Abstract 5387: Investigating the role of p53 in doxorubicin-mediated lysosome membrane permeabilization in breast cancer cells

Abstract

Abstract Background: Doxorubicin (DOX) is a chemotherapeutic characterized by multiple mechanisms. Wild-type p53 (WTp53) may be activated in the presence of either DNA damage or ROS. Upon activation, WTp53 may upregulate the transcription of pro-apoptotic Bcl-2 family members capable of triggering mitochondrial membrane permeabilization or lysosome membrane permeabilization (LMP), ultimately resulting in apoptosis. We have previously reported of DOX-induced LMP between WTp53 and mutant p53 (MUTp53) breast cancer cells. The goals of this study were to establish (1) the role of p53 in the initiation of DOX-mediated LMP and (2) whether p53 is critical to the onset of cytotoxicity. Methods: Studies were conducted using DOX at a range of concentrations and durations in both WTp53 (MCF-7 and MDA-MB-361) and MUTp53 (MDA-MB-231 and T47D) breast cancer cells. LMP was demonstrated using fluorescence microscopy as the cytosolic release of a 10 kDa AlexaFluor-488 Dextran conjugate that was preloaded into lysosomes. Viability was determined by MTT. Cytotoxicity was determined by trypan blue. Experiments were conducted in the presence of pifithrin-α p53 inhibitor to establish the importance of p53 towards DOX cytotoxicity. Results: Significant LMP was determined as early as 0.5 h in both of the WTp53 cell lines at 10 μM. MDA-MB-361 cells demonstrated a greater degree of LMP with 1 μM DOX showing significant LMP by 2 h, an effect that was not observed in MCF-7 cells until 6 h. The earliest onset of notable LMP in MUTp53 MDA-MB-231 cells occurred at 6 h with 10 μM DOX only. T47D cells did not demonstrate notable LMP at this time. By 24 h, both WTp53 cell lines demonstrated LMP across all of the tested concentrations of DOX, while both MUTp53 cell lines only demonstrated notable LMP for 10 μM DOX. At this same time, notable reductions in cell viability for the tested range of concentrations were observed for all cell lines except for T47D cells. Overall, the inhibition of p53 reversed notable LMP in both MCF-7 and MDA-MB-361 cells across all of the tested conditions. Similarly, reduced viability was reversed following the inhibition of p53. Conclusions: These results indicate a role of p53 in LMP in WTp53 cells. Furthermore, the combination of p53 and LMP seem necessary for the induction of cytotoxicity in WTp53 cells. Under circumstances of MUTp53 expression, cells promote a pro-oncogenic response with a lesser potential for LMP, MMP, and cytotoxicity. Citation Format: Rachel Nicoletto, Clyde M. Ofner. Investigating the role of p53 in doxorubicin-mediated lysosome membrane permeabilization in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5387.