorubicin (DOX). Other reported mechanisms include redox cycling for reactive oxygen species (ROS) production, which is getting recognized for a larger role than previously recognized. In addition, ROS production may be implicated in lysosome membrane permeabilization (LMP) and subsequent cell death. We have previously reported DOX-induced LMP in breast cancer cells. The goal of this study is to establish the role of LMP in the cytotoxic process and whether ROS are the initiator of this LMP. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 h exposure periods in MCF-7 and MDA-MB-231 breast cancer cells. Intracellular localization of DOX was examined by fluorescence microscopy using lysosome and nuclear markers. ROS production was determined by the DCFDA assay using a fluorescence plate reader. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Mitochondrial membrane potential (MMP) was determined by JC-1 assay. Viability was determined by MTT. Cathepsin B activity will be determined by Z-Arg-Arg-AMC cathepsin B substrate. Experiments will be conducted in the presence of E-64 cathepsin B inhibitor and Tiron superoxide scavenger to establish the importance of LMP and ROS towards DOX cytotoxicity. Results: Nuclear uptake was seen in both cell lines as early as 2 h at a concentration of 10 μM. Furthermore, lysosome accumulation of DOX, although small, was visible by 2 h in MDA cells, increasing in degree with time and concentration. ROS levels were found to be highest at 2 h in MCF-7 cells, increasing by up to 100% of the untreated control, and occurring concurrently with LMP at higher concentrations. Preliminary results suggest much lower ROS levels in MDA-MB-231 cells. In MCF-7 cells, ROS production decreased at 6 h, but increased by 24 h with concurrent increases in LMP. Although less than the MCF-7 cells, MDA cells demonstrated notable LMP for the first time at 5 and 10 μM conditions, occurring concurrently with MMP. By 24 h, viability was substantially reduced for both cell lines, with MCF-7s demonstrating a greater reduction in viability. Significant MMP was not observable until 24 h in MCF-7 cells. Conclusions: The early and strong ROS occurring with LMP suggest a previously unrecognized DOX cytotoxic combination in MCF-7 cells. LMP does not appear to result from lysosomal accumulation of DOX. A reduced role of ROS and LMP may account for the lesser cytotoxicity in MDA-MB-231 cells. Both ROS and LMP appear to be necessary for cytotoxicity in MCF-7 cells. Citation Format: Rachel E. Nicoletto, Clyde M. Ofner. Investigating the roles of reactive oxygen species and lysosomes in the cytotoxicity of doxorubicin in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB103.