ML-SA5-Induced Cytotoxicity and Alterations in Lysosomal and Mitochondrial Activity in Triple-Negative Breast Cancer Cells

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that expresses neither estrogen receptors, progesterone receptors, nor HER2; therefore, limiting the current available treatments. Recent evidence shows that the lysosomal cation channel TRPML1 is overexpressed in TNBC cells whereas minimal in normal cells hence can be targeted for anticancer therapy. The objective of the present study is to determine whether Mucolipin synthetic agonist 5 (ML-SA5), a small-molecule TRPML1 agonist, selectively induces cell death in MDA-MB-231 cells and to elucidate the cellular pathways involved in this process Methods: This study was conducted in MDRL 1 and 2 Lab, Ziauddin University, Clifton campus and this is in-vitro experimental study completed in 8 months. The cytotoxicity of MDA-MB 231 cells exposed to various doses of ML-SA5 was studied over a different time duration using the MTT assay. Cell death was further analyzed by propidium iodide and annexin V-FITC staining. Changes in mitochondrial and lysosomal activity and intracellular reactive oxygen species were evaluated. Results: ML-SA5 induced dose-dependent cytotoxicity with IC₅₀ values in the lower micromolar range (6.8 μM). PI staining indicated also confirmed cell death, with minimal apoptosis. Mitochondrial staining revealed altered morphology and compromised function. Lysosomal labeling showed enlarged lysosomes suggesting impaired lysosomal integrity. A significant rise in ROS levels was observed, indicating oxidative stress. Conclusion: The activation of TRPML1 by ML-SA5 leads to increased oxidative stress and damage to mitochondria and lysosomes, resulting in cell death in TNBC cells.