Abstract 2954: Investigating the role of lysosome permeabilization for doxorubicin cytotoxicity in breast cancer cells

Background: The nuclear cytotoxic pathway of doxorubicin (DOX) is well established. Other mechanisms include reactive oxygen specie (ROS) interactions and ceramide destabilization of lipid membranes. Damage to lysosome membranes can induce apoptosis and cell death. Recent evidence of DOX induced lysosome permeabilization raises the possibility of released lysosomal enzymes triggering a mitochondrial apoptosis. The hypothesis of this study is that lysosome membrane permeabilization (LMP) precedes mitochondrial apoptosis resulting from DOX in two breast cancer cell lines. Methods: Studies were conducted using DOX concentrations ranging from 0.1 μM to 10 μM for 2, 6, and 24 hr exposure periods in MCF-7 and MDA-MB-231 breast cancer cell lines. Viability was determined by the MTT procedure. Intracellular localization and accumulation of DOX was examined by fluorescence microscopy. DNA strand breaks were detected using the TUNEL assay. LMP was demonstrated by cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. MMP was determined by the JC-1 assay using fluorescence microscopy. JC-1 aggregates in mitochondria, producing a detectable red fluorescence. When mitochondria depolarize, the dye is released and fluorescence shifts to green. Fluorescence was detected at 529 nm (green) and 590 nm (red). Intensity was quantified using ImageJ. Data was expressed as the ratio of red to green fluorescence and normalized to the untreated control. Results: By 2 hr, both MCF-7 and MDA-MB-231 cell lines demonstrated nuclear uptake as well as DNA strand breaks present in 11 to 16 % of the total treated cell population. Lysosome uptake of DOX was detectable but small. LMP was a notable 50% in MCF-7 cells whereas MDA-MB-231 cells displayed a small 22% LMP. Mitochondrial membrane permeabilization (MMP) was detectable but small in both cell lines when compared to the untreated control. By 6 hr, DNA strand breaks increased to 23 and 24%, and MMP was similar in both cell lines. However, LMP was substantial at 80% of MCF-7 cells but only 34% of MDA-MB-231 cells for the 10 μM concentration. The 5 μM concentration also showed a large difference of about 80% and 22%, respectively. At 24 hr, DNA strand breaks were 26 and 33%, and MMP was similar with red to green fluorescence ratios of 0.9 and 0.7 for MCF-7 and MDA-MB-231 cells. However, the LMP difference between the two cell lines continued and the cytotoxicity showed differences of viabilities of 60% or below for 3, 5, and 10 μM DOX in the MCF-7 cells, but 60% viability only at 10 μM for the MDA-MB-231 cells. Conclusions: The nuclear uptake and DNA breaks corroborate the expected nuclear pathway of cytotoxicity. The greater lysosome permeabilization and greater cytotoxicity in the MCF-7 cell line suggests a role of lysosomes in DOX cytotoxicity. These results do not exclude the possibility of a lysosome triggered mitochondrial apoptosis, which might represent an alternate pathway of DOX cytotoxicity. Acknowledgements: Financial support is appreciated from the Milton Lev Memorial research fund. Citation Format: Rachel E. Nicoletto, Mohammed M. Alvi, Clyde M. Ofner. Investigating the role of lysosome permeabilization for doxorubicin cytotoxicity in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2954.